aek table of contents · kulturmanagement.(kukm) ... d-99407 weimar or via e-mail to...

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AEKTable of Contents

■ Welcome................................................................................................................................................................................... 5..

■ General.Information................................................................................................................................................................. 6

■ Information.for.Presentations.and.Poster................................................................................................................................ 7

■ AEK.Member.Page.................................................................................................................................................................... 8

■ Time.Table................................................................................................................................................................................ 9

. . ■ Program.Wednesday,.March,.20th.2013................................................................................................................................. 9

. . ■ Program.Thursday,.March,.21st.2013.................................................................................................................................... 10

. . ■ Program.Friday,.March,.22nd.2013......................................................................................................................................... 11

■ Abstracts.Short.Talks.............................................................................................................................................................. 12

. . ■ Symposium.1:.Invasion.and.metastasis:.a.different.“point.of.view”.................................................................................... 12

. . ■ Symposium.2:.Targeting.heterogeneous.Breast.Cancer...................................................................................................... 14

. . ■ Symposium.3:.Dissecting.cancer.genomes.and.epi-genomes.............................................................................................. 15

. . ■ Symposium.4:.Modelling.of.oncogenic.pathways.on.the.way.to.clinical.pay-off?................................................................ 16

. . ■ Symposium.5:.Closing.on.the.ultimate.culprits:.the.tumor.cells.and.its.neighbourhood..................................................... 17

. . ■ Symposium.6:.Targeted.therapy.–.melanoma.a.success.story?.......................................................................................... 20

■ Abstracts.Posters.................................................................................................................................................................... 21

. . ■ Section.1:.Tumor.genetics.and.heterogeneity..................................................................................................................... 21

. . ■ Section.2:.Oncogenes/tumor.suppressor.genes..................................................................................................................27

. . ■ Section.3:.Invasion.and.metastasis.................................................................................................................................... 34

. . ■ Section.4:.Tumor.environment.and.inflammation............................................................................................................... 44

. . ■ Section.5:.Systems.biology.and.cancer.............................................................................................................................. 50

. . ■ Section.6:.Novel.approaches.to.tumor.therapy.................................................................................................................. 53

. . ■ Section.7:.Free.topics..........................................................................................................................................................67

■ List.of.Invited.Speakers.and.Main.Authors..............................................................................................................................76

■ Notes...................................................................................................................................................................................... 80

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AEKAEK AEK

AEK

5

AEKWelcome

Dear AEK-Members, colleagues, ladies and gentlemen,

on.behalf.of.the.program.committee.it.is.my.great.pleasure.to.announce.the.17th.AEK.International.Cancer.Congress.to.be.held.in.Heidelberg.March.20.–.22,.2013...

The.AEK.International.Cancer.Congress.is.organized.by.the.“Abteilung.Experimentelle.Krebsforschung”.(AEK).within.the.German.Cancer.Society..It.is.now.a.tradition.that.the.International.AEK.Congress.will.be.jointly. subsidized.by. the.“Deutsche.Krebsgesellschaft”.and. the.Deutsche.Krebshilfe.. This.provides.us.

with.an.excellent.opportunity.for.assembling.a.scientific.program.with.highly.renowned.international.and.national.scientists.presenting.and.discussing.latest.data.and.future.perspectives.of.different.hot.topics.in.cancer.research...

The.topics.of.this.forthcoming.conference.will.include.latest.news.from.the.human.cancer.genome.and.epigenome.projects,.mo-delling.of.oncogenic.pathways,.tumor.heterogeneity.particularly.related.to.breast.cancer,.oncogenic.tumor.cell.regulation,.inva-sion.and.metastasis,.and.the.steadily.increasing.appreciation.of.the.tumor.microenvironment.with.its.tumor-stroma.interaction,.inflammasome,.and.exosome.action..Malignant.melanoma.will.be.discussed.as.an.example.of.novel.treatment.modalities..As.key.not.speakers.and.particularly.devoted.to.these.topics,.we.are.happy.to.announce.Mina.Bissell,.Lawrence.Berkeley.National.Laboratory,.Berkley.and.Bob.Weinberg,.Whitehead.Institute.for.Biomedical.Research,.Cambridge.MA..

In.addition,.two.central.poster.sessions.as.well.as.plenary.sessions.including.selected.short.talks.from.abstract.submissions.will.provide.excellent.platforms.for.all.participants.and.young.scientists.in.particular.to.present.and.discuss.their.results.in.a.highly.stimulating.and.scientifically.top-class.environment..As.the.AEK.strongly.encourages.young.scientists.to.attend.this.important.meeting,.travel.bursaries.for.young.researchers,.selected.from.abstract.submitters,.are.in.the.planning.as.are.prizes.for.the.best.posters.

The.congress.will.take.place.in.Heidelberg,.Germany‘s.oldest.university.town.and.the.town.with.a.longstanding.tradition.for.AEK.Congresses..The.venue.place.will.be.the.spectacular.EMBL.Advanced.Training.Centre.with.its.outstanding.architecture.inspired.by.DNA‘s.double.helix.-.a.landmark.and.scientifically.uniquely.stimulating.surrounding.for.this.occasion..

Similarly.important.for.the.success.of.the.Congress.is.your.interest.and.participation..I,.therefore,.would.like.to.cordially.invite.you.to.our.17th.International.AEK.Congress.and.very.much.looking.forward.to.welcome.you.at..this.occasion.in.Heidelberg..

With.best.regards

Prof..Dr..Petra.BoukampCongress.Chairman

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AEKAEK AEKGeneral Information

Registration fees

Early.Registration.untilFebruary.7,.2013

Permanent.Ticket

Late.Registration.from.February.8,2013.

Permanent.Ticket

Member.AEK 150,00.e 190,00.e

Non-Member 240,00.e 280,00.e

Student.** ..70,00.e ..90,00.e

Get.together. . . 45,00.e /ticket

Opening hours registration desk

Wednesday,.March.20th. 08:00.–.19:00Thursday,.March.21st. 08:00.–.18:30Friday,.March.22nd. 08:00.–.16:30

Terms and Conditions

The. fees. for. participating. in. the. scientific. program. are. charged. in. the. name. and. on. account. of. the. Deutsche. Krebsgesell-schaft.(DKG)..In.accordance.with.§.4/22a.of.the.German.sales.tax.act.the.above.fees.are.tax.exempt..The.tax.ID.of.the.DKG.is.DE258657754..

The.fees.for.the.social.get-together.and.the.handling.fees.are.charged. in.the.name.and.on.account.of.the.KONGRESS-.UND..KULTURMANAGEMENT.(KUKM)..The.tax.ID.number.is.DE158265638..

Once.you.have.sent.the.completed.registration.form,.your.registration.is.binding..Further.information.on.prices.and.deadlines.can.be.found.on.www.aek-congress.org..Reduced.fees.can.only.ne.applied,.after.an.adequate.certification.has.been.presen-ted. to.KUKM..This. can.be.submitted.by. fax.+49.3643.2468-31,.by.mail. to.P.O..Box.36.64,.D-99407.Weimar.or.via.e-mail. [email protected].

In.case.of.cancellation.or.non-participation,.there.will.be.no.money.refund..In.the.event.of.a.change.of.registration.with.KUKM.a.processing.fee.of.15.E.is.charged..The.charges.for.rebooking.are.15.E..

By.registering,.the.participant.agrees.that.he/she.will.not.make.any.claims.toward.the.host.if.the.conference.in.consequence.of.political,. commercial.or.natural. forces.will.be.cancelled.or.altered. in. its.program..Neither. the.host.nor.KONGRESS-.UND..KULTURMANAGEMENT.GMBH.can.be.made.liable.for.lost.items,.accidents.or.damages.to.persons.and.things,.whatever.the.cause..Participants.(and.the.person.accompanying).to.the.conference.and.the.social.events.attend.at.their.own.risk.and.responsibility.

** Reduced fees can only be applied, after an adequate certification has been presented to KONGRESS- UND KULTUR-MANAGEMENT GMBH. This can be submitted by fax +49 3643 2468-31, by mail to P.O. Box 36 64, D-99407 Weimar or via e-mail to [email protected].

AEK

7

AEKInformation for Presentations and Poster

Presentation and Short Talks

Please.be.prepared.for.short.talk.(10.minutes).and.discussion.(5.minutes)..Presentations.must.be.brought.to.the.preview.room,.either.on.a.CD-Rom.or.on.a.USB.memory.stick.at.least.1.hour.before.the.beginning.of.your.session..The.lecture.hall.is.equipped.with.projection.hardware.and.a.computer..In.order.to.avoid.any.delays.in.the.order.of.events,.the.use.of.personal.notebooks.is.not.possible.

Poster

The.maximum.size.for.posters.is.DIN.A0,.upright.position.814.mm.(with).×.1189.mm.(height).There.will.be.a.poster.board.provided.for.each.poster..The.abstract.number.published.in.the.scientific.program.will.be.attached.to.the.upper.left.corner..Material.to.mount.your.poster.will.be.available.on.site,.using.your.own.material.to.mount.your.poster.is.not.permitted.

All.posters.should.be.mounted.by.10.00.a.m..on.Wednesday,.March.20th..As.the.space.allows.for.all.posters.to.be.presented.throughout.the.entire.meeting.we.would.very.much.appreciate.all.posters.to.remain.in.place.until.the.end.of.the.Conference,.Friday,.March.22nd.

Posters.that.are.not.taken.down.by.Friday,.March.22nd,.5:15.p.m..will.be.disposed.of.if.not.claimed.

There.will.be.two.different.poster.sessions:

Poster session 1 (guided) | March 20th, 2013 – 3:00 p.m. to 5:30 p.m

Please.be.prepared.for.a.short.essence.(2.minutes).and.discussion.(5.minutes).in.front.of.your.poster.during.the.guided.poster.session...Based.on.this.presentation,.the.three.best.posters.will.be.selected.for.poster.awards!

Poster session 2 (free) | March 21st, 2012 – 3:30 p.m. to 5:00 p.m.

Free.poster.session.to.ensure.sufficient.exchange.between.the.poster.presenters.

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AEKAEK AEKAEK Member Page

AEK member assembly

The.AEK.member.assembly.is.scheduled.for.Thursday,.March.21st,.6.30.-.7.30.p.m.

Benefits of AEK Membership

•. Being.member.of.a.large.interdisciplinary.group.of.scientists.in.experimental.and.translational.cancer.research

•. Reduced.fees.for.future.AEK.Congresses.and.German.Cancer.Congresses.(forthcoming.DKK.2014)

•. Privileged.contact.to.more.than.900.members.via.www.krebsgesellschaft-aek.de

•. Corporate.membership.in.the.European.Association.for.Cancer.Research.(EACR).http://www.eacr.org/

•. Free.member.journal.FORUM

•. Opportunities.to.help.shaping.future.AEK.congresses.and.activities

•. All.AEK.members.are.eligible.to.apply.for.EACR.Fellowships.and.Awards

•. Application.for.membership.can.be.downloaded.from.http://krebsgesellschaft.de/wub_mitgliedschaft,78241.html

•. If.you.have.any.question.or.need.more.information.please.contact:

Katrin.Hackl.Deutsche.Krebsgesellschaft.e..V.Kuno-Fischer-Straße.8D-14057.Berlin

. +49.30.3229329-36. +49.30.3229329-22

. [email protected]

AEK

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AEKTime Table Wednesday, March, 20th 2013

08:00 – 10:00 Registration

10:00 – 10:15 Welcome Petra.Boukamp,.Otmar.Wiestler,.Rainer.Engers

10:30 – 12:00 Symposium 1: Invasion and metastasis: a different “point of view” – Imaging cancer invasion and therapyChairs:.Rainer.Engers,.Matthias.Dobbelstein

10:30.–.11:00 Targeting.cancer.invasion.and.therapy.resistancePeter.Friedl

11:00.–.11:30 Seeing.is.believing.is.understanding:.real-time.microscopy.of.brain.tumor.progression.and.re-sponse.to.therapiesFrank.Winkler

11:30.–.11:45 Anti-tumor.effects.of.peptide.hormone.analogs.in.pheochromocytoma.cell.lines.and.preclinical.tumor.imagingMartin.Ullrich

11:45.–.12:00 Bevacizumab.treatment.of.HER2.expressing.xenograft.prevents.uptake.of.fluorescent.labeled.Trastuzumab:.Correlation.of.in.vivo.fluorescence.imaging.with.multispectral.3D.ultramicroscopyMichael.Dobosz

12:00 – 13:30 Lunch Break

13:30 – 15:00 Symposium 1 continued: Invasion and metastasis: a different “point of view” – Tracing metastatic cancerChairs:.Nils.Cordes,.Rainer.Engers

13:30.–.14:00 Circulating.tumor.cells.in.cancer.patients:.Biology.and.clinical.implicationsKlaus.Pantel

14:00.–.14:30 Metastasis.from.a.radiation.oncologist.point.of.viewAmir.Abdollahi

14:30.–.14:45 A.complex.interplay.between.CD44,.the.ECM,.G-Protein-coupled.receptors.and.Receptor.Tyrosine.Kinases.in.tumor.progression.Véronique.Orian-Rousseau

14:45.–.15:00 Few.genes.are.associated.with.the.capability.of.pancreatic.ductal.adenocarcinoma.cells.to.grow.in.the.liver.of.nude.rats.Hassan.Adwan

15:00 – 17:30 Poster Session 1 and Coffee

17:30 – 19:00 Symposium 2: Targeting heterogeneous Breast CancerChairs:.Lisa.Wiesmüller,.Roland.Moll

17:30.–.18:00 Triple.negative.breast.cancer:.Subtypes,.molecular.targets,.and.therapeutic.apporachesJennifer.Pietenpol

18:00.–.18:30 Receptor.tyrosine.kinases.in.breast.cancer:.from.mechanisms.to.therapeutic.targetingNancy.Hynes

18:30.–.18:45 EZH2.regulates.the.heterogenic.composition.of.basal-like.breast.cancer.cell.populationsIttai.Ben-Porath

18:45.–.19:00 Development.of.inhibitors.targeting.p130Cas.as.novel.therapeutics.for.breast.cancerJoerg.Kumbrink

19:30 Welcome Reception “Wine & Cheese“

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AEKAEK AEKTime Table Thursday, March, 21st 2013

08:30 – 10:00 Symposium 3: Dissecting cancer genomes and epi-genomesChairs:.Martin.Lipp,.Peter.Lichter

08:30.–.09:00 Dissecting.the.genome.complexity.underlying.pediatric.brain.tumorsPeter.Lichter

09:00.–.09:30 Probing.the.cancer.methylomeStephan.Beck

09:30.–.09:45 Global.DNA.methylation.signature.of.HPV-related.oropharyngeal.squamous.cell.carcinomaJochen.Hess

09:45.–.10:00 UVA/UVB-induced.epigenetic.regulation.of.tumor.suppressor.and.miRNA.gene.expressionRuediger.Greinert

10:00 – 10:15 Coffee break

10:15 - 11:30 Hunting the elusive oncogene: a stroke of good luck Chair:.Bernd.GronerBob.Weinberg

11:30 – 12:30 Cancer Award12:30 – 14:00 Lunch Break14:00 – 15:30 Symposium 4: Modelling of oncogenic pathways on the way to clinical pay-off?

Chairs:.Klaus-Peter.Janssen,.Reinhold.Schäfer14:00.–.14:30 Genome.analysis,.computer.modelling.and.personalized.cancer.treatment

Ralf.Herwig14:30.–.15:00 Reverse.engineering.transcriptional.networks.in.cancer.cells

Reinhold.Schäfer15:00.–.15:15 DNA-methylome.analysis.of.mouse.intestinal.adenoma.identifies.a.tumour-specific.signature.that.

is.partly.conserved.in.human.colon.cancerMarkus.Morkel

15:15.–.15:30 Strong.negative.feedback.from.Erk.to.Raf.confers.robustness.to.MAPK.signallinRaphaela.Fritsche

15:30 – 17:00 Poster Session 2 and coffee

17:00 – 18:30 Symposium 5: Closing in on the ultimate culprits: the tumor cells and its neighourhood– Regulatory faults in the tumor cellChairs:.Michael.Schwarz,.Martin.Eilers

17:00.–.17:30 Targeting.Myc.and.N-Myc.for.Tumor.TherapyMartin.Eilers

17:30.–.18:00 Oncogene-mediated.alterations.in.chromatin.conformationMark.Rubin

18:00.–.18:15 Galectin-8.as.a.new.modulator.of.K-Ras-induced.signaling.in.carcinoma.cellKlaudia.Giehl

18:15.–.18:30 Degradation.of.macrophage-colony.stimulating.factor.receptor,.c-Fms,.in.epithelial.cancer.cells.is.enhanced.by.induction.of.autophagyAlexandra.Koch

18:30 – 19:30 AEK Member Assembly

20:00 Social Get-together

AEK

11

AEKTime Table Friday, March, 22nd 2013

08:30 - 10:00 Symposium 5 continued: – The many faces of the tumor environmentChairs:.Jürgen.Becker,.Holger.Kalthoff

08:30.–.09:00 Cytoprotective.signalling.pathways.in.tissue.repair.and.cancerSabine.Werner

09:00.–.09:30 Remodeling.and.homeostasis.of.the.extracellular.matrix:.implications.for.fibrotic.diseases.and.cancer.Janine.T..Erler

09:30.–.09:45 Remodelling.of.the.lymphoma-stroma.interface.within.secondary.lymphoid.organs.involves..fibroblastic.reticular.cells.and.dendritic.cellsUta.Elisabeth.Höpken

09:45.–.10:00 A.genetic.mouse.model.to.identify.the.role.of.the.immune.adapter.protein.MyD88.in.colorectal.cancerAnne.Holtorf

10:00 - 10:30 Coffee break

10:30 - 12:00 Symposium 5 continuedChairs:.Klaus.Bosslet,.Winfried.Wels

10:30.–.11:00 Extracellular.trafficking.of.oncogenes.–.therapeutic.and.diagnostic.implicationsJanusz.Rak

11:00.–.11:15 Humanized.tumor.mice.–.a.new.model.to.investigate.and.improve.cancer.therapyAnja.Kathrin.Wege

11:15.–.11:30 Towards.multi-scale.modelling.of.the.patient.individual.tumor.microenvironmentNiels.Grabe

11:30.–.12:00 Immunogenic.cell.death.fingerprinting.for.the.detection.of.adjuvants.in.cancer.therapyOliver.Kepp

12:00 - 13:30 Lunch Break

13:30 - 14:30 Key-note Lecture: Why don’t we get more cancer? A proposed role of the microenvironment in restraining cancer progression Chair:.Petra.BoukampMina.Bissell

14:30 - 14:45 Short Break14:45 – 16:15 Symposium 6: Targeted therapy – melanoma a success story?

Chairs:.Cornelia.Mauch,.Jens.Hoffmann14:45.–.15:15 BRAF.Inhibitors.and.beyond.

Meenhard.Herlyn15:15.–.15:45 Natural.circulating.plasmacytoid.dendritic.cells:.Key.for.antitumor.immunity?

Jolanda.de.Vries15:45.–.16:00 Preclinical.study.of.combined.non-viral.gene-.and.chemotherapy.of.malignant.melanoma

Wolfgang.Walther

16:15 Poster Prizes and concluding remarks

12

AEKAEK AEKAbstracts Short TalksSymposium 1: Invasion and metastasis: a different “point of view” – Imaging cancer invasion and therapy

Symposium 1 - Short talk 0016

M. Ullrich.1,2,.C. Ziegler.2,.R. Bergmann.1,.J. Pietzsch.3,1,.M. Ehrhart-Bornstein.2,.A. V. Schally.4,.G. Eisenhofer.2,.S. Bornstein.2

1. Helmholtz-Centre.Dresden.Rossendorf,.Institute.of.Radiopharmaceutical.Cancer.Research,.Dresden,.Germany.

2. University.Hospital.Carl.Gustav.Carus,.Department.of.Medicine.III,.Dresden,.Germany.

3. Technical.University.Dresden,.Department.of.Chemistry.and.Food.Chemistry,.Dresden,.Germany.

4. University.of.Miami.Miller.School.of.Medicine,.VA.Medical.Center.and.Dep..of.Medicine,.Div..of.Endocrinology.and.Hematology-Oncology,.Miami,.United.States

Anti-tumor effects of peptide hormone analogs in pheochromocyto-ma cell lines and preclinical tumor imaging

Pheochromocytoma. (PHEO). is. a. rare. but. potentially. lethal. neuroen-docrine.tumor.arising.from.catecholamine.producing.chromaffin.cells..Available.treatment.strategies.are.limited.and,.if.the.tumor.has.metas-tasized,. not. very. effective.. The. abundant. expression. of. peptide. hor-mone.receptors.on.endocrine.tumor.cells.allows.specific.targeting.by.highly.effective.anti-tumor.peptide.analogs..The.present.study.focuses.on.the.preclinical.evaluation.of.potential.therapies.in.the.treatment.of.pheochromocytoma.targeting.peptide.hormone.receptors.

Our. in. vitro. evaluation. of. peptide. hormone. receptor. expression. on.the.mouse.pheochromocytoma.(MPC).cell.line.and.the.more.malignant.mouse.tumor.tissue-derived.(MTT).cell. line.was.based.on.RT-PCR.and.immunohistological.analysis..Based.on.these.data,.we.evaluated.the.ef-fects.of.cytotoxic.peptide.hormone.analogs.on.cell.viability,.apoptosis.and.necrosis.on.MPC.and.MTT.cells..For.in.vivo.studies,.we.established.a.subcutaneous.mouse.model.of.MPC-derived.PHEO.and.multimodal.tumor.imaging.using.PET,.MRI,.and.CT,.fluorescence.and.bioluminescenceOur.data.revealed.significant.anti-tumor.effects.mediated.by.the.cyto-toxic.peptide.hormone.analogs.AN-162.and.AN-238.targeting.somatosta-tin.receptor.2.(SSTR2),.by.the.antagonist.Cetrorelix.and.the.cytotoxic.analog.AN-152.targeting.luteinizing.hormone-releasing.hormone.recep-tor. (LHRH-R). as. well. as. by. the. antagonist. MIA-602. targeting. growth.hormone-releasing.hormone.receptors.(GHRH-R).on.MPC.cells..Similar.anti-tumor.effects.were.evidenced.for.AN-152.and.MIA-602.also.on.the.more.aggressive.MTT.cells..Using.our.newly.established.mouse.model,.we.were.able.to.visualize.the.growth.of.MPC.cell-derived.subcutaneous.pheochromocytomas.in.vivo.by.multimodal.molecular.imaging.including.SSTR2.PET..Additionally,.ex.vivo.tumor.characterization.demonstrated.unaltered. peptide. hormone. receptor. expression. during. in. vivo. tumor.growth.in.mice.

Our.current.investigation.provides.strong.evidence.for.a.possible.future.treatment.of.malignant.pheochromocytoma.using.targeted.peptide.hor-mone.receptor.therapy.

Symposium 1 - Short talk 0109

M. Dobosz.1,.S..Strobel.1,.W..Scheuer.1

1. Roche.Diagnostics,.ma.Research.and.Early.Development,.Discovery.Oncology,.Penzberg,.Germany.

Bevacizumab treatment of HER2 expressing xenograft prevents uptake of fluorescent labeled Trastuzumab: Correlation of in vivo flu-orescence imaging with multispectral 3D ultramicroscopy.

Bevacizumab. (Avastin),. an. anti-vascular. endothelial. growth. factor.(VEGF).antibody,.has.been.approved.for.the.treatment.of.different.so-lid. tumors.. However,. clinical. evidence. reveals. that. neutralizing. VEGF.causes.resistance.and.thus.new.anti-angiogenic.modalities.are.needed.to.improve.progression.free.survival.of.cancer.patients..It.has.been.de-monstrated.that.bevacizumab. induces.normalization.of.tumor.vessels.thereby.allowing.more.efficient.delivery.of.chemotherapy.and.antibo-dies..Based.on.recent.data.this.has.been.questioned.by.others..There-fore,. a. detailed. understanding. of. the. molecular. and. cellular. mode. of.action. of. anti-VEGF. therapy. could. provide. a. rational. for. combination.therapies.with.other.targeted.agents.and.chemotherapy..Furthermore.such. knowledge. would. also. facilitate. the. optimization. of. treatment.schedules.

We. applied. non-invasive. near-infrared. optical. imaging. in. combination.with.ex.vivo.multispectral.fluorescence.ultramicroscopy.to.monitor.dif-ferent.aspects.of. tumor.morphology,.antibody.penetration.and. treat-ment. responds. in. an. orthotropic,. Her2. overexpressing. breast. cancer.xenograft..Ultramicroscopy.provides.the.possibility.for.three-dimensi-onal.acquisition.of.multiple.tumor.parameters.on.a.cellular.resolution..This. new. imaging. method. bridges. the. gap. between. macroscopic. and.microscopic.imaging.modalities.and.opens.up.a.new.three-dimensional.and.quantitative.insight.into.tumor.biology..Both.imaging.technologies.were.used.to.analyze.the.therapeutic.treatment.effect.of.bevacizumab.on.the.tumor.vasculature.and.how.this.pretreatment.influences.the.pe-netration.behavior.of.fluorescent.labeled.trastuzumab.

Our. results. reveal. that. vessel. normalization. induced. by. bevacizumab.treatment. leads.to.a.reduced.deposition.of.trastuzumab.in.the.tumor.tissue.

AEK

13

AEKAbstracts Short TalksSymposium 1 continued: Invasion and metastasis: a different “point of view” – Tracing metastatic cancer

Symposium 1 continued - Short talk 0066

V. Orian-Rousseau.1,.A..Matzke.1,.K..Fuchs.1

1. Karlsruhe.Institute.for.Technology,.Institute.for.Toxicology.and.Genetics,.Eggenstein-Leopoldshafen,.Germany.

A complex interplay between CD44, the ECM, G-Protein-coupled re-ceptors and Receptor Tyrosine Kinases in tumor progression

A.member.of.the.CD44.family.of.transmembrane.glycoproteins,.namely.CD44v6.collaborates.with.Receptor.Tyrosine.Kinases.such.as.Met.and.VEGFR-2.(Orian-Rousseau.et.al.,.2002;.Tremmel.et.al.,.2009).. In.both.cases,.the.co-receptor.CD44v6.fulfills.two.distinct.functions..The.ecto-domain.of.CD44v6.is.required.for.the.presentation.of.the.ligands.to.the.RTKs..Our.data.indicate.that.CD44v6.is.able.to.bind.both.HGF.and.VEGF.in.order. to.promote. the.activation.of. their. respective. receptors,.Met.and.VEGFR-2.. In.addition,.the.cytoplasmic.domain.of.CD44v6. in.asso-ciation.with.ERM.(Ezrin-Radixin-Moesin).proteins.and.the.cytoskeleton.is.necessary.for.the.activation.of.signaling.originating.from.activated.receptors.(Orian-Rousseau.et.al.,.2007).The.specificity.of.the.co-receptor.function.of.CD44v6.for.Met.and.VEG-FR-2.allowed.the.identification.of.peptides.that.can.block.their.activati-on.in.vitro.and.in.vivo..These.peptides.have.been.used.in.highly.metasta-tic.rat.and.human.pancreatic.carcinoma.models.where.they.drastically.inhibit.growth.of.tumors.and.metastatic.spreading.of.the.cancer.cells..Most.excitingly,.the.CD44v6.peptides.eliminate.already.established.pan-creatic.cancer.metastases.suggesting.a.role.of.CD44v6.in.the.survival.of.metastatic.cells.in.distant.organs.A. completely. different. function. of. CD44. relates. to. its. ability. to. bind.components.of.the.extracellular.matrix.such.as.hyaluronan.(HA)..Bin-ding.of.CD44.to.HA.controls.the.activation.of.the.chemokine.receptor.CXCR4.(Fuchs.et.al.,.submitted)..The.ligand.of.this.chemokine.receptor,.namely.CXCL12.plays.a.pivotal. role. in.vascularization.and. in.directing.metastatic.cells. to.target.organs..CXCL12. induces.a.complex.between.CD44.and.CXCR4.that.is.necessary.for.the.formation.of.blood.vessels..In.stark.contrast.small.HA.fragments.(sHA).prevents.this.complex.formati-on.thereby.inhibiting.neo-vascularization.Our.experiments.suggest.a.key.function.of.CD44.isoforms.in.the.regula-tion.of.the.tumor.environment,.of.angiogenesis.and.of.metastasis.upon.binding.to.hyaluronan.and.collaborating.with.G-protein-coupled.recep-tors.like.CXCR4.and.RTKs.such.as.Met.and.VEGFR-2.Orian-Rousseau,.V.,.Chen,.L.,.Sleeman,.J.P.,.Herrlich,.P.,.and.Ponta,.H..(2002)..CD44.is.required.for.two.consecutive.steps.in.HGF/c-Met.signa-ling..Genes.Dev.16,.3074-3086.Orian-Rousseau,.V.,.Morrison,.H.,.Matzke,.A.,.Kastilan,.T.,.Pace,.G.,.Herr-lich,. P.,. and. Ponta,. H.. (2007).. Hepatocyte. growth. factor-induced. Ras.activation.requires.ERM.proteins.linked.to.both.CD44v6.and.F-actin..Mol.Biol.Cell.18,.76-83.Tremmel,.M.,.Matzke,.A.,.Albrecht,.I.,.Laib,.A.M.,.Olaku,.V.,.Ballmer-Hofer,.K.,.Christofori,.G.,.Heroult,.M.,.Augustin,.H.G.,.Ponta,.H.,.et.al..(2009)..A.CD44v6.peptide.reveals.a.role.of.CD44.in.VEGFR-2.signaling.and.angio-genesis..Blood.Fuchs.K,.Hippe.A,.Schmaus.A,.Homey.B,.Sleeman.J,.Orian-Rousseau.V..Opposingeffects.of.high.molecular.weight.HA.(hHA).and.small.Ha.frag-ments.(sHA).on.CXCR4activation.via.CXCL12..Submitted

Symposium 1 continued – Short talk 0021

H. Adwan.1,.E..Eyol.1,.T..Hielscher.1,.M..R..Berger.1

1. German.Cancer.Research.Center,.Toxikology.and.Chemotherapy,.Heidelberg,.Germany.

Few genes are associated with the capability of pancreatic ductal adenocarcinoma cells to grow in the liver of nude rats

Purpose:. Owing. to. aggressiveness. and. chemo-resistance,. pancreatic.ductal.adeno-carcinoma.(PDAC). is.characterised.by.a.poor.prognosis..At.the.advanced.stage,.which.is.characterised.by.metastasis.into.adja-cent. organs,. especially. the. liver,. all. systemic. treatment. approaches.have.failed,.so.far..To.address.this.disease.specific.dilemma.we.aimed.to.establish.animal.models,.which.mimic.liver.metastasis.of.PDAC..These.models.can.be.also.used.for.identifying.new.specific.tumor.markers,.as.well.as.serving.as.tools.for.potential.therapeutic.approaches.

Methods:.From.a.panel.of.sixteen.pancreatic.cancer.cell.lines,.two.hu-man.(Suit2-007.and.Suit2-013).and.a.rat.(ASML).cell.line.were.selected.for.their.property.to.grow.in.the.liver.of.male.RNU.rats.and.mimic.liver.metastasis.of.PDAC..By.serial.transplantation.the.take.rate.was.impro-ved.until. they.showed.reliable.and.reproducible.growth.as.orthotopic.xenografts.For.better.monitoring.of.metastatic.tumor.growth.in.vivo,.all.three.pan-creatic.cancer.cell.lines.were.stably.transfected.with.eGFP.and.lucifera-se.marker.genes..In.addition,.the.mRNA.expression.profile.of.13.human.PDAC.cell.lines.was.analyzed.by.BeadChip.array.analysis.

Results:.Three.orthotopic.xenograft.models,.which.mimic.liver.metasta-sis.of.PDAC,.were.successfully.established.in.male.RNU.rats.following.intra-portal. implantation.The.models.vary. in. their.aggressiveness.and.macroscopic.growth..The.BeadChip.array.analysis.showed.that.only.33.genes.and.5.signalling.pathways.were.identified.as.significantly.associated.with.the.ability.of.the. cell. lines. to. grow. initially. and/or. consistently. in. rat. liver.. Only. a.minority.of. these.genes. (osteopontin,.matrix.metalloproteinase-1.and.insulin-like.growth.factor.1).has.been.intensively.studied.and.shown.to.be.closely.related.to.cancer.progression..The.function.of.the.remaining.30.genes.ranges.from.moderate.to.poorly.investigated,.and.their.func-tion.in.cancer.progression.is.still.unclear.

Conclusions:.The.ensuing.three.pancreatic.cancer.liver.metastasis.mo-dels.vary.in.their.aggressiveness.and.macroscopic.growth..They.will.be.used.for.preclinical.evaluation.of.new.therapeutic.approaches.aiming.at.the.genes.identified.

14

AEKAEK AEKAbstracts Short Talks Symposium 2: Targeting heterogeneous Breast Cancer

Symposium 2 – Short talk 0044

I. Ben-Porath.1,.R..Granit.1

1. The.Hebrew.University.of.Jerusalem,.Department.of.Developmental.Biology.and.Cancer.Research,.Jerusalem,.Israel.

EZH2 regulates the heterogenic composition of basal-like breast cancer cell populations

Question:. The. mechanisms. controlling. intratumoral. cellular. hetero-geneity.are.poorly.understood..Studies.in.recent.years.have.supported.the. notion. that. differentiation-like. events. may. occur. within. tumors,.potentially. generating. cellular. heterogeneity.. Cells. possessing. stem/progenitor. properties. could. drive. such. events.. Basal-like. breast. can-cers,.an.aggressive.and.poorly.differentiated.form.of.the.disease,.have.been.linked.in.their.overall.phenotype.to.normal.progenitor.cells.in.the.breast..These.tumors.typically.contain.cells.that.express.markers.of.va-rious.mammary.lineages..Whether.this.heterogeneity.reflects.functional.differences.between.cell.populations.is.unknown.

Methods.and.Results:.We.have. found.that.basal-like.tumors.contain.a.subpopulation. of. “bi-lineage”. cells,. which. express. both. luminal. and.basal.markers,.and.represent.a.minority.of.the.tumor.cells..We.found.that. the. EZH2. oncogene,. which. encodes. the. catalytic. subunit. of. the.Polycomb.repressive.complex.2,.promotes.bi-lineage.identity:.silencing.of.EZH2.led.to.the.reduction.of.the.numbers.of.bi-lineage.cells.within.the. breast. cancer. cell. population,. while. its. overexpression. increased.their.numbers..In.contrast,.the.GATA3.tumor.suppressor.gene,.which.en-codes.a.master.transcription.factor.promoting.luminal.differentiation,.acts.to.suppress.bi-lineage.identity,.counteracting.the.activity.of.EZH2..We.found.that.bi-lineage.cells.express.genes.active.in.normal.progenitor.cells,.possess.increased.sphere.formation.capacity.and.display.increa-sed.tumorigenic.capacity.

Conclusions:.Our.findings.indicate.that.bi-lineage.cells.possess.a.primi-tive.phenotype.that.is.advantageous.for.tumorigenesis,.and.reveal.that.the.interplay.between.EZH2.and.GATA3.can.determine.the.composition.of. basal-like. breast. cancer. cell. populations.. These. findings. provide. a.novel.view.of.the.intratumoral.cellular.heterogeneity.within.basal-like.breast.cancers.and.its.regulation.


J. Kumbrink.1,.S..Soni.2,.N..Sailer.3,.K..H..Kirsch.1

1. Boston.University.School.of.Medicine,.Biochemistry,.Boston,.United.States.

2. Mount.Sinai.School.of.Medicine,.Developmental.and.Regenerative.Biology,.New.York,.United.States.

3. University.of.Munich,.Biochemistry,.Munich,.Germany.

Development of inhibitors targeting p130Cas as novel therapeutics for breast cancer.

Aggressive.breast.cancers.are.characterized.by.elevated.levels.of.the.adapter.protein.p130Cas.(Crk-associated.substrate)..High.p130Cas.ex-pression.in.primary.breast.tumors.is.associated.with.increased.rate.of.relapse.and.poor.response.to.tamoxifen.treatment.of.recurrent.disease,.and.doxorubicin.resistance..p130Cas.promotes.the. integration.of.pro-tein. complexes. involved. in. signaling. pathways. in. response. to. growth.factors,. integrins,.and.estrogen..While.p130Cas.exerts.no. intrinsic.ki-nase.activity,.its.activation.depends.majorly.on.upstream.kinases.that.phosphorylate.its.substrate.domain.(SD)..The.SD.contains.15.YxxP.mo-tifs,. which. upon. phosphorylation. recruit. the. small. adapters. Crk. and.Nck,.resulting.in.the.activation.of.downstream.signaling.crucial.for.cell.migration.and.adhesion.

We.developed.a.decoy.approach.to.block.endogenous.p130Cas.function.by.ectopic.expression.of.a.constitutively.phosphorylated.SD.(Src*/SD,.an. attenuated. Src. kinase. domain. (Src*). with. no. oncogenic. potential,.fused. to. the. p130Cas. SD).. Ectopically. expressed. Src*/SD. competes.with. endogenous. p130Cas. for. SD. interacting. proteins. thus. limiting.the. activity. of. p130Cas. in. integrating. signaling. complexes.. The. Src*/SD.construct.was.shown.by.us. to.attenuate.the.MAPK/ERK.and.PI3K/Akt.survival.pathways,.reduce.cell.migration,.induce.apoptosis,.and.im-portantly.re-sensitize.MCF-7-derived.tamoxifen-resistant.breast.cancer.cells.(TAM-R).to.tamoxifen..Moreover,.a.dominant.Src*/SD.phenotype,.characterized.by.reduced.lamellipodia.formation.and.membrane.ruffling.as.well.as.tighter.cell.cluster.formation,.emerged..The.fact.that.not.all.of.the.YxxP.motifs.in.the.SD.are.phosphorylated.by.Src.or.required.for.Crk.and.Nck.binding.supports.the.hypothesis.that.shorter.SD.fragments.are.sufficient.to.inhibit.breast.cancer..Therefore,.deletion.constructs.of.the.p130Cas.SD.region.fused.to.the.Src*.domain.were.generated,.stably.transduced.into.TAM-R.cells,.and.assessed.for.their.inhibitory.potenti-al..The.previously.defined.Src*/SD.phenotype.was.used.to.validate.the.decoy.function.of.the.fusion.proteins..By.this.approach,.the.SD.region.responsible.for.mediating.the.phenotypic.changes.was.so.far.narrowed.down. to. five. tyrosine. motifs.. Importantly,. the. same. region. was. also.functional.in.LM2.breast.cancer.cells,.a.highly.lung.metastasis.causing.derivative.of.MDA-MB-231.cells.

In. vivo.experiments. to. test. the.activity.of. the. shortest. inhibitory.SD.fragment.on.breast.cancer.progression.in.an.orthotopic.mouse.model.are.currently.in.preparation..These.experiments.encompass.mammary.fat.pad. injections.of.breast.tumor.cells.expressing.the.smallest.func-tional.SD.fragment.and.monitoring.of.tumor.growth.and.metastasis.for-mation.of.these.xenografts.by.bioluminescence.imaging..The.long-term.goal.is.to.develop.small-inhibitory.compound(s).targeting.this.fragment.for.future.clinical.applications.

AEK

15

AEKAbstracts Short Talks Symposium 3: Dissecting cancer genomes and epi-genomes


E. Kostareli.1,2,.D..Holzinger.3,.O..Bogatyrova.4,.T..Hielscher.5,.G..Wichmann.6,.M..Keck.7,.B...Lahrmann.8,.N..Grabe.8,.C..Flechtenmacher.9,C..Schmidt.4,.T..Seiwert.7,.G..Dyckhoff.1,.A..Dietz.6,.D..Hoefler.3,.M..Pawlita.3,.A..Benner.5,.F..Bosch.1,.P..Plinkert.1,.C..Plass.4,.D..Weichenhan.4,.J..Hess.1,2

1. University.Hospital.Heidelberg,.Department.of.Otolaryngology,.Head.and.Neck.Surgery,.Heidelberg,.Germany.

2. DKFZ-ZMBH.Alliance,.Junior.Research.Group.Molecular.Mechanisms.of.Head.and.Neck.Tumors,.Heidelberg,.Germany.

3. German.Cancer.Research.Center.(DKFZ),.Division.of.Genome.Modifications.and.Carcinogenesis,.Heidelberg,.Germany.

4. German.Cancer.Research.Center.(DKFZ),.Division.of.Epigenomics.and.Cancer.Risk.Factors,.Heidelberg,.Germany.

5. German.Cancer.Research.Center.(DKFZ),.Division.of.Biostatistics,.Heidelberg,.Germany.

6. University.Hospital.Leipzig,.Department.of.Otorhinolaryngology,.Leipzig,.Germany.

7. The.University.of.Chicago,.Department.of.Medicine,.Chicago,.United.States.

8. BIOQUANT,.Hamamatsu.Tissue.Imaging.and.Analysis.Center.(TIGA),.Heidelberg,.Germany.

9. University.Hospital.Heidelberg,.Institute.of.Pathology,.Heidelberg,.Germany.

Global DNA methylation signature of HPV-related oropharyngeal squamous cell carcinoma

Head. and. neck. squamous. cell. carcinoma. (HNSCC). is. one. of. the. most.prevalent.and.lethal.cancers.worldwide,.and.recently.high-risk.types.of.human.papilloma.virus.(HPV).have.been.associated.with.an.increasing.number.of.oropharyngeal.squamous.cell.carcinoma.(OPSCC)..Strikingly,.patients.with.HPV-positive.OPSCC.are.highly.curable.with.ionizing.radi-ation.and.have.better.survival.as.compared.to.HPV-negative.patients..In.the.past,.several.groups.unraveled.an.obvious.difference.in.the.ge-netic. landscape.with.significantly.more.mutations. in.HPV-negative.as.compared.to.HPV-positive.tumors,.supporting.the.hypothesis.that.the.better.clinical.outcome.of.patients.with.HPV-positive.tumors.is.simply.a. reflection.of. less.genetic. aberrations.at. the. time.of. treatment..We.focused.our.attention.on.HPV-dependent.alterations. in. the.gene.pro-moter.methylome,.since.its.chemically.stable.nature.makes.this.epige-netic.modification.an.attractive.novel.molecular.biomarker.for.disease.staging. and. prognosis,. while. its. physiological. reversibility. makes. it. a.potent.druggable.target.for.translational.head.and.neck.oncology..App-lying.an.array-based.approach.to.monitor.global.changes.in.CpG.island.hypermethylation.between.HPV-negative.and.HPV-positive.OPSCCs,.we.identified. a. specific. pattern. of. differentially. methylated. regions. that.critically.depends.on.the.presence.of.viral.transcripts..HPV-related.al-terations.were.confirmed.for.the.majority.of.candidate.gene.promoters.by.mass.spectrometric,.quantitative.methylation.analysis..Genes.ALD-H1A2,.OSR2,.GATA4,.GRIA4.and.IRX4.showed.highly.significant,.inverse.correlation.between.promoter.hypermethylation.and.transcript.levels..Interestingly,.Kaplan.Meier. analysis. revealed.a. significant. correlation.between.the.ALDH1A2low.OSR2low.GATA4high.GRIA4high.IRX4high.pro-moter.methylation.pattern.and.improved.survival.in.three.independent.patient. cohorts.. ALDH1A2. protein. levels. determined. by. immunohisto-chemistry.on.tissue.microarrays.confirmed.the.association.with.the.cli-nical.outcome..In.summary,.our.study.highlights.specific.alterations.in.global.gene.promoter.methylation.in.HPV-driven.OPSCCs.and.identifies.a.signature,.which.predicts.the.clinical.outcome.in.OPSCCs.


R. Greinert.1,.I..P..Chen.1,.A..Faust.1,.S..Henning.1,.S..Moertl.2,.A..Kraemer.2,.V..Beate.1

1. Elbekliniken.Stade/Buxtehude,.Mol..Cell.Biology,.Buxtehude,.Germany.2. Helmholtz.Zentrum.München,.Radiation.Biology,.Neuherberg,.Germany.

UVA/UVB-induced epigenetic regulation of tumor suppressor and miRNA gene expression.

Question:.Skin.cancer.is.the.most.frequent.cancer.in.the.white.populati-on.worldwide..The.main.risk.factor.for.this.development.is.overexposure.to.solar.and.artificial.UV-radiation..UVA.-.and.UVB.radiation.have.recen-tly.been.classified.by. IARC.as.“know. to.be.carcinogenic. in.humans”.UV-radiation.causes.a.number.of.pre-mutagenic.DNA-lesions.which.are.able.to.induce.genetic.mutations.leading.to.skin.cancer.(basal.cell.car-cinoma.(BCC),.squamous.cell.carcinoma.(SCC).and.malignant.melanoma.(MM)..However,.research.of.the.last.ten.years.has.increased.evidence.that.also.epigenetic.modifications.play.an.important.role.in.the.etiolo-gy.of. skin. cancer..Epigenetic.modifications.are.mainly. introduced.via.DNA-methylation,.histone-modifications.(acetylation,.methylation,.ubi-quitination,.etc.).and.post-translational. regulation. through.the.action.of.micro-RNAs.

Methods:.To.evaluate.whether.UV-radiation.is.able.to.induce.epigenetic.changes.in.UVA-.(and.UVB).irradiated.human.keratinocytes.we.have.in-vestigated.differential.gene.expression.of.certain.tumor.suppresor.and.miRNA.genes..HaCaT.cells.have.been.chronically.irradiated.with.UVA.for.10.and.15.weeks.(200kJ/m²/week)..This.irradiation.regime.transformed.human.HaCaT.cells.to.be.tumorigenic. in.the.nude.mice.leading.to.the.development.of.SCC.in.xenografts..Epigenetic.changes.in.UVA-irradiated.HaCat.cells.and.and.tumor-derived.cells.were.then.detected.by.methyla-tion-specific.RT-PCR.and.ChIP-techniques..Using.micro-array.techniques.and.RT-PCR.we.furthermore.studied.differential.expression.of.UVB/UVA-irradiated.primary.human.keratinocytes.6.hrs.after.irradiation..

Results:.Chronic.UVA.irradiation.silenced.p16.expression.via.promoter.CpG-island.hypermethylation.and.histone-methylation..This.repression.of. p16. was. also. found. in. tumor-derived,. re-cultivated,. cells,. although.different.epigenetic.modifications.where.found.on.the.histone.level..Our.micro-array.experiments.furthermore.show.that.UVA-.(and.UVB-).irradi-ation.of.human.primary.keratinocytes.introduces.pronounced.changes.in.miRNA.expression..Most.interestingly,.miR-23b.expression.is.remar-kably.increased.after.UVA-irradiation,.which.might.introduce.premature.differentiation. in.epidermal.stem.cells..Other.differentially.expressed.miRNAs.target.genes.which.are.known.to.be.involved.in.skin.carcinoge-nesis.(e.g..PTCH,.E-Cadherin).

Conclusion:.Our.results.show.for.the.first.time.that.UV-radiation.indu-ces.epigenetic.modifications.in.human.cells.and.that.these.changes.are.introduced.in.genes.and.cellular.pathways.which.are.highly.relevant.for.skin.cancer.induction.and.progression.

16

AEKAEK AEKAbstracts Short Talks Symposium 4: Modelling of oncogenic pathways on the way to clinical pay-off?


M. Morkel.3,1,.C..Grimm.1,.L..Chavez.1,.M..Vilardell.1,.S..Tierling.2,.B..Timmermann.1,.J..Walter.2,.M..Schweiger.1,.H..Lehrach.1,.R..Herwig.1,.B..Herrmann.1,.1. Max-Planck-Institut.für.mol..Genetik,.Berlin,.Germany.2. Universität.des.Saarlandes,.Saarbrücken,.Germany.3. Charité.–.Universitätsmedizin.Berlin,.Mol..Tumor.Pathology.–.

Tumorsystembiologie,.Berlin,.Germany.

DNA-methylome analysis of mouse intestinal adenoma identifies a tumour-specific signature that is partly conserved in human colon cancer

Aberrant.CpG.methylation.is.a.universal.epigenetic.trait.of.cancer.cell.genomes..However,.human.cancer.samples.or.cell.lines.preclude.the.in-vestigation.of.epigenetic.changes.occurring.early.during.tumour.deve-lopment..Here,.we.have.used.MeDIP-seq.to.analyse.the.DNA.methylome.of.APCMin.adenoma.as.a.model.for.intestinal.cancer.initiation,.and.pre-sent.a.list.of.more.than.13,000.recurring.differentially.methylated.re-gions.(DMRs).characterizing.intestinal.adenoma.of.the.mouse..We.show.that.Polycomb.Repressive.Complex.(PRC).targets.are.strongly.enriched.among.hypermethylated.DMRs,.and.several.PRC2.components.and.DNA.methyltransferases.were.up-regulated.in.adenoma..We.further.demons-trate.by.bisulfite.pyrosequencing.of.purified.cell.populations.that.the.DMR.signature.arises.de-novo.in.adenoma.cells.rather.than.by.expansi-on.of.a.pre-existing.pattern.in.intestinal.stem.cells.or.undifferentiated.crypt.cells..We.found.that.epigenetic.silencing.of.tumour.suppressors,.which. occurs. frequently. in. colon. cancer,. was. rare. in. adenoma.. Quite.strikingly,.we.identified.a.core.set.of.DMRs,.which.is.conserved.between.mouse.adenoma.and.human.colon.cancer,.thus.possibly.revealing.a.glo-bal.panel.of.epigenetically.modified.genes.for. intestinal.tumours..Our.data.allows.to.draw.a.distinction.between.early.conserved.epigenetic.alterations.occurring.in.intestinal.adenoma.and.late.stochastic.events.promoting.colon.cancer.progression,.and.may.facilitate.the.selection.of.more.specific.clinical.epigenetic.biomarkers.


R. Fritsche.1,.F..Witzel.1,2,.A..Sieber.1,.R..Herr.3,.N..Schmidt.1,.S..Braun.3,.T..Brummer.3,.C..Sers.1,.N..Blüthgen.1,2

1. Charite.University.Hospital,.Pathology,.Berlin,.Germany.2. Humboldt.University,.Institute.of.Theoretical.Biology,.Berlin,.Germany.3. Centre.for.Biological.Systems.Analysis.(ZBSA),.Albert-Ludwigs-

University.of.Freiburg,.Freiburg,.Germany.

Strong negative feedback from Erk to Raf confers robustness to MAPK signallin

Protein. levels.within.signal.transduction.pathways.vary.strongly.from.cell. to.cell.. For.example,. it.has.been. reported. that.concentrations.of.the.terminal.kinase.in.MAPK.signalling,.Erk,.varies.about.4-fold.between.clonal.cells.under.the.same.conditions..In.the.present.study,.we.analy-sed.how.signalling.pathways.can.still.process.information.quantitatively.despite. strong.heterogeneity. in.protein. levels.. Mathematical. analysis.of. isolated. phosphorylation-dephosphorylation. cycles. predicts. that.phosphorylation.of.a.signalling.molecule.is.proportional.to.the.protein.concentration..We.systematically.perturbed.the.protein.levels.of.Erk.in.human.cell.lines.by.siRNA..We.found.that.the.steady-state.phosphory-lation.of.Erk.is.very.robust.against.perturbations.of.Erk.protein.level,.suggesting.that.there.are.mechanisms.that.provide.robustness.to.the.pathway. against. protein. fluctuations.. Using. mathematical. modelling,.we.identified.three.potential.mechanisms.that.may.provide.robustness.against.fluctuating.protein. levels:. 1.. kinetic.effects. (saturation.of. the.activating.kinase.Mek),.2..transcriptional.negative.feedbacks.and.3..ne-gative.feedbacks.on.the.post-translational.level..By.experimental.ana-lysis.of.the.systems,.which.included.analysis.of.effects.of.overexpres-sing.Mek,.measuring.transcript.levels.of.negative.feedback.regulators,.and. application. of. generic. inhibitors. of. transcription. and. translation,.we.could.exclude.kinetic.effects.and.transcriptional.negative.feedback.as. mechanisms. of. robustness.. By. analysing. a. panel. of. cell. lines. we.found.that.cells.are.robust.as.long.as.the.signal.passes.through.Raf-1..In.contrast,.cells.where.the.pathway.is.activated.by.a.mutation.in.B-Raf.loose.robustness..Detailed.molecular.analysis.of.the.system.shows.that.a.single.post-translational.feedback.to.Raf.mediates.robustness..Thus,.robustness.is.provided.through.a.fast.post-translational.mechanism.alt-hough.variation.of.Erk.levels.occurs.on.a.timescale.of.days.

AEK

17

AEKAbstracts Short Talks Symposium 5: Closing in on the ultimate culprits: the tumor cells and its neighbourhood


K. Giehl .1,.S.-J..Barnard .1,.A..Porr .2,.S..Chakraborty .1,.A..Menke .1

1. Justus-Liebig-Universität.Giessen,.Molekulare.Onkologie.solider.Tumore,.Giessen,.Germany.

2. Universität.Ulm,.Ulm,.Germany.

Galectin-8 as a new modulator of K-Ras-induced signaling in carci-noma cells

Monomeric. Ras. GTPases. act. as. molecular. switches. in. signaling. pa-thways. important. for. cell. growth,. differentiation,. and. survival.. Onco-genic.mutant.Ras.proteins.are.commonly.found.in.human.tumors,.with.mutant.K-Ras.being.most.prevalent..These.activating.point.mutations.directly. contribute. to. malignant. transformation. by. arresting. K-Ras. in.the.active,.GTP-bound.state..The.spatial.organization.of.the.farnesyla-ted.K-Ras.in.specific.nanoclusters.of.the.membrane.is.highly.important.for.accurate.signal.transduction,.but.the.mechanisms.underlying.K-Ras.nanoclustering.are.mainly.unknown..Galectin-3,.a.β-galactoside.binding.protein. that. contains. a. carbohydrate-recognition-binding. domain. as.well.as.a. farnesyl-binding.pocket,.has.been.shown.to. regulate.K-Ras-GTP. nanocluster. formation. as. well. as. K-Ras-induced. signal. transduc-tion.(Shalom-Feuerstein.et.al.,.Cancer.Res..2008,.68:6608)..By.in.vitro.binding.and.co-immunoprecipitation.studies.we.identified.the.tandem-repeat.Galectin-8.as.a.new.binding.partner.for.K-Ras..Subcellular.loca-lization.studies.revealed.that.K-Ras,.Galectin-3.and.the.two.isoforms.of.Galectin-8,.namely.Galectin-8.long.and.short,.are.localized.at.the.cell.membrane..Sequence.analyses.document.that.the.short.isoform.lacks.exon.11.which.encodes.for.42.amino.acids.localized.in.the.hinge.region.of.Galectin-8..Most.of.the.analyzed.lung.and.pancreatic.adenocarinoma.cell.lines.express.Galectin-3.and.one.or.two.of.the.Galectin-8.isoforms..Lung.adenocarcinoma.cell.lines.with.mutated.K-Ras.exhibit.considerably.less.amounts.of.Galectin-3.protein.than.cell.lines.with.wild-type.K-Ras..Since.forced.expression.of.oncogenic.EGFP-K-Ras(V12).inhibits.the.ex-pression.of.Galectin-3.on.protein.and.mRNA.level.without.affecting.the.expression.of.Galectin-8,.we.suggest.that.K-Ras.modifies.the.expression.of.its.binding.partner.Galectin-3..Downregulation.of.the.Galectin-8.ex-pression.by.siRNA.results. in.an.enhanced.phosphorylation.of.PKB/Akt.and.p42/44.MAPK..In.summary,.we.have.identified.Galectin-8.as.a.new.binding.partner.for.oncogenic.K-Ras..Our.results.further. indicate.that.the. two. identified.Galectin-8. isoforms.modulate.K-Ras. induced.signal.transduction.and.thus.might.affect.K-Ras-induced.malignant. transfor-mation.[This.work. is.supported.by.the.Universitätsklinikum.Giessen.und.Mar-burg.Forschungsförderung.and.the.LOEWE.Center.UGMLC.]


A. Koch .1,.S..C..Niemann-Seyde.1,.B..Breyer .1,.S..Saran.1,.D..D..H..Tran .1,.T..Tamura .1

1. Medizinische.Hochschule.Hannover,.Physiologische.Chemie.OE.4310,.Hannover,.Germany.

Degradation of macrophage-colony stimulating factor receptor, c-Fms, in epithelial cancer cells is enhanced by induction of autophagy.

Nutrient.deprivation,.hypoxia.or.oxidative.stress.is.a.general.feature.of.solid.tumors..In.order.to.survive,.the.cells.activate.proteolytic.degra-dation.processes.such.as.autophagy.to.remove.stress-induced.damage.and. recycle. nutrients.. However,. it. is. still. poorly. understood. in. which.cellular.context.autophagy.promotes.or.suppresses.survival.of.cancer.cells.

The.receptor.of.macrophage-colony.stimulating.factor.(M-CSF),.c-Fms,.has.been.connected.with.enhanced.metastasis.and.invasiveness.in.bre-ast.cancer..However,.c-Fms-signals.in.epithelial.cancer.cells.are.not.well.examined.

We.detected.the.transcript.of.c-Fms.and.M-CSF.in.nine.out.of.ten.dif-ferent.epithelial.cancer.cell.lines,.but.the.protein.is.hardly.detectable.except.in.BeWo.cells.that.do.not.express.M-CSF..Half-life.of.exogenously.expressed. c-Fms. in. HeLa. or. A431. cells. is. less. than. 1. h,. and. the. cells.contain.C-terminal.fragments.of.c-Fms.of.about.80,.75,.55,.and.50.kDa.suggesting.that.the.protein. is.rapidly.degraded..Aminoacid.starvation.(EBSS),.an. inducer.of.autophagy,. led.to.depletion.of.full. length.c-Fms.in. mouse. embryo. fibroblasts. stably. expressing. c-Fms. and. transiently.transfected.HEPA1-6.and.HeLa.cells. in.which. tyrosine.phosphorylated.c-Fms.breakdown.products.were.accumulated.within.4.h..Lysosomal.in-hibitor. chloroquine. accumulated. the. 55. kDa. fragement.. Furthermore,.c-Fms.partially.co-localized.with.autophagosome.marker.LC3.in.cancer.cells..In.BeWo.cells.in.the.absence.of.M-CSF,.the.c-Fms.cleavage.pattern.was.similar.to.that.in.HeLa,.A431.or.HEPA1-6.cells..The.fragments.were.still.present.after.treatment.with.monensin.or.Brefeldin.A.that.prevent.transport.to.the.plasma.membrane,.suggesting.that.cleavage.occurs.wi-thout.endocytosis,.and.from.intracellular.sources..Moreover,.we.found.the.50.kDa.fragment,.potentially.the.soluble.intracellular.domain.of.c-Fms,.being.phosphorylated.on.tyrosine.and.localized.in.the.nucleus.as.shown.by.subcellular.fractionation.and.confocal.microscopy..Moreover,.we. mapped. a. putative. nuclear. localization. signal. to. aminoacids. 676-678..A.GFP-fusion.protein.of.the.c-Fms-intracellular.domain.(ICD).was.phosphorylated.on.tyrosine.and.mediated.phosphorylation.of.Erk-1/2.or.STAT3/5.with.intensities.differing.from.ligand-activated.wild.type.c-Fms,.and.also.induced.expression.of.a.known.c-Fms.target.gene,.ccl2.

These.data.suggest.that.proteolytic.processes.such.as.autophagy.may.have.the.potential.to.modulate.receptor.tyrosine.kinase.signals.in.epi-thelial.cancer.cells.

18

AEKAEK AEKAbstracts Short Talks Symposium 5 continued: Closing in on the ultimate culprits: the tumor cells and its neighbourhood


U. E. Höpken.3,.A..Rehm.1,.M..Gätjen.1,.A..Mensen.2,.K..Schradi.3,.K..Gerlach.1,.V..Begay.4,.A..Leutz.4,.M..Lipp.3,.B..Dörken.5,1

1. Max-Delbrück-Center.for.Molecular.Medicine,.Department.of.Hematology,.Oncology.and.Tumor.Immunology,.Berlin,.Germany.

2. Charite-Universitätsmedizin.Berlin,.Institute.of.Medical.Immunology,.Berlin,.Germany.

3. Max-Delbrück-Center.for.Molecular.Medicine,.Tumor-.and.Immunogenetics,.Berlin,.Germany.

4. Max-Delbrück-Center.for.Molecular.Medicine,.Department.of.Cell.Differentiation.and.Tumorigenesis,.Berlin,.Germany.

5. Charite-Universitätsmedizin.Berlin,.Department.of.Hematology,.Oncology.and.Tumor.Immunology,.Berlin,.Germany.

Remodelling of the lymphoma-stroma interface within secondary lymphoid organs involves fibroblastic reticular cells and dendritic cells

Lymphoma.cell.survival.and.progression.is.critically.dependent.on.no-dal.access.and.specific.microanatomical.localization.within.secondary.lymphoid.organs.(SLO)..The.tumor.stroma.in.SLOs.consists.of.different.cell.types.including.cells.of.mesenchymal.origin,.cells.of.the.innate.and.adaptive.immune.response,.cells.of.the.vasculature,.but.also.of.the.ex-tracellular.matrix..Here,.we.dissect.the.survival-promoting.contribution.of.two.major.stroma.cell.populations,.fibroblastic.reticular.cells.(FRC).and.dendritic.cells.(DC),.respectively.

We.applied.an.oncogene-driven. transgenic.mouse.model,. the.Em-Myc.strain,. which. mimics. several. aspects. of. Myc-induced. aggressive. hu-man. B-cell. lymphoma.. Lymphoma. B-cells. derived. thereof. were. used.for.adoptive.transfer.experiments.and.progression.and.localization.of.lymphoma.B.cells.were.analyzed..Genetically-engineered.mouse.strains.for.a.transcription.factor,.homeostatic.cytokines.and.DCs.were.used.to.probe.environmental.factors.upon.adoptive.lymphoma.cell.transfer.

Em-Myc.lymphoma.localized. in.the.T.cell.zone.in.close.proximity.to.a.chemokine. providing. cell. population,. the. gp38+. FRCs.. Moreover,. this.myofibroblast-derived.cell.population.provided.prosurvival.signals,.fo-remost.the.hedgehog.ligand.Indian.hedgehog..Other.stroma.cell.popula-tions,.such.as.T.cell.zone-localized.DCs,.were.also.found.to.intermingle.closely.with.infiltrating.lymphoma.B.cells..Functionally,.transfer.of.Em-Myc.B.cells.in.DC-depleted.mice.delayed.lymphoma.growth.considerably..In.vitro,.co-culture.of.primary.lymphoma.cells.with.DCs.counteracted.the.inherent.apoptosis.program.of.tumor.cells..In.gene.expression.analysis.of.tumor-derived.DCs,.we.obtained.a.pattern.of.cytokines.indicative.of.a.transformation.toward.a.putatively.tolerogenic.milieu..This.observation.could.be.corroborated.in.cytometric.bead.arrays..Furthermore,.we.iden-tified.the.transcription.factor.C/EBPß.as.a.principal.regulator.of.shaping.a.(pro-).inflammatory,.tumor-permissive.microenvironment.within.SLOs.Within. nodal. tumor. cell. niches,. lymphoma. cells. initiate. a. reciprocal.crosstalk.with.FRCs.and.DCs,.thus.effectuating.synthesis.and.release.of.growth-promoting.factors.and.inflammatory.cytokines..


A. Holtorf .1,.B..Holzmann.1,.K.-.P..Janssen.1

1. Klinikum.rechts.der.Isar.der.TU.München,.Chirurgische.Klinik.und.Poliklinik,.München,.Germany.

A genetic mouse model to identify the role of the immune adapter protein MyD88 in colorectal cancer

Introduction:.Chronic. inflammation.of. the.colon. is.a.well.documented.trigger.of.colon.tumorigenesis..However.the.underlying.molecular.and.cellular.mechanisms.remain.unclear..Previous.studies.highlighted.a.cri-tical.role.of.the.innate.immune.system,.especially.pattern.recognition.receptors.from.the.Toll-like.receptor.(TLR).family,.in.colon.cancer..The.adapter. protein. Myeloid-differentiation. factor. 88. (MyD88). is. shared.between.several.TLRs.and.Interleukin-1.receptor.family..MyD88-defici-ency.has.been.found.by.us.and.others.to.protect.mice.from.intestinal.cancer.formation.in.genetic.models.for.colon.carcinogenesis..The.mou-se.model.presented.here.allows. tissue-specific.expression.of.MyD88,.and.thereby.dissection.of.the.complex.interaction.between.tumor.and.immune.system.during.intestinal.carcinogenesis.

Material. and.Method:. Insertion.of.an.“intron-gene-trap”.flanked.with.loxP.motifs.into.the.first.exon.of.the.Myd88.gene.locus.leads.to.global.inactivation. of. Myd88. expression. (MyD88stp/stp),. faithfully. phenoty-ping.a.global.gene.knock-out..Tissue-specific.re-expression.of.MyD88.in.mice.is.mediated.based.on.the.Cre-recombinase..Breeding.of.MyD88stp/stp.mice.with.LysMCre.or.pvillin-Cre.mice. leads. to. tissue-specific.ex-cision. of. the. “intron-gene-trap”,. retaining. endogenous. regulation. of.gene. expression.. MyD88. expression. and. successful. reconstitution. of.TLR-signaling.was.detected.in.either.myeloid.cells.(MyD88MYEL).or.in-testinal.epithelial.cells.(MyD88IEC)..Subsequently,.these.animals.were.mated.with.established.genetic.mouse.models.for.human.colon.cancer.(pvillin-KrasV12G.and.Apc1638N).

Results:.Global.MyD88.deficiency.significantly.decreased.development.of.tumors,.induced.by.either.oncogenic.KRAS.or.loss.of.function.of.the.tumor.suppressor.Apc..Re-expression.of.MyD88. in. intestinal.epithelia.and.tumor.cells.only.partially.restored.tumor.growth..Moreover,.activa-tion.of.the.MAPK/cMyc.pathway.was.independent.of.MyD88.expression.in.intestinal.epithelia.and.tumors..On.the.other.hand,.reconstitution.of.MyD88.expression.in.myeloid.cells.triggered.tumor.development.indi-stinguishable.from.parental.Apc1638N.mice.

Conclusion:.Our.preliminary.data.indicate.that.MyD88-mediated.signa-ling.in.myeloid.cells,.but.not.in.epithelial-derived.cancer.cells,.is.crucial.for.intestinal.tumor.development.

AEK

19

AEKAbstracts Short Talks Symposium 5 continued II: Closing in on the ultimate culprits: the tumor cells and its neighbourhood

Symposium 5 continued II – Short talk 0006

A. K. Wege.1,.K..Schardt.2,.A..Krömer.3,.E..M..Jung.4,.O..Ortmann.1,.G..Brockhoff.1

1. University.of.Regensburg,.FHK.St..Josef,.Regensburg,.Germany.2. University.of.Regensburg,.Institute.of.Pathology,.Regensburg,.

Germany.3. University.of.Regensburg,.Department.of.Surgery,.Regensburg,.

Germany.4. University.of.Regensburg,.Institute.of.Radiology,.Regensburg,.

Germany.

Humanized tumor mice – a new model to investigate and improve cancer therapy

The.immunological.impact.on.antibody.based.anti-cancer.therapies.re-mains. incompletely.understood.due.to. the. lack.of.appropriate.animal.models.for.in-vivo.analysis..To.overcome.this.limitation,.we.generated.a.novel.humanized.tumor.mouse.(HTM).model.by.concurrent.transplanta-tion.of.human.hematopoietic.stem.cells.and.human.breast.cancer.cells.in.neonatal.NOD-scid.IL2.receptor.null.mice..Five.weeks.after.intrahe-patic.transplantation.a.functional.human.immune.system.developed.in.all.organs.and.in.addition.tumor.cells.were.detectable.in.lung.and.bone.marrow.(early.dissemination)..In.the.age.of.three.months,.solid.tumors.associated.to.liver.(and.spleen).or.tumor.cell.effusions.were.detecta-ble.. Interestingly,. BT474. cells. (originally. isolated. from. a. solid. tumor).resulted. in. solid. tumor. formation. and. weak. dissemination. whereas.SK-BR-3.originally. isolated. from.pleural. tumor.effusion. reappeared. in.tumor.ascitis.accompanied.by.metastases.in.these.mice..High.resoluti-on.ultrasound.(HRU).in.combination.with.contrast.enhanced.ultrasound.(CEUS).and.color-coded.elastography.enabled.the.early.detection.and.characterization.of.small.liver.lesions.in.HTM.in.vivo..Tumor.growth.was.accompanied.by.specific.T.cell.maturation.and.tumor.cell.specific.T-cell.proliferation..In.addition,.Natural.Killer.cell.accumulation.and.activation.was.observed.in.HTM.which.was.further.enhanced.upon.IL-15.treatment.facilitating.the.possibility.of.immune.cell.modulation.in,.e.g..antibody-dependent.cell-mediated.cytotoxicity.(ADCC).based.immunotherapeutic.approaches.in.HTMs..Furthermore,.T.cell.receptor.(TCR).analyses.indica-ted.a.nearly.full.human.TCR.repertoire.in.spleen,.liver.and.lymph.nodes.including.some.dominantly.expressed.TCR.chains.The.neonatal.transplantation.enabled.the.combined.transfer.of.major.hi-stocompatibility.complex.(MHC).mismatched.tumor.cells.together.with.human. hematopoietic. stem. cells. resulting. in. a. solid. coexistence. and.interaction. without. evidence. for. rejection.. Overall,. humanized. tumor.mice.represent.a.powerful.in-vivo.model.that.permits.the.investigation.of.the.human.immune.defence.against.cancer,.in.particular.with.respect.to.antibody.based.targeted.therapies.and.might.reveal.new.strategies.to.overcome.therapy.failure.in.non.responder.patients.

Symposium.5.continued.II.–.Short.talk.0123

N. Grabe.1,2,3,.T..Sütterlin.1,3,.K..Safferling.1,3,.C..Ernst.1,3,.I..Zörnig.1,.D..Jäger.1,3,.N..Halama.1,3

1. National.Center.for.Tumor.Diseases,.Medical.Oncology,Heidelberg,.Germany.


3. University.Heidelberg,.BIOQUANT,.TIGA.Center,.Heidelberg,.Germany.

Towards multi-scale modelling of the patient individual tumor micro-environment

One.of.the.key.issues.preventing.durable.therapy.success.for.the.indi-vidual.cancer.patient.is.a.lack.of.understanding.of.the.tumor.microenvi-ronment..It.is.the.battlefield.of.cancer.where.immune.cells.interact.with.stroma.and. tumor.cells. in.a. time.dynamic,. self-organizing.processes..From. a. systems. biologist’s. points. of. view. these. complex. actions. can.in.future.only.be.understood.when.analyzing.the.situation.individually.for.each.patient.and.using.computational.modeling.to.bridge.the.data.acquired.at.different.scales.by.different.methods..We.briefly.present.how.such.models.could.be.constructed.on.the.level.of.the.individual.patient.by.integrating.multiple,.complementary.tech-nologies.such.as.histology,.whole-slide.imaging,.cytokine.profiling.and.multi-agent.modeling..First,.we.show.how.multi-agent.systems.can.be.used.to.model.spatial.T-cell.movement.in.chemotactic.fields.at.the.ex-ample.of.our.own.graphical.multi-scale.modeling.environment.EPISIM..We.then.show.how.to. integrate.semi-automated.histology,.automated.whole-slide.imaging.(WSI).and.image.processing.for.allowing.the.precise.localization. and. robust. quantification. of. intra-tissue. cell. and. expres-sion.profiles..We.precisely.monitor.the.spatial.patterns.of.T-cell.profiles.(CD3,. CD8,. CD45). in. the. tumor. microenvironment. in. a. colorectal. pa-tient.cohort.and.show.how.to.predict.from.this.the.patients’.responses.to.chemotherapy..50-plex.assays.on.micro-dissected.tissues.are.then.used.to.generate.spatially.resolved.cytokine.profiles.of.the.microenvi-ronment.of. individual.patients..From.this,.network.modeling.based.on.protein-interaction.networks.derives.frequent.“signature-cytokine-net-works”.(SCNs).(e.g.. IL4-IL9-CSF2-IL2)..These.SCNs.cluster.patients.by.descriptive.cell-cell.communication.networks.and.indicate.potential.key.cytokines.for.therapeutic.interventions..Linking.then.cytokine.signaling.to.protein-protein.interaction.networks.uncovers.candidate.causal.rela-tions.behind.the.SCNs.at.the.intracellular.scale.and.points.to.relevant.immune.cell.populations..This.in.turn.further.yields.potential.individual.intracellular.drug.candidates.as.well.as.potential.combination.therapies.

20

AEKAEK AEKAbstracts Short Talks Symposium 6: Targeted therapy – melanoma a success story?


W. Walther.1,.D..Kobelt.1,.J..Aumann.1,.G..Freundt.1,.M..Schmidt.2,.I..Fichtner.1,.D..Behrens.1,.P..Schlag.3,1

1. Max-Delbrück-Center.,.Berlin,.Germany.2. MOLOGEN,.Berlin,.Germany.3. Charite.Comprehensive.Cancer.Center,.Berlin,.Germany.

Preclinical study of combined non-viral gene- and chemotherapy of malignant melanoma

Question:.For.successful.gene.therapy.safe.and.efficient.vectors.in.com-bination.with.efficient. therapeutic.options.are.essential..Among.non-viral.vectors.the.MIDGE.vector.technology.(MOLOGEN).represents.the.smallest.vector.system.for.transient.gene.transfer..We.analysed,.if.this.vector.can.be.employed.for.efficient.non-viral.gene.therapy.

Methods:. In. vitro. gene. transfer. was. performed. using. equimolar. lipo-fection. and. electroporation.. To. analyse. vector. load. and. distribution.fluorescently.labelled.vector.molecules.were.used.and.assessed.by.flu-orescence.microscopy,.FACS.and.qPCR..Transgene.expression.and.distri-bution.was.analysed.by.luminescence.assays.(Luc),.FACS.(GFP),.qRT-PCR.(Luc,. hTNF-alpha). and. ELISA. (hTNF-alpha).. Cytotoxicity. of. hTNF-alpha.and.vindesine.was.monitored.with.MTT..LDH-release.and.caspase.8,.9,.3/7.activation.assays.were.used.to.analyse.cell.death..For. intratumo-ral. in. vivo.gene. transfer.of.xenotransplanted.melanoma,. jet-injection.was.used.. In.vivo.vector.distribution.and.gene.expression.was.analy-sed.by.q(RT)-PCR,.ELISA.and.IHC..For.combination.therapy.experiments.vindesine.was.given. i.v..24h.after.gene. transfer.once.a.week.. In.vivo.apoptosis.was.shown.by.TUNEL-assay.

Results:. This. preclinical. gene. therapy. study. is. aiming. at. the. local.non-viral. sensitization.gene. therapy.of.malignant.melanoma.by.using.the.MIDGE.vector..Our. in.vitro.experiments.showed.superiority.of.the.MIDGE. vector. compared. to. plasmid. vectors. regarding. luciferase. and.hTNF-alpha. expression.. In. different. human. melanoma. cell. lines. gene.expression. increased.up.to.10-fold.(transfection).and.>>100-fold.(electroporation).compared.to.the.parental.plasmid..For.the.therapeutic.sensitization.approach.we.combined.the.MIDGE.based.hTNF-alpha.gene.transfer.with.vindesine.treatment.in.vitro.and.in.vivo..The.in.vitro.hTNF-alpha.gene.expression. led.to.up.to. 10-fold.reduction.of. the.vindesine.IC50..This.is.due.to.an.accelerated.and.increased.activation.of.caspases.leading.to.apoptosis..The.combined.in.vivo.gene.transfer.of.the.hTNF-alpha.encoding.MIDGE.vector.and.vindesine.treatment.significantly.re-duced.the.tumor.growth.in.xenotransplanted.A375.and.patient-derived.tumors..Regarding.safety,.the.MIDGE.vector.had.low.systemic.leakiness.and.fast.clearance.after.intratumoral.jet-injection.

Conclusion:.The.MIDGE.vector.demonstrated.great.potential.for.clinical.application.in.gene.therapy.for.treatment.of.melanoma..This.will.repre-sent.the.basis.for.a.clinical.phase.I.trial.starting.early.2013

Notes:

AEK

21

AEKAbstracts Posters Section 1: Tumor genetics and heterogeneity

Poster P-0002

O. Harismendy.1,.L..Bao.1,.S..Kotsopoulos.2,.S..Rozenzhak.1,.J..Olson.2,.E..Funk.2,.M..Nakano.1,.B..Crain.1,.S..Pond.3,.K..Messer.1,.R..Schwab.1,.M..S..Chee.3,.K..A..Frazer.1

1. University.of.Colifornia.San.Diego,.Moores.UCSD.Cancer.Center,.La.Jolla,.United.States.

2. RainDance.Technologies.Inc.,.Lexington,.MA,.United.States.3. Prognosys.Biosciences,.La.Jolla,CA,.United.States

Identification of Low Prevalence Somatic Mutations in Heterogene-ous Tumor Samples

High.throughput.sequencing.enables.the.digital.measurement.of.each.allele. in. a. DNA. sample.. This. provides. an. ideal. method. to. interrogate.mutations.present. in.heterogeneous. samples. such.as. solid. tumors. in.which. clonal. selection. or. contamination. with. stroma. can. hinder. the.identification.of.important.somatic.mutations..We.developed.an.ultra-deep.targeted.sequencing.(UDT-Seq).assay.to.screen.42.cancer.genes.via. microdroplet-based. PCR. (RainDance. Technologies). and. direct. se-quencing.of.the.amplicons.on.the.Illumina.GA..This.UDT-Seq.library.in-terrogates.~86.kb.of.DNA.located.in.cancer.mutational.hotspots.(87%.of.all.COSMIC.database.entries).and.~23.kb.located.in.exons.sequenced.in.HapMap.samples.for.the.assay.calibration.and.performance.evalua-tion..We.devised.a.statistical.filtering.of. the.mutations.by.using.both.experimental.estimation.of.the.sequencing.error.rate.and.training.with.a. calibration. sample.. We. measured. the. performance. of. our. assay. by.processing.4.blends.of.4.HapMap.samples,.interrogating.158.SNPs.with.known.prevalence.in.each.blend..The.sensitivity.and.specificity.of.our.method.is.>88%.and.>99%.respectively.for.mutations.present.at.1%.or.greater..We.next.interrogated.4.cancer.samples.(xenografts,.2.of.which.with.matching.primary.samples).from.4.different.fresh.frozen.tissues..We.were.able.to.detect.and.validate.low-prevalence.somatic.mutations.in.all.samples.of.which.some.are.well-known.driver.mutations..Finally,.we.analyzed.the.robustness.of.the.detection.and.prevalence.measure-ment.after.performing.whole.genome.amplification.and.show.that.WGA.leads.to.an.underestimation.of.the.mutant.allele.for.mutations.present.at.5%.prevalence.or.lower..Featuring.a.streamlined.sample.preparation.to. interrogate. a. large. number. of. bases,. this. assay. is. well. suited. for.clinical.applications.to.study.clonal.selection.in.cancer.progression.or.treatment.with.sub-optimal.heterogeneous.cancer.samples.

Poster P-0003

V. Taly.1,.D..Pekin.2,.S..Kotsopoulos.3,.X..Li.3,.I..Atochin.3,.H..Gang.3,.E..Funk.3,.D..Le.Corre.1,.L..Benhaim.4,.L..Benhaim.4,.J..B..Hutchinson.3,.D..R..Link.3,.H..Blons.4,.P..Laurent-Puig.4

1. Universite.Paris.Descartes,.Translational.Research.and.Microfluidics,.Paris.Cedex.06,.France.

2. Universite.de.Strasbourg,.Institut.de.Science.et.d’Ingenierie.Supramoleculaires.(ISIS),.Strasbourg,.France.

3. RainDance.Technologies.Inc.,.Lexington,.MA,.United.States.4. Universite.Paris.Descartes,.INSERM.UMR-S775,.Paris.Cedex.06,.

France

Quantitative detection of circulating tumor DNA by droplet-based digital PCR

By.segregating.individual.target.DNA.molecules.into.millions.of.aqueous.droplets. acting. as. independent. microreactors,. our. procedure. allows.for. extremely. precise,. sensitive,. and. fast. quantification. of. mutated.genes.. The. sensitivity. of. the. procedure. was. confirmed. by. measuring.1/200,000. dilution. of. KRAS-mutated. cell-line. DNA. in. a. background. of.wild-type.DNA..Furthermore,.plasma.of.more.than.50.patients.with.me-tastatic.CRC.were.tested..Our.procedure.enabled.detection.of.the.target.mutation.and. the.wild-type.DNA,.and. thus,.measurement.of. the. total.amplifiable. DNA.. DNA. concentration. in. the. plasma. samples. varied. by.two.orders.of.magnitude.and.was.not. correlated. with. the.proportion.of. mutated. DNA,. which. varied. from. 42%. to. 0.1%.. By. using. a. duplex.analysis.to.detect.either.of.the.two.most.frequent.mutations.of.KRAS.(G12D.and.G13D).and.the.wild-type.DNA,.the.expected.mutation.(known.by. primary. tumor. characterization). was. detected. in. 16. out. of. the..19.samples..Two.samples.had.a.low.amount.of.amplifiable.DNA.leading.to.an.inconclusive.result..Five.samples,.positive.for.the.G13D.mutation,.were.also.tested.for.the.G12D.mutations.and.were.negative..Moreover,.we.tested.54.plasma.samples.from.patients.with.metastatic.cancer.with.known.KRAS.status.(mutated.or.not).in.a.multiplex.format.allowing.the.simultaneous.analysis.of. the.seven.more. frequent.mutations.of.KRAS.or. the. wild-type. sequence.. Among. the. mutated. samples,. 13. out. the..19.mutated.ones.were.positive.and.32.of. the.non-mutated.ones.were.negatives..Our.results.demonstrate.that.our.digital.PCR.method.enables.non-invasive.detection.of.KRAS. in.plasma.of.patients.with.metastatic.CRC. with. high. sensitivity. and. high. specificity.. We. anticipate. that. the.method.will.be.employed.in.multiple.applications.in.the.clinic,.including.diagnosis,.cancer.recurrence.monitoring,.and.treatment.management.

22

AEKAEK AEKAbstracts Posters Section 1: Tumor genetics and heterogeneity

Poster P-0042

C. Blassl.1,.H..Schneck.1,.F..Meier-Stiegen.1,.T..Fehm.1,.H..Neubauer.1

1. Universitäts.Frauenklinik.Tübingen,.AG.Tumorprogression,.Tübingen,.Germany.

Analysing The Mutational Status Of PIK3CA In Circulating Tumor Cells From Metastatic Breast Cancer Patients

Background:. The. frequently. altered. phosphatidylinositol-3-kinase.(PI3K)/Akt. signaling. pathway. is. involved. in. the. regulation. of. cellular.processes. required. for. breast. carcinogenesis.. The. aim. of. the. project.was. to. develop. a. method. to. identify. hotspot. mutations. in. the. PIK-3CA.gene.in.circulating.tumor.cells.(CTC).of.metastatic.breast.cancer.(metBC).patients..

Methods:.From.44.enrolled.CTC-positive.metBC.patients.a.total.number.of.57.peripheral.blood.samples.were.analysed.by.CellSearch®..Genomic.DNA.of.enriched.CTCs.was.isolated,.amplified.and.analyzed.for.PIK3CA.mutations. in. exons. 9. and. 20. which. lead. to. E542K,. E545K. or. H1047R.amino.acid.changes.which.result.in.increased.PI3K.activity..The.muta-tions. were. detected. by. using. SNaPshot-methodology. comprising. PCR.amplification.and.single.nucleotide.primer.extension..

Results:. SNaPshot. analysis. was. established. using. genomic. DNA. from.different.breast.cancer.cell. lines.and.then.successfully.transferred.to.investigate.blood.samples.and.single.cells..Overall,.twelve.hotspot.mu-tations.in.either.exon.9/E545K.(6/12,.50%).or.exon.20/H1047R.(6/12,.50%).could.be.determined.within.9.out.of.57.(15.8%).blood.samples.from.7.out.of.44.(15.9%).patients;.CTC.counts.ranged.from.1-9748..PIK-3CA.variants.E542K,.E545G.and.E545A.were.not.detected.

Conclusions:. Analysing. the. PIK3CA. genotype. of. CTCs. has. clinical. re-levance. with. respect. to. drug. resistance,. e.g.. against. HER2-targeted.therapy..The.herein.described.approach. including.SNaPshot. technolo-gy.provides.a.simple.method.to.characterize.hotspot.mutations.within.CTCs.enriched.from.peripheral.blood.and.can.be.easily.adopted.for.ana-lysing.further.therapeutically.relevant.SNPs..

Poster P-0067

K. Nöske.1,.B..Falkowska-Hansen.1,.P..Boukamp.1

1. DKFZ,.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany.

Regulation of stem cell maintenance in tumour-derived human epi-dermis

The.human.epidermis.is.maintained.by.a.balance.of.stem.cell.mainte-nance.and.proliferation.with.subsequent.differentiation..Homeostasis.is.achieved.by.asymmetric.division.either.of.the.stem.cell.itself,.to.yield.one.stem.cell.and.one.transit.amplifying.cell,.or.by.the.stem.cell.proge-ny..To.test.which.mechanism.holds.true.in.the.human.epidermis,.we.use.an.in.vivo-like.long-term.organotypic.culture.model..This.allows.for.stem.cells.to.be.labelled.during.a.pulse.period.and.to.be.followed.for.several.weeks.(chase)..Thereby,.the.slowly.cycling.stem.cells.establish.as.label-retaining.cells.(LRCs).in.the.basal.layer.and.can.be.investigated.for.their.mode.of.division.by.using.markers.specific.for.e.g..asymmetric.division.LRCs,.the.potential.stem.cells,.are.not.restricted.to.normal.epidermis.but.similarly.establish.in.epithelia.derived.from.epidermal.tumour.cells.(HaCaT-ras.II4.cells)..We.use.malignant.HaCaT-ras.II4.cells.to.isolate.vi-tal.LRCs.with.a. in.vivo. labelling.technique..The.cells.were.transduced.with.an.inducible.H2B-GFP.Tet-Off.construct.that.by.addition.of.Doxycy-cline.allows.for.the.selection.of.LRCs..The.isolated.LCRs.will.be.analy-sed.regarding.their.molecular.characteristics.(RNA.expression.profile).and.their.functional.requirements.for.successful.stem.cell.division.and.maintenance..With.this.we.hope.to.gain.insight.into.the.mechanistic.and.regulatory.properties.of.stem.cells.in.the.human.epidermis.and.in.ma-lignant.epithelia.

AEK

23


Poster P-0069

C. Leufke.1,.J..Leykauf .1,.D..Krunic.1,.A..Jauch .2,.P..Boukamp.1

1. German.Cancer.Research.Center,.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany.

2. University,.Institute.of.Human.Genetics,.Heidelberg,.Germany

The Telomere Profile Distinguishes Two Classes of Genetically Di-stinct Cutaneous Squamous Cell Carcinomas

Non-melanoma. skin. cancer. is. the. most. frequent. cancer. worldwide.with. increasing. frequency. and. aggressiveness. especially. for. cutane-ous. squamous. cell. carcinomas. (SCCs). under. immunosuppression.. So.far,.molecular.mechanisms.are.still.insufficiently.understood..With.UV-irradiation.as.one.of.the.known.major.carcinogens,.UV-indicative.mu-tations.and.chromosomal.aberrations.are.prominent..Telomeres.as.the.physical.ends.of.chromosomes.are.essential.to.guarantee.chromosomal.integrity..Genomic. instability.caused.by.telomere.erosion.or.aberrant.telomere. distribution. are. important. mechanisms. of. tumorigenesis.To.check.whether. this. is.also. the.case. for.SCC.development,.we.first.established.telomere. length-profiles. in.skin.sections.and.even.in.nor-mal,.healthy.skin.we.identified.discrete.epidermal.sites.of.evenly.short.telomeres..In.precancerous.actinic.keratosis.(AKs),.SCCs.and.basal.cell.carcinomas. (BCCs). we. identified. two. telomere. phenotypes:. i). tissue-wide,.evenly.short.telomeres.(homogeneous).and.ii).longer.telomeres.with.high.differences.in.length.(heterogeneous)..This.suggests.for.two.modes.of.initiation..While.tumor.histotype,.location,.patient.gender.or.age.failed.to.distinguish.the.two.telomere.phenotypes,.we.saw.a.trend.for.higher.degree.of.aberrant.p53.and.cyclin.D1.expression.with.long/heterogeneous. telomeres. in. SCCs.. Instead,. telomerase. seems. not. to.play.a. role. for. the.dichotomy..Additionally,.we.could.find.a. correlati-on. for. short/homogeneous. telomeres. with. a. simple. karyotyp. and. for.long/heterogeneous. length.with.a.more.complex.karyotyp..SCCs.from.renal.transplant.recipients.showed.long/heterogeneous.telomeres.and.a.complex.karyotyp,.suggesting.for.a.selective.advantage.of.this.sub-typ. under. immunosuppression.. As. a. second. telomere. dependent,. but.telomere. length-independent,.mechanism.for.genomic. instability. ,.we.investigated.changes.in.telomere.distribution..We.identified.a.progres-sion-dependent.increase.in.telomere.associations.in.AKs.(17/17).and.ad-ditional.telomeric.aggregates.(TAs). in.SCCs.(24/32).and.BCCs.(30/31).Thus,.telomere.distribution.appeares.a.general.pathological.feature.of.UV-dependent.skin.cancer.and.may.serve.as.promising.tumor-therapeu-tic.target.

Poster P-0070

R. Warta.1,2,.J..Chaisaingmongkol.3,.O..Popanda.3,.E..Herpel.4,.C..Plass.3,.P..Schmezer.3,.G..Dyckhoff.2,.C..Herold-Mende.1,2

1. Heidelberg.University.Hospital,.Division.of.Neurosurgical.Research.,.Heidelberg,.Germany.

2. Head.and.Neck.Surgery,.University.of.Heidelberg,.Department.of.Otorhinolaryngology,.Heidelberg,.Germany.

3. German.Cancer.Research.Center.(DKFZ),.Division.of.Epigenomics.and.Cancer.Risk.Factors,.Heidelberg,.Germany.

4. Institute.of.Pathology,.University.Hospital.Heidelberg,.NCT.Tissue.Bank,.Heidelberg,.Germany

CSPG4 protein expression is regulated by DNA methylation and a ne-gative influence factor for patient survival

Objective:. HNSCC. biology. is. strongly. influenced. by. various. genetic.alterations.that.appear. in.early. tumor.stages..Moreover.there. is.now.increasing.evidence.that.epigenetic.changes.like.DNA.methylation.also.contribute. significantly. to. malignant. transformation.. The. expression.level.of.markers.of.undifferentiated.cells.might.be.accurate.prognostic.indicators.in.cancer..Interestingly,.there.are.hints.that.chondroitin.sul-fate.proteoglycan.4.(CSPG4).is.expressed.on.normal.neural.stem.cells.and.cancer.stem.cells.of.gliomas.and.its.expression.is.associated.with.a.poor.prognosis.and.resistance.to.treatment..Therefore.we.hypothesize.that.CSPG4.is.a.good.candidate.HNSCC.marker,.however.a.systematic.analysis.of.the.role.of.CSPG4.in.HNSCC.is.still.missing.

Methods:. We. performed. an. in-depth. analysis. of. CSPG4. expression. in.a.panel.of.HNSCC.tumors.and.normal.controls..Therefore.we.used.im-munohistochemical.staining,.qPCR.and.in.silico.expression.analysis.of.public.available.expression.microarray.datasets..The.DNA-methylation.status.of.a.CpG-Island. in.CSPG4.promoter. in.HNSCC. tumors.was.ana-lyzed.by.the.highly.innovative.MassARRAY.technique.and.validated.by.5-AZA.treatment.of.two.HNSCC.cell.lines..Finally.the.CSPG4.expression.data.and.the.methylation.status.of.the.CSPG4.promoter.were.correlated.to.the.survival.prognosis.of.the.HNSCC.patients.

Results:.The.expression.analysis.revealed.a.dramatic.CSPG4.mRNA.ove-rexpression.in.HNSCC.tumors.and.an.increased.CSPG4.protein.expres-sion. in.a.subgroup.of.HNSCCs..Next.we. identified.hypomethylation.of.the.CSPG4.promoter.in.HNSCC.which.correlated.with.high.CSPG4.mRNA.and.protein.expression..This.was.confirmed.by.5-AZA.treatment.of.two.HNSCC.cell.lines..Finally.we.found.that.high.CSPG4.expression.and.low.promoter.methylation.are.significantly.associated.with.an.adverse.pro-gression-free.and.overall.survival.

Conclusion:.In.this.first.in-depth.study.of.CSPG4.in.HNSCC.we.revealed,.that.overall.as.well.as.progression-free.survival.of.HNSCC.patients.is.lin-ked.to.tumor.CSPG4.promoter.methylation.and.protein.expression..We.found.that.CSPG4.expression.is.often.dramatically.increased.in.HNSCC.tumors,.a.result.consistent.with.our.detection.of.generally.lower.CSPG4.promoter.methylation..These.data.suggest.that. in.HNSCC.tumor.cells,.demethylation.at.the.CSPG4.promoter.contributes.to.increased.CSPG4.expression..In.support.of.this.possibility,.we.found.that.treating.HNSCC.cells.harboring.a.highly.methylated.CSPG4.promoter.with.demethylati-on.agents.caused.CSPG4.re-expression.In.conclusion.our.findings.support.our.initial.hypothesis.that.CSPG4.is.an.important.player.in.HNSCC.biology..

24

AEKAEK AEKPoster P-0077

D. Belharazem.1,.J..Kunanz.2,.A.-.K..Henne.3,.P..Kienle.3,.P..Ströbel.4

1. Institut.of.Pathology,.Mannheim,.Germany.2. Institut.of.Surgery,.Mannheim,.Germany.3. Institut.of.surgery,.Mannheim,.Germany.4. Institut.of.Pathology.,.Göttingen,.Germany.

Loss of imprinting of isulin-like growth factor 2 (IGF2) in colon carci-nomas activates the cell cycle and proliferation genes

Introduction:.The.insulin-like.growth.factor.2.(IGF2).gene.is.regulated.by.imprinting.in.normal.tissues..The.loss.of.imprinting.(LOI).results.in.the.bi-allelic.expression.of.IGF2..Its.increased.activity.has.been.associ-ated.with.many.cancers.including.colorectal.cancer.(CRC).To.investigate.the.significance.of.IGF2.in.CRC,.normal.tissue.and.tumor.biopsies.from.400.patients.were.analyzed.and.correlated.with.clinical.outcome.and.the.KRAS.and.PIK3.status.

Methods:.The. IGF2.820.G/A.gene.polymorphism.and. imprinting.status.were. examined. by. restriction. fragment. length. polymorphism. of. DNA.derived. from. normal. tissues. and. tumors. then. of. RNA. from. heterozy-gous.cases.. IGF2.protein.analysis.as.well.as.an.RNA.microarray.were.performed.

Results:. Forty-one. percent. of. the. cases. were. heterozygous.. LOI. was.detectable.in.both.normal.tissues.and.tumors.in.60%.of.cases..Tumors.with.LOI.had.significantly.higher. IGF2.protein. levels..Gene.expression.analysis. revealed. significance. of. cell. cycle. progression. and. mitosis.genes.in.LOI..

Conclusion:.IGF2.LOI.is.a.common.and.early.change.in.patients.with.CRC.and.in.normal.tissues,.provides.possibly.a.preneoplastic.change..Howe-ver.increased.IGF2.protein.levels.as.well.as.a.different.cell.cycle.specific.gene.expression.profile.were.detected.in.tumors.with.LOI.Future.studies.must.be.performed.to.find.out,.whether.such.subsets.of.tumors.differ.in.their.response.to.chemotherapy.or.alternative.thera-pies.

Poster P-0078

D. Belharazem.1,.A..Schäffer.2,.A..Marx.3,.P..Ströbel.2

1. Institut.of.Surgery,.Mannheim,.Germany.2. Institut.of.Pathology.,.Göttingen,.Germany.3. Institut.of.Pathology,.Mannheim,.Germany

Loss of imprinting of IGF2 is associated with gastrointestinal stromal tumors (GIST)

Introduction:.Gastrointestinal.stromal.tumors.(GISTs).are.the.most.com-mon.mesenchymal.tumors.of.the.human.gastrointestinal.tract.that.may.occur.in.the.entire.gastrointestinal.tract.most.commonly.in.the.stomach.or.small.intestine.The.insulin-like.growth.factor.2.(IGF2).stimulates.cell.proliferation.and.is.one.of.imprinted.genes,.in.which.only.the.paternal.allele.is.expressed..When. loss.of. imprinting.(LOI).occurs,.the.maternal.allele.may.also.be.expressed.The. regulation. of. gene. imprinting. occurs. at. CpG. enriched. imprinting.control.regions.(ICR),.a.differentially.methylated.region.(DMR).that.is.variably.methylated.depending.upon.parent.of.origin.In.this.work.we.investigated.the.IGF2.loss.of.imprinting.status.in.GISTs..Furthermore. IGF2.expression. level.was.analysed.and. the.variation.of.ICR.methylation.pattern.was.determined.

Methods:.IGF2.gene.polymorphism.rs680.A/G.was.analyzed.in.113.tumor.samples.using.an.allele.specific.PCR..The.loss.or.the.retention.of.imprin-ting.(LOI/.ROI).was.determined.using.RNA.from.heterozygous.samples.IGF2. mRNA. and. protein. expression. were. quantified. in. LOI. and. ROI.groups.using.real.time.PCR.and.ELISA..The.antiapoptotic.genes.c-FLIP.and.BIRC3.expression.were.also.examined..The.CpG.methylation.patter.was.analyzed.using.bisulfite.conversion.and.sequencing.

Results:. Thirty. percent. of. the. GISTs. were. heterozygous. at. the. rs680.A/G.. Seventy-three. percent. of. the. tumors. showed. loss. of. imprinting.followed.by.an. increased. IGF2. level..An.over.expression.of.apoptosis.Inhibitor.genes.BIRC3.and.c-FLIP.were.identified.in.LOI.cases..Analysis.of.the.IGF2/H19.imprinting.control.region.(ICR).revealed.marked.hyper-methylation.at.the.ICR3.and.ICR7.in.LOI.patients.

Conclusion:. Gastrointestinal. stromal. tumors. present. frequently. IGF2.loss. of. imprinting. with. a. hypermethylation. of. ICR3. and. 7.. That. could.have. the.potential. to. indicate.persons.at. risk. in. screening.programs..Selective.inhibitions.of.the.increased.c-FLIP.and.BIRC3.levels.in.LOI.pa-tients.could.represent.a.promising.therapeutic.approach.for.this.subset.of.tumors.with.IGF2.LOI.

Abstracts Posters Section 1: Tumor genetics and heterogeneity

AEK

25


Poster P-0099

V. Vafaizadeh.1,.D..Löscher.1,.V..von.Manstein.1,.T..Darvishi.1,.M..Weitmann.1,.C..M..Yang.1,.B..Groner.1

1. Georg-Speyer-Haus,.Frankfurt.am.Main,.Germany

The functional interplay between activated Stat5 and Stat3 in bre-ast cancer formation and progression and the therapeutic effects of targeted inhibition

Breast.cancer.is.the.most.common.cause.of.cancer.related.mortality.in.women.worldwide..Genetic.heterogeneity,.epigenetic.plasticity.and.the.origin.of.tumor.initiating.cells.(TICs).are.contributing.to.diverse.courses.of.the.disease.in.individual.patients.and.differences.in.the.response.to.therapeutic. interventions.. Epithelial. mesenchymal. transition. (EMT). is.thought.to.be.an.early.event.in.the.metastatic.process.and.induces.cells.with. properties. resembling. TICs.. The. reverse. process,. mesenchymal.epithelial.transition.(MET),.can.be.observed.at.sites.of.metastatic.colo-nization..Distinct.cytokine.mediated.milieus.in.the.tumor.microenviron-ment.can.contribute.to.these.phenotypic.changes..We.are.studying.the.functional.interplay.between.two.main.mediators.of.cytokine.signaling.in.the.mammary.gland,.the.signal.transducers.and.activators.of.tran-scription.Stat5.and.Stat3..They.play.central.roles. in.the.regulation.of.EMT.and.MET.and.cause.dynamic.changes.in.the.tumor.cell.epigenome..The.simultaneous.activation.of.both.Stats.has.been.detected. in.more.than.70%.of.the.ER+HER2-,.human.luminal-A.breast.cancer.subtype.We.established.a.novel.mouse.tumor.model.by.orthotopic.transplantati-on.of.mammary.stem.cells.(MaSCs).stably.expressing.an.activated.Stat5.variant..Transgenic.grafts,.persistently.expressing.P-Stat5,.undergo.pre-cocious.alveologenesis.in.virgin.animals.and.do.not.regress.properly.in.the.post-lactational. involution. stage.. Stat5.activation. counteracts. the.activation.of.Stat3,.which. is. required.as.a.pro-apoptotic. factor.during.involution.in.wild.type.glands..The.simultaneous.activity.of.both.Stat.pro-teins,.and.probably.epigenetic.alterations.induced.by.the.activity.of.the.estrogen.and.progesterone.receptors,. led.to. the. formation.of.ER+PR+.adenocarcinomas,. resembling. the.human. luminal-A.breast.cancer.sub-type..These. tumors.could.be.serially. transplanted.and.contain.a.small.fraction.of.Lin-CD24lowCD44high.TICs..We.compared.the.gene.expression.patterns.of.tumor.tissues.and.nontransformed,.P-Stat5.transgenic.grafts.to. identify. potential. Stat3. and. Stat5. target. genes. for. instrumental. in.oncogenesis..The.expression.of.some.ER.and.PR.signaling.downstreams,.such.as.amphiregulin,. Igf2,.Wnt4,.RANKL,.as.well.as.the.expression.of.some.regulators.of.EMT/MET,.Snail,.Slug.and.Klf4,.were.detected.in.the.tumor.cells..In.addition,.we.defined.the.reciprocal.effects.of.both.Stat.proteins.on.histone.marks.associated.with.gene.expression.or.repressi-on.in.two.human.luminal.tumor.cell.lines..Stat3.seems.to.induce.the.B-cell.lymphoma.gene.6,.Bcl6,.whereas.Stat5.seems.to.repress.its.activity..Our.tumor.model.allows.the.identification.of.new.genetic.and.epigenetic.biomarkers,.which.are.necessary.for.a.better.diagnosis.and.treatment.of.luminal.breast.cancer.and.prediction.of.the.risk.of.relapse.

Poster P-0106

M. Rivera.1,.C..Sers.2,.P..M..Schlag.2,.I..Fichtner.1,.J..Hoffmann.3

1. MDC,.Experimental.Pharmacology,.Berlin,.Germany.2. Charité.-.Universitätsmedizin,.Berlin,.Germany.3. EPO.GmbH,.Experimental.Pharmacology,.Berlin,.Germany

ColoNet - A Systems Biology Approach for Integrating Molecular Di-agnostics and Targeted Therapy in Colorectal Cancer. Establishment of a new cohort of patient derived early xenograft models

Background:.KRAS.mutations.occurring.in.up.to.40%.of.the.colon.car-cinoma. patients. are. exclusion. criteria. for. EGFR-directed. therapy. and.until.now.the.only.biomarkers.that.reached.the.clinics..However,.among.KRAS-wildtype.patients.only.roughly.30%.of. the.patients.show.a.po-sitive.response.and.further.reliable.biomarkers.are.urgently.needed.A.cohort.of.29.colon.carcinoma.PDX.(patient.derived.xenografts).was.extensively.characterized.regarding.its.sensitivity.towards.therapy.and.molecular. characteristics. with. the. aim. to. find. predictive. biomarkers..First.correlation.analyses.suggest.that.the.EGFR.ligands.amphiregulin.and. epiregulin. play. an. important. role. in. resistance. towards. EGFR-di-rected.therapies..In.order.to.validate.the.data.obtained.with.this.disco-very.cohort,.a.new.cohort.of.early.colon.carcinoma.PDX.was.established.and.characterized.

Results:.In.a.period.of.2.years.samples.from.fresh,.post.operative.colon.carcinoma. tissue. from. 39. patients. were. transplanted. subcutaneously.into. the. flank. of. immunodeficient. mice.. After. three. successful. in. vivo.passages.the.model.was.declared.as.engrafted..These.tumors.were.trans-planted.to.a. larger.group.of.mice,.which.were.randomized.and.treated.with.standard.of.care.chemotherapy.and.targeted.therapies.erlotinib,.ce-tuximab.and.avastin.to.assess.the.tumor.sensitivity.towards.these.drugs.In.the.case.of.18.patients.the.samples.showed.engraftment.and.could.be.characterized..The.time.for.the.first.engraftment.was.between.4.weeks.and.3.months,. further.passages.showed.the.tendency.to.grow.faster..The.response.rate.of.the.tumors.was.heterogeneous.and.comparable.to.reported.response.rates.in.clinical.trials..10/18.tumors.were.resistant.to.cetuximab.and.11/18.models.were.resistant.to.erlotinib.

Outlook:.This.newly.established.cohort.of.PDXs.will.be.used.to.validate.the.results.obtained.with.the.discovery.cohort..The.tumors.are.current-ly. characterized. for. the. most. relevant. cancer. related. mutations. and.also. the.expression.of.EGFRs.and. their. ligands..Our. focus.will. be. the.identification.of.additional.resistance.mechanisms.in.the.KRASwt.sub-group.which.is.resistant.to.the.treatment..The.data.will.be.analyzed.to.improve.the.understanding.of.EGFR.function.and.resistance.mechanisms.towards.EGFR-targeting.substances.This.well.characterized.xenograft.panel.can.be.used.for.further.studies.to.test.novel.therapies.or.combinations.for.colon.cancer.Our.results.show,.that.these.PDXs.in.early.passages.maintain.the.mor-phology.and.biology.of.the.patient.tumor,.reflect.the.heterogenous.re-sponse.rates.as.observed.in.the.patients.and.can.be.used.as.validated.preclinical.tool.for.translational.medicine.projects.

26

AEKAEK AEKAbstracts Posters Section 1: Tumor genetics and heterogeneity

Notes:Poster P-0116

T. Redmer.1,.Y..Welte.1,.R..Schäfer.2,.C..R..Regenbrecht.1,3

1. Institute.of.Pathology,.Tumor.stem.cells,.Berlin,.Germany.2. Institute.of.Pathology,.Molecular.tumor.pathology,.Berlin,.Germany.3. Laboratory.of.Functional.Genomics.(LFGC),.Berlin,.Germany.

Understanding the cellular plasticity of melanoma initiating cells

Tumor.initiating.cells.represent.a.cellular.sub-fraction.of.solid.and.hae-matological.tumors.that.is.responsible.for.the.growth.of.a.given.tumor..These.cells.are.identifiable.by.specific.signatures.of.marker.molecules..Targeting.of.those.molecules.by.neutralizing.antibodies.or.small.mole-cule.inhibitors.represent.new.promising.tools.in.cancer.therapy..Currently,. tumor. initiating.cells. in.malignant.melanoma.have.been. lin-ked.to. the.expression.of. the.neural.crest.stem.cell.marker.CD271.and.the.pentaspan.glycoprotein.and.glioma.stem.cell.marker.CD133..We.have.analysed.11.melanoma.cell. lines.including.9.primary.melanoma.derived.A375. and. MeWo. for. protein. expression. of. both. markers.. A. strong. ex-pression.of.CD271.was.observed.in.5.cell.lines.but.CD133.expression.was.present.only.in.2.out.of.11..However,.these.two.cell.lines.comprised.both.CD271. and. CD133. expressing. cells.. Flow. cytometry. and. immunofluore-scence.revealed.strong.variations.in.the.percentage.of.both.cellular.sub-sets.ranging.from.60.to.15.%.of.CD271.and.0.-.3.%.of.CD133.expressing.cells,.respectively..The.high.CD271/.CD133.ratio.found.in.melanoma.cell.cultures.may.suggest.the.derivation.of.CD133.positive.cells.from.CD271+.sub-sets.. A. change. of. the. melanoma. cell. phenotype. from. CD271+. to.CD133+.may.either.proceed.stepwise.including.an.intermediate.cell.state.or.immediately.by.a.down-regulation.of.CD271..Indeed,.the.shRNA.medi-ated.knock-down.of.CD271.did.not.contribute.to.an.increase.in.CD133.cell.surface.expression..Thus.it.seems.very.likely,.that.the.transition.needs.the. presence. of. CD271.. We. additionally. found. both. cellular. fractions.CD271+. and. CD133+. contiguous. to. melanoma. cells. bearing. a. CD271+/CD133+.phenotype..This.intermediate.cell.state.was.further.detected.in.CD271high. cell-derived. xenograft. tumors.. Furthermore,. CD271. expres-sing.cells.were.convertible.and.gave.rise.to.CD133.positive.cells.in.vitro.by.changes.in.the.growth.factor.environment..In.addition,.the.isolation.of.the.CD271+.fraction.from.melanoma.cell.cultures.that.initially.compri-sed.both.subsets.demonstrated.the.plasticity.of.melanoma.cells..Within.3. days. of. culture,. a. small. fraction. (~. 4. %). of. intermediate. cells. and.CD133+.cells. (0.8.%).had. formed..Taken.together,.our.results.suggest.a.high.plasticity.of.melanoma.initiating.cells.that.in.response.to.certain.growth.factors.either.supplemented.with.the.medium.or.secreted.by.the.tumor.stroma.can.undergo.phenotypic.changes..Thus,.therapeutic.inter-ventions.at.least.for.the.malignant.melanoma,.requires.more.than.one.molecular.marker.to.specifically.target.the.CD271.positive.sub-fraction.e.g..by.a.neutralizing.antibody.or.by.an.antibody-tagged.drug..Inhibiti-on.of.CD271-triggered.signalling.events.is.potentially.given.by.the.small.molecule.inhibitor.Ro.08-2750.that.specifically.targets.the.CD271.ligand.NGF.thereby.preventing.its.binding.to.the.receptor.

AEK

27

AEKAbstracts Posters Section 2: Oncogenes/tumor suppressor genes

Poster P-0001

U. Nitsche.1,.S..Leis.1,.C..Späth.1,.M..Maak.1,.F..G..Bader.1,.H..Friess.1,.K.-.P..Janssen.1

1. Klinikum.rechts.der.Isar,.Chirurgische.Klinik.und.Poliklinik,.München,.Germany

Molecular interactions between two pathways in colon cancer

Question:. Estimation. of. recurrence. risk. is. essential. for. patients. with.colon.cancer.in.order.to.initiate.individual.therapeutic.regimes..In.pre-ceding.work,.we.have.shown.that.even.subtle.division.of.TNM.groups.or.a.classification. including.additional.clinical.markers.has. reached.a.functional.limit.to.predict.prognosis..Today,.biomarkers.play.an.increa-sing.role.for.risk.calculation.of.patients.with.cancer..We.have.identified.a.marker.panel,.which.includes.several.gene.mutations.and.expression.levels..It.divides.colon.cancer.into.three.described.molecular.subtypes.and.thus.stratifies.patients.into.different.risk.groups.The. aim. of. this. study. was. to. investigate. the. tumor. suppressor. gene.SASH1.(SAM.and.SH3.domain.containing.1).and.the.metastasis-promoting.gene.MACC1.(metastasis.associated.in.colon.cancer.1)..In.the.cited.mar-ker.panel,.expression.levels.of.these.two.genes.individually.predicted.the. prognosis. of. patients,. and. were. inversely. correlated. with. each.other..Here,.we.characterize.colorectal.cancer.cell.lines.regarding.the.functional.connection.between.SASH1.and.MACC1.

Methods.and.Results:. In. line.with.previous.patient.data,.RNA-interfe-rence. experiments. in. SW480. cells. revealed. an. inverse. regulation. of.SASH1.and.MACC1.after.transcript-specific.knock.down..No.protein.bin-ding.among.each.other.was.detected.by. immunoprecipitation,.sugge-sting. no. regulatory. processes. by. direct. or. indirect. SASH1. and. MACC1.protein.interactions..To.investigate.differences.of.RNA.stability.as.ano-ther.possible.mechanism.of.interaction,.transcripts.of.SASH1.and.MACC1.were.quantified.by.real-time.PCR..In.SW480.cells,.novel.RNA.synthesis.was. inhibited. by. Actinomycin. D.. The. time-dependent. decline. of. tran-scripts. revealed. reduced. RNA. stability. of. MACC1. compared. to. SASH1..However,.RNA.stability.of.MACC1.was.independent.of.SASH1.expression.levels,. also.making. this.way.of. interaction.unlikely.. Finally,. global. in-hibition.of. intracellular.proteasomal.protein.degradation.by.a.specific.inhibitor.(MG132).with.consecutive.immunoblotting.resulted.in.accumu-lation.of.SASH1.protein,.but.not.MACC1.protein..Proteasome-dependent.regulation.of.SASH1.may.play.a.role.in.the.control.of.MACC1.

Conclusion:.Declining.expression.of.the.tumor.suppressor.gene.SASH1.is.linked.to.overexpression.of.the.metastasis-associated.gene.MACC1.by.complex.cellular.mechanisms..This.results. in.an. increased.recurrence.risk.for.patients.with.colon.cancer.

Poster P-0005

B. Gyorffy.1,.E..V..Broude.2,.A..Chumanevich.3,.J..F..Catroppo.4,M..Chen.2,.I..B..Roninson.2

1. Semmelweis.University,.Ist.Dept.of.Pediatrics,.Budapest,.Hungary.2. University.of.South.Carolina,.Department.of.Drug.Discovery.and.

Biomedical.Sciences,.South.Carolina.College.of.Pharmacy,.Columbia,.United.States.

3. Senex.Biotechology.Inc.,.Columbia,.United.States.4. University.of.South.Carolina,.Department.of.Pathology,.Microbiology.

and.Immunology,.School.of.Medicine,.Columbia,.United.States

Role of CDK8 gene complex in breast cancer progression and treat-ment outcome

The. oncogenic. transcription-regulating. kinase. CDK8. is. a. mediator. of.chemotherapy-induced. tumor-promoting. paracrine. activities;. higher.CDK8.expression.is.associated.with.shorter.relapse-free.survival.(RFS).in. breast. cancer. (Porter. et. al.,. PNAS,. 2012).. Immunohistochemical.analysis.of.CDK8.protein. in. tissue.arrays. from.a. total.of.320.normal,.hyperplastic,. benign. and. malignant. breast. samples. showed. that. the.average.CDK8.staining.intensity.is.significantly.higher.in.tumor.cells.of.invasive.ductal.carcinomas.of.the.breast.than.in.other.tissues..We.have.investigated. the.expression.of.CDK8,. its. isoform.CDK19,. their.binding.partner. CCNC. and. two. other. components. of. the. CDK8. module. of. the.transcriptional.Mediator.complex,.MED12.and.MED13.(CDK8.gene.com-plex).using.microarray.data.from.3,491.breast.cancers..CDK8.expression.was.positively.correlated.with.the.expression.of.CDK19,.CCNC.and.MED13.but. negatively. correlated. with. MED12.. CDK8. expression. was. strongly.increased.in.ER-.relative.to.ER+.breast.cancers.and.inversely.correla-ted.with.the.expression.of.ESR1.(ER-alpha)..An.inverse.correlation.for.ESR1.expression.has.also.been.observed.with.CCNC.and.CDK19,.but.not.with.MED13.or.MED12..We.have.analyzed.RFS.correlations.of.the.CDK8.gene.complex.using.as.a.positive.controlTOP2A,.a.proliferation.marker.found.earlier.to.be.the.strongest.negative.predictor.of.RFS.(Gyorffy.et.al.,.Breast.Cancer.Res.Treat,.2010)..Higher.expression.of.TOP2A.was.a.strong.predictive.marker.of.shorter.RFS.among.both.treated.and.untre-ated.ER+.patients.but.not.among.ER-.patients..In.contrast,.CDK8.showed.a. weaker. correlation. with. shorter. RFS. among. systemically. untreated.ER+.patients,.no.correlation.among.untreated.ER-.patients,.but.a.very.strong.correlation.with.poor.RFS.among.the.treated.patients,.both.ER+.and. ER-.. These. findings. indicate. that. CDK8. expression. is. associated.with. treatment. failure. in.breast.cancer,. in.agreement.with. its. role. in.chemotherapy-induced. tumor-promoting. paracrine. activities.. In. con-trast,.MED12.showed.the.opposite.prognostic.correlation,.higher.levels.of.MED12.being.associated.with.longer.RFS,.especially.in.the.untreated.patients..This.positive.prognostic.correlation.of.MED12.can.be.explained.by.its.inhibitionof.the.TGFbpathway,.an.effect.distinct.from.its.transcrip-tional.activity. in. the.Mediator.complex.(Huang.et.al.,.Cell,.2012)..Our.findings.warrant.further.investigation.and.the.potential.use.of.the.CDK8.gene.complex.as.diagnostic.markers.predictive.of.therapy.outcome.

28

AEKAEK AEKAbstracts Posters Section 2: Oncogenes/tumor suppressor genes

Poster P-0009

A. Honegger.1,.J..Leitz.1,.J..Bulkescher.1,.K..Hoppe-Seyler.1,.F..Hoppe-Seyler.1

1. German.Cancer.Research.Center,.Molecular.Therapy.of.Virus-Associated.Cancers,.Heidelberg,.Germany.

Silencing of Human Papillomavirus (HPV) E6/E7 Oncogene Expression Affects both the Contents and Amounts of Exosomes Released from HPV-positive Cancer Cells

Question:.The.human.papillomavirus.(HPV).E6/E7.oncogenes.play.a.cru-cial.role.for.HPV-induced.carcinogenesis..Exosomes.are.small.vesicles.of.endosomal.origin.that.are.secreted.by.many.cells,. including.tumor.cells..Here,.we.investigated.whether.silencing.of.endogenous.HPV.E6/E7.expression.may.influence.the.contents.or.amounts.of.exosomes.re-leased.from.HPV-positive.cancer.cells.

Methods:.Exosome.purification.and.characterization,.RNA.interference

Results:.We.found.that.exosomes.secreted.from.HeLa.cells.are.enriched.for. Survivin. protein.. RNA. interference. studies. revealed. that. mainte-nance.of.both.intracellular.and.exosomal.Survivin.amounts.was.strongly.dependent.on.continuous.E6/E7.expression..This.indicates.that.intracel-lular.HPV.activities.are.translated.into.visible.alterations.of.exosomal.protein.contents..Besides.Survivin,.HeLa.exosomes.contain.additional.members.of.the.inhibitor.of.apoptosis.protein.(IAP).family.(XIAP,.c-IAP1,.Livin)..In.contrast,.we.did.not.obtain.evidence.for.an.exosomal.location.of.the.HPV.E6.and.E7.oncoproteins..Moreover,.we.found.that.silencing.of.HPV.E6/E7.expression.led.to.a.significant.increase.in.exosome.amounts.released.from.HeLa.cells..This.effect.was.associated.with.the.reinduc-tion. of. p53,. stimulation. of. the. p53. target. genes. TSAP6. and. CHMP4C.which.can.enhance.exosome.production,.and.induction.of.senescence.

Conclusion:.Taken.together,.our.results.show.that.silencing.of.HPV.E6/E7.oncogene.expression.profoundly.affects.both. the.composition.and.amounts. of. exosomes. secreted. by. HPV-positive. cancer. cells.. This. in-dicates.that.HPVs.can.induce.molecular.signatures.in.exosomes.which.may.affect.intercellular.communication.and.could.be.explored.for.dia-gnostic.purposes.

Poster P-0019

E. Zeller.1

1. Institut.für.experimentelle.und.klinische.Pharmakologie.und.Toxikologie,.Abteilung.Toxikologie,.Tübingen,.Germany

Long non-coding RNA Gtl2/Meg3 and its role in hepatic cancer

Carcinogenesis.is.a.progressive.multistep.progress.comprising.various.genetic.alterations.such.as.mutations. in.tumor.suppressor.genes.and.oncogenes..Non-genotoxic.carcinogens.(NGCs).induce.tumor.formation.by. mechanisms. other. than. changes. in. the. underlying. DNA. sequence..Phenobarbital. (PB). is. a. classic. non-genotoxic. carcinogen. leading. to.perturbations.in.gene.expression.and.DNA.methylation..Recently,.non-coding.RNAs.from.the.Dlk1-Dio3.cluster.were.identified.as.potential.bio-markers.for.mouse.liver.tumor.promotion.caused.by.PB..One.gene.within.this.imprinted.region.is.named.Gtl2/Meg3..In.humans,.loss.of.MEG3.ex-pression.is.found.in.various.primary.tumors,.but.not.in.hepatoblastoma,.a.malignant.liver.neoplasm.occurring.in.infants.and.children.

We.noted.that.Meg3.is.overexpressed.in.PB-promoted.mouse.liver.tu-mors.as.well.as.in.the.majority.of.human.hepatoblastoma.samples.con-taining. activating. CTNNB1-mutations.. In. murine. PB-treated. liver. only.one.of.the.three.transcript.variants.mentioned.in.the.NCBI.database.was.expressed..This.transcript,.however,.could.not.be.detected.in.cell.cul-ture..Thus.a.Meg3.expression.vector.was.created.and.stably.transfected.into.two.different.mouse.hepatoma.cell.lines.leading.to.several.sublines.with.strong.Meg3.expression..We.showed.that.Meg3.overexpression.led.to.p53.protein.accumulation.and.activation..Additionally,.apoptosis.was.affected.by.Meg3..Furthermore,.we.compared.Meg3.transfected.and.un-transfected.cells.concerning.putative.targets.and.signaling.pathways.

AEK

29


Poster P-0024

M. Laible.1,.M..Faelth-Savitski.1,.L..Kacprzyk.1,.S..Blaich.1,.H..Sültmann.1

1. DKFZ/NCT,.Cancer.Genome.Research,.Heidelberg,.Germany

Functional analysis of TMPRSS2:ERG fusion gene variants in prostate cancer cell lines

The.TMPRSS2-ERG.fusion.gene.is.found.in.~50%.of.all.prostate.cancers..This.fusion.event.results.in.high.expression.of.the.transcription.factor.ERG.as.it.is.set.under.the.control.of.the.androgen.responsive.TMPRSS2.promoter..Fusion.positive.tumors.show.a.clearly.altered.gene.expressi-on.pattern.and.therefore.represent.a.distinct.subtype.of.prostatic.tu-mors..However,.it.is.not.yet.well.understood.which.role.the.fusion.event.plays. in.tumor.formation.and.whether. it.confers.a.growth.advantage..Recent.studies.have. indicated. the. involvement.of.ERG. in. the.process.of.EMT..Furthermore,.several.variants.of.the.TMPRSS2:ERG.(T/E).fusion.gene. have. been. described.. Within. the. ERG. positive. subgroup. tumors.with.a.T/E.VI.variant.showed.a.more.aggressive.phenotype.than.tumors.carrying.other.T/E.variants..To.study.differences. in. the.molecular.ef-fects.of.T/E.variants,.we.chose.to.compare.three.T/E.variants.by.induci-ble.overexpression.in.the.prostate.cell.lines.LNCaP.and.RWPE1.Successful.overexpression.was.confirmed.by.Western.blot.and.the.func-tionality.of.the.ORFs.was.evaluated.by.measuring.mRNA.levels.of.known.ERG.downstream.targets..A.set.of.genes.deregulated.in.T/E.tumors.were.selected. from. a. previous. expression. profiling.. mRNA. levels. of. these.genes.was.measured.after. induction.of.ERG. in. the.cell. lines..Most.of.the.candidate.genes.were.regulated.in.the.same.direction.as.expected.based.on.the.microarray.data,.indicating.a.good.concordance.of.the.in.vitro.model.with.the.regulatory.function.of.ERG.in.prostate.tumors..Fur-ther.cell.culture.experiments.showed.a.decrease.in.viability.upon.ERG.overexpression.in.LNCaP.and.RWPE1..Accompanying.this.drop.in.viabili-ty,.LNCaP.cells.exhibited.strong.morphological.changes,.characterized.by.loss.of.cell-cell.adhesion..In.summary,.we.have.created.a.model.for.studying.induction.of.EMT-like.characteristics.by.the.ERG.transcription.factor..Further.experiments.will.focus.on.the.differences.found.between.the.T/E.variants.

Poster P-0055

C. Panke.1,.D. Weininger.1,.F..Schelter.1,.A..Haas.2,.T..Schlothauer.2,.S..Bader.2,.S..Ranjan.2,.H.-P..Josel.3,.U..Baer.1,.H..Burtscher.1,.O..Mundigl.2,.M..Grote.2,.U..Brinkmann.2,.C..Sustmann.1

1. Roche.Diagnostics.GmbH,.Pharma.Research.And.Early.Development.(pRED).–.Discovery.Oncology,.Penzberg,.Germany.

2. Roche.Diagnostics.GmbH,.Pharma.Research.And.Early.Development.(pRED).–.Large.Molecule.Research,.Penzberg,.Germany.

3. Roche.Diagnostics.GmbH,.Professional.Diagnostics,.Penzberg,.Germany

Bispecific antibodies for FACS-based quantitation of oncogenic cell surface proteins

Absolute.quantitation.of.cell.surface.receptor.levels.is.not.only.a.prere-quisite.for.understanding.the.biology.of.oncogenic.signaling.networks.but.it.is.also.of.great.importance.for.the.analysis.of.drug-antigen.expo-sure.and.for.the.definition.of.minimal.residual.diseases..Flow.cytome-try. is. the.gold.standard. for. fast.and.accurate.quantitation.of.cellular.protein.levels..However,.it.requires.fluorescently.labeled.antibodies.as.well. as. calibration. standards.. A. critical. step. for. quantitation. still. re-mains.the.production.of.suitable.detection.antibodies.with.a.precisely.defined.ratio.of.antigen.binding.sites.to.fluorophores..Problems.often.arise.as.consequence.of. inefficient.and.unspecific. labeling.which.can.influence.antibody.properties..Additionally,.the.number.of.incorporated.fluorophores.usually.resembles.a.Gaussian.distribution,.requiring.a.spe-cial.normalization.step.for.quantitation..To.address.these.problems,.we.constructed.different.mono-.and.bivalent.bispecific.antibodies.with.bin-ding.site(s).for.different.oncogenic.receptor.tyrosine.kinases.and.with.an. additional. hapten-binding. scFv. fusion.. As. antigens. of. interest. we.chose.the.receptor.tyrosine.kinases.cMet,.ErbB2/Her2.and.ErbB3/Her3..These. receptors. are. prominent. oncogenes. and. therefore. pursued. as.drug.targets.in.the.clinic..For.detection.of.these.receptors.we.covalently.coupled.the.fluorophore.Cy5.to.the.respective.hapten.which.was.quanti-tatively.bound.by.the.bispecific.antibody..A.panel.of.tumor.cell.lines.was.assessed. for.absolute. receptor.expression. levels.of. the.antigens.and.the.data.was.set.in.relation.to.mRNA,.gene.count.and.immunoblot.data..We.could.reproducibly.quantify.these.receptors.and.omit.the.otherwise.required.normalization.step..Furthermore,. these.antibodies.were.also.used.for.quantitation.of.proteins.in.intracellular.vesicles,.using.confocal.microscopy..In.summary,.this.approach.significantly.improves.receptor.quantitation. thereby.providing. the.basis. for.understanding.oncogenic.tyrosine.kinase.receptor.signaling.networks.

30


Poster P-0062

R. Mejias-Luque.1,.M..Vieth.2,.A..Jung.3,.M..Gerhard.1

1. Institut.für.medizinische.Mikrobiologie,.Immunologie.und.Hygiene,.Technische.Universität.München,.München,.Germany.

2. Institut.für.Pathologie..Klinikum.Bayreuth.GmbH,.Bayreuth,.Germany.3. Pathologisches.Institut,.Medizinische.Fakultät..Ludwig-Maximilians-

Universität.München,.München,.Germany

Mutual regulation of STAT3 and Sox2 in gastric cancer

Gastric.cancer.is.one.of.the.most.common.causes.of.cancer.related.de-ath.worldwide,.which.is.mainly.due.to.late.diagnosis.and.poor.treatment.options.. Infection.with.H..pylori.and.genetic.alterations. influence.the.risk.of.developing.gastric.tumours..However,.the.molecular.mechanisms.involved.in.gastric.carcinogenesis.are.still.not.fully.understood.

Constitutive. STAT3. activation. is. a. key. event. in. the. development. and.progression.of.gastric.cancer.since.it.promotes.gastric.tumorigenesis.in.both.human.adenocarcinomas.and.mouse.models..Apart.from.its.role.in. cancer,.STAT3.has.been.also. reported. to. regulate. self-renewal.and.pluripotency.of.embryonic.stem.cells,.binding.to.enhancer.sequences.of.target.genes.along.with.the.pluripotency.transcription.factors.Oct4.and.Sox2..Of.these,.Sox2.activates.the.expression.of.gastric.differenti-ation.markers,.suggesting.that.it.may.control.differentiation.of.gastric.epithelial.cells..On.the.other.hand,.Sox2.has.been.also.identified.as.an.adult.gastric.stem.cell.marker.in.mice..Since.altered.expression.of.Sox2.has.been.observed.in.gastric.tumors,.we.aimed.at.studying.a.possible.crosstalk.or.interaction.of.STAT3.activation.and.Sox2.in.gastric.tumori-genesis

To.firstly.assess.the.degree.and.pattern.of.Sox2.expression.in.relation.to.STAT3.activation,.we.analysed.the.expression.of.Sox2.and.p-STAT3.in.31.gastric.tumor.samples,.and.observed.that.32.3%.of.the.cases.showed.co-expression.of.both.transcription.factors..Furthermore,.we.detected.increased. levels.of.Sox2. in.tumors.from.gp130F/F.mice.with.hyperacti-vated. STAT3.. Interestingly,. Sox2. and. p-STAT3. were. also. co-expressed.in.gastric.cancer.cell. lines,.suggesting.that.STAT3.could.be.regulating.Sox2.expression.in.the.stomach..Unexpectedly,.no.changes.in.Sox2.pro-tein. expression. were. detected. after. transfecting. gastric. cancer. cells.with.different.STAT3.constructs..However,.Sox2.transcriptional.activity.increased.in.a.dose.dependent.manner.after.STAT3.wt.or.constitutively.active.STAT3.transfection..On.the.other.hand,.we.also.observed.that.in.turn.Sox2.increased.STAT3.transcriptional.activity,.suggesting.that.both.transcription.factors.can.regulate.each.other.in.gastric.cancer..Further.experiments.will.be.carried.out.in.order.to.elucidate.the.molecular.me-chanism.by.which.Sox2.and.p-STAT3.regulate.each.other.and.the.effect.on.target.genes.important.for.the.development.and.progression.of.gas-tric.tumors.

Poster P-0087

W. Roos.1,.C..Barckhausen.1,.E..Jöst.1,.S..Naumann.1,.M..Christmann.1,.B..Kaina.1

1. Institute.of.Toxicology,.University.Medicine.Mainz,.Mainz,.Germany.

Malignant melanomas: role of MGMT and pro-caspase-8 in their in-trinsic resistance to alkylating agents

Malignant.melanoma.has.a.dismal.prognosis.because.of.its.intrinsic.re-sistance.to.therapy..First.line.chemotherapeutics.include.the.methyla-ting.agents.dacarbazine.(DTIC).and.temozolomide.(TMZ)..Using.a.panel.of.eleven.cell.lines.we.showed.that.TMZ.induces.primarily.apoptosis.in.melanoma.cells..The.MGMT.activity. in.the.cell. lines.varied.between.0.and.1100.fmol/mg.protein,.and.MGMT.activity.of.the.cell.lines.correlated.with.the.level.of.resistance.to.TMZ..Additionally,.MGMT.inactivation.sen-sitized.while.MGMT.transfection.protected.melanoma.cells..This.shows.that. O6-methylguanine. (O6MeG). is. the. critical. DNA. lesion. triggering.apoptosis.in.melanoma.cells..One.cell.line,.derived.from.a.patient.sub-jected.to.DTIC.therapy,.exhibited.a.high.level.of.resistance.to.TMZ.not.because.of.MGMT.expression.but.because.of. lack.of.MSH2.and.MSH6.mediated.mismatch.repair.(MMR)..As.there.is.no.relationship.between.MGMT.or.MMR.and.therapeutic.outcome,.the.data.points.to.additional.mechanisms. whereby. melanomas. acquire. resistance. to. these. drugs..Melanoma.cell. lines.differing. in. their.p53.status.do.not.differ.signifi-cantly.in.their.apoptosis.response.to.TMZ..Our.data.show.that.p53.wild-type.melanoma.cells.respond.to.O6MeG.by.up-regulating.the.Fas/CD95/Apo-1.receptor.–.this.however.does.not.lead.to.the.activation.of.the.apo-ptosis. cascade.. Furthermore,. the. data. show. that. melanomas. prevent.death.receptor.triggered.apoptosis.by.silencing.pro-caspase-8..The.role.of.pro-caspase-8.in.melanoma.resistance.was.verified.by.expression.of.dominant-negative.FADD,.siRNA.knockdown.of.pro-caspase-8.or.stimu-lation. with. a. Fas/CD95/Apo-1. activating. antibody.. A. single. treatment.with. interferon-β. (IFN-β). reactivated. pro-caspase-8. and. sensitized.melanoma.cells.to.TMZ..The.expression.of.pro-caspase-8.could.further.be. enhanced. by. additional. pre-treatment,. in. combination. with. IFN-β,.with. the. HDAC. inhibitor. valproic. acid. (VPA),. which. caused. significant.sensitization.in.vitro.and.in.a.xenograft.mouse.model..The.data.suggest.approaches.for.abrogating.intrinsic.drug.resistance.by.inhibiting.MGMT.and.reactivating.pro-caspase-8.expression.during.therapy.of.malignant.melanoma..Work.is.supported.by.DFG.KA724.and.DFG.RO3617.

AEK

31


Poster P-0102

V. Begay.1,.K..Zimmermann.2,.B..Cirovic.1,.S..Kaufer.1,.U..Leser.2,3,.A..Leutz.1,3,4

1. Max.Delbrück.Center.for.molecular.medicine,.Cell.differentiation.and.tumorigenesis,.Berlin,.Germany.

2. Institute.for.Computer.Science,.Berlin,.Germany.3. Humbold-University,.Berlin,.Germany.4. Institute.of.Biology.Brandenburg.center.for.Regenerative.Therapies,.

Berlin,.Germany.

Deregulated expression of the C/EBPbeta LIP isoform predisposes to tumorigenesis: implication for the tumor microenvironment

The. CCAAT. enhancer. binding. protein. beta. (C/EBPb),. a. leucine. zipper.transcription. factor,. controls. proliferation,. differentiation,. apoptosis.and.senescence.in.several.cell.types.and.is.involved.in.immunity,.female.reproduction,.bone,.glucose.and.fat.metabolism..Three.C/EBPb.isoforms.(LAP*,.LAP,.and.LIP).that.differ.in.N-terminal.length.and.gene.regulatory.capacity.are.generated.by.alternative.translation.initiation.from.a.sin-gle.intronless.mRNA..Expression.of.the.truncated.C/EBPb.LIP.isoform,.lacking.the.transactivation.domain,.is.regulated.by.the.rapamycin.sen-sitive.TORC1.complex.and.the.RNA.binding-protein.CUGBP1..C/EBPb.LIP.is.thought.to.dominantly.interfere.with.the.differentiation.inducing.and.proliferation. arresting. members. of. the. C/EBP. family.. Upregulation. of.LIP.has.been.found.in.many.human.tumor.biopsies..Here,.we.show.that.deregulated.expression.of.the.C/EBPb.LIP. isoform.from.its.own. locus.enhances.tumor.development.in.mesenchymal.and.epithelial.tissues.in.mice,.with.B-NHL. lymphoma.being.the.prevalent.tumor..Expression.of.C/EBPb.isoforms.was.high.in.lymphoma.cells.of.wild.type.(WT).and.LIP.mutant.mice,.but.low.in.CD19+.B.cells.sorted.before.tumor.onset.from.all.genotypes..The.LIP:LAP.ratio.was.dramatically.increased.in.lymphomas.compared.to.CD19+.B.cells,.suggesting.that.increase.in.LIP.isoform.ex-pression.contributes.to.disease.development.or.maintenance.also.in.WT.mice..Deregulated.C/EBPb.LIP.expression.leads.to.a.reduced.apoptotic.rate.in.both.B.cells.before.tumor.onset.and.lymphoma..Since.the.incre-ase.of.the.LIP:LAP.ratio.is.under.the.control.of.mTOR.and.chronic.activa-tion.of.mTOR.leads.to.tumorigenesis,.gene.expression.profiling.analysis.was.conducted.on.lymphoma.found.in.LIP.heterozygous.mice.and.in.WT..The.preliminary.data.revealed.that.expression.of.cytokines.and.chemo-kines.are.altered.in.C/EBPb.LIP.mutants,.suggesting.a.potential.change.in.the.tumor.microenvironment..Our.results. imply.that. imbalanced.C/EBPb.LIP.isoform.expression.is.a.key.event.in.various.types.of.tumors.

Poster P-0102

A. Jou.1,.C..Shao.2,.E..Orlova.1,.C..Herold-Mende.3,.P..Rados.4,.P..Lichter.1,.K..Freier.3,.M..Gross.1

1. DKFZ,.Molecular.Genetics,.Heidelberg,.Germany.2. Bioquant,.Heidelberg,.Germany.3. Heidelberg.University.Hospital,.Heidelberg,.Germany.4. Universidade.Federal.do.Rio.Grande.do.Sul,.Porto.Alegre,.Germany.

Functional analysis on the importance of stem cell-associated tran-scription factor SOX2 in the pathogenesis of HNSCC

Head. and. neck. squamous. cell. carcinoma. (HNSCC). is. the. 6th. most.common.solid.malignant. tumour,.affecting.more. than.900000.people.worldwide.per.year..The.5-year.survival. rate.of.55%.did.not. improve.in. the. last.years..Among.the.genetic. imbalances.reported. for.HNSCC,.amplifications.of.chromosomal.regions.3q26.were.associated.with.bad.prognosis..In.previous.work.of.our.group,.it.was.observed.that.in.40.oral.squamous.cell.carcinoma,.11.had.amplification.of.3q26.. In.all.of.those.11.carcinomas,.a.small.region.of.0,85.Mbp.was.detected.as.a.common.amplified.region.with.SOX2.as. the.only.candidate.gene. located..SOX2.is.a.transcription.factor.that.is.involved.in.the.stabilization.of.embryo-nic.stem.cells.in.their.pluripotent.state.and.in.reprogramming.somatic.cells..It.was.implicated.in.proliferation,.apoptosis.and.migration.in.se-veral.types.of.cancer;.however,.the.role.of.SOX2.in.HNSCC.is.not.clear..We.performed.SOX2.knock.down.in.the.HNO223.tongue.carcinoma.cell.line..Microarray.analysis.identified.altered.expression.of.714.genes.after.SOX2.knock.down.with.258.upregulated.and.456.downregulated..Preli-minary.data.of.the.functional.profile.showed.immune.response.as.the.most.frequently.altered.biological.process..Others.biological.processes.found.deregulated.are.apoptosis.and.cell.proliferation..Confirmation.of.this.data.by.other.methods.is.currently.being.done..Upon.SOX2.knock.down,. cells. showed. decrease. in. S. phase. (36%). compared. to. control.cells.(62%).after.treatment.with.doxorubicin..More.functional.studies.will.be.performed.to.further.elucidate.the.role.of.SOX2.in.HNSCC.

32


Poster P-0122

J. Winkler.1,.K..Holzer.1,.E..Eiteneuer.1,.C..Sticht.2,.J..Lorenzo.3,.W..Roth.1,.R..Geffers.4,.N..Gretz.2,.K..Breuhahn.1,.P..Schirmacher.1,.S. Singer.1


2. University.of.Heidelberg,.Medical.Faculty.Mannheim,.Medical.Research.Center,.Mannheim,.Germany.

3. University.Hospital.Heidelberg,.Medical.Biometrics.and.Informatics,.Heidelberg,.Germany.

4. Helmholtz.Centre.for.Infection.Research,.Braunschweig,.Germany

Regulation and function of the nuclear transport factor Cellular Apo-ptosis Susceptibility (CAS) in hepatocellular carcinoma

Eukaryotic.cells.fundamentally.rely.on.a.highly.efficient.and.selective.nucleocytoplasmic.transport.system..Proteins.of.the.karyopherin-beta.family.including.importins.and.exportins.represent.an.essential.part.of.the.nucleocytoplasmic.transport.machinery..Growing.evidence.suggests.that.aberrant.expression.and.function.of.nuclear.transport.factors.can.contribute.to.cancer.formation..Here,.we.studied.the.function.and.regu-lation.of.the.exportin.Cellular.Apoptosis.Susceptibility.(CAS).in.hepato-cellular.carcinoma.(HCC).

Own.expression.profiling.data.of.karyopherin.beta. family.members. in.HCC.and.non-tumorous.liver.tissue.samples.revealed.that.CAS.is.one.of.the.most.significantly.deregulated.nuclear.transport.factors.in.HCC..We.could.validate.overexpression.of.CAS.in.HCC.on.mRNA.and.protein. le-vel.by.using.qRT-PCR.and.immunohistochemistry,.respectively.and.also.found.a.correlation.with.tumor.de-differentiation.(r=0.471;.p<0.01)..CAS.depletion.by.RNAi.in.HCC.cell.lines.(HLE.and.HLF).significantly.decrea-sed.tumor.cell.viability,.proliferation,.migration,.and.invasion.and.led.to.an.increase.in.cell.death..Based.on.microarray.data.we.could.identify.the. X-linked. inhibitor. of. apoptosis. (XIAP). as. a. potential. downstream.target.of.CAS.and.direct.knockdown.of.XIAP.partially.phenocopied.the.effects.observed.upon.CAS.depletion..Furthermore,.XIAP.and.CAS.mRNA.expression. levels. were. correlated. in. HCC. patients. (r=0.438,. p<0.01).supporting. the. in.vivo.relevance.of. the.findings..Finally,.we.observed.that.CAS.expression.is.correlated.with.nuclear.accumulated.p53.in.HCC.tissue.samples.(r=0.422,.p<0.01).and.hypothesized.that.CAS.could.be.a.repression.target.of.wild-type.p53..Indeed,.p53.induction.in.HepG2.cells.by.using.Nutlin.or.Camptothecin.led.to.reduced.CAS.mRNA.and.protein.levels.

Our.data.suggest.that.CAS.is.required.to.maintain.a.malignant.pheno-type.in.HCC.cells.and.that.the.pro-survival.effects.of.CAS.are.partially.linked.to.XIAP..In.the.light.of.previous.studies.describing.pro-apoptotic.functions.of.CAS.under. stressed.conditions. the. role.of.CAS.seems. to.be.context-dependent,.which.may.affect.its.potential.as.a.therapeutic.target.

Poster P-0129

I. Augustin.1,.V..Goidts.1,.A..Bongers.1,.G..Kerr.1,.B..Radlwimmer.1,.C..Hartmann.1,.C..Herold-Mende.1,.A..von.Deimling.1,.G..Reifenberger.1,.M..Boutros.1

1. DKFZ,.Signaling.and.Functional.Genomics,.Heidelberg,.Germany

The Wnt secretion factor Evi/Gpr177 promotes glioma tumorigenesis

Malignant.astrocytomas.are.highly.aggressive.brain.tumors.with.poor.prognosis..Despite.multimodal.therapy,.including.surgical.resection.fol-lowed. by. combined. radio-. and. chemotherapy,. the. median. survival. of.glioblastoma.patients.has.remained.as.low.as.12-14.months.throughout.the. past. decade.. While. a. number. of. structural. genomic. changes. and.dysregulation. of. signaling. pathways. in. gliomas. have. been. described,.the.identification.of.biomarkers.and.druggable.targets.remains.an.im-portant.task.for.novel.diagnostic.and.therapeutic.approaches..Recent.evidence.supports.the.involvement.of.canonical.and.non-canonical.Wnt.signaling. in. glioma. development. and. malignant. progression.. Evi/Wls/Gpr177,.a.highly.conserved.seven-pass.transmembran.protein,.is.a.core.component.of.the.Wnt.signaling.pathway.. It. is.an.essential.Wnt.cargo.receptor.required.for.pan-Wnt.protein.secretion,.affecting.both.canoni-cal.and.non-canonical.signaling..Here,.we.show.that.Evi.is.tumor-stage.independently.overexpressed.in.astrocytic.gliomas.suggesting.that.Evi.is.involved.in.the.earliest.stages.of.glioma.tumorigenesis..Depletion.of.Evi.expression.in.glioma.and.glioma-derived.stem-like.cells.led.to.decre-ased. cell. proliferation. and. apoptosis.. Correspondingly,. transcriptome.profiling. identified. a. strong. transcriptional. downregulation. of. genes.associated.with.cell.cycle.regulation.after.Evi.silencing..Migration.expe-riments.revealed.a.reduced.capacity.for.migration.of.glioma.cells.upon.Evi. silencing. and. Evi. depletion. also. reduced. tumor. growth. of. human.glioma. cells. after. xenotransplantation. in. mice.. We. further. show. that.Evi.overexpression.is.sufficient.to.promote.downstream.Wnt.signaling..Taken.together,.our.study.identifies.Evi.as.an.essential.regulator.of.gli-oma.tumorigenesis,.and.offering.novel.therapeutic.options.to.interfere.with.the.aberrant.regulation.of.growth.factors.at.the.site.of.production.

AEK

33


Notes:Poster P-0131

A. Saalfrank.1,.T..Flisikowska.1,.S..Leuchs.1,.S..Eser.2,.E..Wolf.3,.D..Saur.2,.A..Schnieke.1

1. TU.München,.Lehrstuhl.Biotechnologie.der.Nutztiere,.Freising,.Germany.

2. TU.München,.Klinikum.Rechts.der.Isar.II,.München,.Germany.3. LMU.München,.Molecular.Animal.Breeding.and.Biotechnology,.

München,.Germany

The pig as a model for human cancer

Cancer. is. a. leading. cause. of. human. death. and. morbidity. worldwide..Most.animal.models.of.solid.cancers.are.in.rodents,.particularly.geneti-cally.engineered.mice..However,.mice.differ.significantly.from.humans.in.size,.lifespan,.physiology,.anatomy.and.diet,.limiting.their.usefulness.for.some.pre-clinical.studies..Pigs.are.increasingly.recognised.as.a.va-luable.adjunct.to.biomedical.research..Our.aim.is.to.provide.a.series.of.genetically-defined. pigs. that. model. serious. and. common. human. can-cers..These.will.allow.new.diagnostic.and.therapeutic.strategies.to.be.investigated.at.human.scale,.and.longitudinal.studies.under.conditions.that.mimic.the.human.patient..Here.we.report.gene-targeted.pigs.car-rying.mutations.in.two.key.tumour.suppressor.genes:.adenomatous.po-lyposis.coli.(APC).and.tumour.protein.53.(TP53).

APC.plays.a.vital.initiating.role.in.both.sporadic.colorectal.cancer.(CRC).(1).and.the. inherited.predisposition.to.colorectal.cancer,. familial.ade-nomatous.polyposis.(FAP).(2)..We.generated.gene-targeted.cloned.pigs.carrying. nonsense. mutations. in. APC. at. sites. orthologous. to. human.germline. mutations. responsible. for. FAP.. At. one. year. old. the. APC1311.mutation.resulted. in. >60.polyps. in. the.colon.and.rectum..Histological.and.molecular.analysis.showed.that.the.porcine.model.recapitulates.all.major.features.of.early.stage.human.FAP.(3).

Somatic.mutations.affecting.p53. function.are.present. in.most.human.cancers,.and.germline.TP53.mutations.are.responsible.for.Li-Fraumeni.multiple.cancer.syndrome..A.survey.of.somatic.p53.mutations.reveals.R175H.as.the.most.frequent.missense.mutation.in.many.sporadic.human.cancers.(4)..We.created.gene-targeted.pigs.carrying.a.latent.TP53R167H.mutant.allele.orthologous.to.human.mutant.TP53R175H.that.can.be.ac-tivated.by.Cre.recombination.to.model.oncogenic.mutant.p53.found.in.sporadic.human.cancers.(5).

We.are.confident.that.both.these.pig.models.will.make.significant.con-tributions.to.human.oncology.

References:•. Powell,.S.M.,.et.al..APC.mutations.occur.early.during.colorectal.tumo-

rigenesis..Nature.359,.235–237.(1992)•. Kinzler,. K.W.,. et. al.. Identification. of. FAP. locus. genes. from. chromo-

some.5q21..Science.253,.661–665.(1991)•. Flisikowska,.T.,.et.al..A.Porcine.Model.of.Familial.Adenomatous.Poly-

posis..Gastroenterology,.143,.1173–1175.(2012)•. Petitjean,. A.,. et. al.. Impact. of. mutant. p53. functional. properties. on.

TP53.mutation.patterns.and.tumor.phenotype:. lessons. from.recent.developments. in. the. IARC. TP53. database.. Hum. Mutat. 28,. 622–629.(2007)

•. Leuchs,.S..et.al..Inactivation.and.Inducible.Oncogenic.Mutation.of.p53.in. Gene. Targeted. Pigs.. PLosOne. 7(10):. e43323.. doi:10.1371/journal.pone.0043323

34

AEKAEK AEKAbstracts Posters Section 3: Invasion and metastasis

Poster P-0008

S. Neuber.1,.M..Meyer.1,.V..Wischmann.1,.R..Moll.1,.A. Schmidt.1

1. Philipps.University.of.Marburg,.Institute.of.Pathology,.Marburg,.Germany

Plakophilin 3 is phosphorylated at Y195 by reactive oxygen species (ROS)-activated c-Src kinase but not by growth factors

An.essential.component.of.desmosomal.adhesion.comprises.the.three.related.plakophilins.(PKP1.to.PKP3)..PKPs.are.desmosomal.plaque.pro-teins.that.are.also.thought.to.be.involved.in.particular.signaling.events.and.possible.also.in.malignant.transformation..Dynamics.of.the.turno-ver.of. junctional.complexes.are.often.fundamentally.disturbed.during.the.processes.of.tumorigenesis,.invasion.and.metastasis.of.tumor-cells..Modifications.especially.of.tyrosine.residues.are.of.importance.for.the.regulation. of. the. junctional. and. cytoskeletal. remodeling. and. may. be.deregulated.in.malignant.processes..Here.we.analyze.a.modification.of.PKP3.by.a.specific.tyrosine.phosphorylation.related.to.some.of.these.aspects.

Taking. advantage. of. available. information. for. PKP3. in. phosphotomic.databases,.we.generated.antibodies.that.specifically.detect.Tyr-phos-phorylated.PKP3.at.Y195.that.may.be.of.special.interest.for.PKP3.regu-lation..These.antibodies.were.employed.for.the.immunobiochemical.and.immunohistochemical.analysis.of.the.respective.Tyr-phosphorylation.of.PKP3.and.the.identification.of.the.responsible.kinase.

Tyr-phosphorylation. of. PKP3. appears. only. transiently. under. normal.conditions.in.epithelial.cells..Treatment.of.cells.with.phosphatase.inhibi-tor.pervanadate.revealed.that.PKP3.is.actually.phosphorylated.at.Y195..To.identify.conditions.that.lead.to.Y195.modification.we.incubated.cells.with.different.growth.factors.(EGF,.IGF1,.HGF).which.did,.however,.not.lead.to.a.specific.phosphorylation.of.Y195,.excluding.some.receptor.ty-rosine.kinases.to.be.responsible..Using.specific.inhibitors.of.Src-family.kinases.on.cultured.cells.we.were.able.to.narrow.responsible.kinases.down.to.Src-kinase.acting.at.Y195.of.PKP3..As.a.proof,.isolated.Src.ki-nase.was.able.to.phosphorylate.recombinant.PKP3.polypeptide.at.Y195.in.in-vitro.assays..When.looking.for.conditions.that.trigger.Y195-phos-phorylation.by.Src-kinase,.we.identified.ROS.as.one.cause..Incubation.of.cells.with.hydrogen.peroxide.led.to.a.rapid.phosphorylation.at.Y195.that.could.be.eliminated.when.Src-kinase.inhibitors.were.additionally.deplo-yed..Finally,.we.detected.Y195-phosphorylation.immunohistochemically.in.tumor.areas.of.poorly.differentiated.prostate.adenocarcinomas.

In.conclusion.we.demonstrate.for.the.first.time.that.PKP3.is.an.additi-onal. junctional. substrate. of. Src-kinase.. Nevertheless,. further. studies.are.needed.to.completely.understand.the.functional.relevance.of.this.mechanism.on.non-neoplastic.and.neoplastic.conditions.

Poster P-0017

E. Molnár.1,.T..Garay.1,.J..Tímár.1,.W..Berger.2,.W..Klepetko.3,.B..Hegedüs.1

1. Semmelweis.University,.2nd.Department.of.Pathology,.Budapest,.Hungary.

2. Medical.University.of.Vienna,.Institute.of.Cancer.Research,.Vienna,.Austria.

3. Medical.University.of.Vienna,.Department.of.Thoracic.Surgery,.Vienna,.Austria

Correlation of cell migration, proliferation and cytokinesis in cancer cells – revisiting the go or grow hypothesis

Background:. The. interplay. of. cell. migration. and. proliferation. in. solid.tumors.is.a.pivotal.factor.in.determining.the.metastatic.potential.that.is.largely.responsible.for.tumor.deaths..The.go.or.grow.hypothesis.postu-lates.that.tumor.cells.show.either.high.migration.or.proliferation..In.our.study.we.investigate.on.a.large.series.of.tumor.cell.lines.whether.this.assumption.stands.for.human.cancer.cell.cultures.

Materials. and. methods:. Thirty-five. human. cancer. lines. derived. from.mesothelioma.(12),.melanoma.(13).and.lung.cancer.(10).were.subjected.to.three-days-long.videomicroscopy..Cell.motility.and.proliferation.was.measured. by. the. expected. number. of. cell. divisions. and. migrated. di-stance.per.day.

Results:.Both.migration.and.proliferation.showed.a.large.variation..The.migration.distance.ranged.from.10.to.300.micron.per.day..Interestingly.mesothelioma. showed. by. far. the. highest. average. migration. (160. mi-cron).while.melanoma.and.lung.cancer.cell.were.significantly.slower.(80.micron.and.50.micron,.respectively)..The.highest.averaged.proliferati-on.was.found.in.mesothelioma.(0.80).while.melanoma.and.lung.cancer.had. rates.of.0.68.and.0.65. respectively..No.significant.changes.were.found.in.the.average.duration.of.a.cytokinesis..Surprisingly,.we.found.a.significant.positive.correlation.between.proliferation.and.migration.in.melanoma.and.in. lung.cancer.. In.contrast,. in.mesothelioma.there.was.no.correlation.between.these.two.cellular.processes..Since.the.FAK/SRC.signaling. is. an. important. pathway. regulating. cell. migration,. activati-on.of.FAK.and.SRC.kinases.were.measured.by.phosphorylation.speci-fic.immunoblot.assay..Interestingly,.activation.of.FAK.showed.a.strong.tendency.to.be.higher.in.the.six.faster.melanomas.compared.to.the.six.slower.lines..In.contrast.activation.of.SRC.was.essentially.equal.in.fast.and.slow.cells..Importantly,.a.significant.negative.correlation.was.found.between.duration.of.cytokinesis.and.migration.distance. in.melanoma..This.finding.suggests.that.instead.of.the.competition.between.migration.and.proliferation. for. the.finite.amount.of.available.ATP,. the.ability. to.rapidly.reorganize.the.cytoskeleton.determines.cytokinesis.length.and.migration.distance.

Conclusions:.We.demonstrate.that.human.cancer.cells.do.not.defer.pro-liferation. for.migration..As.a.matter.of. fact. regarding.one.single. cell.cytokinesis.and.migration.are.separated.temporally.but.on.the.level.of.a.cell.population.–.and.this.is.the.case.in.tumors.–.cells.migration.and.proliferation.occurs.simultaneously..Of.note,.tumor.cells.derived.from.various.organs.differ.in.terms.of.the.simultaneous.regulation.of.migra-tion.and.proliferation..Furthermore.our.observation.would.suggest.that.highly.proliferative.tumors.may.display.significant.invasion.of.the.sur-rounding.normal.tissue.

AEK

35

AEKAbstracts Posters Section 3: Invasion and metastasis

Poster P-0023

S. Kaduthanam.1,2,.S..Gade.1,.M..Meister.2,.J..Brase.1,.M..Johannes.1,.A..Warth.3,.H..Dienemann.4,.P..Schnabel.3,.F..Herth.4,.H..Sültmann.1,.T..Muley.2,.R..Kuner.1

1. DKFZ,.Cancer.Genome.Research,.Heidelberg,.Germany.2. Thoraxklinik,.STF,.Heidelberg,.Germany.3. University.of.Heidelberg,.Pathology,.Heidelberg,.Germany.4. Thoraxklinik,.Surgery,.Heidelberg,.Germany

Serum miRNAs associated with early relapse in operable lung aden-ocarcinoma patients

microRNAs.(miRNA).are.short.non-coding.RNAs.that.emerge.as.a.new.class.of.markers.for.diagnosis.and.prognosis.in.non-small-cell.lung.can-cer.(NSCLC)..miRNAs.cannot.only.be.stably.quantified.in.tissues.but.as.well.in.body.fluids.like.serum..However,.little.is.known.about.circulating.miRNAs.as.prognostic.markers.in.lung.adenocarcinoma.patients.

The.aim.of.this.study.was.to.identify.novel.miRNAs.as.prognostic.mar-kers.involved.in.disease.recurrence.in.serum.and.tissue.of.patients.with.early-stage.NSCLC.

Serum.miRNAs.were.screened.using.qRT-PCR.based.arrays.comparing.adenocarcinoma.patients.(n=.40).with.and.without.recurrence.2.years.after. surgery.. Selected. miRNAs. associated. with. disease. recurrence.were. validated. in. the. sera. of. an. independent. patient. cohort. (n=. 114).and.analysed.in.46.fresh-frozen.matched.lung.adenocarcinoma.tissues.Two.circulating.miRNAs.were.identified.in.the.screening.and.confirmed.in.the.validation.cohort.to.be.increased.in.sera.of.early-stage.NSCLC.pa-tients.suffering.from.recurrence.within.2.years..Elevated.levels.of.one.of.the.two.miRNAs.were.exclusively.observed.in.the.group.of.high-risk.patients. diagnosed. for. operable. adenocarcinoma.. The. differentiation.between.NSCLC.patients.with.low.and.high.risk.for.recurrence.was.im-proved.by.accounting.for.both.the.identified.miRNA.and.tumour.stage..The.expression.of.this.miRNA.in.NSCLC.tissues.did.not.reveal.an.associ-ation.with.metastatic.spread.as.was.observed.for.the.circulating.form.in.the.sera.of.early-stage.NSCLC.patients..Currently,.we.aim.to.identify.the.source.of.the.elevated.serum.miRNA.in.formalin-fixed,.paraffin.embed-ded.(FFPE).tissue.of.NSCLC.patients.using.miRNA.in-situ.hybridization.In.conclusion,.circulating.miRNAs.were. found.to.be.associated.with.a.high.risk.of.recurrence.in.early-stage.lung.adenocarcinoma.patients.and.may.serve.as.putative.non-invasive.prognostic.markers.

Poster P-0031

A. Bethge.1,.U..Schumacher.2,.G..Wedemann.1

1. University.of.Applied.Sciences.Stralsund,.Institute.for.Applied.Computer.Science,.Stralsund,.Germany.

2. University.Medical.Center.Hamburg-Eppendorf,.Institute.for.Anatomy.and.Experimental.Morphology,.Hamburg,.Germany

Computer Simulation of the Metastatic Progression

The.process.of.metastasis.formation.is.still.subject.of. intense.discus-sion.and.even.established.models.differ.considerably. in.several.basic.details.and.conclusions.drawn.from.them..In.addition,. it. is.difficult.to.evaluate.these.different.models.for.their.clinical.relevance..Therefore.a. computer. model. was. developed. which. permits. comparison. of. such.models.quantitatively.with.clinical.data..It.can.also.be.used.to.predict.the.outcome.of.treatment.interventions.

The.computer.model.is.based.on.a.discrete.event.simulation.procedure..The.growth.of.the.primary.tumour.is.described.via.analytical.functions,.while.a.rate.function.models.the.intravasation.events.of.the.primary.tu-mour.and.its.metastases..Events.describe.the.behaviour.of.the.emitted.malignant.cells.until.the.formation.of.new.metastases.

The. computer. model. allows. investigating. various. different. questions,.such. as:. Are. metastases. able. to. metastasize?. Is. metastasis. an. early.or.a.late.event?.Do.metastases.grow.faster.or.slower.than.the.primary.tumour?.Do.metastases.undergo.a.dormancy.phase.before.they.start.to.grow.out.into.a.macro.metastasis?.Furthermore,.the.effects.of.different.treatments,.e.g..resection.of.the.primary.tumour,.radionuclide.therapy.or.radioembolization,.can.be.simulated.and.analysed.

Comparing.the.simulation.results.with.clinical.or.experimental.data.may.help.to.answer.those.questions.asked.above..For.instance,.the.compa-rison. of. the. simulated. data. with. the. clinical. data. of. a. hepatocellular.carcinoma.revealed.that.late.metastases.are.of.no.clinical.relevance.for.the.patient’s.outcome.if.he.is.left.untreated..Only.the.first.metastases.seeded.from.the.primary.tumour.contribute.significantly.to.the.tumour.burden.and.thus.cause.the.patient’s.death.(Bethge.et.al..PLoS.ONE.7(4):.e35689,.2012).

36


Poster P-0035

S. Schultz.1,.H..Neubauer.1,.H..Bartsch.2,.K..Petat-Dutter.2,.B..Kootz.3,.M..Bonin.3,.H..Seeger.1,.S..Kahlert.4,.U..Vogel.5,.T..Fehm.1,.K..Sotlar.2

1. University.Womenshospital,.AG.Tumorprogression,.Tuebingen,.Germany.

2. University,.Pathology,.Munich,.Germany.3. University,.biology,.Tuebingen,.Germany.4. University,.Womenshospital,.Munich,.Germany.5. University,.Pathology,.Tuebingen,.Germany

Hsa-miR-199a-5p is functionally relevant in progression from ductal carcinoma in situ to invasive breast tumours

A.fundamental.and.clinically.important.step.during.the.breast.tumouri-genesis.is.the.transition.from.ductal.carcinoma.in.situ.to.invasive.ductal.carcinoma..At.this.point,.cancer.cells.gain.the.potential.to.spread.and.metastasize.. Since. the. molecular. characteristics. of. this. step. still. re-main.poorly.understood.an.improved.knowledge.of.the.transition.from.pre-invasive.to.invasive.breast.cancer.will.pave.the.way.for.novel.pre-ventative. and. therapeutic. strategies.. We. analyzed. miRNA. expression.profiles. in. 15. matched. pairs. of. ductal. carcinoma. in. situ. and. invasive.ductal.carcinoma.areas.isolated.by.laser.capture.microdissection.from.formalin.fixed.and.paraffin.embedded.breast.cancer.tissues.using.Illu-mina.miRNA.BeadChip.microarray.platform..Differential.expression.of.two.candidate.miRNAs.-.hsa-miR-199a-5p.and.hsa-miR-214.-.was.further.investigated.by.quantitative.RT-PCR. in.additional. independent.sample.pairs.from.25.breast.cancer.patients..Invasion.assays.were.performed.in.combination.with.knock.down.of.hsa-miR-199a-5p.

In.total,.matched.pair.microarray.analysis.revealed.33.significantly.dif-ferentially.expressed.miRNAs. (p-value. <0.05)..Expression.of.hsa-miR-199a-5p. is. upregulated. in. invasive. ductal. carcinoma. according. to. our.microarray.data.-.a.result.which.we.were.able.to.validate.by.qRT-PCR.in.independent.sample.pairs..Hsa-miR-214.could.not.be.validated..Knock.down.of.hsa-miR-199a-5p. in. invasive.MDA-MB-231.and.TMX2-28.breast.cancer.cells.using.a.specific.inhibitor.significantly.reduced.invasiveness.by.approx..73%.and.71%,.respectively.(both.p-value.<0.05).

For.the.first.time,.we.identified.candidate.‘progression’.miRNAs.which.are.differentially.expressed.between.ductal.carcinoma.in.situ.and.inva-sive.ductal.carcinoma.using.laser.capture.microdissection.and.microar-ray.analysis.on.FFPE.breast.cancer.tissues..Hsa-miR-199a-5p.could.be.validated. in.an. independent.sample.cohort,. is. influencing. in.vitro.cell.invasiveness. and. may. therefore. be. a. potential. drugable. regulator. of.tumour.progression.and.invasion.in.breast.cancer.

Poster P-0036

N. Taylor.1,.K..Lacy.2,.F..Nestle.2,.C..Wells.1

1. King’s.College.London,.Division.of.Cancer.Studies,.London,.United.Kingdom.

2. King’s.College.London,.Institute.of.Dermatology,.London,.United.Kingdom

P-21 Activated Kinase in Melanoma

Melanoma.is.a.malignant.form.of.skin.cancer.that.represents.only.5%.of.total.skin.cancer.cases,.however,.it.is.the.most.lethal.form,.accounting.for.75%.of.deaths..The.primary. treatment.of.early.stage.disease.via.surgical.excision.has.a.5.year.survival.rate.of.98%..However,.metastatic.disease.has.a.drastically.reduced.5.year.survival.rate.of.15%,.primarily.due.to.the.lack.of.effective.and.successful.treatments..Therefore,.the.prevention.of.metastasis.in.melanoma.patients.would.dramatically.im-prove.overall.patient.survival..p21-activated.kinases.(PAKs).are.a.family.of.serine/threonine.protein.kinases,.containing.6.isoforms,.which.have.a.variety.of.different.downstream.effects.including.cell.cycle.progres-sion,.the.inhibition.of.apoptosis,.and.regulation.of.the.actin.cytoskele-ton..Overexpression.of.specific.PAK.isoforms.has.been.found.in.a.wide.variety.of.human.cancer.cell. lines.such.as.breast,.prostate.and.colon.cancer..The.level.of.PAK.expression.and.involvement.in.melanoma.has.not.been.previously.investigated..Therefore,.as.PAKs.have.a.functional.link.to.both.cell.motility.and.proliferation,.they.may.provide.therapeu-tic.targets.for.the.prevention.of.melanoma.progression.and.metastasis..This.study.has.investigated.the.level.of.all.6.PAK.isoforms.in.a.variety.of.melanoma.cell. lines. (A-375,.WM-115,.SK-MEL-2.and.SK-MEL-28). from.primary.and.metastatic.origins,.compared.to.melanocytes.of.both.adult.and.neonatal.origin..Following. this,. the.comparative.cell. invasiveness.was.quantified.using.both.2D.(Invadopodia.Assay).and.3D.(Spheroid.As-say.in.Collagen.I.matrix).invasion.assays..Additionally,.cell.motility.was.assessed.using.timelapse.microscopy..From.these.experiments,.we.have.shown.for.the.first.time.that.multiple.PAK.isoforms.are.overexpressed.in.melanoma.cell.lines.when.compared.to.melanocytes..Of.these,.two.PAK.isoforms. (PAK1. and. PAK4). show. direct. and. inverse. expression. trends.with.cell.invasiveness.indicating.a.potential.key.role.for.these.kinases.in.melanoma.progression..Further.experiments.are.underway.to.investi-gate.the.involvement.of.PAK1.and.PAK4.in.the.invasiveness.and.progres-sion.of.melanoma.

AEK

37


Poster P-0041

C. M. Yang.1,.T..Chiba.1,.V..Vafaizadeh.1,.B..Groner.1

1. Georg-Speyer-Haus,.Frankfurt.am.Main,.Germany.

Reprogramming of tumor cells: signaling events and phenotypes

Development. and. differentiation. of. embryonic. stem. cells. are. accom-panied.by.a.loss.of.lineage.potential.and.increasing.functional.restric-tions..This.process.is.reversible.and.somatic.cells.can.be.reprogrammed.into.induced.pluripotent.stem.cells.(iPSC).by.the.action.of.four.defined.transcription.factors.(Oct3/4,.Sox2,.Klf4.and.Myc).or.by.the.combined.action.of.the.miR302-367.cluster.and.Hdac2.inhibition..Reprogramming.can.also.be.used.to.affect.the.transformation.phenotypes.of.tumor.cells.and.serve.as.a.potential.therapeutic.strategy..The.miR302-367.cluster.can.suppress.tumorigenicity.and.induce.apoptosis.in.cancer.cell.lines..We.are.investigating.the.molecular.mechanisms.which.confer.the.repro-gramming.potential.of. the.miR302-367.cluster. in. tumor.cells.and.are.assessing.the.potential.therapeutic.benefit..

The.Akt.serine/threonine.kinase.seems.to.play.a.key.role.in.the.repro-gramming.of.tumor.cells.through.its. interactions.with.the.reprogram-ming. factors. Sox2. and. Oct3/4.. These. interactions. enhance. the. tran-scriptional.activity.of.Sox2.and.Oct3/4.and.the.reprogramming.process..Akt.is.also.a.key.regulator.of.EMT.(epithelial.to.mesenchymal.transiti-on),.an.early.step.in.the.metastatic.cascade,.through.inhibition.of.GSK3.and. activation. of. NFkB/Snail. signaling.. In. addition,. Akt. regulates. cell.proliferation,.apoptosis.and.cell.migration.

MET.(mesenchymal.to.epithelial.transition).can.be.observed.during.the.early.phases.of.the.reprogramming.process.of.somatic.cells..E-cadherin.is.a.key.regulator.of.MET.and.can.replace.the.function.of.Oct3/4.in.repro-gramming..MET.is.increased.by.up-regulation.of.E-cadherin.and.down-regulation.of.the.EMT.associated.transcription.factors.Snail,.Slug.and.Twist..These.factors.reduce.cell-cell.adhesion.and.increase.the.migrati-on.potential.by.suppression.of.p63..

As.a.model. for. reprogramming. induced.suppression.of. tumorigenicity.and.metastasis,.we.are.using.U87MG.glioblastoma.cancer.cells..We.in-troduced.the.miR302-367.cluster,.encoded.by.a.lentiviral.gene.transfer.vector,.into.U87MG.cells.and.expressed.the.miRNA.in.a.conditional.fa-shion..We.are.monitoring.the.consequences.miR302-367.induction.with.respect.to.their.effects.on.the.up-.or.downregulation.of.signal.transduc-tion.pathways,.cell.growth,.invasiveness,.colony.formation,.drug.sensiti-vity,.radiation.sensitivity,.and.tumor.formation.in.mice.

Poster P-0050

M. Kovacheva.1,.M..Zepp.1,.S..Berger.2,.M..Berger.1

1. DKFZ,.Toxicology.and.Chemotherapy,.Heidelberg,.Germany.2. Central.Institute.for.Mental.Health,.Molecular.Biology,.Mannheim,.

Germany

Bone sialoprotein II and integrin alpha v beta 3 as potential targets in treatment of breast cancer skeletal metastasis

Metastasis.renders.breast.cancer.virtually.incurable..Therefore,.deciphering.the.molecular.mechanism.of.this.process.would.help.to.improve.prognosis.for.affected.patients.

We.examined.the.impact.of.bone.sialoprotein.II.(BSP.II).and.its.interaction.partner.integrin.alpha.v.beta.3.(ITGB3).on.bone.metastasis.by.using.a.con-ditional. breast. cancer. cell. line. in. a. nude. rat. model.. Combination. of. RNA.interference.(RNAi).with.the.tetracycline-controlled.transcription.activati-on.(tet).system.was.used.to.conditionally.knockdown.either.BSP.or.ITGB3.within.generated.MDA-MB.-231.subclones..In.addition,.all.generated.cell.lines.were.equipped.with.reporter.genes.for.red.fluorescent.protein.mCherry.and.firefly.luciferase.to.follow.tumor.progression.and.osteolytic.lesions.in.vivo.Quantification.of.the.knockdown.efficiency.and.functional.tests.were.per-formed.after.six.days.with.or.without.doxycycline..ITGB3.mRNA.concentra-tions.were.decreased.to.15.–.27.%.in.three.respective.cell.clones,.which.re-sulted.in.a.corresponding.reduction.in.protein.concentrations.by.19.–.40%..In.cell.lines.interfering.with.BSP.II.expression,.protein.concentrations.were.decreased.by.22.–.86%.

Next,. the. transgenic. cell. clones. were. subjected. to. a. series. of. functional.tests.for.tumor.progression..The.ITGB3.knockdown.resulted.in.inhibition.of.migration.up.to.87.%.(48h).and.up.to.22%.inhibition.of.proliferation.in.the.respective. cell. clones.. The. decrease. in. BSP. II. expression. led. up. to. 90%.reduced.proliferation.and.spontaneous.migration.(48h),.and.up.to.81%.inhi-bition.of.colony.formation.

Subsequently,.the.respective.clones.were.used.to.determine.the.function.of.BSP.II.in.bone.metastasis.using.an.animal.model.for.site-specific.osteolytic.lesions.in.nude.rats..As.quantified.by.bioluminescence.imaging,.tumor.size.was.significantly.reduced.upon.BSPII.knockdown,.leading.to.full.remission.of.bone.metastasis.in.some.cases..When.analysing.two.independent.BSPII.cell.clones,.two.or.three.weeks.of.persistent.BSPII.knockdown.led.to.a.reduction.of.soft.tissue.lesions.up.to.86%.and.a.reduction.in.skeletal. lesions.up.to.92%.compared.to.the.respective.controls.

In.conclusion,.the.conditional.knockdown.of.ITGB3.resulted.in.inhibition.of.migration.and.proliferation.in.one.of.the.cell.clones..The.miRNA-mediated.knockdown.of.BSP.impaired.the.metastatic.properties.of.the.cell.clones.in.vitro.and.in.vivo..Therefore,.we.assume.that.both,.ITGB3.and.BSP.II,.could.be.potential.targets.for.future.breast.cancer.therapies.

38


Poster P-0052

K. Krull.1,.M..Hoffmann.1,.H..E..Gabbert.1,.R..Engers.1,2

1. Institut.für.Pathologie,.Universitätsklinikum.Düsseldorf,.Düsseldorf,.Germany.

2. Zentrum.für.Pathologie,.Zytologie.und.Molekularpathologie,.Neuss,.Germany

Effects of TMPRSS2-ERG gene fusion on transformation, proliferati-on, migration and invasion

Prostate.cancer.(PCa).is.one.of.the.most.frequently.diagnosed.cancers.in. men.. The. biological. behaviour. of. PCa. varies. dramatically,. ranging.from.insignificant.tumors,.requiring.no.therapy,.to.highly.aggressive.and.metastatic.tumors,.leading.to.death.within.a.relatively.short.period.of.time..The.molecular.mechanisms,.determining.the.aggressive.behaviour.have.not.been.elucidated.yet.

Since.about.50%.of.all.PCas.have.been.found.to.harbour.fusions.between.the.prostate-specific.TMPRSS2-gene.and.one.gene.of.the.oncogenic.ETS.family.members,.especially.the.ETS-related-gene.ERG,.we.investigated.the. functional. role.of. the.most. frequent.TMPRSS2-ERG-fusion.variant.(T1/E4).on.oncogenic.transformation,.cell.proliferation,.migration,.and.invasion..As.the.benign.prostate.epithelial.cell.line.BPH-1.could.not.be.stably.transfected.by.various.methods,.HEK293-cells.were.used.instead.Stable. overexpression.of. the. T1/E4. TMPRSS2-ERG-fusion. variant. (ΔN-ERG). in.HEK293.cells.significantly.stimulated. invasion,.as.determined.by.the.Matrigel.invasion.assay..In.search.for.the.underlying.mechanism.we.found.that.ΔN-ERG.stimulated.secretion.of.the.invasion.promoting.matrix. metalloproteinase. Pro-MMP-9. and. simultaneously. reduced. se-cretion. of. the. Pro-MMP-9. inhibitor. TIMP-1.. In. contrast,.ΔN-ERG. ove-rexpression. did. not. affect. cell. proliferation. and. migration.. Moreover,.overexpression.of.ΔN-ERG.was.not.sufficient.to.induce.oncogenic.trans-formation.in.vitro..Finally,.an.influence.of.ΔN-ERG.on.the.Tiam1-Rac.si-gnalling.pathway.was.not.observed.

In.summary,.ΔN-ERG,.resulting.from.TMPRSS2-ERG.gene.fusion,.signi-ficantly.induces.invasion.of.HEK293.cells.by.affecting.the.balance.bet-ween.MMPs.and.TIMPs,.but.has.no.effect.on.proliferation.and.migration..Moreover,.ΔN-ERG.is.not.sufficient.to.induce.oncogenic.transformation.in.vitro.

Poster P-0060

M. Berning.1,.L..Schardt.1,.P..Boukamp.1

1. DKFZ,.Heidelberg,.Germany

Squamous Cell Carcinoma Models

In.vitro.human.skin.models.are.valuable.research.tools.holding.the.po-tential.to.mimic.skin.cancer.initiation.and.progression..Most.skin.mo-dels. use. natural. polymer. hydrogels. as. dermal. equivalents,. which. do.not.resemble.the.human.dermis.neither.in.its.ECM.composition.nor.in.its. structure.. Here,. we. describe. a. self-assembly. matrix. composed. of.human.ECM.and.structural.collagen.fibers.produced.by.human.dermal.fibroblast.as.a.novel.basis.for.skin.cancer.models.

The.cell-derived.matrix.based.organotypic.cultures.(cdmOTC).are.scree-ned. in.co-culture.with.human.keratinocytes.and.several. immortalized.keratinocytes.of.the.HaCaT. in.vitro.skin.cancer.model..Human.kerati-nocytes. regenerate. fully. differentiated. and. long-term. vital. epithelia..The.immortalized.keratinocytes.(HaCaT),.the.benign.Ha-Ras.transfected.keratinocytes.(HaCaT.A5),.the.malignant.Ha-Ras.transfected.keratino-cytes. (HaCaT. II-4). and. the.metastatic. keratinocytes. (HaCaT.RT3). are.followed.up.to.6.weeks. in.co-culture..Their. invasive.phenotype. in.cd-mOTCs.strongly.resemble.the.phenotypes.seen.upon.tumor.cell.trans-plantation.

Furthermore,.the.cdmOTC.model.can.be.applied.for.drug.screenings..It.is.known.that.immunosuppressive.drugs.especially.cyclosporine.A.(CsA).increase.the.risk.of.squamous.cell.carcinoma.(SCC).by.65-100.times..In.line.with.a.more.aggressive.behaviour.upon. immunosuppression,.CsA.treatment.of.the.immortal.but.non-tumorigenic.HaCaT.keratinocytes.in.cdmOTCs.triggers.an.invasive.phenotype.

The.potential.of. cell-derived.matrices. is. still. not. tapped..We.succeed.in. building. fibroblast-derived. matrices,. which. can. be. used. as. dermal.equivalents.resembling.more.features.of.the.human.dermis.in.vivo.and.can.be.used. for. investigations. in. tissue.homeostasis.and.skin.cancer.invasion.

AEK

39


Poster P-0063

H. Peterziel.1,.J..Danner.1,.A..Danner.2,.S..Barbus.2,.H.-.K..Liu.3,.P.-.N..Pfenning.4,.P..N..Harter.5,.M..Mittelbronn.5,.W..Wick.4,.G..Schütz.3,.B..Radlwimmer.2,.P..Lichter.2,.J..Hess.1,.P..Angel.1

1. German.Cancer.Research.Center.Heidelberg,.Signal.Transduction.and.Growth.Control,.Heidelberg,.Germany.

2. German.Cancer.Research.Center.Heidelberg,.Molecular.Genetics,.Heidelberg,.Germany.

3. German.Cancer.Research.Center,.Molecular.Biology.of.the.Cell.I,.Heidelberg,.Germany.

4. University.of.Heidelberg.,.Neuro-Oncology,.Heidelberg,.Germany.5. Neurological.Institute/Edinger.Institut,.Goethe.University,.Frankfurt/

Main,.Germany.

Regulation and function of the mucin-like glycoprotein podoplanin in glioma

Podoplanin.(PDPN).is.a.mucin.like.type-1.transmembrane.protein.which.is.highly.expressed.in.tumors.of.different.origin,.including.squamous.cell.cancers,.germinal.neoplasia.and.tumors.of.the.central.nervous.system..Recently,.we.found.strong.overexpression.of.PDPN. in.human.astrocy-tic.brain.tumors,.specifically.in.primary.glioblastoma.multiforme.(GB)..Regulation.of.PDPN.expression.involves.increased.PI3.kinase.activati-on,.subsequent.activation.of.protein.kinaseB/Akt.and.the.downstream.transcription.factor.AP-1..Activation.of.this.pathway.is.mediated.by.loss.of.PTEN.function,.consistently,.mice.lacking.Pten.exhibit.elevated.Pdpn.protein.levels.in.the.subventricular.zone.of.the.brain..Additionally,.PDPN.expression.is.subject.to.negative.transcriptional.regulation.by.promoter.methylation.in.human.GB.and.in.glioma.cell. lines..In.patients.high.ex-pression.of.PDPN.is.associated.with.poor.prognosis..Silencing.of.PDPN.results.in.a.decrease.in.proliferation,.migration.and.invasion.of.glioma.cell.lines.in.vitro.and.slows.down.the.migration.in.orthotopic.brain.sli-ces..Consistently,.tumor.formation.after.injection.of.PDPN.knock-down.cells.into.SCID.mice.is.significantly.reduced.compared.to.tumors.after.injection.of.parental.cells..These.data.highlight.a.functional.role.of.PDPN.in.glioma.progression.and.suggest. targeting.of.PDPN.expression.as.a.promising.strategy.for.the.treatment.of.patients.with.primary.GB.

Poster P-0068

H. Bühler.1,.C..Hoberg.2,.P..NguemgoKouam.2,.I..Pisarenko.2,.B..Priesch-Grzeszkowiak.2,.I..Adamietz.1

1. Universitätsklinikum.Marienhospital,.Klinik.für.Strahlentherapie.und.Radioonkologie,.Herne,.Germany.

2. Universitätsklinikum.Marienhospital,.Institut.für.Molekulare.Onkologie,.Strahlenbiologie.und.Experimentelle.Strahlentherapie,.Herne,.Germany

Zoledronic acid inhibits the motility of cancer stem cells from the human breast cancer cell line MDA-MB 231.

Question:. Various. effects. on. tumor. cells. have. been. described. for.zoledronic.acid..However,.only.limited.data.exist.regarding.its.influence.on.the.motility.of.tumor.cells..Since.migration.is.a.decisive.step.in.me-tastasis,.we.examined.whether.zoledronic.acid.reduces.the.motility.of.tumor.cells,.which.may.lead.to.less.aggressive.metastasis.

Methods:.The.hypothesis.of.tumor.stem.cells.postulates.that.recurrences.may.occur.from.cancer.stem.cells.rather.than.from.“normal,”.somatic.tumor.cells..We.investigated.the.effects.of.zoledronic.acid.on.stem-like.progenitor.cells.obtained.via.the.formation.of.spheroids.from.the.human.breast.cancer.cell.line.MDA-MB.231.

Results:. Stem-cell. properties. were. verified. by. measurement. of. high.CD44.expression.and.an.absence.of.CD24.expression..Time-resolved.vi-deography.showed.that.zoledronic.acid.strongly.reduced.the.migration.of. cancer. stem. cells.. Cellular. velocity. was. reduced. by. 61%. following.exposure.to.1.µM.zoledronic.acid,.and.by.82%.after.exposure.to.10.µM.zoledronic.acid..Accumulated.distance.traveled.by.the.cells.was.reduced.by.60%.and.79%.after.exposure.to.1.µM.and.10.µM.zoledronic.acid,.re-spectively..The.remaining.cellular.motility.led.to.very.little.change.in.di-stance,.with.cellular.activity.appearing.more.as.“stepping.on.the.spot.”

Conclusions:.This.suppression.of.cancer.stem.cell.motility.could.poten-tially.contribute.to.the.clinical.benefits.that.recent.studies.have.descri-bed.following.the.adjuvant.administration.of.zoledronic.acid.in.breast.cancer.

40


Poster P-0075

D. Weissinger.1,.K..Tagscherer.1,.S..Macher-Göppinger.1,2,.N..Wagener.3,.A..Haferkamp.4,.W..Roth.1,2

1. German.Cancer.Research.Center,.Molecular.Tumor.Pathology,.Heidelberg,.Germany.

2. University.of.Heidelberg,.Pathology,.Heidelberg,.Germany.3. University.of.Heidelberg,.Urology,.Heidelberg,.Germany.4. University.of.Frankfurt,.Urology,.Frankfurt,.Germany

Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma

Question:.Overexpression.of.Decoy.Receptor.3.(DcR3),.a.soluble.mem-ber. of. the. tumor. necrosis. factor. receptor. superfamily,. is. a. common.event. in. several. types.of.cancer.. In. renal.cell. carcinoma. (RCC),.DcR3.overexpression. is.associated.with. lymph.node.and.distant.metastasis.as.well.as.a.poor.prognosis..However,.the.functional.relevance.of.DcR3.overexpression.in.renal.cancer.is.unknown.

Methods:.Cell.culture,.siRNA.transfection,.ectopic.transgene.expressi-on,.Luciferase.assays,.migration.assays,.invasion.assays,.adhesion.as-says,.ex.vivo. tissue.technique,. in.vivo.experiments,.Western.blotting,.quantitative.RT-PCR,.immunohistochemistry.

Results:.In.our.studies.we.show.that.DcR3.strongly.promotes.adhesion,.migration,.and.invasiveness.of.RCC.cell.lines.in.vitro..In.a.mouse.xeno-graft.model,.this.phenotype.is.confirmed.in.vivo..Further,.we.identified.the.signaling.pathway.regulating.DcR3.expression.in.RCC..The.expressi-on.of.DcR3.is.regulated.in.a.PI3K/AKT-dependent.manner.involving.the.transcription.factor.NFAT..This.mechanism.is.demonstrated.in.vitro.as.well.as.in.an.ex.vivo.model.of.RCC.using.slice.culture.of.freshly.obtained.human.RCC.tissue.

Conclusion:.Our.results.identify.DcR3.as.a.key.driver.of.tumor.cell.disse-mination.and.designate.DcR3.as.a.promising.marker.for.rational.therapy.of.renal.cell.carcinoma..

Poster P-0082

A. Pervaiz.1,.M..R..Berger.1,.H..Adwan.1

1. German.Cancer.Research.Center,.Toxikology.and.Chemotherapy,.Heidelberg,.Germany.

Role of Platelet-derived CXC chemokine ligand 7 in Colorectal Cancer cells

Introduction:.Platelet-derived.CXC.chemokine.ligand.(CXCL).7.has.been.found.to.be.associated.with.chronic.inflammation.and.tumor.progressi-on,.but.its.role.in.colorectal.cancer.growth.remains.unclear.

Methodology.and.Results:.1. We.injected.the.rat.colorectal.CC531.cancer.cells.via.the.portal.vein.

into.rat.liver.. After.selected.time.points.(3,.6,.9,.12.and.21.days),.the.cancer.cells.

were.re-isolated.and.the.expression.profile.of.chemokines.was.stu-died.by.micro-array.analysis..Compared.to.control.CC531.cells,.CXCL7.was.initially.among.the.>150-fold.down-regulated.genes..Thereafter,.the.expression.of.this.gene.in.CC531.cells.regained.its.normal.level.within.3.weeks.of.further.growth.in.rat.liver.

2. Human.samples.of.colorectal.cancer.were.also.investigated.to.iden-tify. the.expression.of.CXCL7..A.positive.correlation.was. found.bet-ween.CXCL7.and.some.other.chemokines.

3. In.further.experiments,.rat.colorectal.cancer.cells.CC531.were.trans-fected.with.200.nM.siRNA.or.negative.control..After.24,.48.and.72.h.of.treatment,.the.cells.were.isolated.and.mRNA.expression.of.CXCL7.was.determined.by.RT-PCR..The.effect.of.CXCL7.knockdown.on.the.proli-feration.of.CC531.cells.was.investigated.by.MTT.assay..Directional.cell.migration.and.invasive.behavior.of.the.cells.through.cell.matrix.was.examined.by.the.Scratch.Assay.as.surrogate.for.the.cells’.migration.and.‘wound.healing’..In.this.part.of.study,.we.have.examined.the.role.of.CXCL7.in.cell.proliferation.and.migration.of.colorectal.cancer.cells.

Exposure.to.siRNA.species.reduced.the.mRNA.expression.of.CXCL7.from.24. to72h.with.maximum.reduction.at.24h. (82%)..Following. the.post-transfection.period,.there.was.a.significant.decline.in.cell.proliferation.up.to.30,.39.and.23%.for.24,.48.and.72h.respectively..Reduced.directio-nal.migration.of.the.cells.through.the.cellular.matrix.was.also.observed.in.the.knockdown.CC531.cells.

Conclusion:. Initially. reduced. expression. of. CXCL7. in. CC531. cells. re-isolated.from.rat.liver.might.contribute.to.decrease.the.motility.of.the.colorectal. cancer. cells,. thus. support. their. homing. to. the. liver.. High.expression.of.CXCL7.contributes.to.cell.proliferation.and.migration.of.colorectal.cancer.cells.in.vitro,.which.is.in.line.with.the.increased.ex-pression.of.this.gene.shortly.after.the.cells’.homing.into.rat.liver..Based.on.these.preliminary.results,.we.will.also.study.the.effect.of.CXCL7.in.a.liver.cancer.metastasis.model.to.evaluate.an.anticipated.correlation.with.colorectal.cancer.progression.

AEK

41


Poster P-0093

T. Berger.1,.R..Kaufmann.2,.J..Clement.1,.J..Eberhardt.3,.C..Malessa.2,.U..Settmacher.2,.A..Hochhaus.1,.K..Wagner.1

1. Jena.University.Hospital.,.Dept..of.Hematology/Oncology,.Jena,.Germany.

2. Jena.University.Hospital,.Dept..of.General,.Visceral.and.Vascular.Surgery,.Jena,.Germany.

3. ALS.Automated.Labsolutions.GmbH,.Jena,.Germany

Hepatocellular carcinoma derived circulating epithelial cells – detec-tion and characterization

Question:.Hepatocellular.carcinoma.(HCC).has.a.high.rate.of.recurrence.and. a. low. rate. of. 5-year. survival.. Disseminated. circulating. epithelial.tumor.cells. (CETCs).are.discussed. to.play.an. important. role. in. tumor.progression.and.their.number.might.reflect.the.aggressiveness.of.the.tumor..The.aim.of.the.study.was.to.identify.suitable.cell.surface.mar-kers. for. the. identification.and.selection.of. those.cells.and. to. further.characterize.them.on.single.cell.level.

Methods:.The.HCC.cell.lines.HepG2.and.Hep3B.were.cultivated.in.RPMI.1640. with. 10. %. FCS.. Primary. cells. from. HCC. patients’. samples. were.propagated. in. AmnioMAXTMII-Complete.. The. cell. surface. proteins. asi-aloglycoprotein.receptor.1.(ASGPR.1),.epithelial.cell.adhesion.molecule.(EpCAM).and.epithelial.glycoprotein.mucin.1.(MUC.1).were.evaluated.as.markers.for.the.detection.of.CETCs..The.specificity.of.the.selected.an-tigens.was.verified.by.immunohistochemistry.and.FACS.analysis..Blood.samples.from.45.patients.scheduled.for.liver.transplantation.were.analy-zed.for.the.occurrence.of.epithelial.antigen.positive.cells..Viable.EpCAM-positive.cells.confirmed.by.propidium. iodide.negativity.were.prepared.by.the.CellCelector.(ALS.Jena).and.analyzed.by.single.cell.real-time.PCR.

Results:.ASGPR.1,.EpCAM.and.MUC.1.proteins.could.be.detected.in.the.established.cell.lines,.primary.cells.as.well.as.in.FFPE.HCC.tissue.sec-tions.showing.characteristic.staining.intensity.and.distribution..Analysis.of. peripheral. blood. samples. from. HCC. patients. also. revealed. the. oc-currence.of.cells.expressing.one.or.more.of.the.investigated.proteins.specifically..EpCAM.turned.out.to.be.the.most.reliable.marker.(detecta-ble.in.81.%.of.all.samples).with.a.broad.and.patient.specific.range.bet-ween.200.and.140.000.cells.per.ml.blood..Single.cell.expression.analysis.revealed.a.high.expression.rate.of.the.housekeeping.genes.RPL37A.and.EEF1A1.in.cell.line.cells.(71/74.cells)..In.viable.EpCAM-positive.cells.the.expression.of.RPL37A.and.EEF1A.could.be.detected.in.36/85.cells.

Conclusions:.The.determination.of.EpCAM-positive.cells.in.the.periphe-ral.blood.of.HCC.patients.can.contribute.to.the.evaluation.of.prognosis,.risk.of.recurrence.and.therapy.response..The.successful.preparation.of.single.cells.is.a.promising.tool.for.a.further.characterization.of.the.cells.on.molecular.level.

Poster P-0101

M. Dahlmann.1,.U..Sack.2,.P..Herrmann.3,.M..Lemm.4,.I..Fichtner.4,.P..M..Schlag.1,5,3,.U..Stein.1,3

1. Max-Delbrück-Centrum,.Surgical.Oncology,.Berlin,.Germany.2. Max-Delbrück-Centrum,.Immune.Regulation.and.Cancer,.Berlin,.

Germany.3. Charité.Universitätsmedizin,.Surgical.Oncology,.Berlin,.Germany.4. Max-Delbrück-Centrum,.Experimental.Pharmacology,.Berlin,.

Germany.5. Charité.Universitätsmedizin,.Charité.Comprehensive.Cancer.Center,.

Berlin,.Germany

shRNA mediated knock down of S100A4 in colorectal carcinomareduces metastasis formation in vivo

Colon.carcinoma,.even.after.the.excision.of.the.primary.tumor,.is.still.a.major.cause.of.death.due.to.its.high.potential.of.metastasis.formati-on..The.small.calcium.binding.protein.S100A4.has.been.found.to.be.an.indicator.for.metachronous.metastasis.formation..Its.expression.level.positively.correlates. to. the.metastatic.potential.of.human.colon.can-cer,.where.it.promotes.cell.motility.and.invasion,.and.negatively.affects.the.survival.of.colon.cancer.patients..A.therapeutic.decrease.of.S100A4.expression.in.patients.could.therefore.result.in.less.metastasis.forma-tion.and.increased.survival.rates..In.the.present.study,.we.used.shRNA.plasmids.to.specifically.inhibit.S100A4.expression.in.the.colorectal.car-cinoma.cell.line.HCT116.and.observed.a.clear.reduction.of.cellular.migra-tion.and.invasion.in.vitro,.to.about.45%.and.55%,.respectively,.while.proliferation.of.the.cell.lines.was.not.affected..Intrasplenical.transplan-tation. of. either. S100A4. knock-down. or. control. cells. in. mice. reduced.the.formation.of.liver.metastases,.together.with.S100A4.reduction..We.also.evaluated.the.therapeutic.potential.of.systemically.applied.shRNA.plasmids.via.repeated.hydrodynamics-based.tail.vein.injection.of.plas-mid.DNA,.expressing.either.specific.S100A4-shRNA.or.unspecific.control.shRNA..Mice.with.intrasplenically.transplanted.HCT116-LUC.cells.showed.a. 3-fold. decrease. in. metastasis. formation. in. the. liver,. when. treated.with.S100A4-shRNA.plasmids..The.average.tumor.size.of.treatment.and.control.group.was.comparable,.but.showed.a.decrease.of.S100A4.and.MMP-9.expression.levels.to.60%.and.20%,.respectively,.when.treated.with.S100A4-shRNA.plasmids..The.developed.liver.metastases.showed.a.similar.reduction.in.S100A4.expression,.while.the.decrease.in.MMP-9.ex-pression.was.less.pronounced.(50%.and.60%,.respectively)..In.summa-ry,.the.knockdown.of.S100A4.via.systemic.application.of.plasmid.DNA,.expressing.gene.specific.shRNA,.resulted. in.a.decrease.of.metastasis.formation.in.a.xenograft.mouse.model.of.colon.cancer.

42


Poster P-0103

P. Nguemgo Kouam.1,.I..Pisarenko.1,.A..Kochanneck.1,.B..Priesch-Grzeszkowiak.1,.H..Bühler.1,.I..Adamietz.1

1. Universitätsklinikum.Marienhospital,.Institut.für.Molekulare.Onkologie,.Strahlenbiologie.und.experimentelle.Strahlentherapie,.Herne,.Herne,.Germany

Influence of Slit2/ROBO1 expression on the migration of irradiated glioblastoma cells.

Question:. Glioblastoma. is. a. highly. invasive. primary. brain. tumor.. The.prognosis. is. poor. despite. of. intensive. radio-chemo. therapy.. Recent.data.indicate.that.irradiation.might.enhance.the.motility.of.glioblasto-ma.cells. and. thereby.promote. local. recurrences.. In. this. study.we. in-vestigated.whether.or.not.slit2/ROBO1.signaling.has.an.impact.on.the.modulation.of.cellular.migration.by.therapeutic.irradiation..New.studies.have.shown. that. this.axon-guidance.system.plays.a. role. in.migration.and.invasiveness.of.different.tumor.cells.

Methods:. The. experiments. were. performed. on. two. different. human.glioma.cell.lines.(U-87.MG,.U-373.MG)..In.addition.slit2.or.ROBO1.over-expressing. clones. were.established. by. transfection. of. the. respective.gene..The.cells.were.irradiated.with.0,.0.5,.2,.or.8.Gy.photons..Expressi-on.of.slit2.and.ROBO1.was.determined.by.western.blotting.and.qRT-PCR..Motility. parameters. (velocity,. accumulated,. and. euclidean. distance).were.obtained.by.time-laps.videography.

Results:.Wild.type.expression.of.slit2.was.moderate.in.U-373.and.very.low.in.U-87.cells..In.contrast.ROBO1.was.high.in.U-87.and.low.in.U373.cells..The.expression.was.markedly.affected.by.irradiation,.however.dif-ferently.in.both.cell.lines..The.migration.of.both.cell.lines.was.enhanced.after. irradiation,. particularly. after. low. doses.. Migration. of. all. trans-fected.clones.was.reduced.by.50%..Irradiation.enhanced.the.motility.of.both.U-373.clones.slightly.but.decreased.it.strongly.for.the.U-87.clones..The.migration.of.U-87-slit2.cells.was.reduced.after.0.5,.2,.and.8.Gy.by.19%,.11%,.and.12%.respectively..The.migration.of.U-87-ROBO1.cells.was.reduced. after. 0.5. Gy. by. 50%. but. slightly. increased. after. 2. or. 8. Gy,.although.the.euclidean.distance.was.reduced.too.by.20%.

Conclusions:.Slit2.and.ROBO1.are.differently.expressed.in.glioblastoma.cells.and.can.be.influenced.by.irradiation..Slit2/ROBO1.are.involved.in.the.cellular.motility.as.shown.by.the.transfected.clones..Irradiation.in.the. range. of. a. fractionated. radio. therapy. enhances. the. migration. of.glioblastoma.cells,.however.strengthened.Slit2/ROBO1.signaling.might.counteract.this.potentially.malignant.side.effect.

Poster P-0112

B. George.1,2,.G..Acuña.Sanhueza.1,2,.L..Faller.2,.P..Bayo.Zaera.1,2,.C..Simon.2,.P..Plinkert.2,.J..Hess.1,2

1. DKFZ,.Experimental.Head.and.Neck.Oncology,.Heidelberg,.Germany.2. University.Hospital.Heidelberg,.Department.of.Otolaryngology,.

Heidelberg,.Germany

Regulation and function of MYBBP1A in the establishment and main-tenance of cellular senscence

Background:.Myb-binding.protein.1a.(MYBBP1A).is.a.predominantly.nu-cleolar.transcriptional.regulator.involved.in.rDNA.synthesis..Recent.stu-dies. identified. a. direct. physically. and. functional. interaction. between.MYBBP1A.and.distinct.nuclear.transcription.factors,. including.p53.and.RELA. both. of. which. represent. key. players. in. the. establishment. and.maintenance.of.cellular.senescence.

Objectives:.The.project.aims.to.determine.the.expression.of.MYBBP1A.protein.in.tumor.samples.and.to.analyze.its.correlation.with.biomarkers.and.regulators.of.apoptosis.and.cellular.senescence,.as.well.as.to.inve-stigate.the.role.of.MYBBP1A-dependent.signaling.and.gene.regulatory.networks.in.these.processes.

Methods:.Immunohistochemical.staining.of.tissue.sections.derived.from.mouse. tumor. models. and. head. and. neck. cancer. patients.. Functional.assays.using.genetically.modified.tumor.cell.lines.with.MYBBP1A.gain-of-function. and. loss-of-function.. Expression. analysis. and. functional.characterization.of.key.regulators.of.apoptosis.and.cellular.senescence.in.tumor.cell.lines.and.a.Mybbp1a.knockout.mouse.model.

Results:.Global.gene.expression.profiling.with.samples.derived.from.an.orthotopic. xenograft. mouse. model. revealed. reduced. MYBBP1A. tran-scription. in. recurrent. as. compared. to. primary. tumors.. We. confirmed.significantly. reduced. MYBBP1A. protein. levels. on. tissue. sections. of.recurrent. as. compared. to. primary. tumors. by. immunohistochemistry,.and. also. found. aberrant. MYBBP1A. protein. levels. in. tumor. samples. of.HNSCC.patients..Functional.characterization.by.ectopic.overexpression.or.gene.silencing.revealed.a.dual.role.of.MYBBP1A.in.tumor.cell.lines,.as.its.expression.correlates.with.accelerated.cell.growth.while.it’s.silen-cing.induced.apoptosis.and.elicited.increased.migration.and.invasion.

Conclusions:.We.provide.experimental.evidence.that.MYBBP1A.serve.as.an.important.molecular.switch.in.the.regulation.of.tumor.cell.physiology.with.regard.to.proliferation,.cellular.apoptosis.and.motility.

AEK

43


Poster P-0124

N. Grabe.1,2,3,.K..Safferling.1,2,3,.K..Westphal.3,.C..Ernst.1,3,.A..Zeppenfeld.1,3,.N..Halama.1,3,.D..Jäger.1,3

1. National.Center.for.Tumor.Diseases,.Medical.Oncology,.Heidelberg,.Germany.


3. University.Heidelberg,.BIOQUANT,.TIGA.Center,.Heidelberg,.Germany

Systems biological analysis of collective cell migration in 3D tissue models for understanding cancer invasion

Collective.migration.is.essential.for.morphogenesis.and.wound.repair.in.healthy.tissue.as.well.as.for.tissue.invasion.in.cancer..In.such.migration.cells.move.in.supracellular,.cadherin.mediated.units.opposed.to.isola-ted. ameoboid. or. mesenchymal. movements.. Understanding. collective.migration.in.healthy.epithelia.is.a.prerequisite.for.dissecting.and.analy-zing.invasion.in.carcinoma..We.have.set.up.a.novel,.easily.reproducible.3D.in.vitro.wound.model.of.epidermal.regeneration..The.model.is.based.on.commercial.tissue.cultures.and.can.thus.be.reproducibly.manufac-tured.in.larger.quantities..Using.for.the.first.time.virtual.microscopy.in.combination.with.a.novel.fluorescent.time-lag.staining.we.could.capture.the.cellular.intra-tissue.streams.during.reepithelialization..Furthermore.the.quantification.of.Ki67.positive.cells.as.well.as.differentiation.mar-ker.and.cell.junction.analysis.enabled.us.to.develop.a.conceptual.model.showing.how.proliferation,.differentiation.and.collective.cell.migration.are. integrated. in.reepithelialization.by.a.novel.“extending.shield.me-chanism”..Keratinocyte.proliferation.thereby.occurred.in.a.concentric.traveling.wave.spatially.lacking.behind.the.extending.epidermal.tongue.(EET).and.producing.most.of.the.new.cells..Quantifying.cell.polarization.showed.that.these.cells.collectively.migrated.towards.the.wound.edge..Inside.the.intact.or.reconstructed.tissue,.cells.moved.in.the.basal.layer.underneath.a.fixed.suprabasal.layer.and.accumulated.in.the.suprabasal.layer.of.the.EET.in.form.of.a.shield..Shield.extension.thereby.appeared.driven. by. massive. collective. cell. migration. from. unwounded. tissue.regions..We.then.characterized.our.model.by.a.systematic.analysis.of..50. cytokines. using. a. multiplex. proteomic. bead. array.. Temporal. inve-stigation.of. the.phosphorylation.status.of.migration.and.proliferation.determining.molecules.of.the.MAPK-.and.JAK-STAT-pathway.enabled.us.to.connect.cytokine.secretion.with.intracellular.signaling.in.wound.he-aling..We.discovered.Erk1/2,.p38.and.STAT3.as.cell.fate.determining.key.players.as.well.as.potential.cytokines.responsible.for.triggering.these.pathways.. Our. results. link. the. mechanism. of. collective. epithelial. mi-gration.which.occurs.during.wound.healing.in.an.orchestrated.manner.with.the.underlying.signal.transduction.pathways.based.on.epithelial-mesenchymal.crosstalk..In.cancer.invasion.this.mechanism.of.collective.migration. is. hijacked. by. the. tumor. in. an. emergent. malignant. exploit.of.the.intrinsically.healthy.processes.of.epithelial.morphogenesis.and.repair.

Poster P-0126

A. El-Heliebi.1,.P..Sedlmayr.2,.T..Kroneis.2,.E..Zoehrer.1,.J..Haybaeck.1,.K..Fischereder.3,.K..Kampel-Kettner.3,.R..Zigeuner.3,.B..Huppertz.2,.C..Lackner.1

1. Medical.University.of.Graz,.Institute.of.Pathology,.Graz,.Austria.2. Medical.University.of.Graz,.Institute.of.Cell.Biology,.Histology.and.

Embryology,.Graz,.Austria.3. Medical.University.of.Graz,.Department.of.Urology,.Graz,.Austria.

Genetic and cytopathological characterization of circulating non-haematological cells in renal cancer

Introduction:.Malignant.tumors.may.release.tumor.cells.into.the.blood.either. singly. (circulating. tumor. cells,. CTCs). or. as. cellular. aggregates.(tumor.cell.microemboli,.CTM)..Detection.of.CTCs.is.an.important.pro-gnostic.factor..However.studies.of.CTCs/CTMs.in.renal.cell.cancer.(RCC).are.limited..We.aimed.to.isolate.so.called.circulating.non-haematologic.cells.(CNHCs).-.putative.CTCs/CTMs.-.in.patients.with.RCC.and.characte-rize.these.cells.by.using.cytopathological,. immunological.and.genetic.analyses.

Methods:.Whole.blood.of.40.patients.with.malignant. (30).and.benign.(10).renal.tumors.was.subjected.to.ScreenCell®.size.filtration..Epithelial.tumor.cells.are.mostly.larger.than.leukocytes.and.are.trapped.on.the.fil-ter..Putative.CTCs/CTMs.were.classified.according.to.cytomorphological.criteria.and.analyzed.by.immunocytochemistry.using.antibodies.against.carbonic. anhydrase. IX. (CAIX,. a. RCC. tumor. marker),. the. endothelial.marker.CD31.and.the.leukocyte.marker.CD45..Potential.CTCs.and.CTMs.were.isolated.from.the.filters.by.laser.capture.microdissection,.DNA.was.extracted,. subjected. to. whole. genome. amplification. and. analyzed. by.array.comparative.genomic.hybridization.(array-CGH)..The.DNA.of.the.respective.primary.tumor.tissue.served.as.control.

Results:.CNHC.clusters.–.putative.CTMs.–.were.negative.for.CD45,.posi-tive.for.CD31.(85%),.while.only.6%.were.CAIX.positive..Putative.CTCs.were. negative. for. CD45. and. 56%. were. positive. for. CAIX.. Array-CGH.analysis.showed.genomic.DNA.imbalances.in.17%.of.isolated.CNHC.clu-sters,.which.did.not.match.the.aberrations.of.the.primary.tumors.

Conclusions:.A.high.fraction.of.CNHC.clusters,.putative.CTMs,.captured.by. ScreenCell. ®. filtration. seem. to. be. endothelial. cells.. On. the. other.side.a.high. fraction.of. the.single.CAIX.positive.CNHCs.may.represent.true.CTCs..Although.speculative,.the.differences.in.genetic.aberrations.between.CTMs.and.the.respective.primary.tumors.might.be.explainable.by.tumor.heterogeneity..For.patients.with.RCC,.cytomorphological.clas-sification.alone.seems.to.be.not.sufficiently.specific.to.identify.CTCs/CTMs..Reliable.detection.should.be.confirmed.by.molecular.biological.methods.

44

AEKAEK AEKAbstracts Posters Section 4: Tumor environment and inflammation

Poster P-0007

F. S. B. Subtil.1,.J..Hänze.2,.A..Arenz.1,.U..Seay.3,.D..Hasan.3,.B..Bischoff.1,.O..R..Gottschald.3,.C..Fournier.4,.G..Taucher-Scholz.4,.M..Scholz.4,.W..Seeger.3,.F..Rose.1,.R..Engenhart-Cabillic.1

1. Philipps-University,.Department.of.Radiotherapy.and.Radiooncology,.Marburg,.Germany.

2. Philipps-University,.Department.of.Urology.and.Pediatric.Urology,.Marburg,.Germany.

3. Universities.of.Giessen.&.Marburg.Lung.Center.,.(UGMLC),.Gießen,.Germany.

4. GSI,.Helmholtzzentrum.für.Schwerionenforschung,.Darmstadt,.Germany

Comparison of carbon-ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells

Radiotherapy. resistance. is. a. commonly. encountered. problem. in. can-cer.treatment..In.this.regard,.stabilization.of.endothelial.cells.and.re-lease.of.angiogenic.factors.by.cancer.cells.contribute.to.this.problem..Here,.employing.human.lung.adenocarcinoma.(A549).cells,.the.effects.of. carbon-ion. and. X-ray. irradiation. on. the. angiogenic. response. were.compared.

A549.cells.were.irradiated.with.biological.equivalent.doses.for.cell.sur-vival. of. either. carbon-ions. (LET. 170. keV/µm,. energy. of. 9.8. MeV/u. on.target).or.X-rays.and.injected.with.Matrigel.into.BALB/c.nu/nu.mice.to.generate.a.plug,.allowing.quantification.of.angiogenesis.by.blood.vessel.enumeration.. The. expression. of. angiogenic. factors. (VEGF,. PlGF,. SDF-1.and.SCF).was.assessed.at. the.mRNA.and.secreted.protein. levels.em-ploying. real. time. RT-PCR. and. ELISA.. Signal. transduction. mediated. by.stem.cell.factor.(SCF).was.assessed.by.phosphorylation.of.its.receptor.c-Kit..For.inhibition.of.SCF/c-Kit.signaling.a.specific.SCF/c-Kit.Inhibitor.(ISCK03).was.used.

Irradiation.of.A549.with.X-rays. (6.Gy).but.not. carbon-ions. (2.Gy). re-sulted. in. a. significant. increase. in. blood. vessel. density. (control. 20.71.±.1.55,.X-ray.36.44.±.3.44,.carbonion.16.33.±.1.03)..Concordantly,.irra-diation.with.X-rays.but.not.with.carbon-ions.increased.the.expression.of. SCF,. and. subsequently. caused. phosphorylation. of. cKit. in. endothe-lial.cells..ISCK03.treatment.of.A549.cells.irradiated.with.X-rays.(6.Gy).resulted.in.a.significant.decrease.in.blood.vessel.density.(X-ray.36.44.±.3.44,.X-ray/ISCK03.4.33.±.0.71)..These.data.indicate.that.A549.cells.irradiated.with.Xrays.but.not.carbon-ions.promote.angiogenesis.

The.present.study.provides.evidence.that.SCF.is.an.X-ray-induced.me-diator.of.angiogenesis.in.A549.cells,.a.phenomenon.that.could.not.be.observed.with.carbon-ion.irradiation..Thus,.in.this.model.system.evalu-ating.angiogenesis,.carbon-ion.irradiation.may.have.a.therapeutic.ad-vantage..This.observation.should.be.confirmed.in.orthotopic.lung.tumor.models.

Poster P-0030

K. Zieger.1,.D..Behrens.2,.I..Fichtner.2,1

1. Max-Delbrück-Center.for.Molecular.Medicine,.Berlin,.Germany.2. Epo.GmbH.,.Berlin,.Germany.

The impact of cancer-associated fibroblasts (CAFs) on the progressi-on of lung carcinoma xenografts

Background:. Several. experimental. studies. have. provided. strong. evi-dence,. that. the. tumor. microenvironment. contributes. significantly. in.progression,.metastasis.and.angiogenesis.of.different.carcinomas..The.major.cell.population.in.the.tumor.stroma.are.cancer-associated.fibro-blasts.(CAFs)..CAFs.are.phenotypically.and.functionally.distinguishable.from.their.normal.counterparts.through.the.expression.of.the.surface.markers.CD90,.PDGFR,.fibroblast-activated.protein.(FAP),.and.the.fila-ment-associated. protein. alpha-smooth-muscle. actin. (-SMA).. Resident.CAFs.are.mainly.responsible.for.production.of.extracellular.matrix.pro-teins,.metalloproteinases.and.growth.factors..The.activated.tumor.stro-ma.generates.a.switch.from.the.“normal”.extracellular.matrix.(ECM).to.a.pro-malignant.microenvironment.supporting.the.proliferation.of.the.tumor. cells..Due. to. the. close. crosstalk. with. tumor. cells,. CAFs.act. as.synergistic.bystanders.and.promote.cancer.progression.The.aim.of.our.work.was.to.develop.methods.for.the.isolation,.ex.vivo.cultivation,.and.characterisation.of.cancer-associated.fibroblasts.from.patient-derived.lung.cancer.xenografts...To.understand.the.function.of.cytokines. in. the. communication. between. cancer. cells. and. CAF’s,. co-cultivation.experiments.have.been.performed..and.differential.cytokine.expression.analyzed.Methods:. CAFs. from. 3. different. patient-derived. lung. carcinoma. xeno-grafts. were. isolated. with. enzymatic. and. mechanical. techniques.. CAF’s.were.propagated.in.cell.culture.until.stable.growth..The.expression.of.the.characteristic.markers.for.CAF’s.were.confirmed.by.FACS.analysis.(rele-vant.markers:.CD90,.FAP,.a-SMA).and.IHC.(PDGFR)..Co-cultivation.of.CAFs.with.tumors.cells.was.performed.in.special.trans-well-systems..Changes.in.gene.and.protein.expression.of. the. interesting.cytokines. IL-6,.TGFβ,.SDF1β.and.Cox-1.was.measured.with.ELISA.and.real-time.PCR.methods.Results:.Methods.for.the.isolation.of.CAFs.and.their.cultivation.under.in-vitro/.ex-vivo.conditions.were.successfully.established..We.were.able.to.show,.that.up.to.90%.of.all.lung.carcinoma-associated.fibroblasts.were.positive. for. CD90,. but. expression. of. a-SMA. and. FAP. varied. between.tumor.models..Co-cultivation.experiments.demonstrated.a. significant.stimulation.of.tumor.cell.proliferation.by.CAF.co-culture..Analysis.of.the.cell. culture. supernatants. revealed. a. constitutive. secretion. of. murine.IL-6,.TGFβ,.SDF-1β.and.Cox-1.by.CAF’s.but.only.rarely.from.tumor.cells..Real-time.PCR.of.both:.tumor.cells.with.and.without.CAF’s.demonstra-ted.an.upregulation.of.the.corresponding.mRNAs.in.the.CAF’s.only.Conclusion:. We. have. shown,. that. tumor. stroma. of. lung. cancer. xeno-grafts.contains.approx..30%.activated.fibroblasts.despite.of.the.spe-cies.difference..The.CAF’s.can.be.grown.ex.vivo.and.express.a.panel.of.relevant.markers..CAF’s.secrete.cytokines.that.significantly.stimulate.progression. of. tumor. growth.. These. findings. demonstrate,. the. CAF’s.could. contribute. to. the. tumor. progression. by. generation. of. a. favou-rable.microenvironment..We.have.shown.that.mouse.CAF’s.can.secrete.cytokines.supporting.the.growth.of.human.tumor.cells..These.findings.demonstrate.that.the.sufficient.activation.of.CAF’s.could.have.impact.on.the.engraftment.of.patient-derived.cancer.material.in.mice..Our.system.can.further.be.used.as.tool. for. insights. into.the.comprehensive.com-munication.between.cancer.cells.and. their.microenvironment.and. for.the.development.of.new.therapeutics.interrupting.this.communication.

AEK

45

AEKAbstracts Posters Section 4: Tumor environment and inflammation

Poster P-0034

T. Brunner.1,.O..Al-Assar.1,.F..Demiciorglu.1,.M..M..Gaspar-Carvalho.1,.S..Lunardi.1,.W..G..McKenna.1,.R..Muschel.1

1. Oxford.University,.Gray.Institute.for.Radiation.Oncology.&.Biology,.Oxford,.Germany.

Contextual regulation of pancreatic cancer stem cell phenotype and radioresistance by pancreatic stellate cells

Question:.Progression.of.pancreatic.ductal.adenocarcinoma.(PDAC). is.promoted. by. dysmoplasia. induced. by. pancreatic. stellate. cells. (PSC)..Epithelial.mesenchymal.transition.(EMT).is.a.process.known.to.contri-bute. to. this. progression. and. shares. many. characteristics. with. caner.stem. cells. (CSC). hypothesis.. We. investigated. the. role. of. PSC. in. EMT.and. CSC. processes. and. the. effect. this. has. on. the. radioresponse. and.tumortumorigen[b1].ecity.of.pancreatic.cancer.cells..We.used.TGFβ.in-hibition.to.control.both.processes.and.identified.several.novel.factors.that.can.be.used.as.therapeutic.agents.

Methods:.Three-dimensional.(3D).in.vitro.and.xenograft.in.vivo.models.were.used.to.measure.the.radioresponsiveness.and.tumortumortumor-tumorigenicity.of.pancreatic.cancer.cells.as.a.function.of.PSC.presence..The. expression. of. EMT. and. CSC. markers. was. assayed. using. different.techniques.in.both.models..A.proteome.profiling.screened.for.novel.fac-tors.regulated.by.PSC.under.3D.conditions.

Results:.We.demonstrated.that.pancreatic.cancer.cells.co-cultured.with.PSC.possess.an.enhanced.CSC.phenotype.and.radioresistance..Further-more,.the.expression.of.several.EMT.and.CSC.markers.supported.enhan-ced.processes.in.our.models.and.that.translated.into.remarkable.in.vivo.tumortumortumorigenicity.. The. use. of. multi-dose. TGFβ. neutralising.antibody. inhibited. the. EMT. and. CSC. processes,. sensitized. pancreatic.cancer.cells.to.radiation.and.reduced.in.vivo.tumorigenicitya..A.prote-ome.screen.identified.multiple.novel.factors.that.were.regulated.by.PSC.in.three.pancreatic.cancer.cell.line.spheres.

Conclusions:. These. results. are. critical. in. highlighting. the. role. of. PSC.in.tumor.progression.and.radioresistance.by.manipulating.the.EMT.and.CSC.processes..TGFβ.is.one.of.the.factors.that.play.an.important.role.in.the.different.processes.mediated.by.PSC.targeting.of.which.implies.better.therapeutic.outcome.

Poster P-0043

M. Tham.1,.K..Sobel.1,.H..Stammer.1,.P..Boukamp.1

1. German.Cancer.Research.Center.(DKFZ),.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany

Wnt/β-catenin-activated stromal fibroblasts: A decisive role for tumor-stroma cross-talk in human skin carcinogenesis

Human. skin. squamous. cell. carcinomas. (SCCs). are. the. second. most.frequent.skin.cancer.type.and.numbers.are.even.increasing.due.to.im-munosuppressed.transplant.recipients,.who.contract.SCCs..Underlying.mechanisms. of. SCC. development. are. not. fully. understood,. but. invol-ve. the.misregulation.of.signaling.pathways. including.aberrant.Wnt/β-catenin.activation,.which.is.a.hallmark.of.many.cancers..In.particular,.previous. studies. have. demonstrated. the. translocation. of. β-catenin.to. nuclei. of. SCC. tumor. cells.. By. analyzing. primary. SCCs,. we. show. in.addition.to.Wnt-deregulation. in. tumor.cells.also.Wnt-activation. in. the.tumor-underlying.stroma.and.particularly.in.fibroblasts..In.vitro,.HaCaT.keratinocytes,.a.model.for.pre-malignant.SCCs,.respond.only.weakly.to.direct. Wnt. pathway. activation. and. show. no. functional. consequences..However,.in.tissue-like.organotypic.co-cultures.(OTCs).with.human.der-mal.fibroblasts,.which.respond.strongly.to.Wnt.3a,.HaCaT.keratinocytes.show. a. significant. increase. in. proliferation.. To. determine. the. critical.mediators.in.this.fibroblast-epithelial.cross-talk,.we.performed.a.global.gene.expression.analysis.of.Wnt.3a-stimulated.fibroblasts,. identifying.that. fibroblasts. secrete. high. amounts. of. the. cytokines. interleukin-8..(IL-8).and.chemokine.(C-C.motif).ligand.2.(CCL2)..Blocking.IL-8.and.CCL2.in.OTCs.prevents.the.Wnt-induced.increase.in.HaCaT.cell.proliferation,.showing.that.both.chemokines.are.indeed.the.responsible.factors..Addi-tionally,.fibroblasts.express.matrix.metalloprotease.1.(MMP1).upon.Wnt.3a.stimulation,.which.assists.keratinocytes.in.modulating.the.extracel-lular.matrix..Thus,.these.findings.suggest.a.Wnt/β-catenin.pathway-de-pendent.fibroblast-epithelial.cross-talk.and.demonstrate.a.novel.role.of.Wnt.signaling.in.the.microenvironment.of.developing.SCCs,.underlining.how.misregulation.influences.tumor.progression.

46


Poster P-0053

S. Mamlouk.1,2,.J..Kalucka.3,.R..Pal.Singh.2,.K..Franke.2,.A..Muschter.2,.A..Langer.2,.C..Jakob.4,.M..Gassmann.5,.G..Baretton.4,.B..Wielockx.2

1. Charité,.Pathology,.Berlin,.Germany.2. Emmy.Noether.Research.Group,.University.of.Technology,.Dresden,.

Pathology,.Dresden,.Germany.3. University.Clinic.Erlangen,.Nephrology.and.Hypertension,.Erlangen,.

Germany.4. University.of.Technology,.Dresden,.Pathology,.Dresden,.Germany.5. University.of.Zürich,.Institute.of.Veterinary.Physiology,.Vetsuisse.

Faculty.and.Zurich.Center.for.Integrative.Human.Physiology.(ZIHP),.Zürich,.Germany

Loss of Prolyl Hydroxylase-2 in Myeloid Cells and T-lymphocytes Im-pairs Tumor Development

The.tumor.microenvironment.plays.a.pivotal.role.during.cancer.develop-ment.and.progression..The.balance.between.suppressive.and.cytotoxic.responses.of.the.tumor.immune.microenvironment.has.been.shown.to.have.a.direct.effect.on.the.final.outcome.in.various.human.and.expe-rimental. tumors.. Recently,. we. demonstrated. that. the. oxygen. sensor.HIF-prolyl.hydroxylase.2.(PHD2).plays.a.detrimental.role.in.tumor.cells,.driving.systemic.growth.and.metastasis.in.mice..

Question:.In.the.current.study,.we.were.interested.to.find.out.whether.ablation.of.PHD2.in.the.hematopoietic.system.can.influence.tumor.de-velopment.in.vivo..

Methods:.We.used.a.syngenic.tumor.model.(Lewis.lung.carcinoma).in.dif-ferent.conditional.PHD2.knockout.mice.directed.to.certain.subtypes.of.the.hematopoietic.system..The.analysis.of.the.obtained.tumors.included.immunohistochemistry,.FACS.and.gene.profiling.

Results:.Our.experiments. show. that.PHD2.knockout. in. the.entire.he-matopoietic.system.led.to.a.significant.decrease.in.tumor.volume,.ge-nerated.by.an.overall.down.regulation.of.pro-.as.well.as.anti-tumoral.cytokines..Intriguingly,.this.imbalance.resulted.in.enhanced.cell.death.but.also.improved.proliferation.of.tumor.cells..Moreover,.we.were.able.to. confine. the.complex.phenotype. to. the.crosstalk.of.PHD2-deficient.myeloid.cells.and.T-lymphocytes.

Conclusion:.Taken.together,.our.findings.reveal.a.multifaceted.role.for.PHD2.in.several.hematopoietic.lineages.during.tumor.development.and.may.have.important.implications.for.the.development.of.tumor.thera-pies.in.the.future.

Poster P-0079

V. Jung.1,.M..Saar.1,.J..Kamradt.1,.K..Schmitt.2,.G..Vitale.3,.M..Stöckle.1,.G..Unteregger.1

1. Universität.des.Saarlandes,.Klinik.für.Urologie,.Homburg,.Germany.2. Universität.des.Saarlandes,.Institut.für.Pathologie,.Homburg,.

Germany.3. University.Milan,.Auxologic.Institute.Italy,.Milan,.Italy

Microarray expression analysis of cancer-associated fibroblasts and normal fibroblasts in prostate cancer

Introduction:. There. is. strong. evidence. that. the. tumor. microenviron-ment. contributes. to. the. normal. prostate. development. as. well. as. to.proliferation,.invasion.and.migration.in.prostatic.carcinoma..Especially.fibroblasts.surrounding.epithelial.tumor.cells.seem.to.play.a.pivotal.role.during.EMT.and.tumor.invasion..Several.strategies.were.undertaken.to.describe. reliable. biomarkers. for. cancer. associated. fibroblasts. (CAF)..Using.fibroblasts. from.patients.undergoing. radical.prostatectomy. the.expression.profile.of.CAF.was.analysed.using.microarrays.and.compared.to. non-cancer. associated. fibroblasts. of. the. same. patient. (NAF).. To.monitor.the.stability.of.gene.expression.profile.during.cell.culture.we.quantified.the.expression.levels.of.some.CAFs.specific.genes.like.FAP,.aSMA,.PDGFRβ.and.TGF-β.between.passages.1.to.7.

Material. and.methods:. Tissue.pieces. from.cancerous.and.cancer. free.areas.of.7.prostates.were.cultivated.for.selection.of.CAF.and.NAF,.re-spectively..Total.RNA.was.isolated.using.RNeasy.Kit.(Qiagen).according.to.manufactures.recommendation..For.microarray.expression.analysis.750ng.biotinylated.RNA.per.sample.was.hybridized.to.Illumina.HT-12v4.0.Expression.BeadChips..All.primers.and.kits.used.for.the.qRT-PCR.expres-sion.analysis.of.CAF-related.genes.during.cell.culture.were.purchased.from.Applied.Biosystems.

Results:.Using.microarray.analysis.we.found.a.significant.overexpres-sion.of.the.following.genes.in.fibroblasts.associated.to.prostate.cancer.tissue:.MAP3K1.(a.MAP.kinase.associated.to.stress.and.NFkB-dependent.pathways),. NBPF11. (neuroblastoma. breakpoint. family. member. 11),.SCARB1. (regulates. transport. of. HDL. thus. increasing. the. intracellular.Cholesterol.level)..The.expression.of.the.plasminogen.receptor.S100A1.and.SNAPC2.was.decreased..Our.qRT-PCR.analysis.offers.a.constant.ex-pression.of.SDF1,.PDGFRß,.TGF-ß.and.βSMA.during.culture.whereas.the.expression.levels.of.the.FAP.gene.decreased.dramatically.from.p1.to.p7.

Conclusion:.This.is.the.first.comprehensive.expression.analysis.of.NAF/CAF. in. prostate. cancer.. Enhanced. expression. (MAP3K1,. SCARB1). may.influence.cell.movement.and.the.level.of.cholesterol.for.androgen.syn-thesis. in. fibroblasts.. Since. macrophage. migration. into. tumor. cells. is.controlled.by.S100A10.CAFs.are.able.to.control.this.process,.too..Thus,.our.results.provide.strong.evidence.that.these.changes.in.gene.expres-sion.in.CAFs.can.contribute.to.functional.alteration.of.adjacent.prostate.cancer.cells..It.is.very.important.to.note.that.during.serial.cultivation.of. fibroblast. the. expression. profile. of. CAF-related. genes. undergoes.changes.as.a.consequence.to.the.particular.in.vitro.culture.

AEK

47


Poster P-0084

M. Nici.1

1. German.Cancer.Research.Center,.Heidelberg,.Germany.

Differential regulation of the IL-6 signaling pathway in HaCaT A5 be-nign tumor keratinocytes and fibroblasts

Tumor.progression.is.regulated.by.a.crosstalk.between.different.cell.ty-pes.within.the.tumor.and.its.surrounding.tissue..The.activated,.progres-sion.promoting. tumor.microenvironment. is. initially. induced.by.a.net-work.of.tumor.derived.growth.factors.(cytokines).that. induce.cellular.responses.in.tumor.and.stromal.cells..In.a.tumor.transplantation.model.of.HaCaT.skin.squamous.cell.carcinomas.(SCC).we.could.demonstrate.the.functional.contribution.of.an.IL-6.regulated.growth.factor.network.to.tumor.progression.In.response.to.ligand.binding,.the.IL-6R.activates.the.JAK/STAT.signaling.pathway.in.both.stromal.fibroblasts.and.tumor.cells..However,.pathway.activation.results.in.the.induction.of.different.target.genes.and.triggers.different.cellular.responses.in.both.cell.types..With.the.determination.of.the.cellular.response.of.IL-6.stimulation.in.the.HaCaT-ras.A5.SCC.and.fibroblast.cells.we.were.able.to.establish.and.validate.a.mathematical.model.for.JAK/STAT.signaling.Comparison.of.the.STAT3.dynamics.showed.a.similar.trend.but.the.total.amount.of.activated.STAT3.proteins. in.HaCaT-ras.A5. is.3.5.to.5.times.higher.than.in.fibroblasts.Further.analyses.of.the.proliferation.in.both.celltypes.after.IL-6.stimu-lation.showed.that.IL-6.promotes.proliferation.in.HaCaT-ras.A5.whereas.the.proliferation.is.not.affected.in.fibroblasts.The.correlation.between.higher.STAT3.activation.and.increased.prolife-ration.in.HaCaT-ras.A5.lead.us.to.the.assumption.that.STAT3.is.involved.in.promoting.proliferation.We.could.confirm.this.by.inhibiting.STAT3.activation.with.a.STAT3.inhibi-tor.which.led.to.a.decreased.level.of.proliferation.In.conclusion,.we.could.clearly.show.that.the.activation.and.more.preci-sely.the.total.amount.of.activated.STAT3.plays.a.major.role.in.HaCaT-ras.A5.proliferation.Analyzing.the.differential.signaling.networks.and.phenotypical.changes.of. keratinocytes. and. fibroblasts. will. create. the. basis. for. therapeutic.targeting.of.the.tumor.promoting.IL-6.effects.in.tumor.cells.and.cells.of.the.tumor.microenvironment.

Poster P-0086

T. Pusterla.1,.J..Németh.1,.I..Stein.2,.L..Wiechert.1,.D..Knigin.2,.S..Marhenke.3,.T..Longerich.4,.V..Kumar.5,.B..Arnold.6,.A..Vogel.3,.A..Bierhaus.5,.E..Pikarsky.2,.J..Hess.7,8,.P..Angel.1,7,8

1. German.Cancer.Research.Center,.Signal.Transduction.and.Growth.Control,.Heidelberg,.Germany.

2. Hebrew.University-Hadassah.Medical.School,.Jerusalem,.Israel.3. Medical.School.Hannover,.Department.of.Hepatology,.Hannover,.

Germany.4. University.Hospital.Heidelberg,.Institute.of.Pathology,.Heidelberg,.

Germany.5. University.Hospital.Heidelberg,.Department.of.Medicine.I.and.

Clinical.Chemistry,.Heidelberg,.Germany.6. German.Cancer.Research.Center,.Molecular.Immunology,.Heidelberg,.

Germany.7. German.Cancer.Research.Center,.Molecular.Mechanisms.of.Head.and.

Neck.Tumors,.Heidelberg,.Germany.8. University.Hospital.Heidelberg,.Otolaryngology,.Head.and.Neck.

Surgery,.Heidelberg,.Germany.

RAGE is a key regulator of oval cell activation and inflammation-associated liver carcinogenesis in mice

The.Receptor. for.Advanced.Glycation-End.products. (RAGE). is.a.multi-ligand.receptor.and.member.of.the.immunoglobulin.superfamily..RAGE.is.mainly.involved.in.tissue.damage.and.chronic.inflammatory.disorders,.sustaining.the. inflammatory.response.upon.engagement.with.damage.associated.molecular.pattern.molecules. (DAMPs).such.as.S100.prote-ins.and.HMGB1..Enhanced.expression.of.RAGE.and.its.ligands.has.been.demonstrated.in.distinct.tumors.and.several.studies.support.its.crucial.role.in.tumor.progression.and.metastasis.by.still.unknown.mechanisms.Here.we.show.that.RAGE.supports.hepatocellular.carcinoma.formation.in.the.Mdr2-/-.mouse.model,.a.prototype.model.of.inflammation-driven.HCC.formation,.which.mimics.the.human.pathology..Mdr2-/-.Rage-/-.(dKO).mice.developed.smaller.and.fewer.HCCs.than.Mdr2-/-.mice..Interestingly,.although.in.pre-neoplastic.Mdr2-/-.livers.RAGE.ablation.did.not.affect.the.onset.of. inflammation,.pre-malignant.dKO.livers.showed.reduced.liver.damage.and.fibrosis,.in.association.with.decreased.oval.cell.activation..Oval.cells.expressed.high.RAGE.levels.and.displayed.reduced.proliferati-on.upon.RAGE.silencing..Moreover,.stimulation.of.oval.cells.with.HMGB1.promoted.an.ERK1/2-Cyclin.D1-dependent.oval.cell.proliferation.in.vitro..Finally,.genetic.and.pharmacologic.blockade.of.RAGE.signaling.impaired.oval.cell.activation.in.an.independent.mouse.model.of.oval.cell.activati-on,.the.Choline.Deficient.Ethionine-supplemented.(CDE).dietary.regime..Conclusions:.Our.data.have. identified.a.novel. function.of.RAGE. in. re-gulating.oval. cell. activation.and. tumor.development. in. inflammation-associated.liver.carcinogenesis.

48


Poster P-0095

A. Otto.1,.C..Janzon.2

1. TU.München,.IMETUM,.Garching,.Germany.2. Klinikum.Leverkusen,.Abteilg.Onkologie/Hämatologie,.

51373.Leverkusen,.Germany

Metabolic heterogeneity of tumor cells in a variable microenviron-ment

The.supply.of.nutrients.in.a.tumor.depends.on.the.microenvironment,.i.e..the.interaction.with.stroma.cells.and.the.distance.from.a.blood.ves-sel..The.question.is.how.variable.concentrations.of.metabolites.in.a.pH.gradient.will.affect.tumor.cell.growth.and.metabolism.

For.a.quantitative.analysis,.the.breast.cancer.cell.line.MCF-7.was.cultiva-ted.in.“low.growth”.medium.(Dulbecco’s.MEM.with.2%.FCS).containing.tumor-relevant. concentrations. of. glucose,. glutamine. and. lactate. and.set.at.different.pH.(6.6.-.7.4)..Growth.was.determined.by.a.cell.count..Metabolic.activity.was.measured.using.a.tetrazolium-based.(mitochond-rial).dehydrogenase.assay.(WST),.by.kinetic.analysis.of.pyruvate.kinase.and.lactate.dehydrogenase.activities,.as.well.as.by.determining.glucose.consumption.and.lactate.production.

The.results.show:1).Tumor.cell. growth.and.metabolic. activity.do.not. correlate..At. limi-

ting.nutrient.concentrations,.the.combination.of.0.1.mM.glutamine./.2.5.mM.glucose.sustains.higher.cell.viability.than.combinations.with.other. concentrations.. Moreover,. at. the. enzymatic. level,. there. is. a.particularly. high. activity. of. mitochondrial. dehydrogenases. and. py-ruvate.kinase..This. suggests.a. shift. to.more.oxidative.metabolism..Lactate.can.enhance.mitochondrial.dehydrogenase.activity.and.cell.growth,.but.only.when.glucose.is.limiting.

2).Cell.growth.and.metabolic.activities.are.highly.sensitive.to.the.extra-cellular.pH..At.an.extracellular.pH.7.4,.there.is.high.metabolic.varia-bility.depending.on.the.metabolite.concentrations.and.combinations,.while.at.an.acidic.pH.(6.6),.these.tumor.cells.become.almost.indepen-dent.of.variable.levels.of.glucose.and.glutamine.for.both.growth.and.the.metabolic.activity.tested..Such.cells.produce.very.little.lactic.acid.compared.to.those.growing.at.pH.7.4.

These.quantitative.results.constitute.the.basis.for.developing.a.meta-bolic. interaction.analysis.using.mathematical.algorithms,.which.could.predict.metabolic.activity.and.survival.of.tumor.cells.depending.on.their.microenvironment..This.would.also.serve.the.evaluation.of.clinical.me-tabolic.imaging.technologies.such.as.MRT.and.PET..

Poster P-0097

A. De Ponti.1,.T..Pusterla.1,.I..Stein.2,.E..Pikarsky.2,.J..Hess.1,3,.P..Angel.1

1. DKFZ.Heidelberg,.Signal.Transduction.and.Growth.Control,.Heidelberg,.Germany.

2. Hebrew.University-.Hadassah.Medical.School,.Department.of.Pathology.and.the.Lautenberg.Center.for.Immunology,.Jerusalem,.Israel.

3. University.Hospital.Heidelberg,.Department.of.Otolaryngology,.Head.and.Neck.Surgery,.Heidelberg,.Germany

RAGE signaling in oval cell activation during liver damage

The.receptor.for.advanced.glycation.end-products.(RAGE).is.a.pattern-recognition.receptor,.able.to.bind.different.classes.of.molecules.relea-sed.during.tissue.damage.and.inflammation.(such.as.HMGB1.and.seve-ral.calcium-binding.S100.proteins)..Rage.engagement. in. inflammatory.conditions.and.in.cancer.upregulates.the.receptor.itself.and.activates.different. pro-inflammatory. responses. and. promotes. tumor. develop-ment.. In. the. liver,.Rage.has.been.shown. to.play.an. important. role. in.chronic.inflammation.and.damage..We.previously.demonstrated.that.in.the. Mdr2. knock. out. mouse,. a. model. of. inflammation-associated. HCC.development,.Rage.ablation. impairs.tumor.development.and.causes.a.delay. in.the.onset.of. liver.damage.and.fibrosis..This.phenotype.is.as-sociated.with.a.reduced.activation.of.oval.cells,.the.hepatic.progenitor.cells.involved.in.liver.regeneration..We.found.that.primary.oval.cells.and.BMOL.cells.(an.oval.cell.line).express.high.levels.of.Rage..In.BMOL.cells.stimulation.by.HMGB1.promotes.an.ERK1/2-cyclin.D1.dependent.cell.pro-liferation..In.accordance,.Rage.knock-down.reduces.BMOL.cell.prolifera-tion,.and.in.vivo.blockade.of.the.receptor.signaling.by.means.of.injection.of.the.decoy.receptor.sRAGE.reduces.oval.cell.activation.

In.order.to.analyse.the.role.played.by.RAGE.in.oval.cell.activation,.we.are.feeding.Rageflx.mice,.a.conditional.knock-out.mouse.line,.with.a.Choline.Deficient. Ethionine-supplemented. diet. (CDE),. a. dietary. regime. which.induces.extensive.oval.cell.activation..Oval.cells.isolated.from.CDE-fed.Rageflx.mice.will.be.recombined.in.vitro.by.means.of.transduction.with.a.lentiviral.vector.carrying.a.Cre.recombinase..On.parallel,.another.vector.carrying.an.shRNA.will.be.used.to.knock-down.Rage.in.BMOL.cell.line..In.Rage-positive.and.Rage-deleted.cells,.we.will.then.characterize.cel-lular.responses.(proliferation,.migration,.apoptosis,.differentiation.and.cytokines.release).and.signalling.pathways.promoted.by.RAGE.ligands.

AEK

49


Poster P-0100

A. Schwenzer.1,.F. Saupe.1,.I..Gasser.1,.O..Lefebvre.1,.M..Marko.1,.T..Hussenet.1,.M..Kammerer.1,.G..Christofori.2,.G..Orend.1

1. INSERM.Unit.1109,.Strasbourg,.France.2. University.of.Basel,.Basel,.Switzerland.

Tenascin-C (TNC) promotes tumor angiogenesis through downregula-tion of WNT inhibitor DKK1

The.extracellular.matrix.molecule.tenascin-C.(TNC).is.a.major.compo-nent.of.the.cancer.specific.matrix.and.high.TNC.expression.is.linked.to.bad.prognosis.in.several.cancers.but.the.underlying.molecular.mecha-nisms.are.poorly.understood.

To.delineate.TNC’s.functions.in.cancer,.we.established.the.first.model.in.which.high.expression.of.TNC.in.cancer.was.recapitulated.in.a.stochastic.model.with.an.intact.immune.system.through.transgene.expression.in.RipTag2.mice.and.compared.sporadic.neuroendocrine.tumor.formation.in.abundance.or.absence.of.TNC..These.studies.provide.a.comprehensive.view.on.the.roles.of.TNC. in.tumor.progression.and.revealed.that.TNC.enhances. survival,. proliferation,. carcinoma. formation,. angiogenesis.and.lung.micrometastasis..In.these.tumors.DKK1.expression.correlated.inversely.with.TNC..Importantly,.we.used.in.vitro.models.to.show.that.a.TNC.substratum.indeed.leads.to.DKK1.downregulation.and.induces.Wnt.signaling.in.several.cellular.models,.including.endothelial.cells..In.addi-tion.we.showed.that.DKK1. is.a.potent. inhibitor.of.tumor.angiogenesis.and.growth.in.vivo.using.a.tumor.xenograft.model.

In.summary.our.results.suggest.that.TNC.promotes.tumor.angiogenesis.through. downregulation. of. DKK1. in. tumor. and. endothelial. cells.. Thus.targeting.TNC.and/or.downstream.regulators.such.as.DKK1.may.repre-sent.a.strategy.to.counteract.TNC.promoting.tumor.angiogenesis.and.progression.

Poster P-0128

A. Pastula.1,.M..Quante.1

1. Klinikum.rechts.der.Isar.TUM,.München,.Germany.

Mesenchymal cells regulate differentiation and proliferation in 3D models of intestinal crypt culture

Stem. cells. reside. within. special. location. in. the. gut. surrounded. by. a.stromal.microenvironment.consisting.of.distinct.cell.types,.matrix,.and.growth.regulators..The.stem.cell.niche.plays.an.important.role.in.stem.cell.maintenance,.division,.and.differentiation..In.the.intestine,.mesen-chymal.cell. types.such.as.pericryptal.fibroblasts.are. thought. to. form.this.niche.at.the.bottom.of.the.crypts..However.the.detailed.mechanism.of. the. interaction.of.mesenchymal. cells. and. intestinal. stem.cells. are.unknown.so.far.

To.investigate.the.role.of.mesenchymal.cells.in.intestinal.crypt.physio-logy.and.pathology.we.established.three.3D.culture.systems.of.mouse.intestinal.crypts.with.and.without.mouse.intestinal.mesenchymal.cells,.such.as.(1).intestinal.crypt.culture.in.matrigel,.(2).organotypic.cell.cul-ture,.and.(3).ex.vivo.culture.of.intestinal.tissue.fragments..The.effects.of.primary.intestinal.fibroblasts.(pFBs),.carcinoma.associated.fibroblas-ts.(CAFs).and.fetal.esophageal.fibroblasts.(FEFs).were.analysed.

Our.first.results.show.that.pFBs.maintain. intestinal.crypt.growth. in.a.short-term.co-culture.system.without.addition.of.routinely.used.exter-nal.growth.factors..Crypts.are.also.preserved.in.short-.and.long-term.ex. vivo. cultures. of. intestinal. tissue. fragments,. where. mesenchymal.cells.are.present,.indicating.that.this.cell.population.is.essential.for.the.maintenance. of. crypt. homeostasis.. Indeed,. by. using. pFB-conditioned.medium.we.confirm. that.potentially. active.components.are.within.fi-broblast.secretome..Moreover.short-term.crypt-pFB.co-culture.results.in.phenotypic.differences:.40%.of.crypts.are.increased.in.size,.without.budding.and.showing.increased.proliferation.and.decreased.differenti-ation..Similar.effect.was.observed.using.pFBs-conditioned.medium..In-terestingly,.intestinal.crypt.co-culture.with.FEF.and.murine.gastric.CAFs.gave.rise.to.similar.phenotype.of.crypts..We.did.not.observe.that.CAFs.induce.stronger.proliferation.of.crypt.cells.than.other.fibroblasts..The.mesenchymal.cells.express.Wnt.ligands.and.bFGF.but.at.the.same.time.express.BMP.molecules.and.BMP.anatagonists.

The.3D.intestinal.stem.cell.niche.organization.in.vitro.shows.that.diffe-rent.mesenchymal.cells.types.support.crypt.growth.in.a.similar.matter..Mesenchymal.cells.are.likely.responsible.for.the.regulation.of.differenti-ation.and.proliferation.in.the.stem.cell.niche.and.during.tumor.initiation..Nevertheless,.the.detailed.mechanisms.remain.to.be.analysed.

50

AEKAEK AEKAbstracts Posters Section 5: Systems biology and cancer

Poster.P-0040

D. Wuttig.1,.O..Bogatyrova.2,.L..Gu.2,.J..Huang.2,.R..Kuner.1,.C..Baer.2,.M..Faelth.1,.L..Feuerbach.3,.C..Gerhaeuser.2,.D..Weichenhan.2,.T..Schlomm.4,.R..Simon.5,.G..Sauter.5,.C..Plass.2,.H..Sueltmann.1

1. DKFZ/NCT,.Cancer.Genome.Research,.Heidelberg,.Germany.2. DKFZ,.Division.of.Epigenomics.and.Cancer.Risk.factors,.Heidelberg,.

Germany.3. DKFZ,.Division.of.Theoretical.Bioinformatics,.Heidelberg,.Germany.4. University.Medical.Center.Hamburg-Eppendorf,.Prostate.Cancer.

Center.Martini-Clinic,.Hamburg,.Germany.5. Dept..of.Pathology,.University.MedicalCenter.Hamburg-Eppendorf,.

Hamburg,.Germany

miRNA deregulation by aberrant promoter methylation in prostate cancer

Questions:.miRNAs.are.important.regulators.of.gene.expression.and.in-volved. in. tumorigenesis.and.disease.progression..Recent.publications.have. demonstrated. that. promoter. methylation. largely. contribute. to.miRNA.deregulation. in.cancer.. The.aim.of. this. study. is. to. investigate.the. impact.of.promoter.methylation.on.miRNA.expression. in.prostate.cancer.(PCa),.the.most.common.malignant.tumor.in.males.and.the.third.leading.cause.of.cancer-related.deaths.in.Western.countries.

Methods:. Two.genome-wide.miRNA.expression.datasets.of. altogether.58.PCa.and.49.non-malignant.prostate.tissues.generated.by.Next.ge-neration.sequencing.and.quantitative.PCR,.as.well.as.published.studies.were.used.to.define.miRNAs.that.are.consistently.deregulated.in.PCa..Putative. promoter. regions. of. these. miRNAs. were. extracted. from. pu-blished.studies..Promoter.regions.that.are.differentially.methylated.in.matched.genome-wide.DNA.methylation.data.of.the.two.datasets.men-tioned.above.were.identified.by.statistical.and.visual.approaches,.and.technically.validated.by.MassARRAY..Promoter.methylation.and.miRNA.expression.were.monitored.after.treatment.of.PCa.cells.with.5-aza-2‘-deoxycytidine.(5-Aza).using.MassARRAY.and.quantitative.PCR,.respec-tively.

Results:. 22. miRNAs. were. consistently. downregulated. and. 17. consi-stently. upregulated. in. several. PCa. datasets.. For. 28. of. these. miRNAs.(72.%).differentially.methylated.regions.(DMRs).were.detected.within.30.putative.promoter.regions.in.both.genome-wide.datasets.analyzed.(1–8.DMRs.per.miRNA)..Methylation.of.these.DMRs.was.decreased.and.miRNA.expression.increased.(except.for.3.miRNAs).upon.demethylation.treatment.with.5-Aza.indicating.regulation.of.these.transcripts.by.aber-rant.methylation.of.the.putative.promoters..Ongoing.technical.validati-on.by.MassARRAY.using.35.PCa.and.35.non-malignant.prostate.tissues.confirmed.aberrant.methylation.for.82.%.of.the.DMRs.analyzed.so.far,.accompanied.by.an. inverse.correlation.with.the.expression.of.the.re-spective.miRNA..Furthermore,.analysis.of.an.independent.genome-wide.methylation.dataset.(TCGA).confirmed.aberrant.methylation.for.11.of.12.DMRs.covered.by.these.data.

Conclusions:. Taken. together,. our. integrative. study. using. several. in-dependent. large.datasets.demonstrates. that.aberrant.methylation.of.putative.miRNA.promoters.in.PCa.is.frequent.and.contributes.to.miRNA.deregulation.

Funding:.This.study. is.supported.by.the.Federal.Ministry.of.Education.and.Research.(BMBF).and.the.German.Cancer.Aid..

Poster.P-0072

K. Korpis.1,.F..Weber.2,.B..Wünsch.2,.P..J..Bednarski.1

1. University.of.Greifswald,.Pharmacy,.Greifswald,.Germany.2. University.of.Münster,.Pharmacy,.Münster,.Germany

Involvement of apoptosis and autophagy in the tumor cell death caused by new sigma receptor ligands

Background:.Once.described.as.a.subtype.of.opioid.receptor,.sigma.re-ceptors.are.now.accepted.as.a.distinct.class.of.receptors.in.many.tu-mors.and.normal.tissues,.although.no.endogenous.ligand.has.yet.been.identified..There.are.at.least.two.subtypes:.σ

1.and.σ

2.which.are.distin-

guishable.pharmacologically,.functionally.and.by.molecular.size.1.Both.subtypes.modulate.ion.channels.and.neurotransmitter.systems.and.are.involved.in.pathophysiological.processes.like.depression.or.Alzheimer’s.disease.2.Moreover,.σ. receptors.are.over-expressed. in.certain.human.tumor.cell. lines.and.the.expression. levels. in.proliferating.tumor.cells.are.~.10.times.higher.than.in.corresponding.quiescent.tumor.cells..Me-anwhile.σ

2. receptor. ligands. were. developed. as. molecular. probes. for.

diagnostic. imaging.3.σ2.Agonists.have.been.proposed.as.potential.an-

ticancer.drugs.because. they.can. induce. tumor.cell.death.by.multiple.signaling.pathways.like.caspase.independent.apoptosis,.cell.cycle.dis-tribution.and.autophagy..On.the.other.hand.σ

2.antagonists.block.apo-

ptosis..By.contrast,.σ1.antagonists.cause.caspase.dependent.apoptosis.

and. their. agonists. have. neuroprotective. activity.4–5. So. far,. a. comple-te.understanding.of.how.sigma.receptor.ligands.induce.cell.death.has.not.yet.been.established..The.aim.of. this.work.was. to.determine. the.mechanism(s).by.which.newly.synthesized.σ.receptor.ligands.cause.cell.death.in.cancer.cell.lines.over-expressing.sigma.receptors.

Methods:.RPMI.8226.multiple.myeloma.cells.were.treated.with.the.sig-ma. ligand.WMS.34-11. and. the.enantiomer.WMS.34-14.. Flow.cytometry.studies.with.Annexin.V.and.propidium.iodide.were.performed.to.observe.cellular.alterations.due.to.apoptosis..Western.blotting.was.used.to.ana-lyze.for.autophagy.

Results:.Flow.cytometry.with.Annexin.V.and.propidium.iodide.staining.of.RPMI.8226.cells.showed.apoptosis.levels.increasing.from.1.%.in.the.control.to.31.%.in.the.treated.cells.after.48.h..Treatment.of.the.cells.caused. dose-dependent. activation. of. caspase-3,. caspase-8. and. cas-pase-9,.as.determined.by.flow.cytometry..However,.neither.the.broad.spectrum. caspase. inhibitor. z-VAD-FMK. nor. the. apoptosis. inhibitor.2,2β-methylenbis(1,3-cyclohexandion). blocked. apoptosis,. suggesting.that.apoptosis.may.be.caspase.independent..Western.blotting.analysis.revealed.the.processing.of.microtubule-associated.protein.light.chain.3.(LC3),.an.autophagosome.marker,.after.a.24.h.treatment.

Conclusion:.The.results.indicate.that.WMS.compounds.induce.cell.death.by.multiple.mechanisms.

References:1.. Crawford.KW.and.Bowen.WD.:.Cancer.Res..2002,62(1):.313–3222..Vilner.BJ.and.Bowen.WD.:.J.Pharmacol.Exp.Ther..2000,292(3):.900–

9113..Wheeler,.KT.et.al.:.British.J..Cancer..2000,82(6):.1223–12324..Riganas,. S. et. al.:. Bioorganic. &. Medicinal. Chemistry. 2012,20(10):.

3323–33315..Zeng,.C.et.al.:.British.J..Cancer..2012,106(4):.693–701

AEK

51

AEKAbstracts Posters Section 5: Systems biology and cancer

Poster P-0110

D. Dvornikov.1,.P..Lucarelli.1,.M..Schilling.1,.S..Depner.1,.U..Klingmüller.1

1. DKFZ,.Division.Systems.Biology.of.Signal.Transduction,.Heidelberg,.Germany

Dynamic modeling of TGFβ signaling in lung cancer

Early.metastasis.independent.of.tumor.size.is.a.major.medical.problem.in. the.field.of. lung.cancer.. Transforming.Growth.Factor.β. (TGFβ). is.a.potent.inducer.of.epithelial-to-mesenchymal.transition.(EMT),.a.process.that.enhances.cell.motility.and.invasiveness.of.cancer.cells.due.to.ac-quired.contact-free.fibroblast-like.phenotype..TGFβ-induced.EMT.might.be.one.of.the.reasons.of.early.metastatic.spread.of.lung.cancer.cells.In. current. project. we. apply. system. biology. approach. to. comprehen-sively.study.TGFβ/Smad.signal.transduction.in.lung.cancer..To.be.able.to.investigate.the.dynamic.properties.of.the.TGFβ/Smad.signaling.pa-thway.in.more.details,.we.established.an.ordinary.differential.equation.(ODE)-based.mathematical.model,.which.was.calibrated.to.the.experi-mental.data..We.observed.that.treatment.with.different.doses.of.TGFβ.results.in.different.cellular.outcomes..Although,.none.of.the.cell. lines.was.completely.growth. inhibited. in.response.to.TGFβ,.only.H1975.cell.line.revealed.partial.growth.inhibition.when.treated.with.high.doses.of.TGFβ..Additionally,.induction.of.EMT.markers.was.observed.only.when.cell.were.treated.with.high.dose.of.TGFβ.

The. combination. of. experimental. data. generation. and. mathematical.modeling.will.allow.us. to.determine.how.dynamics.of. intracellular.si-gnaling.pathway.is.converted.into.the.cell.fate.decisions.

Poster P-0113

H. Busch.1,.J..Neumann.2,.J..Nascimento-Minardi.3,.R..Köhler.2,.P..Krammer.2,.M..Li-Weber.2,.M..Börries.1

1. DKTK,.Freiburg,.Germany.2. DKFZ,.Heidelberg,.Germany.3. Freiburg.Institute.for.Advanced.Studies.(FRIAS),.Freiburg,.Germany

Functional mapping of transcriptome Profiling predicts Effective-ness of Rocaglamide towards Apoptosis Induction in Cancer Cells

Cancer.is.a.complex.genetic.disease.characterized.by.the.loss.of.tran-scriptional.or/and.translational.control.and.a.disruption.of.mechanisms.controlling.cell.survival.and.death..Cancer.therapy.is.inherently.difficult.due. to. the. large. patient-to-patient. tumor. heterogeneity. and. the. ac-companying.selective.resistance.to.chemotherapeutics..Thus,.a.consi-derable.challenge.in.treatment.is.to.select.suitable.drugs.for.individual.cancer.types..

Recently,.we.have.shown.that.Rocaglamides.(Roc),.a.natural.product.of.medicinal.plant.Aglaia,.induce.apoptosis.in.a.panel.of.human.cancer.cell.lines.in.vitro.as.well.as.in.vivo.and.in.mouse.models..Interestingly,.Roc.shows.no.or.only.minor.toxicity.on.normal.hematologic.cells..Despite.many.years.of.research,.the.molecular.determinants.by.which.Roc.pre-ferentially.kill.tumor.cells.are.largely.unknown..In.this.study,.we.pursue.a.novel.Systems.Biology.motivated.approach.by.linking.cell.function.to.drug.sensitivity..We.compared.the.transcriptome.profile.of.malignant.vs..normal.T.cells.in.response.to.Roc.treatment.and.revealed.how.basal.ac-tivities.of.genes.that.control.certain.signaling.pathways.play.a.decisive.role.in.drug.susceptibilities..

Based.on.this.knowledge,.we.derive.critical.pathways.that.functionally.link.cellular.transcriptomes.to.Roc.effectiveness.in.general..Using.this.pathway-based.Biomarkers.we.examined.samples.from.the.Human.gene.expression.atlas.and.predicted.Roc.responsiveness.in.96.different.tis-sue,.patient.tumor.and.cell.line.samples,.comprising.3567.microarrays.in. total..Our.new.finding.suggests. that. the.pathway-based.Biomarker.may.be.used.for.the.development.of.a.personalized.diagnostic.tool.to.predict.efficacy.of.Roc,.probably.also.other.drugs,.in.cancer.treatment.

52

AEKAEK AEKAbstracts Posters Section 5: Systems biology and cancer

Poster P-0132

M. Assadi Gehr.1,.B..Gierke.2,.J..Lamerz.3,.T..Schindler.3,.M..Venturi.4,.H..Langen.3,.M..Pawlak.2

1. Actelion.Pharmaceuticals.Ltd..,.Allschwil,.Switzerland.2. University.of.Tuebingen,.NMI.Natural.and.Medical.Sciences.Institute,.

Reutlingen,.Germany.3. F..Hoffmann-La.Roche,.Translational.Research.Sciences,.Basel,.

Switzerland.4. F..Hoffmann-La.Roche,.Translational.Medicine.Oncology,.Penzberg,.

Germany.

Multiple marker profiling from FFPET sections using Reverse Phase Protein Arrays: Discriminating sub-types of Non Small Cell Lung Can-cer

Background:.The.gold.standard.to.preserve.the.majority.of.tumor.speci-mens.for.clinical.diagnostics.is.their.fixation.in.formalin.and.paraffin-embedment.(FFPE)..This.procedure.is.routinely.combined.with.immuno-histochemistry.(IHC).but.excludes.them.from.further.protein.analysis.due.to.robust.cross-linking..Reverse.Phase.Protein.Arrays. (RPPA).are.novel.tools.that.allow.the.throughput.analysis.of.small.amounts.of.tis-sues. in. parallel. for. a. multitude. of. signaling. proteins.. While. RPPA. are.well.established.for.usage.of.cryo-preserved.material,.we.demonstrate.a.good.correlation.of.RPPA.and.IHC.analysis.for.FFPE.tumour.samples.

Objectives:. To. demonstrate. clinical. applicability. of. the. RPPA-based.screening. approach,. we. started. to. analyze. FFPE. tumour. samples. of.NSCLC.for.a.comprehensive.set.of.signaling.proteins,.with. the.aim.to.identify.proteins.that.can.discriminate.cancer.sub-types.

Materials.and.Methods:.Relative.expression.and.activation.state.of.150.proteins.representing.key.molecules.of.central.signaling.pathways.were.profiled.in.40.samples.of.NSCLC..Protein.extracts.were.prepared.from.single.FFPE.sections.of.age-matched.adeno.(AC).and.squamous.cell.car-cinoma.(SCC).specimen.(20.samples.per.sub-type)..Crude.extracts.were.spotted.in.a.sample.microarray.format.on.glass.slides..The.arrays.were.stained.with.150.validated.antigen-specific.antibodies.and.fluorescence.dye.conjugates.to.quantify.fluorescence.signal.intensity.

Results:.Cytokeratin.5.and.Napsin.A,.two.already.known.reference.mark-ers.for.NSCLC,.could.be.verified.from.the.RPPA.data.as.being.differen-tially.regulated.between.the.two.subtypes..In.addition,.PAK2.-.the.p21.activated.kinase.2.-.could.be.identified.as.a.new.third.marker.which.is.significantly.up-regulated.in.SCC.

Conclusion:.Comprehensive.data.sets.generated.by.RPPA.from.limiting.FFPE.tumour.sample.material.allow.the.identification.of.differences.in.signalling.networks.directly.on.the.protein.level..The.approach.may.be.a.versatile.tool.in.clinical.research,.translational.medicine.and.pharma-ceutical. drug.development. to. stratify.patient. tumours.and. to.predict.drug.response.

Notes:

AEK

53

AEKAbstracts Posters Section 6: Novel approaches to tumor therapy

Poster P-0004

C. Domschke.1,.Y..Ge.2,.A..Schneeweiss.1,.C..Sohn.1,.P..Beckhove.2,.F..Schütz.1

1. University.Hospital.of.Heidelberg,.Department.of.Gynecology.and.Obstetrics,.Heidelberg,.Germany.

2. German.Cancer.Research.Center.(DKFZ),.Division.of.Translational.Immunology,.Tumor.Immunology.Program,.Heidelberg,.Germany

Treatment of advanced metastasized breast cancer patients with bone marrow-derived tumor-reactive memory T cells in a phase I/II study

Introduction:.The.bone.marrow.of.breast.cancer.patients.harbours.tu-mor-reactive.memory.T.cells.(TCs).with.therapeutic.potential..We.recen-tly.described.the.immunological.effects.of.adoptive.transfer.of.ex.vivo.re-stimulated.tumor-reactive.memory.TCs.from.the.bone.marrow.(BM).of.12.metastasized.breast.cancer.patients.in.a.clinical.phase-I.study..In.this.trial,.adoptive.T.cell.transfer.resulted.in.the.occurrence.of.circula-ting.tumor.antigen-reactive.type-1.TCs..We.here.describe.the.long-term.clinical.outcome.and.its.correlation.to.tumor-specific.cellular.immune.response.in.now.altogether.16.metastasized.breast.cancer.patients.

Methods:.Sixteen.metastatic.breast.cancer.patients.with.pre-existing.tumor-reactive.BM.memory.TCs.were.included.into.the.present.study..The.study.protocol.was.approved.by.the.ethical.committee.of.the.Uni-versity.of.Heidelberg..All. sixteen.patients.had.a.history.of.cytostatic.and/or.hormonal.therapy..The.study.protocol.involved.one.transfusion.of.TCs.which.were.reactivated.in.vitro.with.autologous.dendritic.cells.pulsed.with.lysate.of.MCF-7.breast.cancer.cells.as.source.of.tumor.an-tigens.(Fig..1)..The.presence.of.tumor-reactive.memory.TCs.in.the.peri-pheral.blood.was.analyzed.by.IFN-βEliSpot.assays.

Results:. Tumor-reactive.memory.TCs. in. the.peripheral.blood.were. in-duced.de.novo.in.7/16.patients.(44%).after.adoptive.TC.transfer..The-se.patients.were.considered.immunological.responders.to.the.therapy.(Fig..2)..Positive.adoptive.immunotherapy.(ADI).response.was.observed.significantly.more.often. in.patients.without.bone.metastases. (Fig.. 3;.p=0.0032),. in.patients.with.high. levels.of. tumor-reactive.BM.TCs.pri-or. to. therapy. (data. not. shown;. p=0.036). and. correlated. significantly.with.the.estimated.numbers.of.transferred.tumor-reactive.TCs.(Fig..3;.p=0.0096)..After.the.treatment,.we.observed.an.overall.median.survi-val.of.33.8.months.in.the.total.cohort.with.3.patients.still.being.alive.at.the.time.of.latest.follow-up..Numbers.of.transferred.tumor-reactive.TCs.correlated.significantly.with.the.overall.survival.of.patients.(Fig..4;.p=0.017)..Concomitantly,.patients.with. immunological.ADI.response.in.the.peripheral.blood.presented.with.a.significantly.prolonged.median.survival. of. 58.6. months. compared. to. non-responders. (Fig.. 4;. median.survival.13.6.months;.p=0.009).

Conclusion:. In.metastasized.breast.cancer.patients,.adoptive.transfer.of.BM.TCs.can.induce.the.presence.of.tumor.antigen-reactive.type-1.TCs.in.the.peripheral.blood..Patients.with.immunological.response.after.ADI.show.a.significantly. longer.overall.survival..Patients.with.bone.meta-stases.significantly.less.frequently.respond.to.the.treatment.and,.the-refore,.might.not.be.optimal.candidates.for.ADI..Although.the.present.study.does.not.yet.prove.the.therapeutical.effect.of.ADI,.these.findings.shed. light.on.the.relation.between. immune.response.and.cancer.pro-gnosis.and.suggest.that.transfer.of.reactivated.bone.marrow.TCs.might.bear.therapeutic.potential.

Poster P-0011

L. Agoni.1,.J..Lenz.2,.C..Guha.1,3

1. Albert.Einstein.College.of.Medicine.of.Yeshiva.University,.Pathology,.Bronx.(NY),.United.States.

2. Albert.Einstein.College.of.Medicine.of.Yeshiva.University,.Genetics,.Bronx.(NY),.United.States.

3. Montefiore.Medical.Center,.Radiation.Oncology,.Bronx.(NY),.United.States.

Human Endogenous Retrovirus K (HERV-K)-derived peptides as tar-get for radiation-enhanced immunotherapy in prostate cancer.

Human.endogenous.retrovirus.K.(HERV-K).is.an.intriguing.target.for.de-veloping.novel.antitumor.reagents..HERV-K.was.the.most.recent.retrovi-rus.to.infect.the.human.germline,.and.the.HERV-K.proviruses.present.in.human.DNA.today.still.encode.viral.proteins..The.approximately.25.full-length.HERV-K.proviruses.in.the.human.genome.are.98-99%.identical,.and.each.encodes.some.or.all.of.the.viral.proteins..HERV-Ks.are.known.to.be.transcribed.in.numerous.diseases.including.cancer,.HIV.infection,.and.autoimmune.disorders..Ionizing.Radiation.(IR).increases.the.immu-nogenicity. of. cancer. cells.. We. hypothesized. that. IR. could. reactivate.transcription.of.HERV-K.proviruses..This.could.increase.the.diversity.of.the.antigen.repertoire.presented.by.irradiated.cancer.cells.and.might.thereby.contribute.to.the.increased.immunogenicity.seen.following.IR..It.could.also.provide.novel.targets.for.antitumor.immunotherapies.

To.test.the.effect.of.IR.on.HERV-K.transcription,.12.cancer.cell.lines.from.cancer.sites.commonly.treated.with.radiotherapy.were.analyzed.by.RT-PCR,.qRT-PCR.and.RNA-seq.at.specific.time-points. from.1. to.72.hours.after.varying.doses.of.β-irradiation.(0,.2.5,.5,. 10.and.20.Gy)..HERV-K.transcription. was. identified. in. all. cell. lines. studied. including. spliced.transcripts.for.rec.and.env.in.9.and.all.12.lines,.respectively..IR.increa-sed.HERV-K.transcription.by.at.least.3-fold.in.five.of.the.cell.lines,.three.of.which.were.prostate.cancers..Specific,.active.HERV-K.loci.were.iden-tified.from.polymorphic.nucleotides.among.the.25.loci:.5.different.loci.predominated.in.the.prostate.cancer.cell.lines.tested.and.varied.among.them..Most.of.the.sequenced.transcripts.contained.open.reading.frames.compatible.with.translation.to.proteins..Proteomic.LC-MS/MS.analyses.of.peptides.eluted.from.MHC-I.complexes.are.ongoing.to.test.for.HERV-K-derived.peptides.

These.findings.show.that.HERV-K.is.actively.transcribed.in.cancer.cell.lines..The.IR-stimulated.increase.in.HERV-K.transcription,.particularly.in.prostate.carcinoma,.may.contribute.to.the.increased.immunogenicity.in.response.to.IR.and.offers.an.attractive.target.for.the.development.of.novel.IR-enhanced.immunotherapies.for.cancer.

54

AEKAEK AEKAbstracts Posters Section 6: Novel approaches to tumor therapy

Poster P-0018

A. Orthmann.1,.M..Lemm.2,.N..Bäsler.1,.I..Fichtner.2,.R..Zeisig.1

1. EPO.GmbH,.Experimental.Pharmacology,.Berlin,.Germany.2. Max.Delbrück.Center,.Experimental.Pharmacology,.Berlin,.Germany.

Nanocarrier – a beneficial tool to treat primary tumours with brain metastasis

Effective.opportunities.to.treat.brain.metastasis.of.most.common.tu-mour. types. associated. with. secondary. brain. colonization. like. breast.cancer,.or.melanoma.and.lung.cancer,.are.still.rare.

The.aim.of.the.present.study.was.therefore.to.develop.drug.loaded.li-posomes.to.improve.the.growth.inhibition.of.the.human.primary.breast.cancer.model.MT-3.and.its.brain.metastases.in.the.mouse.To.obtain.fluid.membrane.liposomes,.small.unilamellar,.negatively.char-ged. vesicles. were. prepared,. consisting. of. a. basic. lipid. together. with.DOPE.and.perifosine.to. increase.the.fluidity.. In.addition,.a.specific. li-gand.to.improve.the.direct.effect.on.the.primary.tumour.and.to.enhance.the.active.drug.transport.across.the.blood.brain.barrier.to.increase.the.drug.concentration.in.the.brain,.was.conjugated.to.the.vesicles..There-fore,.the.19mer.angiopeptide.sequence.was.used.as.ligand.to.target.the.LRP.receptor,.expressed.on.the.surface.of.human.MT-3.breast.cancer.cells.and.by.barrier.forming.epithelial.cells.MT3.was.used.as.a.dual.model,.transplanted.s.c..and.in.parallel. intra-cerebrally.to.mimic.brain.metastasis. into.nude.mice,.while.oxaliplatin.(OxP),.a.drug.which.cannot.cross.the.blood.brain.barrier,.was.selected.as.drug.for.encapsulation.The.cytotoxicity.of.the.OxP. liposomes.on.MT-3.cells.was.about.40-45.fold.higher.than.that.of.the.free.drug,.while.epithelial.cells.used.to.si-mulate.a.cell.barrier.in.vitro.(like.the.BBB),.were.more.resistant.to.the.liposomes.Uptake.of. ligand.bearing,.fluid. liposomes.by.MT-3.breast.cancer.cells.was.the.best,.while. ligand.free,. rigid. liposomes.were.taken.up.at. the.lowest.degree..A.corresponding.result.was.determined.for.transcytosis.of.these.liposomes.across.a.tight.epithelial.cell.barrier.formed.by.MDCK.cells.Free.and.liposomal.encapsulated.OxP.was.than.used.to.treat.MT-3.in.the.dual.xenograft.mouse.model.Treatment.with.free.OxP.resulted.in.an.inhibition.of.the.s.c..tumour.by.23%.(T/C)..This.effect.was.further.improved.if.OxP,.encapsulated.in.flu-id.liposomes.(T/C.19.8%)..The.best.inhibition.was.obtained.with.ligand.bearing,.fluid.liposomes.(T/C.11.5%),which.was.significantly.better.than.that.of.the.free.drug.

On. the. other. hand,. treatment. with. free. OxP. had. no. effect. on. brain.tumour. growth,. while. all. liposomal. formulations. clearly. inhibited. the.growth.of.brain.tumours..Best.results.were.obtained.with.ligand.bearing.fluid.OxP.liposomes.with.a.T/C.41%.

We.are.currently.optimizing.the.treatment.schedule.mainly.to.reduce.serious.side.effects.on.the.blood.composition.which.were.observed.in.addition.to.the.convincing.inhibition.effects,.and.to.increase.the.inhibiti-on.of.the.brain.tumour.growth.even.more.to.demonstrate.the.advantage.of.platinum.encapsulating.liposomes.for.the.treatment.of.breast.cancer.derived.brain.metastases.

Poster P-0020

H. Hülsmann.1,.C..Bender.2,.J..Rolff.3,4,.I..Fichtner.4,.R..Herwig.5,.U..Korf.6,.H..Sültmann.1,.R..Kuner.1

1. DKFZ./.NCT,.Cancer.Genome.Research,.Heidelberg,.Germany.2. TRON,.Mainz,.Germany.3. Experimental.Pharmacology.&.Oncology.GmbH,.Berlin-Buch,.Germany.4. Max-Delbrück-Center,.Berlin,.Germany.5. Max.Planck.Institute.for.Molecular.Genetics,.Berlin,.Germany.6. DKFZ,.Molecular.Genome.Analysis.,.Heidelberg,.Germany.

Identification of drug-associated proteins in NSCLC xenograft mo-dels by reverse-phase-protein-arrays

At.present,.most.non-small.cell.lung.cancer.(NSCLC).treatments.are.not.adapted.to.the.individual.response.of.a.patient..The.stratification.of.pa-tients.for.the.most.efficient.response.to.conventional.chemotherapeu-tics.and.targeted.therapies.will. improve.established.therapy.schemes.and.patient’s.perspectives..Our.project.aims.at.unraveling.the.influence.of.specific.signaling.molecules.on.the.response.to.common.NSCLC.drugs.as.conventional.chemotherapy.and.different.novel.EGFR-inhibitors..We.searched.for.predictive.markers.for.the.response.of.NSCLC.tumors.to.certain.drugs.and.novel.combinations.of.treatments,.which.may.be.use-ful.for.therapy.decisions.towards.a.more.efficient.personalized.treat-ment.

We.used. the. reverse-phase.protein.array. technology. (RPPA). to.quan-tify. the. expression. of. 107. proteins. of. cancer-relevant. pathways. (e.g..MAPK,.JAK/STAT,.PI3K/AKT).in.64.patient.derived.NSCLC.xenograft.mo-dels..The.tumor.models.are.characterized.by.different.response.rates.upon.treatment.with.established.chemotherapeutics.(e.g..Gemcitabine,.Paclitaxel,. Carboplatin). and. EGFR-targeted. therapies. (Cetuximab,. Er-lotinib)..Statistical.analysis.indicated.significant.associations.between.the.expression.of.distinct.proteins.before.treatment.and.the.response.to.certain.drugs..Proteins.of.the.ErbB.signalling.pathway.were.differen-tially. expressed. in. Carboplatin. responders. and. non-responders.. MEK1.upregulation.was.observed.upon.Cetuximab. treatment. in. responders..We. revealed. an. association. between. higher. Phospho-SRC. expression.and. increased. Gemcitabine. response. rates.. Based. on. RPPA. analyses.candidate.targets.were.selected.for.validation.experiments..Predictive.targets. for.drug.sensitivity.are.being.analyzed. to. investigate. the.de-pendency. between. target. protein. activity,. downstream. signaling. and.response. to. distinct. drugs. in. tumor. cell. lines. and. xenograft. models..Furthermore,.we.suggest.novel.potent.combinations.of.inhibitors/acti-vators.of.predictive.targets.and.established.drugs

AEK

55


Poster P-0022

Z. Mihály.1,.M..Kormos.1,.B..Györffy.1

1. Semmelweis.University.Budapest,.1..Dept.of.Pediatrics,.Budapest,.Hungary

Meta-analysis of Biomarker Candidates Predicting Survival After Ta-moxifen Treatment

Background:. To. date,. three. molecular. markers. (ER,. PR. and. CYP2D6).have.been.used.in.clinical.settings.to.predict.the.benefit.of.the.anti-es-trogen.tamoxifen.therapy..However,.even.ER,.the.most.effective.of.the-se.can.only.identify.halfofthe.patients.responding.to.endocrine.therapy..Our.aim.was.to.validate.new.biomarker.candidates,.that.predicting.re-sponse.to.tamoxifen.treatment,.by.evaluating.these.in.a.meta-analysis.of.available.microarray.datasets.with.known.treatment.and. follow-up.information.

Methods:.Data.sources:.published.biomarker.candidates.were.identified.in.Pubmed.(2007–2012).and.in.the.2010–2012.ASCO.and.SABCS.abstracts..Breast.cancer.microarray.datasets.were.downloaded.from.GEO.and.EGA..Study.eligibility.criteria,.participants.and.interventions:.offrom.the.bio-marker.candidates,.only.those.identified.or.already.validated.in.a.clini-cal.cohort.were.eligible..From.the.transcriptomic.datasets.relapse-free.survival.data.of.tamoxifen.treated.patients.and.overall.survival.data.of.endocrine.treated.patients.were.used..Of.the.transcriptomic.datasets,.only.patients.with.tamoxifen.treatmentforrelapse-free.survival.and.en-docrine.treatmentfor.overall.survival.were.eligible..Synthesis.methods:.the.raw.microarray.data.was.re-processed.and.integrated.into.two.da-tabases..Relapse.free.survival.(RFS).up.to.5.years.was.used.as.endpoint.in.a.ROC.analysis..In.the.EGA.dataset,.Kaplan-Meier.analysis.was.perfor-med.for.overall.survival.(OS).

Results:. All. together. 60. biomarker. candidates. were. identified.. The.transcriptomic.datasets. included.584.GEO-based.and. 1208.EGA-based.microarrays.. InAmongthese,. the. best. performing. genes. were:. PRG.(RFS:.AUC=0.645,.p=2.3E-7;.OS.HR=0.,59,.p=3.1E-7),.and.SLC7A5.(RFS:.AUC=0.621,.p=9.2E-5;.OS:.HR=1.6.p=3.6E-6)..Further.genes.significant-ly. correlated. to. relapse-free. survival. include. p53. (p=0.0012),. CALM3.(p=0.00019),.BCL2.(p=0.0085),.BTG2.(p=0.00079),.CXCL10.(p=0.0036),.SDF1. (p=0.0009),. HOXB7. (0.0011),. IGF1R. (p=0.0051),. SLC3. (p=0.0057).and.TPM4.(p=0.0027)..None.of.the.other.genes.reached.statistical.si-gnificance.for.relapse-free.survival.

Discussion:.A. limitation.of.our.study. is.the.missing.dosage.and.treat-ment.length.dataforinthe.transcriptomic.datasets..In.summary,.we.per-formed.an.independent.validation.for.biomarker.candidates.predicting.survival.after.tamoxifen.treatment.

Poster P-0029

M. Metzig.1,2,.D..Fuchs.1,2,.S..Macher-Goeppinger.1,2,.P..Schirmacher.2,.H..Gröne.1,.W..Roth.1,2

1. German.Cancer.Research.Center,.Heidelberg,.Germany.2. Institute.of.Pathology,.University.of.Heidelberg,.Heidelberg,.Germany.

Morphology and molecular mechanisms of necroptosis in colorectal carcinoma

Question:.While.cell.death.research.in.the.last.decades.has.mainly.been.focused.on.elucidating.apoptosis,.the.necrosis-like.types.of.cell.death.are.presently.obtaining.recognition. in.both.physiology.and.pathology..Necroptosis,.a.RIP1-dependent.form.of.programmed.necrosis.occurring.under.caspase-deficient.conditions,.has.been.implicated.in.chronic.in-flammation.and.cancer..The.aim.of.our.project.is.to.study.the.subcellular.morphology.and.the.molecular.mechanisms.of.necroptosis.in.colorectal.cancer.

Methods:.Cytotoxicity.and.apoptosis.assay,.FACS.analysis,.siRNA.knock-down,. co-immunopreciptitation. experiments,. stable. and. transient.transfections,.immunohistochemistry,.electron.microscopy.

Results:.We.established.a.cell.culture.model.for.chemotherapy-induced.necroptosis. in. colorectal. cancer.. We. found. that. colorectal. carcinoma.cell.lines.were.sensitized.to.chemotherapy-induced.cell.death.when.co-treated.with.a.caspase.inhibitor..Since.a.RIP1-specific.inhibitor.(necro-statin.1).diminished.this.effect.we.concluded.that.cells.were.dying.due.to.necroptosis..Mechanistic.studies.revealed.that.chemotherapy-indu-ced.necroptosis.requires.the.inhibition.of.caspase.activity.and.occurs.in.a.tumor.necrosis.factor.receptor. 1. (TNFR).and.receptor. interacting.protein. (RIP). dependent. manner.. Moreover,. some. chemotherapeutics.augmented.necroptosis.signaling.by.up-regulation.of.TNFR1.surface.ex-pression.and.de-stabilization.of.inhibitors.of.apoptosis.(IAPs)..In.order.to.investigate.the.biological.significance.of.necroptosis.in.colon.cancer.we. assessed. RIP. expression. in. human. colorectal. carcinoma. samples.by.immunohistochemistry..RIP.was.significantly.less.abundant.in.colon.carcinoma. compared. to. normal. tissue.. Since. the. ultrastructural. mor-phological.phenotype.of.necroptosis.is.unknown,.we.investigated.colon.carcinoma.cells.upon.specific.induction.of.necroptosis.via.electron.mi-croscopy..Interestingly,.in.early.phases.of.necroptosis.the.cells.exhibi-ted.distinct.characteristics.such.as.ER.dilatation.and.fusion.of.enlarged.mitochondria.

Conclusions:.Necroptosis-inducing.agents.could.re-sensitize.colorectal.cancer.cells.to.conventional.chemotherapy..Necroptosis.requires.TNFR1.and.RIP.signaling.and.is.accompanied.by.characteristic.subcellular.mor-phological. features.. RIP. expression. is. down-regulated. in. colon. carci-noma.. Taken. together. our. findings. suggest. that. specific. induction. of.necroptosis.could.represent.a.novel.therapeutic.strategy.in.colorectal.cancer.

56


Poster P-0032

Z. Pénzváltó.1,.A..Lánczky.1,.B..Györffy.1

1. Semmelweis.University,.I.st.Department.of.Pediatrics,.Budapest,.Hungary

Biomarkers of platinum resistance in ovarian cancer

Question:.With.over.15,000.deaths.each.year.in.the.US.and.a.five-year.overall.survival.of.44%.ovarian.cancer.is.the.most.lethal.gynecological.cancer..Besides.surgery,.chemotherapy.protocols.containing.taxol.and.platinum.derivatives.represent.the.primary.treatment.options.for.ova-rian.cancer..However,.platinum.resistant.cancer.recurs.in.approximately.25%.of.patients.within.six.months..Improvement.in.treatment.can.be.achieved. by. identifying. biomarkers. predicting. response. to. platinum.agents..In.our.present.study.we.aimed.to.identify.clinically.applicable.biomarkers.of.therapy.response.

Methods:.We.have.set.up.a.databank.of.publicly.available.ovarian.mi-croarray.datasets.containing.treatment.and.response.information..For.this,.we.searched.GEO.and.TCGA. to. identify.datasets. suitable. for. the.analysis.of.the.effect.of.the.genes.on.therapy.response..We.performed.receiver.operating.characteristic.(ROC).analysis.in.R.for.all.genes.and.then.ranked.them.based.on.the.area.under.the.curve.(AUC).values..We.identified. the. most. promising. candidates. and. compared. these. to. the.available.literature..Then,.eight.genes.were.selected.for.in.vitro.func-tional.validation.including.JRK,.CNOT8,.RTF1,.CCT3,.NFAT2CIP,.MAP2K1,.FUBP1.and.CSDE1..We.preformed.gene.silencing.with.simultaneous.48.h.carboplatin.administration.in.four.ovarian.cancer.cell.lines.(SK-OV-3,.CAOV-3,.ES-2,.and.OVCAR-3)..The.efficacy.of.the.gene.silencing.was.va-lidated.by.qRT-PCR..The.change.in.the.cell.resistance.was.measured.by.MTT.cell.viability.tests.

Results:.We.identified.1267.patients.in.8.datasets.meeting.our.criteria.in.GEO.and.TCGA..The.average.progression.free.survival.is.24.8.months.with.731.progressions..Of.these.patients,.1152.received.a.platinum-based.chemotherapy.and.631.received.taxol.(614.patients.received.both.taxol.and.platinum)..The.ROC.values.of.the.selected.genes.were.JRK.(0.625),.CNOT8.(0.610),.RTF1. (0.620),.CCT3.(0.620),.NFAT2CIP.(0.612),.MAP2K1.(0.611),.FUBP1.(0.608).and.CSDE1.(0.605).The.expression.change.of.JRK.(p=0.0002),.MAP2K1.(p=0.0117).and.CNOT8.(p=0.0036).were.significant-ly.correlated.with.progression.free.survival..JRK.was.not.expressed.in.the.cell.lines..The.silencing.of.RTF1,.FUBP1.and.MAP2K1.caused.signifi-cant.sensitization.in.every.investigated.cell.lines.(p.values.respectively:.0.000565,.6.50006E-07,.3.29E-05).

Conclusions:. We. identified. and. in. vitro. validated. the. effect. of. three.potential.biomarkers,.RTF1,.MAP2K1.and.FUBP1.correlated.to.platinum.resistance.in.ovarian.cancer.

Poster P-0037

C. Herskind.1,.Q..Liu.1,.L..Ma.1,.B..Zhang.1,.C..Kettner.1,.F..Schneider.1,.M..R..Veldwijk.1,.F..Wenz.1

1. Universitätsmedizin.Mannheim,.Medical.Faculty.Mannheim,.Heidel-berg.University.,.Dept..of.Radiation.Oncology,.Mannheim,.Germany.

Biological effect of large dose fractions of ionizing radiation as ap-plied in Intraoperative Radiotherapy (IORT)

Question:.Novel.radiotherapy.techniques.such.as.stereotactic.radiosur-gery.(SRS),.stereotactic.body.radiation.therapy.(SBRT),.high-dose-rate.(HDR),. brachytherapy. boost,. and. intra-operative. radiotherapy. (IORT),.use.a.single.or.very.few,.very.large.dose.fractions.instead.of.conven-tional.small.daily.fractions..Furthermore,.the.increased.time.to.deliver.a.dose.fraction.may.allow.repair.of.sublethal.lesions.during.the.treat-ment..In.IORT,.irradiation.may.interact.with.wound.healing..The.purpose.was.to.study.the.biological.effect.of.high.single.doses.on.the.relative.biologic.effectiveness.(RBE),.repair,.and.wound.healing.in.vitro.

Methods:.Human.MCF7.breast.cancer.cells.and.normal.diploid.skin.fibro-blasts.were.irradiated.with.50.kV.X-rays.from.the.Intrabeam®.machi-ne.(Carl.Zeiss.Surgical.GmbH,.Oberkochen,.Germany). in.a.tumour-bed.phantom,. 10.MeV.electrons,.or.6.MV.X-rays. from.a. linear.accelerator..RBE.for.clonogenic.cell.survival.was.determined.by.the.colony.formati-on.assay;.DNA.double-strand.breaks.(DSB).were.quantified.by.detecting.phosphorylated.histone.γH2AX.foci;.cell.migration.was.quantified.by.the.in.vitro.wound.healing.(scratch).assay.

Results:. The. RBE. of. 10. MeV. electrons. relative. to. 6. MV. X-rays. was. not.significantly.lower.than.1.for.MCF7..However,.50.kV.X-rays.from.the.Intra-beam.machine.showed.an.increased.RBE.(mean.1.35;.95%.c.i.;.1.2-1.5).at.the.clinically.relevant.dose.rate.of.15.1.Gy/min.in.8.1.mm.tumor-bed.depth..The.RBE.decreased.at.the.lower.dose.rate.of.9.8.Gy/min.(12.7.mm.depth).suggesting.a.role.of.repair.of.sublethal.damage.(SLD).during.protracted.irradiation..A.half-life.of.40.min.for.SLD.repair.was.determined.for.MCF7.in. split-dose. experiments. with. 6. MV. X-rays.. DSB. induction. and. repair.monitored.by.γH2AX.foci.showed.a.sub-linear.relation.with.dose.which.persisted.at. later. time.points. (4-24h). indicating. that. saturation.of. the.DSB.repair.system.may.take.place.at.higher.doses..Whereas.a.high.dose.of..12.Gy.of.6.MV.X-rays.strongly.inactivated.colony.formation.of.fibroblasts,.migration.in.the.wound.healing.assay.was.not.inhibited,.and.even.slightly.stimulated,.by.irradiation..The.cytokine.TGF-β1,.which.plays.a.central.role.in.wound.healing,.inhibited.migration.at.concentrations.of.1-10.ng/ml.but.no.interaction.between.irradiation.and.TGF-β1.was.observed.

Conclusions:.In.summary,.biological.effects.of.high.single.doses.as.used.in.IORT.may.differ.from.those.expected.from.studies.with.conventional.fraction. sizes.. Further. biological. studies. of. very. large. dose. fractions.are.warranted.

AEK

57


Poster P-0038

C. Zhang.1,.K..Schönfeld.1,.M..Burger.2,.S..Genßler.1,.C..Sahm.1,.C..Brendel.1,S..Naundorf.3,.M..Odendahl.4,.U..Köhl.5,.T..Tonn.4,.M..Grez.1,.J..P..Steinbach.2,.W..S..Wels.1

1. Chemotherapeutisches.Forschungsinstitut.Georg-Speyer-Haus,. Frankfurt.am.Main,.Germany.2. Institute.for.Neurooncology,.University.Hospital,.Frankfurt.am.Main,. Germany.3. EUFETS.GmbH,.Idar-Oberstein,.Germany.4. German.Red.Cross.Blood.Donation.Service.East,.Dresden,.Germany.5. Hannover.Medical.School,.Hannover,.Germany.

ErbB2/HER2-specific natural killer cells for adoptive cancer immu-notherapy

Significant.progress.has.been.made.over.the.last.decade.towards.rea-lizing.the.potential.of.natural.killer.(NK).cells.for.cancer.immunothera-py..NK.cells.can.respond.rapidly.to.transformed.and.stressed.cells,.and.have. the. intrinsic.potential. to.extravasate.and. reach. their. targets. in.almost.all.body.tissues..In.addition.to.donor-derived.primary.NK.cells,.also.continuously.expanding.cytotoxic.cell.lines.such.as.NK-92.are.being.considered.for.adoptive.cancer.immunotherapy..High.cytotoxicity.of.NK-92.has.previously.been.shown.against.malignant.cells.of.hematologic.origin.in.preclinical.studies,.and.general.safety.of.infusion.of.NK-92.cells.has.been.established.in.phase.I.clinical.trials..To.enhance.their.thera-peutic.utility,.we.genetically.modified.NK-92.cells.to.express.chimeric.antigen.receptors.(CAR).specific.for.different.tumor-associated.surface.antigens.including.ErbB2.(HER2),.EpCAM,.CD20.and.GD2.[1-5]..Such.CAR.were.composed.of.a. tumor-specific.scFv.antibody. fragment. fused.via.hinge.and. transmembrane.domains. to. intracellular. signaling.moieties.such.as.CD3.zeta.chain,.or.composite.fusion.molecules.also.containing.a.costimulatory.protein.domain.in.addition.to.CD3.zeta.

For.development.towards.clinical.applications,.here.a.codon-optimized.second.generation.CAR.was.constructed.that.consists.of.an.ErbB2-spe-cific.scFv.antibody.domain.fused.via.a.linker.to.a.composite.CD28-CD3.zeta.signaling.domain..GMP-compliant.protocols.for.vector.production,.lentiviral. transduction. and. expansion. of. genetically. modified. NK-92.single.cell. clones.were.established..Functional.analysis.of. these.cells.revealed.high.and.stable.CAR.expression,.selective.cytotoxicity.against.ErbB2-expressing.but.otherwise.NKresistant.tumor.cells.of.different.ori-gins.in.vitro,.as.well.as.homing.to.ErbB2-expressing.tumors.in.vivo.and.antimetastatic. activity. upon. intravenous. application. in. murine. model.systems..Ongoing.work.now.focuses.on.the.development.of.these.cells.for.adoptive.immunotherapy.of.ErbB2-positive.glioblastoma..In.additi-on,.we.are.investigating.potential.endogenous.resistance.mechanisms.employed.by.glioblastoma.cells.to.escape.NK.cell.immunosurveillance.1.. Uherek.et.al.,.Blood.100:.1265–1273,.2002.2..Müller.et.al.,.Cancer.Immunol..Immunother..57:.411–423,.2008.3..Sahm.et.al.,.Cancer.Immunol..Immunother.,.61:.1451–1461,.2012.4..Esser.et.al.,.J..Cell..Mol..Med..16:.569–581,.2012.5..Tavri.et.al.,.Mol..Imaging.8:.15–26,.2009.

Poster P-0039

S. Genssler.1,.C..Zhang.1,.M..Burger.2,.C..Sahm.1,.K..Schönfeld.1,.J..P..Steinbach.2,.W..S..Wels.1

1. Chemotherapeutisches.Forschungsinstitut.Georg-Speyer-Haus,. Frankfurt/Main,.Germany.2. University.Hospital,.Institute.for.Neurooncology,.Frankfurt/Main,.. Germany.

Genetically Modified Natural Killer Cells Targeting EGFR Expressing Malignancies

Natural.killer.(NK).cells.are.the.body‘s.first.line.of.defense.against.viral.infections.and.malignant.cells.. In.addition. to.primary.NK.cells,. conti-nuously.expanding.cytotoxic.cell.lines.such.as.NK-92.hold.promise.for.cancer.immunotherapy..The.therapeutic.utility.of.such.cells.can.be.fur-ther.enhanced.by.genetic.modification.with.chimeric.antigen.receptors.(CAR),.which.recognize.antigens.differentially.expressed.on.the.surface.of.tumor.cells..CAR.typically.consist.of.an.extracellular.antigen-specific.single-chain. antibody. fragment. (scFv),. fused. via. a. flexible. hinge. and.transmembrane.region.to.an.intracellular.signaling.moiety.such.as.CD3.zeta.chain.or.a.composite.CD28-CD3.zeta.fusion.molecule..Here.we.ge-nerated.second.generation.CAR.constructs.specific. for. the.epidermal.growth.factor.receptor.(EGFR).and.its.variant.EGFRvIII..Overexpression.and.malignant.activation.of.EGFR.has.been.described.for.many.tumors.of.epithelial.origin..Likewise,.enhanced.EGFR.levels.and.expression.of.the. constitutively. active. EGFR. mutant. form. EGFRvIII. are. a. particular.feature.of.glioblastoma.multiforme.(GBM)..We.developed.a.panel.of.hu-manized.and.codon-optimized.CAR.that.employ.scFv.antibody.fragments.either.recognizing.epitopes.restricted.to.wildtype.EGFR.or.ERGFRvIII,.or.an.epitope.common.to.both.target.receptors..For.signaling.these.CAR.carry.a.composite.CD28-CD3.zeta.domain..Upon.lentiviral.transduction.of.NK-92.cells,.single.cell.clones.were.derived.and.characterized.for.CAR.expression..Functional.analysis.in.vitro.revealed.high.and.specific.cyto-toxicity.of.CAR-expressing.NK-92.cells.towards.otherwise.NK-resistant.tumor.cells.carrying.EGFR.or.EGFRvIII..Ongoing.work.now.aims.at.the.evaluation.of.retargeted.NK-92.cells.in.glioblastoma.xenograft.models.in.NOD/SCID.common.gamma.chain.knockout.mice.

58


Poster P-0049

H. Riffkin.1,.R..Handrick.1,2,.G..Henke.3,.V..Meier.3,.T..Witte.1,.V..Jendrossek.1

1. IFZ.(Tumor.Research)/Essen.University.Hospital,.Molecular.Cell.. Biology,.Essen,.Germany.2. University.of.Applied.Science.Bieberach,.Biberach,.Germany.3. Tübingen.University.Hospital.,.Department.of.Radiation.Oncology,. Tübingen,.Germany.

Molecular targets of Erufosine (ErPC3) action under hypoxia

Background. and. purpose:. Cellular. membranes. are. an. attractive. tar-get. for. the.modulation.of. tumor.growth.and.sensitivity. to.anticancer.treatment.. Our. previous. work. showed. that. membrane-targeted. drug.erufosine. (ErPC3).exerts.potent.antineoplastic.effects.as. single.drug.and.in.combination.with.ionizing.radiation.on.glioblastoma.and.prostate.cancer.cell.lines.in.vitro,.at.least.when.treatment.was.performed.under.normoxic.culture.conditions..Cytotoxic.action.of.ErPC3.was.associated.with.pronounced.inhibition.of.the.survival.kinase.protein.kinase.B.(AKT).and.important.downstream.targets.of.AKT..However,.tumor.hypoxia.is.a.common.feature.of.human.solid.tumors.and.is.considered.as.one.main.biological.factor.promoting.tumor.cell.resistance.to.chemotherapy.and.ionizing.radiation..Therefore,.here.we.aimed.to.analyze.antineoplastic.activity.of.ErPC3-treatment.under.hypoxia.and.the.potential.of.ErPC3.to. inhibit. the. AKT-pathway. under. these. resistance-promoting. culture.conditions.

Methods:.Human.T98G.(glioblastoma).and.PC3.(prostate.cancer).cells.were.treated.with.increasing.concentrations.of.ErPC3.under.normoxic.(21. percent.of.oxygen).and.hypoxic. (1. percent.of.oxygen). conditions..Efficacy.of.ErPC3.was.determined.by.colorimetric.proliferation.assays..Induction.of.cell.death.was.analyzed.using.fluorescence.microscopy.and.flow.cytometry.as.well.as.western.blotting..Activity.of.AKT.and.its.key.downstream.effectors. involved. in.the.ErPC3.response.was.verified.by.western.blot.analysis.using.specific.antibodies.

Results:.Our.findings.showed.high.sensitivity.of.T98G.and.PC3.cells.to.ErPC3. under. normoxia. and. hypoxia.. The. antineoplastic. action. of. the.drug.at.low.oxygen.was.paralleled.by.the.increase.cell.death.induction,.which.was.associated.with.a.significant.decline.of.phosphorylation.and.thus.activation.of.AKT.and.of.some.its.effector.proteins.

Conclusion:.Our.data.demonstrate.that.ErPC3.is.a.hypoxia-active.drug.counteracts.AKT-mediated.survival.signals.also.under.conditions.of. li-mited.oxygen.availability..Our.findings.make.ErPC3.a.promising.drug.for.the. treatment. of. solid. human. tumors. with. deregulated. AKT-signaling.and. high. levels. of. tissue. hypoxia,. such. as. glioblastoma. and. prostate.cancer.

Poster P-0061

C. Lehmann.1,.Y..Kienast.1,.M..Thomas.1,.F..Herting.1,.S..Hart.2,.N..Dimoudis.1

1. Roche,.Pharma.Research.and.Early.Development.(pRED),.Penzberg,. Germany.2. Roche,.Translational.Medicine.Hub,.Singapore,.Singapore.

Impact of CXCL12 – CXCR4/CXCR7 pathways in tumor-related angio-genesis: in vitro and in vivo validation

CXCL12.or.stromal-cell.derived.factor-1.(SDF-1).is.an.β-chemokine.signa-ling.through.its.receptors.CXCR4.and.CXCR7..SDF-1.is.involved.in.multiple.basic.biological.processes.but.is.also.associated.with.cancer.progressi-on..More.specifically,.SDF-1.upregulation.in.cancer.is.thought.to.enhan-ce. tumor. vascularization. by. (1). directly. inducing. sprouting. of. nearby.capillaries,.(2).synergizing.with.VEGF.to.enhance.neo-angiogenesis,.(3).recruitment.of.circulating.endothelial.progenitor.cells..Moreover,.SDF-1.was.reported.to.be.involved.in.resistance.to.anti-angiogenic.treatment..Hence,.we.sought. to.validate.SDF-1. in.vitro.and. in.vivo.as.a.potential.target.to.fight.cancer.related.angiogenesis.First,.we.confirmed.the.mode.of.action.described.for.SDF-1.in.vitro..In.primary. human. endothelial. cells,. SDF-1. led. to. intracellular. ERK. phos-phorylation,.induced.targeted.migration.and.stimulated.sprouting.in.a.3D-spheroid.assay..These.effects.were.completely.abrogated.by.neutra-lizing.monoclonal.antibodies.directed.against.SDF-1.or.CXCR4.as.well.as.the.small.molecule.CXCR4-specific.inhibitor.AMD3100.We.then.investigated.SDF-1’s.potential.impact.on.angiogenesis.in.more.complex.in.vivo.settings..In.a.cornea.pocket.assay,.SDF-1.did.not.indu-ce.vessel.sprouting.and.did.not.show.synergism.with.VEGF..Also,.VEGF.induced.vascularization.was.not.inhibited.by.injecting.SDF-1.and.CXCR7.blocking.antibodies..These.data.were.verified.in.a.subsequent.matrigel.plug.assay,.where.SDF-1.alone.or.SDF-1/VEGF.co-administration.had.no.influence.on.vessel.formation.Furthermore,.we.investigated.the.efficacy.of.SDF-1,.CXCR4.and.CXCR7.blockade.using.three.different.tumor.models.in.vivo..In.two.patient.de-rived.HCC.xenografts,.application.of.an.anti-SDF-1.monoclonal.antibody.alone.had.no.effect.on.tumor.growth..Combination.treatment.with.so-rafenib.did.not. result. in.any.significant.additional.anti-tumor.effects..We.further.studied.the.impact.of.SDF-1,.CXCR4.and.CXCR7.inhibition.in.the.syngeneic.colon.cancer.model.CT26.WT..There.was.neither.detecta-ble.reduction.of.tumor.growth,.nor.any.effect.on.tumor.vascularization.under.any.treatment.

AEK

59


Poster P-0071

S. Chatterjee.1,.C..Wieczorek.1,.J..Schöttle.1,.M..Siobal.1,.Y..Hinze.2,.T..Franz.2,.R..Thomas.3,.B..Neumaier.1,.R..Ullrich.1,4

1. Max.Planck.Institute.for.Neurological.Research,.Cancer.Therapy,.Cologne,.Germany.

2. Max.planck.Institute.for.Biology.of.Aging,.Cologne,.Germany.3. University.of.Cologne,.Department.of.Translational.Genomics,.

Cologne,.Germany.4. Clinic.I.of.Internal.Medicine.and.Center.for.Integrated.Oncology,.

University.Hospital.Cologne,.Cologne,.Germany.

Intermittent anti-angiogenic treatment improves cytotoxic thera-peutic outcome in a NSCLC xenograft model

Hypothesis.and.Aim:.Over-expression.of.VEGF. in.tumors1,2. results. in.a.leaky,.dilated,.saccular.vasculature.leading.to.non-uniform.blood.flow3,4..In.the.past.use.of.higher.drug.doses.to.increase.the.concentration.of.drugs. in. tumors. have. failed. because. of. this. flawed. delivery. system..Anti-angiogenic. agents. targeting. VEGF/VEGFR. passively. prunes. dys-functional. and. leaky. vessels. in. tumors. resulting. in. vessel. normaliza-tion.known.as.the.‘normalization.window’4,5..Hence.here.is.the.rationale.for.elucidating.the.duration.of.normalization.window.in.tumors.during.which.healthy.and.homogeneous.vessels.are.well-fortified.ensuring.ef-fective.and.targeted.drug.delivery.

Methods,.Results:.In.this.approach.we.non.invasively.monitored.tumor.blood.flow.in.response.to.the.VEGFR/PDGFR.inhibitor.Vatalanib.(PTK787).using.[15O]H

2O.positron.emission.tomography.(PET). in.PC9.and.H1975-

NSCLC.murine.xenograft.models.In.contrast,.tumor.blood.flow.decreased.drastically.from.day.4. in.the.controls..Control.tumor.vessels.exhibited.extensive.leakiness.visualized.by.massive.extravasation.of.FITC-dextran..However,.PTK787.treatment.markedly.reduced.leakiness.in.the.tumors..Thus,.VEGFR/PDGFR.inhibi-tion.seems.to.improve.blood.flow.by.tumor.vessel.normalization.in.the.first.week.of.treatment.that.can.be.monitored.by.[15O]H

2O.PET.

We.hypothesized.that.cytotoxic.treatment.starting.within.this.normali-zation.window.may.result.in.improved.drug.concentration.within.the.tu-mor..In.PC9.NSCLC.xenografts.Erlotinib.administered.daily.with.PTK787.pre-treatment. lead. to. fast. and. dramatic. tumor. regression. compared.to. tumors. receiving. Erlotinib. as. a. monotherapy.. Mass-Spectrometric.analysis.of.tumor.lysates.showed.much.higher.Erlotinib.concentration.in.Erlo+PTK.treated.sets.in.contrast.to.Erlo-only.sets..A.long-term.tu-mor.xenograft.study.for.8.weeks.with.Erlotinib.and.intermittent.anti-angiogeneic. therapy. led.to.consistent. tumor.shrinkage.without.onset.of.resistance..

Conclusion:.This.study.might.provide.valuable. insight.about.obtaining.maximum.drug.efficacy.by.optimal.scheduling.of.anti-angiogenic.the-rapy.

References:•. 1. Peter.Carmeliet,.Nature,.December.2005•. 2.Robert.S..Kerbel,.Carcinogenesis,.March.2000•. 3.Rakesh.K..Jain,.Science,.January.2005•. 4. .Dai.Fukumura,.Dan.G..Duda,.Lange.L.Munn.and.Rajesh.K..Jain,.Micro-

circulation,.March.2010•. 5.Frank.Winkler,.Rakesh.K..Jain.et.al,.Cancer.Cell,.December.2004

Poster P-0081

S. Theobald.1,.K..M..Greulich-Bode.1,.P..Boukamp.1,.S..Neidle.2,.S..Ohnmacht.2

1. German.Cancer.Research.Center,.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany.

2. University.College.London,.School.of.Pharmacy,.London,.United.Kingdom

G-Quadruplex ligands: anti-cancer drugs targeting telomeres and telomerase

The. G-quadruplex. structure. is. a. high-order. nucleic. acid. structure,.which. can. be. occur. in. chromosomal. regions. including. oncogene. pro-moter.regions.(e.g..hTERT,.c-myc)..Additionally.and.most.interestingly,.G-quadruplexes.may.be.preferentially.formed.in.the.3’.overhang.region.of.telomeres..Therefore,.ligands.targeting.the.G-quadruplex.are.deve-loped.and.have.been.proposed.to.be.highly.potent.anti-cancer.drugs.These.G-quadruplex.stabilizing.small.molecules.have.been.reported.to.cause. DNA. damage. response,. genomic. instability,. downregulation. of.oncogenes,. deregulation. of.mitosis,. senescence. and.apoptosis..Up. to.now,.there.are.no.potent.molecules.used.in.cancer.treatment..Never-theless,.since.the.rational.behind.G-quadruplex.as.therapeutic.targets.is.extremely.auspicious,.new.G-quadrulex.ligands.are.produced.in.phar-maceutical.institutes.

In.our.study.we.compare.BRACO-19,.a.well.established.G-quadruplex.li-gand,.with.a.more.recent.G-quadruplex. ligand,.namely.Compound.146.(C146),.a.naphthalene.diimide.derivative..We.investigated.the.effects.of.C146.on.immortalized.keratinocytes.carrying.mutated.p53.and.constitu-tively.expressing.the.c-Myc.oncogene,.thus.mimicking.a.preneoplastic.cell.type.(HaCaT-myc):.Changes.in.cell.proliferation,.morphology,.telo-mere.organization.in.3D.interphase.nuclei,.telomere.length,.telomerase.activity,.TRF-2.expression,.cell.cycle.control.and.induction.of.apoptosis.under.treatment.

Interestingly. BRACO-19. showed.unusual. effects. for. a.G-quadruplex. li-gand..Treated.cells.show.no.and.no.influence.on.telomere.length.while.inducing.polyploidy.of.the.treated.cells.In.contrast.C146.treated.cells.show.telomerase.inhibition,.telomere.in-stability.in.a.high.degree.and.induction.of.apoptosis..The.cell.proliferati-on.is.time-.and.dose-dependent.decreased.and.the.IC-50.concentration.from.C146.is.0,5µM.lower.than.the.BRACO-19.concentration.

Our.results.revealed.that.BRACO-19.in.vitro.has.a.cellular.effect.which.seems.not.to.correlate.with.reported.G-quadruplex.effects..C146.did.not.show.these.undesired.effects.but.had.a.vast.impact.on.telomere-rele-vant.mechanisms,. including.the.down.regulation.of.telomerase,.which.play.an.important.role.in.several.types.of.cancer.

60


Poster P-0083

J. Engel.1,.O..Treeck.1,.O..Ortmann.1,.F..Köster.2,.S..Buchholz.1,.S..Seitz.1,B..Kwok.1

1. Medizinische.Universität.Regensburg,.Gynecolog.and.Obstetrics,.Regensburg,.Germany.

2. Medical.University.Schleswig.Holstein,.Campus.Lübeck,.Obstetrics.and.Gynecology,.Lübeck,.Germany.

Targeting of GnRH-receptors on triple negative breast cancers with the cytotoxic GnRH-analog AEZS-125

Introduction:. Receptors. of. gonadotropin-releasing. hormone. (GnRH),.also. regarded. as. luteinizing. hormone-releasing. hormone. (LHRH),. are.found.to.be.expressed. in.triple.negative.breast.cancer.(TNBC),.which.contributes.to.approximately.10.to.20%.of.breast.cancer..Selective.tar-geting.of.the.GnRH.receptors.is.one.of.the.many.approaches.in.breast.cancer.therapy.AEZS-125.is.a.Disorazol-Z.conjugate.linked.to.the.GnRH.receptor.agonist.D-Lys

6-LHRH..Disorazol-Z.is.a.novel.natural.cytotoxic.compound.isolated.

from.the.myxobacterium.Sorangium.cellulosum..Owing.to.the.binding.af-finity.of.the.D-Lys

6-LHRH.peptide.to.the.GnRH.receptors,.it.was.hypoth-

esized.that.AEZS-125.demonstrates.GnRH.receptor.specific.cell.growth.inhibition.when.the.compound.is.directed.to.the.GnRH.receptors.by.the.peptide. ligand. and. the. hybrided. Disorazol-Z. adversely. influences. cell.proliferation.in.the.targeted.GnRH.receptor.positive.cells.

Study.design:.The.expression.of.GnRH.receptors.in.the.TNBC.cell.lines.HCC1806.and.MDA-MB-231.was.evaluated.by.RT-PCR.and.Western.blot..The.fibroblast.cell.line.LTK(-),.which.does.not.express.GnRH.receptors,.served.as.a.negative.control.The.cytotoxic.activities.of.Disorazol-Z.and.AEZS-125.were.investigated.in.LTK(-),HCC1806.and.MDA-MB-231cells..Receptor.blocking.experiments.were.carried.out.to.study.the.receptor.mediated.activity.of.AEZS-125..100mM.Triptorelin,.a.GnRH.agonist.that.has.high.receptor.affinity,.was.used.to.block.the.GnRH.receptors.on.the.HCC1806.cells.and.therefore.may.attenuate.the.binding.of.AEZS-125.to.the.receptors..After.10.minutes.of.Triptorelin.pre-treatment,.the.cells.were.washed,.incubated.in.different.concentrations.of.AEZS-125.for.an-other.10.minutes,.washed.again.and.cultivated.in.drug.free.medium.for.48.hours..Cell.viability.was.analyzed.by.cell.titer.blue.(CTB).assay.

Results:.RT-PCR.and.Western.blot.illustrated.that.GnRH.receptors.were.expressed.in.both.mRNA.and.protein.levels.in.HCC1806.and.MDA-MB-231,.whereas. no. expression. was. detected. in. LTK(-).. Disorazol-Z. showed. a.better. performance. than. AEZS-125. in. cell. growth. inhibition. in. LTK(-),..HCC1806.and.MDA-MB-231.In.the.receptor.blocking.experiments,.AEZS-125.was.noticed.to.effec-tively.inhibit.cell.growth.but.its.anti-proliferation.effect.was.diminished.in.the.presence.of.Triptorelin,.and.a.higher.EC50.was.obtained,.show-ing.that.the.binding.and.uptake.of.AEZS-125.into.the.HCC1806.cells.was.mediated.by.GnRH.receptors.

Conclusions:. The. above. data. demonstrated. that. AEZS-125. underwent.GnRH.receptor.mediated.entry.and.anti-proliferation.activities.in.GnRH.receptor.positive.TNBC.cells..AEZS-125.is.thus.a.candidate.for.targeted.cancer.cell.treatment..Experiments.are.in.plan.to.verify.the.cytotoxic.potential.of.AEZS-125.in.vivo.

Poster P-0088

J. Wegert.1,.S..Michelbach.1,.E..Geißinger.2,.C..Otto.3,.N..Graf.4,.M..Gessler.1.

1. Biocenter,.University.of.Wuerzburg,.Developmental.Biochemistry,.Wuerzburg,.Germany.

2. University.of.Wuerzburg,.Institute.of.Pathology,.Wuerzburg,.Germany.3. Department.of.Surgery,.University.of.Wuerzburg,.Experimental.

Surgery,.Wuerzburg,.Germany.4. Saarland.University.Hospital,.Division.of.Pediatric.Oncology.and.

Hematology,.Homburg/Saar,.Germany.

Primary cell cultures as an in vitro Wilms tumor model

Wilms.tumor.is.one.of.the.most.common.solid.tumors.in.childhood..It.ari-ses.from.undifferentiated.embryonic.kidney.cells.and.most.frequently.presents.as.a.unilateral.and.sporadic.tumor..Functional.analysis.of.gene.candidates.and.testing.of.novel.therapeutics.in.Wilms.tumors.has.been.hampered.by.the.lack.of.in.vitro.model.systems..WT.are.characterized.by.a.spectrum.of.histological.appearances,.but.the.few.published.cell.lines.are.mostly.derived.from.rare.anaplastic.variants,.or.even.non-WT..There.has.been.some.success.in.establishing.primary.cultures,.but.these.are.often.poorly.characterized.or.only.derived.from.infrequent.WT1.mutant.tumors.

We.have.generated.a.set.of.primary.WT.cell.cultures.using.a.simple.cul-tivation.and. testing.protocol..Our. cultures.could.be.established.after.preoperative.chemotherapy.and.irrespective.of.histological.subtypes.or.genetic.alterations..By.immunohistochemistry.these.cells.appear.either.epithelial.or.more.mesenchymal,.the.latter.possessing.a.longer.life.span.of. up. to. 45. population. doublings. before. undergoing. senescence.. The.mesenchymal.cells.can.undergo.chondrogenic,.osteogenic.and.adipoge-nic.differentiation,.reflecting.the.histological.spectrum.typically.found.in.WT..In.parallel.xenotransplantation.experiments.with.serial.passaging.we.could.establish.less.differentiated.blastemal.WT.cells.that.grow.as.spheroids.and.develop.WT.like.histology.in.vitro.

As.the.primary.WT.cells.are.amenable.to.genetic.modification,.they.are.ideal.substrates.to.test.functions.of.genes.known.to.be.deregulated.or.mutated. in.WT.and.to.test.candidates.for.therapeutic.agents..The.set.of.WT.cells.was.used.to.test.chemotherapeutic.drugs.according.to.WT.treatment. protocol. as. well. as. new. cytostatic. agents.. These. cells. re-spond.well.to.substances.used.in.present.chemotherapeutic.regimens..Testing.of.novel.drugs.is.currently.under.way..Our.set.of.cultures.repre-sents.a.novel.and.powerful. tool. for.preclinical. compound. testing.and.further.research.into.Wilms.tumor.biology.

AEK

61


Poster P-0089

H. Adwan.1,.A..Murtaja.1,.M..R..Berger.1

1. German.Cancer.Research.Center,.Toxikology.and.Chemotherapy,.Heidelberg,.Germany

Riproximin is a new plat derived antineoplastic agent with efficacy against experimental pancreatic cancer

Purpose:. Pancreatic. ductal. adenocarcinoma. (PDAC). has. one. of. the.worst.prognoses.of.all.malignancies..At.time.of.diagnosis,.80%.of.pati-ents.with.PDAC.have.unresectable.tumors,.and.conventional.therapies.are.nearly.ineffective.The. relative. poor. efficacy. of. chemo/radiotherapy. against. PDAC. indi-cates.that.the.development.of.new.therapeutic.strategies.is.a.crucial.step.to.improve.the.survival.of.patients.with.this.disease..This.includes.the. modification. of. established. therapies,. the. testing. of. new. targets.and.the.improved.delivery.of.drugs.to.their.target.Riproximin. (Rpx). is. a. new. plant. lectin,. which. was. isolated. from. the.plant.Ximenia.Americana..Riproximin.was.classified.as.a.ribosome. in-activating.protein.(RIP).of.type.II.and.its.anticancer.activity.has.been.recently.demonstrated.in.vitro.and.in.vivo

Methods:.In.vivo.experimentsThe.rat.pancreatic.cancer.cell.line.(ASML).was.selected.for.its.property.to. simulate. liver. metastasis. in. nude. rats,. as. described. before. (Eyol)..Tumor-bearing.rats.were.treated. intravenously.with.different.concen-trations. of. Rpx. as. single. agent. or. in. combination. with. gemcitabine.(GEM).or.dinaline.(DIN).Ripx.was.administered.twice.weekly.at.doses.ranging.from.500µg/kg.to.1500µg/kg..GEM.and.DIN.were.administered.to.animals.for.a.period.of.2.weeks.either.intravenously.at.a.dose.of.50mg/kg.or.perorally.at.a..5.times.weekly.dose.of.10mg/kg.

Results:.The.intravenous.administration.of.Rpx.(1500µg/kg.and.500µg/kg).reduced.the.tumor.growth.significantly.by.61%.and.67%,.respec-tively.(p=0.00174.-p=.0.00024)..The.survival.rate.was.also.significantly.increased.(21.8.days.for.riproximin.treated.versus.17.6.days.in.untreated.control.rats;.P=0.01349).after.the.intravenous.administration.of.Rpx.Rats,.which.were.treated.with.the.high.dose.of.Rpx.showed.no.further.reduction.in.tumor.size,.when.compared.with.rats.treated.with.the.low.dose.

In.comparison,.no.significant.effect.on. tumor.size.or.on.survival.was.observed.after.treatment.with.gemcitabine.or.dinaline.The.combination.therapy.with.Rpx.and.GEM.as.well.as.Rpx.and.DIN.pro-duced.a.significant.reduction.in.mean.tumor.size.when.compared.with.the.corresponding.untreated.control.group.but.it.was.not.superior.over.the.single.therapy.with.Rpx.

Conclusion:.Taken.together.the.results.of.our.in.vivo.experiment.suggest.that.riproximin.has.a.clear.potential.for.pancreatic.cancer.treatment.Further.experiments.are.required.for.optimizing.the.combination.thera-py.with.other.antineoplastic.agents.

Poster P-0089

S. Rösch.1,2,3,.G..Dyckhoff.2,.R..Warta.4,5,.S..Stamova.1,.M..Schnölzer.6,.V..Eckstein.7,.P..Beckhove.1,.C..Herold-Mende.4,1

1. German.Cancer.Research.Center.(DKFZ),.Department.of.Translatio-nal.Immunology,.Heidelberg,.Germany.

2. University.Hospital.Heidelberg,.Department.of.Head.and.Neck.Sur-grey,.Heidelberg,.Germany.

3. University.Hospital.Heidelberg,.Department.of.Experimental.Neuro-surgery,.Heidelberg,.Germany.

4. University.Hospital.Heidelberg,.Experimental.Neurosurgery,.Heidel-berg,.Germany.

5. Universtity.Hospital.Heidelberg,.Department.of.Head.and.Neck.Surgrey,.Heidelberg,.Germany.

6. German.Cancer.Research.Center.(DKFZ),.Depatment.of.Functional.Proteom.Analysis,.Heidelberg,.Germany.

7. University.Hospital.Heidelberg,.Department.of.Internal.Medicine.V,.Heidelberg,.Germany

Identification of tumor stroma-associated antigens as novel targets for immunotherapy in HNSCC

Introduction:.Tumor.growth.is.not.simply.determined.by.malignant.can-cer.cells,.but.also.by.the.tumor.stroma.consisting.of.cancer-associated.fibroblasts. (CAFs).amongst.others..T.cell-based. immunotherapies. tar-geting. the. tumor-promoting. microenvironment. might. be. a. promising.therapeutical.approach.because.these.normal.cells.are.genetically.sta-ble.and.therefore.treatment.would.be.largely. independent.of. immune.escape.variants.but.would.affect.the.whole.tumor.

Methods:.Antigen.identification.was.done.by.proteomics.in.primary.tu-mor.cells.and.corresponding.CAFs. from.head.and.neck.squamous.cell.carcinoma.(HNSCC).by.the.newly.developed.automated.two.dimensio-nal. liquid.chromatography.system.PF2D..Immunogenicity.of.the.resul-ting.fractions.was.tested.with.autologous.patient.blood.by.the.IFN-β-ELISPOT.assay..Protein.composition.of.fractions,.triggering.an.immune.response,.was.further.identified.via.mass.spectrometry.peptide.finger-printing.. For. selected. proteins. peptide. epitopes. have. been. predicted.and. synthesized.. The. potential. use. of. theses. candidate. antigens. to.trigger.an. immune.response.was.confirmed. in.an. independent.HNSCC.patient.cohort.as.well.as.compared.to.healthy.donors.

Results:.Significant.T.cell. responses.were.detected.against.38.out.of.112.protein.fractions.derived.from.primary.HNSCC.tumor.cells.and.CAFs..After.exclusion.of.highly.abundant.cell.type-specific.proteins,.22.tumor-specific,.4.CAF-specific,.and.12.candidate.antigens.present.in.tumor.cells.and.CAFs.were.identified..Validation.of.these.putative.TAAs.revealed.si-gnificant. T. cell. responses. against. 11. out. of. 38. TAAs.. Immunogenicity.of.these.selected.candidate.TAAs.was.confirmed.in.an.independent.set.of.HNSCC.patients. (n=20)..Finally,.analysis.of. immunogenic.TAAs. in.a.cohort. of. 5. healthy. donors. confirmed. tumor-. and. CAF-specific. T. cell.responses.for.10.of.the.identified.TAAs.

Conclusion:.In.conclusion,.based.on.an.innovative.proteomics.approach,.we.successfully.identified.10.novel.tumor-.and.CAF-associated.antigens.in.HNSCC..Future.studies.are.needed.to.examine.the.clinical.relevance.of.these.antigens.in.HNSCC.and.to.corroborate.their.suitability.for.an.immunotherapeutical.use.

62


Poster P-0094

V. v. Manstein.1,.D..Löscher.1,.T...Darvishi.1,.M..Weitmann.1,.J..Weissenberger.2,.B..Groner.1,.V..Vafaizadeh.1

1. Georg-Speyer-Haus,.Frankfurt,.Germany.2. Universitätsklinikum,.Experimentelle.Neurochirurgie,.Frankfurt,.

Germany

Cooperation and Inhibition of Stat3 Activating Kinases in Cancer Cells

The. definition. of. signaling. pathways. and. their. components. in. normal.cells.and.the.deregulation.of.these.pathways.through.genetic.alterations.in.tumor.cells,.has.led.to.the.concept.of.“targeted.therapy”..Drugs.di-rected.against.aberrantly.activated.pathway.components,.mainly.prote-in.kinases,.are.able.to.affect.the.growth.and.survivial.of.tumor.cells.and.have.resulted. in.major. therapeutic.benefits. for. tumor.patients..Based.on.this.principle.new.cancer.drugs.are.being.introduced.at.a.rapid.rate.The.signal.transducer.and.activator.of.transcription.3.(Stat3).has.been.identified.as.a.potential.cancer.drug.target..It.is.transiently.activated.by.cytokines.and.growth.factors.in.normal.cells,.but.persistently.activated.in.many.types.of.cancer..Activation.is.mediated.through.tyrosine.phos-phorylation..The.constant.activation.of.Stat3.induces.the.expression.of.target.genes.which.cause.the.proliferation.and.survival.of.cancer.cells,.as. well. as. their. migration. and. invasive. behavior.. The. downregulation.of.Stat3.by.RNAi.in.tumor.cells.inhibits.their.growth.and.induces.their.apoptosis..Multiple.receptor.and.receptor.associated.tyrosine.kinases,.but.also.cytoplasmic.ones.have.been.shown.to.be.able.to.phosphorylate.Stat3..Stat3.can.e.g..be.activated.by.the.IL6-JAK-Stat.axis,.Src.and.bone.marrow.kinase.x-linked.(Bmx)..Since.many.specific.inhibitors.of.Stat3.activating.kinases.are.available,.it.might.become.therapeutically.useful.to.define.the.upstream.activating.network.in.particular.tumor.entities..For.this.purpose,.we.are.defining.the.molecular.cascades.in.model.sy-stems,.the.murine.glioma.cell.lines.Tu-2449.and.Tu-9648.and.the.human.breast.cancer.cell. lines.MDA-MB-231.and. -468.with. focus.on. the.Bmx.kinase..We.have.observed.Bmx.activation.and.high.levels.of.activated.Stat3.in.the.glioma.cells.We. are. studying. the. hierarchy. and. the. interconnections. of. enzymes.leading. to. the. activation. of. Stat3. in. these. tumor. cell. lines. through.the. downregulation. of. particular. tyrosine. kinase. genes. by. RNAi. and.the. treatment.of. the.cells.with.specific.kinase. inhibitors..The.effects.on.growth.and.morphology.of. the.cells.are.being.used.as.phenotypic.parameters..Addition.of.canertinib,.an.ATP-competitive.Bmx. inhibitor,.to.glioma.cells.for.six.hours,.results.in.the.strong.decline.of.P-Stat3,.in-dicating.that.Stat3.is.a.substrate.of.this.kinase.in.these.cells..However,.48.hours.after. canertinib. addition,. Stat3. phosphorylation.at. tyrosine.residue.705. is.being. restored..We.suggest. that. the. inhibition.of.Bmx.triggers.a.compensatory.mechanisms,.possibly.through.the.relieve.of.negative.feedback.inhibition..Another.tyrosine.kinase.might.become.ac-tivated.and.replaces.the.function.of.Bmx..This.adaptive.resistance.could.possibly.be.based.on.endocrine.remodeling.and.the.secretion.of.auto-crine.factors..We.are.investigating.the.underlying.mechanisms.through.the.combination.of.tyrosine.kinase.inhibitors.and.the.sequestration.of.soluble.factors.by.antibodies..These.experiments.indicate.that.the.in-hibition.of.a.single. target.structure.might.not.be.sufficient. to. induce.lasting. therapeutic. effects. and. that. compensatory. mechanisms. are.activated.in.tumor.cells..The.definition.of.these.cooperating.pathways.and. their. simultaneous. inhibition. will. suggest. effective. combination.therapies.

Poster P-0098

L. Bauer.1,.R..Langer.1,.M..Mandl.1,.K..Becker.1,.J..Slotta-Huspenina.1,.A..Novotny.2,.A..Hapfelmeier.3,.H..Höfler.1,4,.G..Keller.1

1. Technische.Universität.München,.Pathologie,.München,.Germany.2. Technische.Universität.München,.Chirurgie,.München,.Germany.3. Technische.Universität.München,.Medizinische.Statistik.und

Epidemiologie,.München,.Germany.4. TecHelmholtz.Zentrum.München,.Pathologie,.Neuherberg,.Germany.

Role of Notch Signaling for Chemoresistance in Gastric Cancer

Questions:.In.a.recent.study.analyzing.the.prognostic.significance.of.the.expression.of.cancer.stem.cell. (CSC). related.genes. in. residual.gastric.tumor.cells.after.neoadjuvant.chemotherapy,.Wnt.and.Notch.signaling.genes,.among.others,.showed.a.prominent.association.with.survival..The.aim.of.this.study.was.to.assess.selected.genes.for.differential.expressi-on.between.pre-.and.post-therapeutic.tumors..In.vitro.we.investigated.the.impact.of.Notch.activity.on.chemosensitivity.in.gastric.cancer.cell.lines.

Methods:.Expression.of.9.genes.was.compared.between.corresponding.pre-. and. post-therapeutic. tumors. from. patients. treated. with. neoad-juvant. chemotherapy. (CTx). demonstrating. partial. (n=22). or. minimal/no. tumor. regression. (n=22).. mRNA. was. isolated. from. macrodissected.FFPE.tissues.and.gene.expression.was.quantified.by.real.time.PCR.using.TaqMan®. low. density. arrays.. Immunohistochemical. staining. (IHC). for.Notch2.was.performed.on.corresponding.pre-.and.post-CTx.tumors.from.patients.with.sub-total,.partial.or.minimal/no.tumor.regression.(n=22,.each).and.from.patients.not.treated.by.CTx.(n=16).and.evaluated.by.a.semi-quantitative.scoring.system..Chemosensitivity.of.three.gastric.can-cer.cell.lines.to.the.gamma-secretase.inhibitor.DAPT.alone.or.in.combi-nation.with.cisplatin.was.determined.by.XTT.or.colony.formation.assays.

Results:.Differential.expression.analysis.revealed.an.increase.of.NOTCH2.and.POU5F1.from.pre-.to.post-CTx.specimens.in.tumors.with.partial.re-sponse.(p=.002.and..028).and.minimal/non.responding.tumors.(p=.062.and. .002).. In.contrast.a.decrease. in.expression.was.observed. in.both.tumor.groups.for.NOTCH1.(p=.072.and..001)..IHC.analysis.of.Notch2.re-vealed.that.cytoplasmic.staining.intensities.of.tumor.cells.increased.si-gnificantly.in.all.groups.of.CTx-treated.patients.(p=.016,..001.and..017).but.not.in.non-CTx.patients..Treatment.of.gastric.cancer.cell.lines.with..10.µM.DAPT.and.2.µM.cisplatin.led.to.a.synergistic.reduction.of.metabo-lic.activity.in.comparison.to.the.single.drugs.

Conclusions:.The.comparison.of.mRNA.expression.between.correspon-ding.pre-.and.post-CTx.specimens.revealed.alterations.consistent.with.an.enrichment.of.CTx-resistant.tumor.cells..Results.of.Notch2-IHC.sug-gested.an.involvement.of.Notch-signaling.in.chemoresistance,.which.was.further.supported.by.the.synergistic.effect.of.cisplatin.and.the.gamma-secretase.inhibitor.DAPT.on.cancer.cells.in.vitro.

AEK

63


Poster P-0104

I. Pisarenko.1,.P..Nguemgo.Kouam.1,.A..Kochanneck.1,.B..Priesch-Grzeszkowiak.1,.H..Bühler.1,.I..Adamietz.1

1. Institut.für.Molekulare.Onkologie,.Strahlenbiologie.und.Experimentelle.Strahlentherapie,.Herne,.Herne,.Germany.

The impact of two different hyperthermia methods on proliferation and motility of human glioblastoma cells.

Question:.The.use.of.hyperthermia.(HT).to.treat.cancer.in.combination.with. radiotherapy. or/and. chemotherapy. has. been. discussed. for. long..Though.the.molecular.mechanisms.of.HT.are.poorly.understood,.many.studies.exist.that.investigated.the.effects.of.HT.on.various.tumor.cells..The.aim.of.our.study.was.to.examine.the.impact.of.HT.on.proliferation.and.motility.of.glioma.cells..As.cancer.cells.invasion.in.the.surrounding.brain.tissue.is.crucial.for.local.recurrences.in.glioma.patients,.reduced.motility.of.the.glioma.cells.could.significantly.improve.the.therapeutic.results.

Methods:.Two.different.glioma.cell.lines.(U-87.MG,.U-343.MG).were.trea-ted.at.42.ºC.with.either.conventional.HT.in.an.incubator.or.capacitive.HT.in.an.electromagnetic.field.(EM)..The.proliferation.of.the.treated.cells.was.investigated.with.a.colorimetric.cytotoxicity.assay.(MTS.assay)..The.cellular.motility.was.monitored.by.time-laps.videography.and.proteins.associated.with.migration.were.analyzed..In.detail,.the.expression.le-vels.of.fascin,.integrin.beta-1,.focal.adhesion.kinase.(FAK),.and.phospho-FAK.were.quantified.by.western.blotting.

Results:.After.4.days.EM.for.1h.or.2h.per.day,.the.proliferation.of.U-343.cells.was.reduced.by.28%.or.87%.respectively..The.proliferation.of.U-87.cells.was.reduced.by.29%.after.1h.and.by.91%.after.2h.daily.treatment..The.treatment.in.an.incubator.resulted.in.a.decrease.of.proliferation.by.only.20%.for.both.cell.lines.after.2h.daily..Fascin.was.increased.in.both.cell.lines.after.incubation.at.42°C.for.1.h.(by.64%.in.U-87.and.by.49%.in.U-343).and.2.h.(by.17%.in.U-87.by.88%.in.U-343)..In.contrast,.the.expression.of.beta-1. integrin,.FAK.and.pFAK.was.significantly.reduced.after.exposure.of.the.cells.for.2.h.to.EM.and.to.conventional.HT.as.well..The.cellular.velocity.of.U-87.cells.was.reduced.by.13%.after.exposure.to.EM.for.2.h,.while.the.accumulated.distance.was.decreased.by.17%..After.application.of. conventional.HT. for.2.h.no. significant. change. in. these.parameters.could.be.observed..However,.after.conventional.HT.for.only.0.5h.per.day.velocity.and.accumulated.distance.were.decreased.by.16%.and.14%.respectively..The.motility.of.U-343.cells.treated.with.EM.for.1h.or.2h.showed.an.increase.in.velocity.and.accumulated.distance.by.36%.or.24%.respectively,.while.conventional.HT.caused.a.decrease.by.8%.after.1h.and.21%.after.2h.for.both.parameters..However,.the.migration.of.untreated.U-343.cells.is.very.low,.so.that.the.changes.observed.after.HT.are.also.low.in.terms.of.absolute.values.

Conclusions:.The.proliferation.of.both.cell.lines.is.reduced.markedly.af-ter. HT.. The. expression. of. proteins. associated. to. cellular. migration. is.also.reduced.whereas.the.actin-organizer. fascin. is.enhanced..Results.for.cellular.motility.are.inconsistent..Fast.U-87.cells.are.retarded.after.HT,.whereas.slow.U-343.cells.are.accelerated.on.a.low.level..It.has.to.be.noted.that.EM.is.more.effective.than.conventional.HT.

Poster P-0105

G. Gdynia.1,.S..Sauer.2,.M..Enders.3,.J..Kopitz.4,.W..Roth.1

1. Institute.of.Pathology.and.German.Cancer.Research.Center,.University.Hospital,.Heidelberg,.Germany.

2. Metabolic.Center.Heidelberg,.University.Hospital,.Heidelberg,.Germany.

3. Institue.of.Inorganic.Chemistry,.University,.Heidelberg,.Germany.4. Institute.of.Pathology,.University.Hospital,.Heidelberg,.Germany.

HMGB1 controls Warburg metabolism in colon carcinomas

Question:.The.survival.of.colon.cancer.cells.is.dependent.on.an.efficient.cellular.energy.metabolism..Thus.it.is.of.great.importance.to.unravel.the.cellular.mechanisms.that.control.the.break-down.of.energy.substrates..Recently. we. have. shown. that. the. HMGB1. (high. mobility. group. box. 1).protein.induces.a.new.form.of.cell.death.accompanied.by.a.decrease.of.the.cellular.ATP.pool..In.this.study.we.examined.the.impact.of.HMGB1.on.glycolysis,.oxidative.phosphorylation.and.glutaminolysis.

Methods:. Isotopomer.analysis,.metabolic.assays.with. radioactive. tra-cers,.ATP.luciferase.assay,.enzymatic.assays,.western.blot,.immunopre-cipitation,.generation.of.rho.zero.colon.carcinoma.cell.lines,.liposome.transfection,.stable.HMGB1-overexpression,.oxygen.consumption.mea-surements,. colon. carcinoma. tissue. slice. culture. experiments. (vibra-tome),.cytotoxicity.testing.

Results:. Several. colon. carcinoma. cell. lines. (SW480A,. HCT116,. HT29).showed. different. sensitivity. to. HMGB1.. In. all. cell. lines. tested. HMGB1.induced. a. Warburg-like. metabolic. phenotype.. In. detail,. HMGB1. bound.to.cytochrome.c.oxidase.and.potently.inhibited.the.mitochondrial.oxy-gen.consumption..Simultaneously.HMGB1.channeled.the.flux.of.glucose.carbons.away.from.the.mitochondria.towards.to.fermentation.by.spe-cific. inhibition. of. the. tetrameric. form. of. PKM2. (pyruvate. kinase). not.affecting.the.dimeric.PKM2..Colon.cancer.cells.resistant.to.HMGB1.could.preserve.glutaminolysis.whereas.HMGB1.sensitive.cancer.cells.showed.a.significant.decrease.in.glutamine.break-down.upon.HMGB1.treatment..Treatment.with.the.anti-metabolite.L-DON.in.combination.with.glucose.deprivation.restored.the.HMGB1.cytotoxicity..Consistently,.cancer.cells.with.a.low.activity.of.the.key.enzyme.of.glutaminolysis,.malic.enzyme.1,.were.more.susceptible.to.HMGB1.cytotoxicity.

Conclusions:.HMGB1.induces.glucose.fermentation.and.inhibits.the.mito-chondrial.oxygen.consumption.of.colon.cancer.cells.thus.fostering.War-burg-like.metabolism..Cancer.cells.that.cannot.adapt.to.these.metabolic.shift.die..Preservation.of.glutaminolysis.can.rescue.colon.cancer.cells.from.HMGB1.cytotoxicity..This.effect.can.be.reversed.by.the.inhibition.of. glutaminolysis. by. the. glutamine-analogue. L-DON.. Targeting. energy.metabolism.with.both.HMGB1.and.anti-metabolites.might.overcome.the.resistance.of.colon.carcinomas.to.conventional.chemotherapy.

64


Poster P-0107

P. Heffeter.1,.V..Pichler.2,.J..Mayr.2,.G..Hermann.3,.D..Groza.1,.G..Koellensperger.3,.M..Galanski.2,.W..Berger.1,.B..Keppler.2,.C..Kowol.2

1. Institute.of.Cancer.Research.Vienna,.Vienna,.Austria.2. Institute.of.Inorganic.Chemistry,.University.of.Vienna,.Vienna,.

Austria.3. Division.of.Analytical.Chemistry,.University.of.Natural.Resources.and.

Applied.Life.Sciences,.Vienna,.Austria

Targeted drug delivery of oxaliplatin based on maleimide-functiona-lised platinum(IV) complexes.

Therapy.with.platinum(II).compounds.(like.cisplatin.or.oxaliplatin).is.li-mited.by.strong.side.effects,. resistance.development,.and. insufficient.tumor.accumulation..Notably,.albumin,.the.most.abundant.protein.in.hu-man.blood.serum,.is.known.to.accumulate.in.malignant.and.inflammatory.tissues.due.to.the.so-called.enhanced.permeability.and.retention.(EPR).effect..Thus,.aim.of.the.presented.study.was.to.enhance.tumor-targeting.of.platinum.compounds.via.binding.to.an.albumin-affine.moiety.(malei-mide)..To.assure.tumor-specific.drug.release,.a.platinum(IV).prodrug.de-sign.was.used..This.class.of.compounds.is.kinetically.inert.and.only.after.reduction. in.the.specific.milieu.of.the.tumor.tissue.the.corresponding.cytotoxic.platinum(II).compound.is.released.and.exerting.its.anticancer.activity..In.this.study,.the.first.maleimide-containing.platinum(IV).drug.KP2156.was.synthesized.and.chemically.characterized.by.one-.and.two-dimensional.NMR.spectroscopy,.ESI-mass.spectrometry,.and.elemental.analysis..In.addition,.also.the.respective.succinimide.derivative.KP2157.was.prepared,.which.possesses.no.reactivity.towards.albumin..Binding.kinetics.with.albumin.and.cystein.were. investigated.by.RP-HPLC.mea-surements.. Moreover,. SEC-ICP-MS. measurements. using. bovine. blood.serum.were.performed.indicating.binding.of.KP2156.to.the.albumin-con-taining.protein. fraction,.while.KP2157.was.exclusively.detected. in. the.low.molecular.weight.fraction.

To.test.the.impact.of.albumin.binding.on.the.anticancer.activity.of.the.new.compounds.in.vivo.the.murine.CT-26.colon.cancer.model.was.used..Both. platinum. complexes. were. well. tolerated. with. no. significant. loss.of.body.weight.and.had.potent.anticancer.potential. resulting. in.signi-ficantly.reduced.tumor.growth..Notably,.KP2156.displayed.significantly.higher.anticancer.activity. than.KP2157. (p. <0.01.at.day. 15). resulting. in.a.distinctly. lower.mean.tumor.burden.(of.0.38.g.vs..0.18.g. for.KP2157.and.KP2156,. respectively)..Histological.examinations.of. the. tumor. tis-sue.revealed.that.these.effects.are.based.on.strong.cell.death.induction.especially. in.case.of.KP2156..Together,. this.suggests.that.the.albumin.binding.of.KP2156.leads.either.to.prolonged.plasma.half-life.of.the.drug.and/or.a.more.selective.accumulation.in.the.malignant.tissue.due.to.the.EPR.effect.

Poster P-0108

R. Trondl.1,.P..Heffeter.2,.M..Jakupec.1,.W..Berger.2,.B..Keppler.1

1. University.of.Vienna,.Vienna,.Austria.2. Medical.University.of.Vienna,.Vienna,.Austria.

NKP-1339, a first-in-class anticancer drug showing mild side effects and activity in patients suffering from advanced refractory cancer

NKP-1339.is.one.of.the.most.promising.investigational.non-platinum.me-tal.drugs. in.clinical.development.against.solid.malignancies..Recently,.NKP-1339.was.evaluated. in.a.clinical.phase. I. trial. regarding. its.safety,.tolerability,.maximum.tolerated.dose,.pharmacokinetics,.and.pharmaco-dynamics.. The. high. tumor. targeting. potential. of. NKP-1339. is. probably.based.on.delivery.to.tumor.sites.by.serum.proteins.such.as.albumin.and.transferrin.as.well.as.on.the.activation.of.the.compound.in.the.reductive.tumor.milieu..The.reduction.of.ruthenium(III).to.ruthenium(II).is.favor-ed.under.hypoxic.condition.(frequently.occurring.in.solid.tumors).and.is.followed.by.severe.disruption.of.the.cellular.redox.balance.and.induction.of.apoptosis.via.the.mitochondrial.pathway.. In.the.recently.completed.clinical.phase. I. trial.34.patients.with.advanced.solid. tumors.were.en-rolled.for.dose.escalation.[1]..NKP-1339.was.infused.on.day.1,.8,.and.15.of.28-day.cycles..To.gain.further.insight.in.the.mechanism.of.action,.protein.expression.studies.in.cancer.cells.exposed.in.vitro.were.performed..The.maximum.tolerated.dose.of.NKP-1339.was.determined.at.625.mg/m2,.and.the.most.common.drug.related.side.effects.were.nausea,.vomiting,.and.fatigue..A.long.lasting.partial.response.was.observed.in.1.patient.with.a.gastro-intestinal.neuroendocrine.tumor.(NET),.and.7.patients.have.expe-rienced.stable.disease,.including.NET.(2),.non-small.cell.lung.cancer.(2),.colorectal.cancer.(1),.sarcoma.(1),.and.cancer.of.unknown.primary.(1)..Moreover,.NKP-1339.was.found.to.down-regulate.in.cancer.cell.lines.the.ER.chaperon.GRP78,.a.key.regulator.of.the.unfolded.protein.response,.which.is.associated.with.intrinsic.and.chemotherapy-induced.resistance.The.very.limited.side.effects.of.NKP-1339.and.the.activity.observed.in.a.variety.of.tumors.so.far.are.very.promising..Further.clinical.phase.I.drug.combination.studies.and.single.agent.phase.II.studies.are.planned.

Reference:1.. Thompson. D,. Weiss. GJ,. Jones. SF,. Burris. HA,. Ramanathan. K,. Infante.

JR,.Bendell.JC,.Ogden.A,.Von.Hoff.DD,.NKP-1339:.Maximum.tolerated.dose.defined.for.first-in-human.GRP78.targeted.agent..ASCO.Annual.Meeting.Abstract.2012.(Abstract.ID.3033).

AEK

65


Poster P-0111

C. Kuznia.1,.B..Klinger.2,.J..Keil.3,.B..Seliger.4,.C..Falk.3,.R..Schäfer.1,.N..Blüthgen.1,.C..Sers.1

1. Charité.Universitätsmedizin.Berlin,.Institute.of.Pathology,.Molecular.Tumor.Pathology,.Berlin,.Germany.

2. Humboldt.University.of.Berlin,.Institute.for.Theoretical.Biology,.Berlin,.Germany.

3. Hannover.Medical.School,.Integrated.Research.and.Treatment.Center.Transplantation,.Hannover,.Germany.

4. Martin.Luther.University,.Institute.for.Medical.Immunology,.Halle,.Germany.

Molecular Mechanisms of Sorafenib-induced Apoptosis in Cancer Cells

Question:.The.RAS/MEK/ERK.pathway.is.a.key.oncogenic.pathway.invol-ved.in.human.cancer..Point.mutations.in.RAS.genes.resulting.in.consti-tutive.activation.of.RAS.proteins.occur.in.~30%.of.human.cancers..RAS.activation. results. in. an. overactivation. of. signaling. pathways. thereby.inducing.cell.proliferation,. invasion.and.apoptosis. inhibition..We. inve-stigated.the.Raf.kinase.inhibitor.Sorafenib.and.the.MEK.inhibitor.U0126.in.human.embryonal.kidney.cells.transformed.by.oncogenic.HRAS..Both.substances.target.the.same.pathway;.yet,.treatment.with.Sorafenib.in-duces.apoptosis.while.U0126.does.not..A.time-resolved.analysis.of.this.effect.by.protein.and.mRNA.arrays,.revealed.an.involvement.of.DDIT4.(also.known.as.Redd1).and.mTOR.signaling.in.the.differential.action.of.Sorafenib.and.U0126..The.present.study.aims.to.examine.whether.that.observation.also.applies.to.human.cancer.cells.and.to.obtain.an.impro-ved.understanding.of.the.underlying.molecular.mechanisms..

Methods:. We. treated. six. different. renal. carcinoma. cell. lines. with. So-rafenib.or.U0126.and.tested.apoptosis.by.cleaved.caspase-3.measure-ment.via.flow.cytometry..In.addition,.expression.of.DDIT4.was.analyzed.using.Real-Time-PCR..To.further.investigate.the.link.between.DDIT4.and.apoptosis.we.performed.a.siRNA-mediated.knock-down.of.DDIT4.as.well..The. activation. of. mTOR. and. associated. components. of. that. signaling.network.were.examined.using.a.Bio-Plex.assay.and.western.blot..The.activating.transcription.factor.ATF4,.described.in.literature.as.a.regula-tor.of.DDIT4,.was.also.analyzed.using.Real-Time-PCR..

Results:. Four. cell. lines. induced. caspase-3. cleavage. and. a. significant.increase. of. DDIT4. after. Sorafenib. treatment. only.. One. cell. line. could.not. induce.DDIT4,.one.cell. line.did. induce.DDIT4,.but.no.cleaveage.of.caspase-3.. siRNA-mediated. knock-down. of. DDIT4. inhibited. caspase-3.cleavage..A.closer.examination.of.the.mTOR.pathway.using.the.Bio-Plex.assay. revealed. no. differences. in. S6-kinase. phosphorylation. between.cells.with.and.without.DDIT4-mediated.apoptosis..A.significant.upregu-lation.of.ATF4.in.combination.with.an.increased.RNA.level.of.DDIT4.could.only.be.confirmed.in.one.out.of.four.selected.cell.lines..

Conclusions:.These.results.encourage.the.hypothesis.that.the.induction.of.DDIT4.plays.an.important.role.for.apoptosis.induction.by.Sorafenib.in.human.renal.cancer..However,.successful.induction.of.apoptosis.might.be.hampered.by.currently.unknown.mechnisms.interfering.with.DDIT4-upregulation.or.apoptosis-induction.as.a.result.of.mTOR.inhibition.

Poster P-0114

U. Jungwirth.1,2,.J..Gojo.1,.T..Tuder.1,.M..Holcmann.1,.G..Walko.3,.P..Heffeter.1,2,.B..K..Keppler.4,2,.W..Berger.4,2

1. Medical.University.Vienna,.Institute.of.Cancer.Research,.Vienna,.Austria.

2. University.of.Vienna.and.Medical.University.of.Vienna,.Research.Platform.„Translational.Cancer.Therapy.Research“,.Vienna,.Austria.

3. University.Vienna,.Max.F..Perutz.Laboratories,.Vienna,.Austria.4. University.Vienna,.Institute.of.Inorganic.Chemistry,.Vienna,.Austria.

Early steps in KP46-induced cell death: involvement of integrin-me-diated cell adhesion and calpain activity

Investigations.of.metal-based.anticancer.compounds,.especially.of.non-platinum.complexes,.have.increased.in.the.recent.years..In.this.study.we.analyzed.the.mechanisms.underlying.the.anticancer.activity.of.the.gal-lium. complex. KP46,. [Tris(8-chinolinolato)gallium(III)],. which. has. been.successfully.studied.in.a.phase.I.clinical.trial..Within.a.few.hours,.KP46.treatment.of.different. colon.and. lung.cancer. cells. at. low.µM.concen-trations. led.to.cell.contraction.and.detachment.followed.by.cell.death.induction.at.about.8–16.hrs.. This.process.was.accompanied.by.enhan-ced.phosphorylation.of.myosin. light.chain,.which. is.known.to.promote.cell.contraction..Moreover,.altered.protein.expression.of.focal.adhesion.proteins,.such.as.loss.of.integrin.β1.and.talin.were.detected.after.short-term. treatment.. In.accordance.with.disturbed. integrin. integrity,.KP46.inhibited. re-adhesion.of. cells..Mg2+,. a.divalent. ion.promoting. integrin-mediated. cell-matrix. binding,. partially. rescued. cell. attachment. after.KP46. treatment.. Loss. of. integrin. engagement. and. cell-matrix. binding.are.characteristic.for.the.induction.of.anoikis.and.activation.of.pro-apo-ptotic.Bcl-2.family.members..KP46.rapidly.upregulated.the.protein.levels.of.pro-apoptotic.Bax,.Bak.and.Bim.after.short-term.exposure..Further-more,. co-treatment. of. KP46. with. the. cell-permeable. calpain. inhibitor.PD150606.reversed.and.blocked.the.cytotoxic.activity.of.KP46..This. is.the.first.study.demonstrating.disturbance.of.integrin.integrity.as.early.steps.in.cell-death.induction.by.the.anticancer.gallium.compound.KP46.and.indicates.an.important.role.of.calpain.in.mediating.this.process.

66


Poster P-0120

S. Keller.1,.B..Mühlthaler.1,.J..Kneißl.1,.S..Heindl.1,.H..Höfler.1,.B..Luber.1

1. Technische.Universität.München,.Institut.für.Allgemeine.Pathologie.und.Pathologische.Anatomie,.München,.Germany

Analysis of E-cadherin as a biomarker for individual molecular thera-py in gastric carcinoma

The. transition. from.benign. to. invasive.and.metastatic. tumors. is. cha-racterized. by. loss. of. cell-cell. adhesion.. In. gastric. cancer,. the. cell.adhesion.molecule.E-cadherin.is.frequently.mutated.and.consequently.functionally.inactivated..E-cadherin.interacts.with.the.epidermal.growth.factor. receptor. (EGFR). in. a. multicomponent. complex. and. negatively.influences. the. receptor. activation,. whereas. somatic. mutation. of. E-cadherin.is.associated.with.increased.activation.of.EGFR1..The.crosstalk.between.EGFR.and.E-cadherin.is.potentially.relevant.for.tumor.progres-sion.and.response.to.molecular.therapy.with.EGFR-targeted.therapy..A.therapeutic.antibody.that.targets.EGFR.is.the.chimeric.monoclonal.IgG1.antibody.cetuximab.which. is. approved. for.head.and.neck.cancer.and.colorectal.cancer..In.a.recent.phase.III.study.(EXPAND).in.advanced.gas-tric.cancer,.a.combination.therapy.of.cetuximab.with.capecitabine.and.cisplatin.showed.no.benefit.compared.to.treatment.with.chemotherapy.alone.. It.was.suggested. that.because.of. the.heterogeneity.of.gastric.cancer,.further.classification.of.the.disease.may.be.required2.The.aim.of.the.project.is.the.analysis.of.E-cadherin.as.a.biomarker.for.the.prediction.of.the.response.to.cetuximab.in.gastric.cancer.In.a.previous.phase.II.trial.of.cetuximab.plus.oxaliplatin/leucovorin/5-fluorouracil. in. first-line. metastatic. gastric. and. oesophago-gastric.junction.cancer,.we.have.shown.that.patients.with.high.expression.of.E-cadherin. in. their. tumors. have. a. better. overall. survival. than. those.with. low.or.moderate.E-cadherin.expression..Two.missense.mutations.in.CDH1.(D402H.and.A408V).were.detected..The.abundance.and.genetic.mutations.of.E-cadherin.were.hypothesized.to.be.potential.biomarkers.for.the.response.to.EGFR-targeted.therapy3..Furthermore,.we.analyzed.the.effect.of.E-cadherin.expression.and.mutation.on.the.cetuximab.re-sponse.in.gastric.cancer.cell.lines..Three.of.five.gastric.cancer.cell.lines.showed.resistance.to.cetuximab.treatment.which.was.associated.with.the.presence.of.mutations.in.the.E-cadherin.gene.CDH14..The.results.of.both.clinical.and.cell.culture.studies.suggest.that.E-cadherin.may.influ-ence.the.treatment.outcome.of.molecular.therapy.with.cetuximab.by.unknown.mechanisms..Therefore,.we.currently. investigate. the.under-lying.molecular.mechanisms.using.a.model.of.three.gastric.cancer.cell.lines,.two.of.them.reveal.mutations.in.CDH1.(KATOIII:.p.E336E;.MKN45:.p.G274_P277del)..By.overexpression.or.silencing.of.E-cadherin,.we.ana-lyze.the.effect.of.E-cadherin.on.cetuximab.sensitivity.

We.believe.that.our.hypothesis-generating.studies.may.be.clinically.re-levant.and. suggest. to.evaluate. the.potential. biomarker.E-cadherin. in.larger.clinical.trials.1. Bremm.et.al..2008,.doi:.10.1158/0008-5472.CAN-07-15882. Lordick.et.al..2012;.doi:10.1093/annonc/mds4993. Luber.et.al..2011;.doi:.10.1186/1471-2407-11-5094. Heindl.et.al..2012;.doi:.10.1007/s00432-011-1128-4

Poster P-0127

C. Hülsewig.1,.C..Bernemann.1,.M..Götte.1,.L..Kiesel.1,.C..Liedtke.2

1. University.Hospital.Münster,.Department.of.Gynecologics.and.Obstetrics,.Münster,.Germany.

2. University.Hospital.Schleswig-Holstein.Campus.Lübeck,.Department.of.Gynecologics.and.Obstetrics,.Lübeck,.Germany.

Wnt signaling – predictive and prognostic marker of triple negative breast cancer (TNBC)

Background:. Triple.negative.breast. cancer. is. characterized.by.an.un-favorable. prognosis.. While. patients. do. not. benefit. from. endocrine. or.Her2. targeted. therapies. except. chemotherapy,. there. is. an. increased.response.(pCR).to.taxane-/anthracycline-containing.therapy.compared.to.other.breast.cancers..Current. research.suggests.an. increased.Wnt.Pathway.activity.in.TNBC,.while.Wnt.pathway.activity.has.been.shown.to.play.an. important.role. in.breast.cancer.pathogenesis..Previous.re-sults.of.our.group.showed,.that.expression.of.Wnt.pathway.modulator.SFRP1.(secreted.frizzled.related.protein.1).is.associated.with.response.to.current.chemotherapeutics.(paclitaxel,.doxorubicin.and.cisplatin).in.silico.and.in.vitro..

Methods:.The.effects.of.siRNA.mediated.knockdown.of.Wnt.associated.transcription.factor.TCF7L2.(transcription.factor.7.like.2).and.treatment.with.small.molecule.XAV939.(specific. inhibitor.of.Wnt.signaling).were.analyzed. in. TNBC. cell. lines. MDA-MB. 468,. MDA-MB. 231,. HCC. 1806. and.luminal.cell.line.MCF7.on.chemo.sensitivity,.cell.invasion,.cell.migration.and.proliferation..

Results:. TCF7L2. knockdown. cells. tend. to. be. more. resistant. against.above-mentioned.cytostatic.agents,.while.TCF7L2.overexpression.signi-ficantly.leads.to.a.decreased.chemo.therapy.resistance..After.TCF7L2.knockdown. in.MDA-MB.231,.SKBR-3,.MCF-7.cells,. invasion.competency.and.cell.migration.(wound.healing).is.significantly.increased..Treatment.with.Wnt.inhibitor.XAV939.of.TNBC.cell.lines.MDA-MB.231,.MDA-MB.468.and.luminal.cell.line.MCF7.results.in.increased.chemo.sensitivity.to.pa-clitaxel. and. doxorubicin.. However,. XAV939. does. not. affect. invasion-/migration.competency..

Conclusion:.We.suggest.Wnt.Signaling,.TCF7L2.and/or.SFRP1.expression.as.a.novel.makers.of.chemotherapy.sensitivity.to.taxane-,.anthracycline.and/or.platinum-containing.chemotherapy.in.TNBC.and.may.be.used.as.predictive.markers.for.neoadjuvant.chemotherapy..Additionally,.TCF7L2.and.SFRP1.may.serve.as.prognostic.markers.for.TNBC.

AEK

67

AEKAbstracts Posters Section 7: Free topics

Poster P-0010

A. Lánczky.1,.B..Györffy.1

1. Semmelweis.University,.First.Department.Of.Pediatrics,.Budapest,.Hungary.

Developing an online tool for the validation of survival-Associated Biomarkers in non small-cell lung cancer using transcriptomic data of 1,566 patients

In.the.last.decade,.optimized.treatment.for.non-small.cell.lung.cancer.had. lead. to. improved. prognosis,. but. the. overall. survival. is. still. very.short..To.further.understand.the.molecular.basis.of.the.disease.we.have.to.identify.biomarkers.related.to.survival..Here.we.present.the.develop-ment.of.an.online.available.tool.suitable.for.the.real-time.meta-analysis.of. published. lung. cancer. microarray. datasets. to. identify. biomarkers.related.to.survival.

First,. we. searched. the. caBIG,. GEO. and. TCGA. repositories. to. identify.samples. with. published. gene. expression. data. and. survival. informati-on..All.together.1,566.NSCLC.samples.in.9.independent.datasets.were.identified,.85%.have.overall.survival.and.45%.progression.free.survival.info,.one-third.adenocarcinomas.and.one-third.squamous.cell.carcino-mas,.57%.male.and.the.average.overall.survival.is.45.months..Kaplan-Meier.survival.plot,.and.the.hazard.ratio.with.95%.confidence.intervals.and.logrank.P.value.are.calculated.and.plotted.in.R.using.Bioconductor.packages..The.complete.analysis.tool.can.be.accessed.online.at:.www.kmplot.com/lung.

We.demonstrate.the.application.of.this.integrative.analysis.pipeline.by.validating. 22. previously. published. survival. associated. biomarkers. in-cluding. VEGF,. ADAM28,. ANXA3,. CADM1,. CD24,. CD82,. CDK1,. CEA,. cyclin.E,.ERCC1,.EZH2,.HER2,.IFNAR2,.MMP9,.OPN,.P16,.p53R2,.RAD51,.S100A4,.survivin,.XAF1.and.XIAP..Each.of.the.biomarker.candidates.was.investiga-ted.in.the.same.cohort.in.which.it.was.originally.described..Survival.was.best.predicted.by.CDK1.(HR=2.56,.p<1e-16),.CD24.(HR=2.45,.p=3.6e-10).and. CADM1. (HR=0.38,. p=7e-12). in. adenocarcinomas. and. by. Cycline. E.(HR=2.44,.p=4.8e-08).in.all.NSCLC.patients.

In.summary,.we.performed.a.meta-analysis.of.survival-associated.genes.and.established.an.integrated.database.and.an.online.tool.for.future.in.silico.validation.of.new.candidates.

Poster P-0025

D. Sharma.1

1. University.of.Duisburg-Essen,.Department.of.Molecular.Biology,.Essen,.Germany.

Function of the acid sphingomyelinase/ceramide pathway in glio-blastoma cells response to ionizing radiation

The.acid.sphingomyelinase(Asm)/ceramide.system.plays.a.central.role.in. radiation. induced. cell. death.. Ionizing. radiation. (IR). activates. Asm.and.triggers.a.release.of.ceramide.and.the.formation.of.ceramide-en-riched.membrane.platforms.that.serve.to.cluster.membrane.receptors.required. for. apoptosis. induction.. Accordingly,. Asm. deficient. T-and. B-lymphocytes,. murine. embryonic. fibroblasts,. and. endothelial. cells. are.resistant.to.IR..In.the.present.study,.we.analysed.whether.radiosensiti-vity.of.glioblastoma.(A172.and.LN308).cells.correlates.with.Asm.activity.level.upon.IR..In.both.cell.lines.IR.induced.Asm.and.the.release.of.ce-ramide,.yet.at.a.different.extent..Whilst.IR.transiently.activated.Asm.in.radiation-resistant.A172.cells,.Asm.activation.was.sustained.upon.irra-diation.in.highly.sensitive.LN308.cells.finally.leading.to.cell.death..Asm.activation.upon.irradiation.is.independent.of.DNA.damage.as.Asm.was.still.stimulated.in.cells.devoid.of.nuclei.(cytoplasts)..Further.studies.de-monstrated.that.inhibiting.ceramide.consumption.in.A172.cells.by.using.glucosyltransferase.inhibitors.greatly.radiosensitized.cells..Vice.versa,.treatment.of.LN308.cells.with.Asm.inhibitor.fluoxetine.decreased.their.sensitivity.to.IR..IR.resulted.in.rapid.activation.of.acid.sphingomyelinase.(Asm),.release.of.ceramide,.and.formation.of.ceramide.enriched.memb-rane.platforms,.which.are.required.for.IR-induced.activation.of.NADPH.oxidase.and.production.of.reactive.oxygen.species.(ROS)..Inhibition.of.NADPH. oxidase. or. removal. of. intracellular. ROS. reduced. IR-triggered.Asm. activation. and. formation. of. ceramide-enriched. membrane. plat-forms,.suggesting.that.IR-triggered.Asm.activation.is.in.turn.regulated.by.ROS.in.a.positive.feedback.manner..Our.findings.establish.the.Asm/ceramide.system.as.a.target.to.improve.the.therapeutic.efficacy.of.ra-diotherapy.for.glioma.cells.

68

AEKAEK AEKAbstracts Posters Section 7: Free topics

Poster P-0027

T. Maier.1,.H..Schneck.1,.C..Blassl.1,.H..Seeger.1,.T..Fehm.1,.M..Pawlak.2,A..Staebler.3,.H..Neubauer.1

1. Eberhard-Karls-University.of.Tuebingen,.Department.of.Gynecology.and.Obstetrics,.Tuebingen,.Germany.

2. University.of.Tuebingen.,.Natural.and.Medical.Sciences.Institute,.Reutlingen,.Germany.

3. Eberhard-Karls-University.of.Tuebingen,.Department.of.Pathology,.Tuebingen,.Germany.

Pathway analysis of membrane-initiated effects of estrogen -no-rethisterone (E-NET) treated breast cancer cells overexpressing PGRMC1 in a mouse model

Background:.An.increased.risk.for.developing.invasive.breast.cancer.in.postmenopausal. women. during. estrogen-progestin. hormone. therapy.has.been.demonstrated..Progesterone.receptor.membrane.component-1.(PGRMC1). is.overexpressed. in.breast.cancer.and. it.may.contribute. to.tumorigenesis.. In. a. transplantation. model. PGRMC1-overexpressing.MCF-7.cells.(WT-12).and.empty.vector.transfected.MCF-7.cells.(EV).were.engrafted.into.nude.mice.and.resulting.tumor.masses.were.explanted,.fixated. in. formalin. and. embedded. in. paraffin. (FFPE,. Neubauer. et. al.,.Menopause.2012).

Objectives:.Aims.of.this.study.were.to.establish.a.method.for.protein.ex-traction.from.FFPE.tissues.to.set.up.of.reverse.phase.protein.microarray.(RPPM).technology.and.to.analyze.expression.and.phosphorylation.of.PGRMC1-signaling.associated.proteins.

Methods:.Different.methods. for. target. retrieval. such.as. the.usage.of.target. retrieval. solution. (TRS,. DAKO). or. citrate. buffer. combined. with.various.protein.extraction.methods.such.as.Qproteome®.FFPE.Tissue.Kit.(Qiagen).were.applied.using.a.pressure.cooker..Quantity.and.quality.of.protein.fraction.were.tested.with.western.blot.analysis.for.low.back-ground.and.high.signals.for.beta-actin.protein..Proteins.were.isolated.from.tumor.tissues.with.the.extraction.combination.providing.the.best.results,.RPPM.arrays.were.printed.and.expression.and.phosphorylation.levels.of.extra.cellular.signal-activated.kinase.1/2.(ERK1/2).and.protein.kinase.B.(Akt).were.determined.in.a.pilot.study.

Results:.We.selected.the.combination.of.target.retrieval.with.the.EXB.buffer. (Qiagen). and. protein. extraction. with. TRS. lysis. mix. to. isolate.protein.lysates.from.FFPE.tissue..RPPM.analysis.demonstrated.a.highly.significant.up.regulation.of.Akt.in.WT-12.tumors.compared.to.EV.tissues..Phosphorylation.levels.of.Akt.in.WT-12.tumors.treated.with.E-NET.were.approximately.78.%.decreased.in.comparison.to.EV.tumors..ERK1/2.ex-pression.and. its.phosphorylation.were.significantly. lower. in.WT-12.tu-mors.compared.to.EV.tumors.

Conclusion:.We.have.successfully.established.a.method.to.perform.pa-thway.analysis.of.FFPE.tissues.by.RPPM..PGRMC1.signaling.may.include.activation.of.ERK1/2.and.Akt.

Poster P-0048

J. Wiedemann.1,2,.J..Zink.1,.P..Simoniello.1,.M..Stange.1,.G..Alphonse.3,.C..Rodriguez-Lafrasse.3,.C..Fournier.1,.M..Durante.1,4

1. GSI.Helmholtzzentrum.für.Schwerionenforschung.GmbH,.Biophysics,.Darmstadt,.Germany.

2. Technische.Universität.Darmstadt,.Biologie,.Darmstadt,.Germany.3. Université.de.Lyon,.Laboratoire.de.Radiobiologie.Cellulaire.et.

Moléculaire,.Lyon,.France.4. Technische.Universität.Darmstadt,.Physik,.Darmstadt,.Germany.

Inflammation related response to ionizing irradiation in human skin cells

Acute.inflammation,.which.can.cause.fibrosis,.is.one.of.the.most.com-mon.side.effects.after.cancer.radiotherapy..On.the.other.hand.low.dose.of. irradiation. exerts. anti-inflammatory. effects. in. patients. suffering.from.chronic.inflammation..This.indicates.a.complex.dependence.of.the.regulation.of.inflammation.on.radiation.quality,.dose.and.the.interaction.of.irradiated.cells.with.the.tissue.environment..In.general.the.two.major.types.of.cell.death,.apoptosis.and.necrosis,.are.involved.in.processes.re-lated.to.inflammation..In.contrast.to.necrosis,.apoptosis.is.a.controlled.process.which. leads.to.the.elimination.of.the.cells.without.triggering.inflammation..In.this.project.we.want.to.investigate.changes.induced.by.ionizing.radiation.that.are.potentially.related.to.induction.or.inhibition.of.inflammation.in.skin,.i.e..the.occurrence.of.apoptosis.and.the.release.of.cytokines.and.other.factors.

To. investigate.the.occurrence.of.apoptosis.and.necrosis.after. irradia-tion.we.first.used.an. immortalized. (HaCaT).keratinocyte.cell. line.and.primary.keratinocytes.(NHEK)..For.both.cell.types.we.could.not.find.any.evidence. for. the. occurrence. of. primary. apoptosis. after. the. irradiati-on.with.X-ray.or.carbon.ions.even.after.a.high.dose..No.morphological.changes.(DAPI.staining).and.no.expression.of.the.cleaved.form.of.cas-pase.3.(Western.Blot.analysis).could.be.detected..Probably.due.to.the.p53.deficiency.of.HaCaT.cells,.we.observed.continued.proliferation. in.these. cells. exposed. to. a. high. dose,. followed. by. mitotic. catastrophe..The.presence.of. cleaved.caspase.3.and.an.enhanced.amount.of. cells.with.sub.G1.DNA.content.(FACS.analysis).at.later.time.points.indicated.the.induction.of.secondary.apoptosis.as.a.result.of.mitotic.catastrophe..Primary.keratinocytes.(NHEK).do.not.respond.with.mitotic.catastrophe.even.to.high.doses.of. irradiation.but.they.stop.to.proliferate..For. low.X-ray.doses.and.UVB.intensities.cell.cycle.progression.was.transiently.delayed,.but.after.a.few.population.doublings.proliferation.ceased.due.to.contact.inhibition.and.irrespective.of.irradiation.

According.to.our.results,.exposure.to.ionizing.radiation.does.not.induce.primary.apoptosis.in.monolayer.cultures.of.keratinocytes..As.we.expect.that.the.extra-cellular.matrix.and.other.cell.types.resident.in.skin.have.an.impact.on.the.occurrence.of.apoptosis.and/or.the.release.of.inflam-mation-related.factors.we.used.a.human.full.thickness.skin.equivalent.(EFT400;. MatTek).. In. first. experiments. we. found. no. evidence. for. the.occurrence.of.apoptosis.after.irradiation.

These.results.obtained.in.monolayer.and.3D-cultures.suggest.that.apo-ptosis.is.not.the.trigger.for.anti-inflammatory.responses.in.skin.cells..This. work. was. supported. by. the. Deutsche. Forschungs. Gemeinschaft.(DFG;.GRK.1657).EU-PARTNER.(Particle.Training.Network.for.European.Radiotherapy).projects.and.BMBF.(GREWIS,.contract.no..02NUK017A).

AEK

69


Poster P-0051

D. Richter.1,.L..Mack.1,.A..Weber.1,.B..Brill.1,.N..Delis.1,.B..Groner.1

1. Georg-Speyer-Haus.,.Frankfurt,.Germany.

Identification of low molecular weight Stat3 inhibitors through the disruption of peptide and protein interactions in AlphaScreen, BiFC and FRET assays

The.signal.transducer.and.activator.of.transcription.3.(Stat3).is.a.tran-scription.factor.which.regulates.cytokine.and.growth.factor.functions.in.many.cell.types..Inappropriately.high.and.long.lasting.activation.was.ob-served.in.tumor.tissues.and.causes.proliferation,.angiogenesis,.migra-tion.and.immune.evasion.in.tumor.tissues..The.activated.form.of.Stat3.is.required.for.the.survival.of.many.cancer.cells.and.Stat3.inhibition.is.being.considered.as.a.promising.drug.target..We.have.identified.peptide.sequences.which.specifically.bind.to.crucial.domains.of.Stat3.and.inhibit.its.transactivation.function..A.20.amino.acid.peptide.aptamer.sequence.(rS3-PA).recognizes.the.dimerization.domain.of.Stat3.and.a.sequence.derived.from.Pias3.(rPP-C8),.a.Stat3.interacting.protein,.recognizes.the.coiled. coil. domain. of. Stat3.. We. are. looking. for. low. molecular. weight.compounds.which.exhibit.the.same.inhibitory.properties.as.the.peptide.ligands..These.compounds.will.be.selected.from.libraries.by.their.ability.to.bind.to.the.Stat3.domains,.cause.the.release.of.the.peptide.ligands.and.thus.act.as.competitive.inhibitors.of.peptide.protein.interactions..For.this.purpose.we.will.first.use.fluorescence.proximity.based.assays.in.vitro,.e.g..the.Alpha-Screen.(amplified.luminescent.proximity.homo-geneous. assay),. HTR-FRET. (homogeneous. time-resolved. fluorescence.resonance.energy.transfer)..Promising.candidates.will.then.be.verified.in.vivo.by.a.test.system.based.on.BiFC.(bimolecular.fluorescence.com-plementation)..For.this.purpose.stably.transfected.cell.lines.will.be.de-rived.in.which.fragments.of.the.yellow.fluorescent.protein.(YFP).will.be.brought. together. through.the.Stat3.peptide. interactions.and.comple-ment.each.other.to.yield.functional.fluorescence..Candidate.compounds.should.be.able.to.disrupt.these.interactions..We.expect.to.identify.low.molecular.weight.compounds.with.drug.like.properties.that.specifically.interfere.with.the.functional.domains.of.the.transcription.factor.Stat3.and.will.test.them.for.their.therapeutic.potential.in.tumor.cell.lines.and.experimental.animals.

Poster P-0054

K. Greulich-Bode.1,.M..Eryilmaz.1,.D..Krunic.1,.P..Boukamp.1

1. German.Cancer.Research.Center,.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany..

UV-C irradiation causes enhanced telomere distruction in TRF2 transfected HaCaT-Myc cells

Question:. Telomeres,. the. ends. of. linear. chromosomes. are. composed.of. the.highly. repeated.sequence. (TTAGGG)

N.and. the. telomere.binding.

proteins,.e.g..the.shelterin.complex..This.name.already.suggests.a.cen-tral. role.of. this.complex. in.maintaining.chromosomal. /.genomic. inte-grity.. The. telomere. repeat. binding. factors. 1. and. 2. (TRF1,. TRF2). have.been.shown.to.be.key.players.in.this.aspect:.Depletion.of.TRF2.leads.to.telomere.fusion,.stable.overexpression.of.TRF2.by.transfection.leads.to.cell.death..Additionally,.it.has.been.proposed.that.TRF2.is.an.important.protein.in.telomere.organization.in.the.3D.interphase.nucleus..Since.this.organization.is.disturbed.by.UV.irradiation,.it.is.tempting.to.speculate.that.TRF2.expression.has.an.impact.on.UV-induced.effets.on.the.telo-meres.of.eukaryotic.cells.

Methods:.We.therefore.investiganted.HaCaT-Myc.cells,.eg.derivates.of.the.spontaneously.immortalized.keratinocyte.cell.line.HaCaT.as.well.das.HaCaT-Myc-TRF2.cells,.eg.HaCaT-Myc.cells.which.have.been.transfected.with.an.inducible.TRF2.gene.We. monitored. TRF2. expression. by. life. cell. imaging. and. investigated.TRF2.transcipts.by.real.time.PCR..Additionally,.fluorescence.in.situ.hy-bridization.was.conducted.to.determine.the.3D.distribution.of.telomers.in.interphase.nuclei.of.HaCaT-Myc.and.HaCaT-Mcy-TRF2.cells..Southern.blot.analysis.was.performed.to.determine.changes.in.telomere.length..These.parameters.were. investigated.up. to.72.hours.after.UV-C.expo-sure.

Results:.Our.experiments.show.that.TRF2.expression.did.rise.after.UV-C.exposure.accompanied.by.enhanced.telomere.aggregate.formation..The.telomere.legth.did.not.change..Interestingly,.the.HaCaT-Myc-TRF2.cells.showed.a.higher.inducibility.(3-fold).of.telomeric.aggregats.as.compa-red.to.the.HaCaT-Myc.system.(2-fold).after.UV-C.irradiation.Conclusion:.Since.telomere.aggregate.formation.has.been.reported.to.lead.to.genomic.instability.and.chromosomal.aberrations.and.genomic.instability,.this.difference.in.telomere.aggregate.formation.after.UV-C.irradiation.points.to.a.higher.degree.of.genomic.instability.in.the.HaCaT-Myc.cells.additionally.having.incuced.expression.of.TRF2..These.results.additionally.support.and.strengthen.the.hypothesis.that.TRF.2.is.a.key.player.in.telomeric.aggregate.formation.and.hence.in.telomere-related.induction.of.genomic.instability.in.UV-C.irradiated.cells.

70


Poster P-0058

I.-P. Chen.1,.S..Henning.1,.P..Boukamp.2,.A..Faust.1,.R..Greinert.1,.B..Volkemer.1

1. Cell.Biology/Krankenhaus.Buxtehude,.Dermatology,.Buxtehude,.Germany.

2. DKFZ,.Heidelberg,.Germany

Aberrant epigenetic alteration at the tumor suppressor gene p16INK4a could be related to UVA carcinogenesis in human keratinocytes

The.long.wavelength.range.of.the.UV.radiation,.UVA,.has.been.demons-trated.to.be.capable.of. inducing.DNA.damage.and.is.regarded.as.car-cinogen..To.address.the.involvement.of.UVA.in.tumorigenesis.and.skin.cancer.as.well.as.a.possible.contribution.of.epigenetic.regulation.here,.we.have.irradiated.the.human.keratinocytes.with.UVA.and.determined.the.gene.expression.of.tumor.suppressor.genes.and.epigenetic.modu-lators..A.long-lasting.reduction.of.expression.of.the.tumor.suppressor.p16INK4a.was.observed.in.human.keratinocytes,.KH8.2.00.cells,.repeatedly.treated.with.a.heavy.dose.of.UVA..This.repression.of.p16INK4a.was.accom-panied.by.an.epigenetic.alteration:.p16INK4a.promoter.hypermethylation..A.compromised.cell.cycle.regulation.with.a.less.stringent.G1-control.af-ter.DNA.damage.contributable.to.the.p16INK4a.repression.characterized.the. UVA-treated. keratinocytes.. Interestingly,. dysregulation. of. p16INK4a.has.often.been.found.(among.others).in.skin.cancers.such.as.malignant.melanoma.or.squamous.cell.carcinoma..Thus.aberrant.epigenetic.alte-ration. at. the. tumor. suppressor. gene. p16INK4a. could. be. related. to. UVA.carcinogenesis.in.human.keratinocytes..However,.epigenetic.alterations.seemed.to.be.strongly.dependent.on.the.UVA-radiation.regimes,.as.an.acute.UVA-irradiation.did.not.cause.the.same.responses.

Poster P-0064

C. Vannier.1,2,.K..Mock.1,.T..Brabletz.1,3,.W..Driever.2,3,4

1. University.of.Freiburg,.Medical.Center,.Dept..of.Visceral.Surgery,.Freiburg,.Germany.

2. University.of.Freiburg,.Institute.Biology.I,.Developmental.Biology,.Freiburg,.Germany.

3. University.of.Freiburg,.BIOSS.-.Centre.for.Biological.Signalling.Studies,.Freiburg,.Germany.

4. University.of.Freiburg,.Institute.for.Advanced.Studies,.Freiburg,.Germany

ZEB1 DEPENDENT REGULATION OF CELL ADHESION AND CELL BEHAVI-OUR IN EARLY DEVELOPMENT AND CANCER

The. ZEB1. transcription. factor. is. best-known. as. inducer. of. epithelial-mesenchymal. transitions. (EMT). in. cancer. metastasis,. acting. mainly.through.transcriptional.repression.of.CDH1.(coding.for.E-cadherin).and.the.EMT-suppressing.miR-200s..Here.we.show.that.ZEB1.zebrafish.or-thologues,. Zeb1a. and. Zeb1b,. control. cell. adhesion. and. movement. du-ring.gastrulation..The.Zeb1/miR-200.double-negative.feedback. loop. is.conserved.in.teleosts,.but.does.not.contribute.to.gastrulation.control..Loss.and.gain.of.function.analyses.reveal.that.Zeb1.represses.cdh1.ex-pression.to.control.adhesion.and.epiboly.of.deep.cells..Moreover,.Zeb1.acts.as.repressor.of.epcam,.and.thereby.may.contribute.to.control.of.epithelial. integrity. of. enveloping. cells.. A. similar. negative. correlation.between.ZEB1.activity.and.EPCAM.expression.was.found.in.human.pan-creatic.and.breast.cancer.cell.lines,.mediated.through.direct.binding.of.ZEB1.to.the.EPCAM.promoter..Thus,.Zeb1.proteins.represent.key.players.in.the.accurate.regulation.of.cell-cell.adhesion.and.cell.motility.which.is.a.prerequisite.for.the.dynamic.cell.behaviours.during.gastrulation.and.malignant.cancer.progression.

AEK

71


Poster P-0073

A. Legin.1,2,.A..Schintlmeister.3,.M..Jakupec.1,2,.M..Galanski.1,.M..Wagner.3,4,.B..Keppler.1,2

1. University.of.Vienna,.Institute.of.Inorganic.Chemistry,.Vienna,.Austria.

2. University.of.Vienna,.Research.Platform.«Translational.Cancer.Therapy.Research»,.Vienna,.Austria.

3. University.of.Vienna,.Large-Instrument.Facility.for.Advanced.Isotope.Research,.Vienna,.Austria.

4. University.of.Vienna,.Department.of.Microbial.Ecology,.Vienna,.Austria.

Nano-scale secondary ion mass spectrometry (NanoSIMS) as a tool for subcellular distribution studies of platinum-based anticancer compounds

Cisplatin. (cis-diamminedichloridoplatinum(II)). has. maintained. its. firm.position. in.cancer.therapy.for.more.than.three.decades,.still. the.me-chanisms.of.cisplatin.selectivity,.resistance.and.toxicity.are.not.com-pletely.understood.(Boulikas.et.al.,.Oncol..Rep..11:559,.2004)..Insights.into.the.subcellular.localization,.in.particular.the.distribution.over.the.cellular.compartments,.can.yield.valuable. information.pointing.to.the.sites.relevant.for.biological.activity..In.order.to.explore.the.suitability.of.NanoSIMS.for.this.purpose,.we.have.investigated.the.distribution.of.15N-labeled.cisplatin.in.the.human.colon.cancer.cell.line.SW480..Under.the.bombardment.with.a.positively.charged.Cs.primary. ion.beam,.we.obtained.12C14N-,.31P-,.34/32S-ion.maps.reflecting.the.cellular.and.nuclear.(phosphorus-rich.structures).morphology.with.a.remarkable.spatial.re-solution.(Fig..1)..The.combination.of.194/198Pt.and.12C15N/12C14N.ratio.(15N/14N.ratio).was.used.for.cisplatin.imaging..After.24.h.exposure.of.the.cells.to.different.concentrations.of.cisplatin,.we.detected.platinum.both. in.the.cytoplasm.and.nucleus..In.parallel.we.have.quantified.the.amount.of.platinum.associated.with.the.cells.by.means.of.inductively.coupled.plasma.mass.spectrometry.(ICPMS).to.assess.the.NanoSIMS.detection.limit.for.platinum..The.strongest.correlation.was.shown.between.the.Pt.and. S. distribution,. both. in. the. cytoplasm. and. nuclear. regions,. consi-stent.with.binding.of.Pt.compounds.to.thiol-containing.molecules.(e.g..peptides.such.as.glutathione.and.proteins.of. the.thioredoxin.system).(Kasherman. et. al.,. J.. Med.. Chem.. 52:4319,. 2009).. Thus,. this. modern.mass-spectrometry. technique. allows. highly. sensitive. multielemental.analysis. on. a. submicrometer. scale.. NanoSIMS. offers. high. spatial. and.mass.resolution.and.provides.a.powerful.tool.for.subcellular.imaging.of.platinum.in.biological.samples.Fig.. 1.Secondary. ion.maps.of.SW480.cell. treated.with.cisplatin. (24.h,..150.µM):.194Pt-.(A),.32S-.(B).and.31P-.(C)..Thin.arrows.show.the.colocaliza-tion.of.Pt.and.S.rich.structures.in.the.cytoplasm..Bold.arrowheads.show.the.colocalization.of.Pt.with.S.and.P.in.the.nucleolus..Scale.bars.=.1.µm.

Poster P-0076

M. Eryilmaz.1,.K..M..Greulich-Bode.1,.P..Boukamp.1

1. Deutsches.Krebsforschungszentrum.(DKFZ),.Genetics.of.Skin.Carcinogenesis.,.Heidelberg,.Germany.

Influence of ionizing radiation on telomeres and genomic changes

In. our. lab. ionizing. radiation. was. used. to. investigate. chromosomal.changes.and.it.has.been.proposed.that.in.this.process.telomeres.play.a.central.role..Two.different.factors.are.known..One.is.the.telomere.length.and.the.second.a.telomere-length.independent.mechanism.of.genomic.instability,.e.g..altered.telomere.organization. in.3D. interphase.nuclei..When.the.normal.telomere.distribution.in.3D.interphase.nuclei.is.distur-bed,. telomeric.associations.and.telomeric.aggregates.(TAs).are.seen..Strikingly,.TA.induction.is.closely.associated.with.the.establishment.of.new.chromosomal. rearrangements. in. two.different. induction.systems.(c-Myc.and.UV).

To.study.the.3D.telomeric.organization.in.interphase.nuclei,.3D.fluore-scence.in.situ.hybridization.was.performed.by.probing.for.the.telomeric.DNA.. 3D. pictures. were. taken. by. using. epiflouresenz. microscopy. with.deconvolution.algorithm..For.exploration.of.the.chromosomal.composi-tion,.after.irradiation,.metaphases.were.prepared.and.multicolor.fluo-rescence.in.situ.hybridization.(mFISH).was.performed..Further,.telome-rase.activity.and.cell.cycle.distribution.were. investigated.at.different.time.points.

As. previously. reported,. upregulated. c-Myc. expression. induces. TAs..Here,.we.irradiated.HaCaT-Myc.cells.with.UV-C,.which.caused.telomeric.associations.with.highest.numbers.for.TA.formation.48.to.72.hours.post.irradiation..This.was.followed.by.determination.of.chromosomal.aberra-tions.8.to.10.days.after.irradiation.

To.examine.if.irradiation.with.X-rays.causes.similar.changes.in.telomere.distribution,.we.treated.human.fibroblast.with.2Gy..72h.post-irradiation.we.detected.only.a.relatively.low.number.of.TAs,.which.increased.up.to.13d.post-irradiation..In.comparison.after.UV-C.irradiation.the.highest.le-vel.of.TA.were.detected.48–72h.post.irradiation.and.declined.at.further.time.points.

Despite.the.different.time.points.of.TA.formation,.chromosomal.aberra-tions.were.seen.after.both.radiations.types.Noteworthy,.the.time.points.for.maximal.TA.formation.seem.to.differe.between.ionizing.radiation.and.UV-irradiation.Taking. together,. the. mechanism. behind. TA. formation. seems. to. differ.between.UV-C.and.x-ray.and.can.be.seen.as.a.new.indicator.for.radiation.induced.genomic.instability

A B C

72


Poster P-0080

E. Specker.1

1. DKFZ,.Heidelberg,.Germany.

The epidermal stem cell – A new approach to identify stem cells as viable label-retaining cells

Stem. cells. in. the. human. interfollicular. epidermis. are. still. difficult. to.identify,.mainly.due.to.a.lack.of.definitive.surface.markers..Consequent-ly,.it.is.a.major.challenge.to.understand.how.epidermal.stem.cells.are.regulated.to.generate.a.healthy.and.constantly.regenerating.epidermis..Since.stem.cells.rarely.divide,.they.can.be.functionally.identified.as.label.retaining.cells.(LRCs)..In.particular,.it.is.possible.to.label.the.cells.with.nucleotide.analogues. like. IdU. (Iododeoxyuridine).and. to.detect. infre-quently.dividing.cells.with.pulse-chase.experiments..Until.now,.the.iso-lation.of.DNA.labelled.vital.LRCs.is.restricted.due.to.the.need.of.fixation.to.visualize.the.DNA.label..To.bypass.this,.cell-intrinsic.fluorescent.dyes.are.required..With.the.generation.of.a.lentiviral.based.vector.expressing.a.Tet-Off.controlled.histone.2B-GFP.(Tet-Off-H2B-GFP).reporter.gene,.a.new.tool.for.the.detection.and.isolation.of.viable.LRCs.was.established..In.initial.experiments,.the.vector.was.successfully.used.to.infect.human.malignant.keratinocytes.(HaCaT.ras.II-4).

The.aim.is.now.to.analyse.the.regulation.of.human.epidermal.stem.cells.and.to.identify.more.reliable.surface.markers.for.their.unbiased.identi-fication..For.this.latter,.normal.human.keratinocytes.are.infected,.culti-vated.in.organotypic.cultures.(OTCs).and.are.allowed.to.establish.LRCs.during.long-term.regeneration..These.LRCs.will.be.isolated.and.purified.via.FACS.sorting.and.used.for.the.analysis.of.their.expression.profile.and.functional.competence.

Poster P-0085

L. Nevaril.1,.M..Am.oros.Alonso.1,.H.-J..Stark.1,.P..Boukamp.1

1. DKFZ,.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany

Expression profiling of human skin regeneration

Defining.the.regulation.of.epidermal.formation.and.its.transition.to.ho-meostasis.is.essential.for.understanding.of.skin.regeneration..While.the.epidermal.stratification.per.se.has.gained.some.interest.previously,.not.enough.is.still.known.about.the.dynamic.processes.at.different.stages.and.modes.of. the.epidermal.growth..Nor.we.have.an. insight. into. the.accompanying.transcriptional.changes.in.the.dermis..We.employ.a.hu-man.skin.equivalent.model.that.makes.an.extensive.range.of.biological.parameters.experimentally.accessible..This.technology.is.now.coupled.with.transcriptional.profiling.and.modern.statistical.methods.to.disclo-se.the.dynamics.of.gene.expression.in.dermis.and.epidermis..We.thus.aim.at.contributing.to.a.solid.frame.of.knowledge.crucial.for.improved.treatments.in.regenerative.medicine.and.possibly.in.dermato-oncology.

AEK

73


Poster P-0091

M. Jakupec 1,2,.U..Jungwirth 2,3,.S..Valiahdi 1,.S..Abramkin 1,.W..Berger 2,3,M..Galanski 1,2,.B..Keppler 1,2

1. University.of.Vienna,.Institute.of.Inorganic.Chemistry,.Vienna,.Austria.

2. University.of.Vienna.and.Medical.University.of.Vienna,.Research.Platform.„Translational.Cancer.Therapy.Research“,.Vienna,.Austria.

3. Medical.University.of.Vienna,.Institute.of.Cancer.Research,.Department.of.Medicine.I.and.Comprehensive.Cancer.Center,.Vienna,.Austria.

Methyl-substitution yields oxaliplatin derivatives with increased ef-ficacy, tolerability and therapeutic window in vivo

Despite.the.well-known.limitations.of.oxaliplatin,.in.particular.its.limi-ted.spectrum.of.clinical.activity.and. its.adverse.neurological.effects,.attempts.to.improve.these.properties.by.structural.modifications.have.been.scarce..Among.a.variety.of.oxaliplatin.derivatives.carrying.alkyl.substituents.on.the.cyclohexane.ring.developed.by.the.authors,.methyl-substituted.species.have.been. recognized.as.being.superior. to. those.with. larger.substituents.both. in.vitro.and. in.vivo..Therefore,. in-depth.biological. evaluation. was. focused. on. enantiomerically. pure. methyl-substituted.derivatives..In.the.intraperitoneal.L1210.leukemia.model.in.DBA/2J. mice,. the. maximum. tolerated. doses. were. found. to. be. higher.than.that.of.the.parent.drug,.and.the.most.efficacious.derivative.yielded.a.higher.increase.in.life.span.(>200%.vs.152%).than.oxaliplatin.(6.mg/kg/day.×.3).as.well.as.a.higher.number.of.long-term.survivors.(5/6.vs.2/6.individuals).at.a.dose.(9.mg/kg/day.×.3).which.is.well.tolerable.in.the.case.of.the.derivative.but.lethal.in.the.case.of.oxaliplatin.(Abramkin.et.al..J..Med..Chem..10:53,.2010)..In.remarkable.contrast.to.oxaliplatin,.activity.in.the.subcutaneous.CT-26.colon.cancer.model.was.not.only.ob-served.in.immunocompetent.BALB/c.mice,.but.also.in.SCID/BALB/c.mice,.suggesting.a.reduced.dependence.on.immunogenic.cell.death.induction..In.behavioral.experiments,. tumor-free.BALB/c.mice.showed.distinctly.reduced. signs. of. the. neuropathy,. in. particular. the. cold. hyperalgesia,.which.is.typically.dose-limiting.for.oxaliplatin.treatment.(Jungwirth.et.al.,.Mol..Pharmacol..81:719,.2012)..Final.steps.of.preclinical.development.are.currently.being.planned.with.the.intention.of.a.clinical.evaluation.in.the.near.future.

Poster P-00115

R. Kuner 1,2,.N..Kahn 3,.M..Meister 4,2,.T..Muley 4,2,.R..Eberhardt 3,.A..Tufman 5,6,.S..Kaduthanam 1,4,.P..A..Schnabel 7,2,.A..Warth 7,.R..M..Huber 5,6,.M..Thomas 2,8,.F..J..Herth 2,3,.H..Sültmann 1,2

1. German.Cancer.Research.Center,.Unit.Cancer.Genome.Research,.Heidelberg,.Germany.

2. German.Center.for.Lung.Research,.Translational.Lung.Research.Centre.Heidelberg,.Heidelberg,.Germany.

3. Thoraxklinik,.University.of.Heidelberg,.Pneumology.and.Critical.Care.Medicine,.Heidelberg,.Germany.

4. Thoraxklinik,.University.of.Heidelberg,.Translational.Research.Unit,.Heidelberg,.Germany.

5. German.Center.for.Lung.Research,.Comprehensive.Pneumology.Center.Munich,.München,.Germany.

6. Ludwig-Maximilians.University.Munich,.Radiotherapy.and.Radiation.Oncology,.München,.Germany.

7. University.of.Heidelberg,.Institute.of.Pathology,.Heidelberg,.Germany.

8. Thoraxklinik,.University.of.Heidelberg,.Thoracic.Oncology,.Heidelberg,.Germany

Detection of RNA and miRNA biomarkers in bronchial fluids for early diagnosis of non-small cell lung cancer

Early.detection.and. the.assessment.of.efficient. therapy.schemes.are.major. needs. in. lung. cancer. patient. care.. CT. diagnostics. is. presently.of. limited. accuracy. and. increase. the. risk. of. overdiagnosis. and. over-treatment..Diagnostic.and.prognostic.molecular.biomarkers.measured.in.body.fluids.by. less-invasive.techiques.would.be.a.useful.diagnostic.adjunct.

Bronchial.fluids.collected.by.bronchoscopic.microsampling.or.bronchial.lavage.were.screened.for.RNA.and.miRNA.biomarkers.aiming.at.the.stra-tification.of.patients.with.malignant.and.non-malignant.lung.diseases..Patients.(n=.120).were.included.after.significant.findings.upon.CT.scan,.and.the.diagnosis.was.assessed.after.surgery..We.screened.for.biomar-kers.by.using.microarray.and.low-density.arrays,.and.validated.putative.transcriptomic.biomarkers.by.qPCR.technique.

An. increase. of. tumor-associated. genes. could. be. detected. in. endo-bronchial.epithelial.lining.fluid.collected.close.to.the.maligant.nodule..Tenascin-C.was.further.characterized.for.different.abundance.of.splice.variants..Furthermore,.bronchial.fluid.samples.were.analysed.for.more.stable.miRNAs.previously.detected. in.bronchial. lavage.of. lung.cancer.patients.and.controls.

Our. results. indicate. the. presence. of. suitable. biomarkers. in. bronchial.fluids.for.novel.approaches.in.lung.cancer.diagnostics.

74


Poster P-00117

E. Pavez Loriè 1,.G..Bläser 1,.P..Bourkamp 1

1. DKFZ,.Skin.Carcinogenesis,.Heidelberg,.Germany.

UV and the skin; in vitro skin model studies of UVA and UVA+B.

Most.skin.cancers.are.caused.by.long-term.exposure.to.the.sun,.where.UVA.and.UVB.play.a.key.role.in.damaging.DNA.by.inducing.photo-oxida-tive.stress.in.the.cell.and.inducing.CPDs..These.responses.are.assumed.to.disturb.the.telomeres.by.making.them.sensitive.and.damage.them..The. cells. will. also. elicit. different. stress. and. apoptotic. response. me-chanisms,.depending.on.how.damaged.the.cells.are.after.UV.exposure..Former.studies.have.mostly. focused.on. the.effects.of.one.UV.source.(UVA. or. B). in. monolayer. cultures,. epidermal. equivalents. or. collagen.based.in.vitro.skin.models,.that.do.not.resemble.the.in.vivo.situation.very.well.and.are.short. lived.. In. this.study.we.have.used.our.group’s.long-term.scaffold.based.skin.organotypic.cultures.(OTCs).that.attains.a. human. skin. homeostatic. state.. The. irradiated. OTCs. received. either.UVA.or.the.entire.UVA.and.B.(UVA+B).range.and.each.of.these.samples.had. an. internal. control,. which. made. it. possible. to. compare. different.radiation.regimes.as.well.as.see.the.“neighbouring”.effects.in.covered.vs.uncovered.tissue..We.have.examined.the.telomere.length.by.means.of.in.situ.hybridisation,.the.expression.of.Hsp72.and.cleaved.Caspase-3.by.immunofluorescence;.2.hours,.3.days.and.14.days.after.UV.exposure..The.results.show.a.marked.telomere.shortening.3.days.after.UVA.(60J/cm2).irradiation;.this.effect.was.not.exclusive.to.the.UVA.treated.part,.but.could.also.be.seen.in.the.internal.control..This.could.also.be.seen.14.days.after,.although.not.as.severe..On.the.contrary.UVA+B.(60J/cm2.+. 100mJ/cm2).did.not.give.any.change.after.3.days..When.examining.stress. response. via. Hsp72. expression,. we. could. see. an. induction. of.Hsp72.in.the.epidermis.by.UVA.3.days.after.exposure..Also.a.de.novo.expression.of.Hsp72.was.seen.in.the.dermal.compartment..Surprisingly.UVA+B.did.not.cause.a.similar.response..The.apoptotic.response.studied.by.cleaved.Caspase-3.expression.had.no.significant.change.3.days.af-ter.UV.treatment,.but.had.an.induction.after.14.days.in.UVA+B.treated.samples.. In.conclusion,.we.see.that.the.response.to.UV.changes.with.time.and.is.different.depending.on.the.source,.which.makes.the.long-term.OTCs.a.useful.model.to.study.the.effects.of.UV..We.can.also.conclu-de.that.UVA.indeed.causes.telomere.shortening,.which.is.not.restricted.to.the.UV.exposed.area.and.this.effect.seems.to.last.for.several.days..We. also. see. an. induction. of. Hsp72. as. described. by. other. groups. be-fore,.but.we.do.not.see.the.same.effects.when.using.a.combined.UVA+B.source..Additionally.cleaved.Caspase-3.has.also.a.different.expression.with.the.different.UV.regimes..All.this.points.to.the.fact.that.to.really.understand.the.overall.effects.of.UV.on.the.skin,.it.might.be.important.to.rethink.the.strategy.of.basing.studies.on.only.one.UV.source.and.to.use.models.that.can.mimic.the.in.vivo.situation.not.only.in.short.term,.but.also.for.longer.time.

Poster P-00119

P. Scholz 1,.H.-J..Stark 1,.P..Boukamp 1

1. Deutsches.Krebsforschungszentrum,.Genetik.der.Hautkarzino-genese,.Heidelberg,.Germany.

Skin Wound Healing – a Human In Vitro Model

The.ability.to.heal.wounds.is.one.of.the.vital.features.of.skin..In.millions.of.patients.this.function.is.impaired,.leading.to.chronic.wounds.and.thus.to. dramatic. decrease. in. quality. of. life. and. increasing. healthcare. ex-penses..To.find.new.therapy.approaches.for.chronic.wounds.a.thorough.understanding.of.wound.healing.is.essential.

Here.we.present.a.novel.in.vitro.model.for.skin.wound.healing..It.is.based.on.our.new.scaffold-based. long-term.organotypic.co-culture.(scaOTC).model,.containing.human.dermal.fibroblasts. (HDF).and.normal.human.keratinocytes.(NHK).with.the.potential.for.long-term.regeneration..The.cultivation.period.of.more.than.15.weeks.is.crucial.for.two.features.of.the.model.system:.1).it.enables.the.epithelium.to.reach.a.close-to.home-ostatic.state,.from.which.it.can.be.reactivated.by.mechanical.wounding.and.2).it.allows.the.detection.of.potential.stem.cells.as.label-retaining.cells.(LRCs).after.pulse-chase.labeling.with.iodo-deoxyuridine.(IdU).The. NHK. reproducibly. re-epithelialize. the. wound. area. of. 5mm. width.within.3–7.days..However,.the.time.of.wound.coverage,.in.particular.the.early.stage.of.migration.out.of.the.unwounded.area,.depends.on.the.age.of.the.OTC..Immunofluorescence.staining.showed.sequential.expression.of.previously.described.wound-induced.and.migration-related.markers,.e.g..keratin.17,.connexin.26.and.vimentin.

Furthermore,.expression.profiling.of.the.epithelia.in.different.phases.of.wound.healing.process,.namely.initial.phase,.migratory.phase.and.hy-perproliferating.phase,.revealed.regulation.of.genes.involved.in.several.and.partially.overlapping.processes:.innate.immune.response/inflamma-tion,.epithelial-mesenchymal.transition.(EMT),.migration,.invasion.and.angiogenesis..Many.genes.that.were.transiently.upregulated.have.also.been.connected.to.tumorigenesis,.cancer.progression.and.metastasis.LRCs.could.be.observed.in.the.wound.area,.already.in.the.early.stage.of.re-epithelialization.by.anti-.IdU.immunofluorescence.staining..Impor-tantly.a.high.number.of. IdU/Ki67.double-positive.cells.were.detected.in.the.migrating.tongue,.demonstrating.substantial.reactivation.of.the.otherwise.slow-cycling.LRCs..After.the.epithelium.had.reached.suffici-ent.thickness.(phase.of.hyperproliferation),.proliferation.rates.dropped.to.levels.seen.in.the.homeostatic.situation..As.hair.follicles.are.missing,.these.findings.strongly.support.the.role.of.human.interfollicular.epider-mal.stem.cells.in.sustaining.not.only.the.general.long-term.regenera-tion,.but.to.also.strongly.contribute.to.wound.healing.in.the.OTCs.and.presumably.also.in.skin.in.situ.

While.cancer.and.wound.healing.share.obvious.similarities,. the.major.difference.seems.to.be.the.level.of.restriction.that.acts.on.cell.activa-tion..Even. in.OTCs.the.expression.of.cancer-related.genes. is.reverted.after.wound.closure,.thus.being.a.useful.tool.for.future.research.

AEK

75


Poster P-00121

A. Bort 1,.D..Krunic 1,.P..Boukamp 1

1. German.Cancer.Research.Center,.Genetics.of.Skin.Carcinogenesis,.Heidelberg,.Germany

The Influence of Poly(ADP-ribose)polymerase (PARP) on the telo-mere damage response after UV-irradiation

The.termini.of.linear.chromosomes.consist.of.a.repetitive.sequence.ter-med.a.telomere..In.humans.these.sequences.consist.of.5’-TTAGGG-3’.and.are.5.to.15.kb.in.length,.but.become.shorter.with.every.successive.cell.division..The.integrity,.and.especially.the.length,.of.telomeres.play.an.important. role. in.genomic.stability..Due. to. the. replication-associated.shortening.of.telomeres,.cells.cease.to.divide.and.become.senescenct.(the.‚Hayflick.limit‘)..Furthermore,.critically.short.telomeres.triggered.the.DNA.damage.machinery.by.activation.of.ATM,.p53,.Mre11.and.γH2AX..This. response. is.comparable. to. that. induced.by.double-stranded.DNA.breaks.[1,2].However,.the.mechanism.underlying.the.shortening.of.telo-meres.after.DNA.damage.remains.elusive.

We. found. a. significant. decrease. of. telomere. length. in. human. kerati-nocytes.and.fibroblasts.3.days.after.UVA+B.irradiation..In.addition,.we.could.show.an.increase.of.DNA.damage.foci.post.UVA+B.irradiation.in.HaCaT.keratinocytes..In.these.cells.the.shelterin.complex.protein.Pro-tection. Of. Telomere. 1. (POT1). (which. only. recognizes. single-stranded.telomeric.sequences).was.shown.to.be. increased.at.telomeres,.which.suggests.the.presence.of.UV-induced.single-stranded.breaks.within.the.telomere.sequence.

Beneke[3]. et. al.. demonstrated. another. cause. of. telomere. shortening:.pharmaceutical.inhibition/knockdown.of.poly(ADP-ribose)polymerase-1.(PARP-1). led.to.a.rapid.decrease. in. telomere. length. in.HeLa.cells..We.have.yet.to.observe.this.phenomenon.in.HaCaT.cells..However,.a.signi-ficant.increase.of.POT1.after.PARP.inhibition.and.a.significant.decline.of.another.essential.shelterin.complex.protein,.Telomeric.Repeat.binding.Factor.2.(TRF2).was.found..Taken.together,.our.data.suggest.a.role.for.PARP.in.the.shortening.of.telomeres.following.UV-damage.[1]. Di.Fagagna.FD,.Reaper.PM,.Clay-Farrace.L,.Fiegler.H,.Carr.P,.von.Zg-

linicki. T,. A. DNA. damage. checkpoint. response. in. telomere-initiated.senescence,.Nature.2003,.426,.194-8

[2].Herbig.U,.Jobling.WA,.Chen.BPC,.Chen.DJ,.Sedivy.JM,.Telomere.shorte-ning.triggers.senescence.of.human.cells.through.a.pathway.involving.ATM,.p53,.and.p21(CIP1),.but.not.p16(INK4a),.Molecular.Cell.2004,14,.501-13

[3].Beneke. S,. Cohausz. O,. Malanga. M,. Boukamp. P,. Althaus. F,. Bürkle. A,.Rapid.regulation.of.telomere.length.is.mediated.by.poly(ADP-ribose).polymerase-1,.nucleic.acids.research,.2008,.1-9

Poster P-00130

H. Schneck 1,.B..Gierke 2,.A..Nasser 3,.S..Dultz 3,.T..Fehm 1,.M..F..Templin 2,.K..Rehfeldt 3,.M..Pawlak 2,.H..Neubauer 1

1. Universitäts-Frauenklinik,.AG.Tumorprogression,.Tübingen,.Germany.2. NMI.–.Natural.and.Medical.Sciences.Institute.at.the.University.of.

Tuebingen,.Reutlingen,.Germany.3. STRATEC.Biomedical.AG,.Birkenfeld,.Germany.

CTC-Detect – Label free method to detect circulating tumor cells

Background:.Circulating.tumor.cells.(CTC).are.essential.for.establishing.metastasis..Current.detection.systems.are.based.on.enrichment.of.CTC.mediated.by.antibodies.specific.for.EpCAM.followed.by.detection.using.immune.fluorescence.microscopy..This.approach.may.miss.CTC.with.eit-her.no.or.low.expression.of.EpCAM.

Objectives:.In.order.to.capture.EpCAM-negative.or.-low.tumor.cells.we.have.tested.different.antibodies.specific.for.surface.proteins.and.extra-cellular.matrix.(ECM).proteins..Isolated.cells.were.visualized.using.label.free.ellipsometric.imaging.detection.technology.

Materials.and.Methods:.Surface.expression.of.CK8,.CD49f,.CD318,.TROP2.and.AQP5.was.verified.on.different.EpCAM-positive.and.-negative.breast.cancer.and.ovarian.cancer.cell.lines.by.immune.fluorescence.analysis.of.cytospins.and.by.flow.cytometry..Antibodies.and.ECM.proteins.(collagen.types. I.and. IV,.hyaluronic.acid,. laminin,.fibronectin,.vitronectin).were.spotted.onto.glass.slides.and.binding.of.tumor.cells.was.investigated..In.parallel.magnetic.beads.were.coated.with.antibodies. to.enrich. for.EpCAM-negative.tumor.cells.spiked.into.blood.To. image.captured.cells.by.means.of.ellipsometry.we.developed.both.hardware.and.software.housed.in.a.prototype.machine.

Results:.EpCAM-negative.tumor.cells.are.very.efficiently.bound.by.anti-bodies.specific.for.TROP2.and.CD49f..They.also.adhere.to.laminin,.col-lagen.types.I.and.IV,.hyaluronic.acid,.fibronectin.and.vitronectin..CK8,.TROP2,. CD49f. immobilised. on. magnetic. beads. are. able. to. enrich. for.EpCAM-negative.tumor.cells.spiked.into.blood.with.low.background.cell.binding..Label.free.ellipsometric.detection.technology.is.able.to.image.single.cells.with.high.resolution.

Conclusion:.By.targeting.various.cell.surface.and.ECM.proteins.as.cap-ture. molecules. enrichment. of. heterogeneous. tumor. cell. populations.can.be.improved..Label.free.cell.detection.may.be.a.versatile.alternative.to.fluorescence.microscopy.

76

AEKAEK AEKList of Invited Speakers and Main Authors

A

Abdollahi, A. (Invited Speaker Symp. S-1) Heidelberg,.Germany

Adwan, A. (Short talk 0021, Poster P-0089) Heidelberg,.Germany

Agoni, L. (Poster P-0011) Bronx.(NY),.United.States.

Assadi Gehr, M. (Poster P-0132) Allschwil,.Switzerland

Augustin, I. (Poster P-0129) Heidelberg,.Germany

B

Bauer, L. (Poster P-0098) Munich,.Germany

Beck, S. (Invited Speaker Symp. S-3) London,.United.Kingdom

Begay, V. (Poster P-0102) Berlin,.Germany

Belharazem, D. (Poster P-0077) Mannheim,.Germany

Ben-Porath, I. (Short talk 0044) Jerusalem,.Israel.

Berger, T. (Poster P-0093) Jena,.Germany

Berning, M. (Poster P-0060) Heidelberg,.Germany.

Bethge, A. (Poster P-0031) Stralsund,.Germany

Bissell, M. (Key-note Lecture) Berkeley.(CA),.United.States

Blassl, C. (Poster P-0042) Tuebingen,.Germany.

Bort, A. (Poster P-00121) Heidelberg,.Germany

Brunner, T. (Poster P-0034) Oxford,.United.Kingdom

Bühler, H. (Poster P-0068) Herne,.Germany

Busch, H. (Poster P-0113) Freiburg,.Germany

C

Chatterjee, S. (Poster P-0071) Cologne,.Germany

Chen, I.-P. (Poster P-0058) Buxtehude,.Germany

D

Dahlmann, M. (Poster P-0101) Berlin,.Germany

De Ponti, A. (Poster P-0097) Heidelberg,Germany.

De Vries, J. (Invited Speaker Symp. S-6) Nijmegen,.The.Netherlands

Dobosz, M. (Short talk 0109) Penzberg,.Germany

Domschke, C. (Poster P-0004) Heidelberg,.Germany

Dvornikov, D. (Poster P-0110) Heidelberg,.Germany

E

Eilers, M. (Invited Speaker Symp. S-5) Wuerzburg,.Germany

El-Heliebi, A. (Poster P-0126) Graz,.Austria

Engel, J. (Poster P-0083) Regensburg,.Germany.

Erler, J. (Invited Speaker Symp. S-5) Copenhagen,.Denmark

Eryilmaz, M. (Poster P-0076) Heidelberg,.Germany.

F

Friedl, P. (Invited Speaker Symp. S-1) Wuerzburg,.Germany

Fritsche, R. (Short talk 0096) Berlin,.Germany.

G

Gdynia, G. (Poster P-0105) Heidelberg,.Germany

Genssler, S. (Poster P-0039) Frankfurt/Main,.Germany

AEK

77

AEKGeorge, B. (Poster P-0112) Heidelberg,.Germany

Giehl, K. (Short talk 0045) Gießen,.Germany.

Grabe, N. (Short talk 0123, Poster P-0124) Heidelberg,.Germany.

Greinert, R. (Short talk 0057) Buxtehude,.Germany.

Greulich-Bode, K. (Poster P-0054) Heidelberg,.Germany

Gyorffy, B. (Poster P-0005) Budapest,.Hungary.

H

Harismendy, O. (Poster P-0002) La.Jolla,.United.States.

Heffeter, P. (Poster P-0107) Vienna,.Austria

Herlyn, M. (Invited Speaker Symp. S-6) Philadelphia.(PA),.United.States

Herskind, C. (Poster P-0037) Mannheim,.Germany

Herwig, R. (Invited Speaker Symp. S-4) Berlin,.Germany

Holtdorf, A. (Short talk 0090) Munich,.Germany

Honegger, A. (Poster P-0009) Heidelberg,.Germany

Höpken, U. E. (Short talk 0059) Berlin,.Germany

Hülsewig, C. (Poster P-0127) Muenster,.Germany

Hülsmann, H. (Poster P-0020) Heidelberg,.Germany.

Hynes, N. (Invited Speaker Symp. S-2) Basel,.Switzerland

J

Jakupec, M. (Poster P-0091) Vienna,.Austria

Jou, A. (Poster P-0102) Heidelberg,.Germany

Jung, V. (Poster P-0079) Homburg,.Germany

Jungwirth, U. (Poster P-0114) Vienna,.Austria

List of Invited Speakers and Main Authors

K

Kaduthanam, S. (Poster P-0023) Heidelberg,.Germany

Keller, S. (Poster P-0120) Munich,.Germany.

Kepp, O. (Invited Speaker Symp. S-5) Villejuif,.France

Koch, A. (Short talk 0047) Hannover,.Germany.

Korpis, K. (Poster P-0072) Greifswald,.Germany

Kostareli, E. (Short talk 0033) Heidelberg,.Germany

Kovacheva, M. (Poster P-0050) Heidelberg,.Germany

Krull, K. (Poster P-0052) Duesseldorf,.Germany

Kumbrink, J. (Short talk 0026) Munich,.Germany.

Kuner, R. (Poster P-00115) Heidelberg,.Germany

Kuznia, C. (Poster P-0111) Berlin,.Germany

L

Laible, M. (Poster P-0024) Heidelberg,.Germany

Lánczky, A. (Poster P-0010) Budapest,.Hungary

Legin, A. (Poster P-0073) Vienna,.Austria

Lehmann, C. (Poster P-0061) Penzberg,.Germany

Leufke, C. (Poster P-0069) Heidelberg,.Germany.

Lichter, P. (Invited Speaker Symp. S-3) Heidelberg,.Germany

M

Maier, T. (Poster P-0027) Tuebingen,.Germany

Mamlouk, S. (Poster P-0053) Dresden/Berlin,.Germany

Mejias-Luque, R. (Poster P-0062) Munich,.Germany

78

AEKAEK AEKMetzig, M. (Poster P-0029) Heidelberg,.Germany.

Mihály, Z. (Poster P-0022) Budapest,.Hungary.

Molnár, E. (Poster P-0017) Budapest,.Hungary

Morkel, M. (Short talk 0056) Berlin,.Germany

N

Neuber, S. (Poster P-0008) Marburg,.Germany

Nevaril, L. (Poster P-0085) Heidelberg,.Germany

Nguemgo Kouam, P. (Poster P-0103) Herne,.Germany

Nici, M. (Poster P-0084) Heidelberg,Germany

Nitsche, U. (Poster P-0001) Munich,.Germany

Nöske, K. (Poster P-0067) Heidelberg,.Germany

O

Orian-Rousseau, V. (Short talk 0066) Eggenstein-Leopoldshafen,.Germany.

Orthmann, A. (Poster P-0018) Berlin,.Germany

Otto, A. (Poster P-0095) Garching,.Germany

P

Panke, C. (Poster P-0055) Penzberg,.Germany

Pantel, K. (Invited Speaker Symp. S-1) Hamburg,.Germany

Pastula, A. (Poster P-0128) Munich,.Germany

Pavez-Lorié, E. (Poster P-00117) Heidelberg,.Germany

Pénzváltó, Z. (Poster P-0032) Budapest,.Hungary.

Pervaiz, A. (Poster P-0082) Heidelberg,.Germany


Peterziel, H. (Poster P-0063) Heidelberg,.Germany

Pietenpol, J. (Invited Speaker Symp. S-2) Nashville.(TN),.United.States

Pisarenko, I. (Poster P-0104) Herne,.Germany

Pusterla, T. (Poster P-0086) Heidelberg,.Germany

R

Rak, J. (Invited Speaker Symp. S-5) Montreal,.Canada

Redmer, T. (Poster P-0116) Berlin,.Germany

Richter, D. (Poster P-0051) Frankfurt/Main,.Germany

Riffkin, H. (Poster P-0049) Essen,.Germany.

Rivera, M. (Poster P-0106) Berlin,.Germany

Roos, W. (Poster P-0087) Mainz,.Germany

Rösch, S. (Poster P-0089) Heidelberg,.Germany

Rubin, M. (Invited Speaker Symp. S-5) New.York.(NY),.United.States

S

Saalfrank, A. (Poster P-0131) Freising,.Germany

Saupe, F. (Poster P-0100) Strasbourg,.France

Schäfer, R. (Invited Speaker Symp. S-4) Berlin,.Germany.

Schmidt, A. (Poster P-0008) Marburg,.Germany

Schneck, H. ( Poster P-00130) Tuebingen,.Germany

Schulz, P. (Poster P-00119) Heidelberg,.Germany

Schutz, S. (Poster P-0035) Tuebingen,.Germany

Schwenzer, A. (Poster P-0100)

Strasbourg,.France

AEK

79

AEKSharma, D. (Poster P-0025) Essen,.Germany

Singer, S. (Poster P-0122) Heidelberg,.Germany

Specker, E. (Poster P-0080) Heidelberg,.Germany

Subtil, F. S. B. (Poster P-0007) Marburg,.Germany

T

Taly, V. (Poster P-0003) Paris.Cedex.06,.France

Taylor, N. (Poster P-0036) London,.United.Kingdom.

Tham, M. (Poster P-0043) Heidelberg,.Germany

Theobald, S. (Poster P-0081) Heidelberg,.Germany

Trondl, R. (Poster P-0108) Vienna,.Austria

U

Ullrich, M. (Short talk 0016) Dresden,.Germany

V

V. Manstein, V. (Poster P-0094) Frankfurt/Main,.Germany

Vafaizadeh, V. (Poster P-0099) Frankfurt/Main,.Germany

Vannier, C. (Poster P-0064) Freiburg,.Germany

W

Walther, W. (Short talk 0028) Berlin,.Germany

Warta, R. (Poster P-0070) Heidelberg,.Germany.

Wege, A. K. (Short talk 0006) Regensburg,.Germany.

Wegert, J. (Poster P-0088) Wuerzburg,.Germany

Weinberg, B. (Invited Speaker Symp. S-3) Cambridge.(MA),.United.States


Weininger, D. (Poster P-0055) Penzberg,.Germany

Weissinger, D. (Poster P-0075) Heidelberg,.Germany

Werner, S. (Invited Speaker Symp. S-5) Zurich,.Switzerland

Wiedemann, J. (Poster P-0048) Darmstadt,.Germany

Winkler, F. (Invited Speaker Symp. S-1) Heidelberg,.Germany

Winkler, J. (Poster P-0122) Heidelberg,.Germany

Wuttig, D. (Poster P-0040) Heidelberg,.Germany

Y

Yang, C. M. (Poster P-0041) Frankfurt/Main,.Germany

Z

Zeller, E. (Poster P-0019) Tuebingen,.Germany

Zhang, C. (Poster P-0038) Frankfurt/Main,.Germany

Zieger, K. (Poster P-0030) Berlin,.Germany

80

AEKAEK AEKNotes

AEK

81

AEKNotes

82

AEKAEKNotes

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