inhibition of the v-atpase by archazolid a: a new strategy ......small molecule therapeutics...

Small Molecule Therapeutics

Inhibition of the V-ATPase by Archazolid A:A New Strategy to Inhibit EMTHenriette Merk1, Philipp Messer2, Maximilian A. Ardelt1, Don C. Lamb2,Stefan Zahler1, Rolf M€uller3, Angelika M. Vollmar1, and Johanna Pachmayr1

Abstract

Epithelial–mesenchymal transition (EMT) induces tumor-initiating cells (TIC), which account for tumor recurrence,metastasis, and therapeutic resistance. Strategies to interferewith EMT are rare but urgently needed to improve cancertherapy. By using the myxobacterial natural compound Arch-azolid A as a tool, we elucidate the V-ATPase, a multimericproton pump that regulates lysosomal acidification, as a crucialplayer in EMT and identify the inhibition of V-ATPase byArchazolid A as a promising strategy to block EMT. Geneticknockdown and pharmacologic inhibition of the V-ATPase by

Archazolid A interfere with the EMT process and inhibit TICgeneration, as shown by a reduced formation of mammo-spheres and decreased cell motility. As an underlying mecha-nism, V-ATPase inhibition by Archazolid A disturbs the turn-over of E-cadherin: Archazolid abrogates E-cadherin loss duringEMT by interfering with its internalization and recycling. Ourstudy elucidates V-ATPase as essential player in EMT by regu-lating E-cadherin turnover. Archazolid A is suggested as apromising therapeutic agent to block EMT and the generationof TICs. Mol Cancer Ther; 16(11); 2329–39. �2017 AACR.

IntroductionTumor initiating cells (TIC) have tumor-initiating ability and

show increased resistance to chemotherapeutics and, therefore,account for metastasis and tumor recurrence. Therefore, TICslimit therapeutic success and represent major problems incancer therapy (1–3). TIC properties such as self-renewal andinvasiveness have been closely associated with epithelial–mes-enchymal transition (EMT), which is a biological process thatconfers a mesenchymal stem-like phenotype to cells: cancercells become highly malignant and invasive, acquire self-renew-al capacity, and develop elevated resistance toward therapeutics(4–6). Thus, EMT plays a pivotal role in cancer progression,relapse, and metastasis (7).

A hallmark of EMT is the change of structural proteins thatmaintain the cytoskeleton and cell–cell adhesions. Especially theloss of the transmembrane glycoprotein E-cadherin represents acrucial event during EMT as it results in breakdown of cell–cellcontacts and accounts for increasedmotility ofmesenchymal cells(8–10).

E-cadherin is controlled both by transcriptional processesand by endolysosomal internalization and degradation. Tran-scriptional repression of E-cadherin is mediated by transcrip-tion factors such as Snail1/2, ZEB1/2, Slug, and Twist1 (11–16). Internalization of E-cadherin from the cell surface intoendosomes and its recycling back to the surface or its degra-dation in lysosomes is a dynamic process and ensures theformation of adherens junctions and thus cell adhesion(17, 18). The loss of E-cadherin is balanced by the increasedexpression of mesenchymal proteins such as N-cadherin,vimentin, and fibronectin.

Despite or maybe just because of the tremendous contri-bution of cancer stem cells (CSC) and EMT to metastasis,tumor recurrence, and therapeutic resistance, strategies forinterfering with the EMT process are rare. Currently, onlycompounds that interfere with TGFb such as galunisertib andfresolimumab get evaluated in clinical trials for glioblastoma,hepatocellular carcinoma, pancreatic cancer, and melanoma(19, 20). However, the actual evidence for EMT in clinicalspecimens is limited and there are no solid studies linkingEMT to treatment outcomes or patient survival. Thus, findingnovel strategies for targeting the EMT is essential and mightcontribute to find new strategies that can help to improvecancer therapy.

We hypothesized that Vacuolar Hþ-ATPase (V-ATPase) is apromising target to address the EMT. V-ATPase is heavily involvedin the regulation of trafficking by acidifying endosomes andlysosomes. By regulating endolysosomal processes, the V-ATPaseaffects tumor cell hallmarks such as proliferation, cell death, andmotility and pharmacologic inhibition of the V-ATPase hasshown potent anti-cancer and anti-metastatic effects in vitro andin vivo (21–26).

Our goal was to characterize a potential function of theV-ATPase in EMT and to analyze the underlying mechanismin order to judge the inhibition of V-ATPase as a promisingnew strategy to address EMT.

1Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-Uni-versity, Munich, Germany. 2Department of Chemistry, Center for Nanoscience(CeNS), Center for Integrated Protein ScienceMunich (CIPSM)andNanosystemsInitiative Munich (NIM), Ludwig-Maximilians-University, Munich, Germany.3Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centrefor Infection Research and Department of Pharmaceutical Biotechnology, Saar-land University, Saarbr€ucken, Germany.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Johanna Pachmayr, Ludwig-Maximilians-University ofMunich, Butenandtstr. 5-13, Munich 81377, Germany. Phone: 49-89-2180-77175;Fax: 49-89-2180-77170; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0129

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

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on May 13, 2021. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 3, 2017; DOI: 10.1158/1535-7163.MCT-17-0129

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Materials and MethodsCompounds and reagents

Isopropyl-b-D-thiogalactopyranosid (IPTG), 4-hydroxytamox-ifen (4-OH-TX), and poly-(2-hydroxyethyl methacrylate, poly-HEMA)were purchased fromSigma-Aldrich. TGFbwas purchasedfrom PeproTech GmbH. Archazolid A and Concanamycin (27)are myxobacterial natural compound provided by Prof. RolfM€uller (Helmholtz Centre for Infection Research andDepartmentof Pharmaceutical Biotechnology, Saarland University, Saar-brücken, Germany).

Cell cultureImmortalized humanmammary epithelial (HMLE) cells trans-

duced with Twist1-ER were kindly provided by Dr. ChristinaScheel (Helmholtz CentreMunich, 2013) and described by Casasand colleagues (14). HMLE Twist1-ER cells were cultivated inMammary Epithelial Cell Growth Medium (MECGM, Ready-to-use, PromoCell GmbH) supplemented with penicillin/strepto-mycin (PAA Laboratories), and 10 mg/mL blasticidin (Gibco) at37�C and 5% CO2.

Induction of EMT by 4-OH-TXThe fusion protein of Twist1 and the modified hormone-

binding domain of the estrogen receptor (Twist1-ER) was acti-vated by treatment with 4-hydroxytamoxifen (4-OH-TX,20 nmol/L) for 10 days. Before EMT induction, epithelial HMLETwist1-ER cells were pretreated with Archazolid A (1 nmol/L or10 nmol/L) for 24 hours and during EMTArchazolid A (0.1 nmol/L) was added. Mesenchymal HMLE Twist1-ER cells were treatedwith Archazolid A (1 or 10 nmol/L) for 24 hours.

Induction of EMT by TGFbEpithelial HMLE Twist1-ER cells were treated with TGFb

(5 ng/mL) for 12 days. Before EMT induction, epithelial HMLETwist1-ER cells were pretreated with Archazolid A (1 nmol/Lor 10 nmol/L) for 24 hours and during EMT Archazolid A (0.1nmol/L) was added.

Transduction of cellsFor the lentiviral transduction of HMLE Twist1-ER cells with V-

ATPase shRNA MISSION Lentiviral Transduction Particles[Vector: pLKO.1-puro-IPTG 3xLacO; SHC332V-1EA; Clone ID:(1) TRCN0000029559, (3) TRCN0000029561, Sigma-Aldrich]and MISSION 3xLacO Inducible Non-Target shRNA ControlTransduction Particles (SHC332V, Sigma-Aldrich) as a controlwere used according to the manufacturer's protocol. HMLETwist1-ER cells were transduced with a multiplicity of infection(MOI) of one. Successfully transduced cells were selected byadding 0.5 mg/mL puromycin to the medium and puromycinwas also added to the medium during cultivation. For shRNAexpression 1 mmol/L IPTG was added for 4 days. In order toensure V-ATPase downregulation, 1 mmol/L IPTG was constantlyadded.

Cell viability assayCell viability was measured according to Nicoletti and collea-

gues (28) by which the percentage of apoptotic nuclei afterpropidium iodide (PI; Sigma-Aldrich) staining ismeasured. Brief-ly, cells were treated as indicated, harvested, and PI staining was

performed. Subdiploid DNA content was determined by flowcytometry (Becton Dickinson) and considered as apoptotic.

Mammosphere assayAssays were performed as previously described by Dontu

and colleagues with modifications (29). In brief, cell viabilitywas measured by Vi-CELL XR (Beckman Coulter) and 20,000viable cells/well were seeded in ultra-low 12-well attachmentplates, coated with poly-(2-hydroxyethyl methacrylate; poly-HEMA) in MEGM Mammary Epithelial Cell Growth MediumBullet Kit (Lonza) containing B27 (Gibco), 20 ng/mL EGF(PeproTech), 10 ng/mL bFGF (Peprotech) and 1 % methyl-cellulose (Sigma-Aldrich). For secondary mammosphere for-mation, primary spheres were dissociated by Trypsin/EDTAand reseeded at 20,000 viable cells/well. After 7 days, eachwell was imaged by confocal microscopy (LSM 510 Meta,Zeiss), and quantity and size of mammospheres were analyzedby using ImageJ.

Boyden chamber assayA total of 1� 105 cells were suspended inMECGMwithout FCS

and added on top of the Transwell permeable supports (CorningIncorporated). MECGM (negative control, NC) or MECGM with10% FCS (positive control, PC) was added to the bottom of themembrane. Boyden chambers were incubated at 37�C, 5% CO2

for 6hours.Migrated cellswere stainedwith crystal violet. Cells onthe upper side were removed with cotton swabs. Migrated cellswere photographed using Zeiss Axiovert 25 microscope (Zeiss)and Canon 450D camera (Canon), counted and normalized tocontrol cells.

ImmunoblottingImmunoblotting was performed as previously described (30).

The following antibodies were used: E-cadherin, vimentin, clau-din-1, ATP6V0C (NBP1-59654, Novus), fibronectin (Santa CruzBiotechnology), HRP-goat anti-rabbit (Bio-Rad LaboratoriesGmbH), HRP-goat anti-mouse (Santa Cruz Biotechnology). Pro-teins were detected by using the ChemiDoc Touch ImagingSystem (Bio-Rad Laboratories GmbH).

LysoTracker stainingCells were treated as described in figure legends and seeded

on m-slides from ibidi (ibidi GmbH), stained for 60minutes with100 nmol/L LysoTracker dye (Moleculare Probes) and Hoechst33342 (5 mg/mL; Sigma-Aldrich) at 37�C and imaged by confocalmicroscopy without fixation.

Quantitative real-time PCR analysisTotal mRNA was isolated from cell culture samples using

Qiagen RNeasy Mini Kit (Qiagen). Reverse transcription wasperformed with the High-Capacity cDNA Reverse TranscriptionKit (Applied Biosystems) according to themanufacturer's instruc-tions. Real-timePCRwasperformedwith the7300Real-TimePCRSystem (Applied Biosystems). SYBR Green Mix I (Applied Bio-systems) was used for: E-cadherin (forward: 50-CAG CAC GTACAC AGC CCT AA-30, reverse: 50-AAG ATA CCG GGG GAC ACTCA-30), vimentin (forward: 50-CGG CGG GAC AGC AGG-30,reverse: 50-TCG TTG GTT AGC TGG TCC AC-30), N-cadherin(forward: 50-ACA GTG GCC ACC TAC AAA GG-30, reverse: 50-CCG AGA TGG GGT TGA TAA TG-30), fibronectin (50-GCT GACAGA GAA GAT TCC CGA-30, reverse: 50-CCA GGG TGA TGC TTG

Merk et al.

Mol Cancer Ther; 16(11) November 2017 Molecular Cancer Therapeutics2330




GAG AA-30) primer (Metabion) and TaqMan Universal PCRMastermix (Life Technologies Corporation) was used for Taqmangene expression assay for V-ATPase Hs00798308_sH (AppliedBiosystems). GAPDH or Actin were used as housekeeper.Obtained average CT values of target genes were normalized tocontrol as DCT. Changes in expression levels were shown as foldexpression (2DDCT) calculated by the DDCT method (31).

Confocal microscopyCells were treated as indicated, fixed with 4% PFA for 10

minutes, permeabilized with 0.2% Triton X-100, and blockedwith 0.2% BSA in PBS. Primary antibodies were diluted in 0.2%BSA and incubated overnight at 4�C. Secondary antibodies werealso diluted in 0.2% BSA and incubated for 45 minutes at roomtemperature. Subsequently, cells were mounted with FluorSaveReagent and analyzed with Leica-SP8 confocal microscope(LeicaMicrosystems). The following antibodies or dyeswereused:E-cadherin, vimentin, N-cadherin (Cell Signaling), E-cadherin(HECD1, Invitrogen), Lamp-1 (Developmental Studies Hybrid-oma Bank), Hoechst 33342 (5 mg/mL; Sigma-Aldrich), AlexaFluor 488 goat anti-rabbit, Alexa Fluor 546 (Invitrogen). Imageswere either taken using Leica-SP8 confocal microscope or ZeissLSM 510 Meta confocal laser microscope.

E-cadherin internalization assayHMLE Twist1-ER cells were cultivated as described for 10 days.

Onday 10, cells were reseeded into ibidim-slides, washedwith ice-cold PBS, and slides were kept at 4�C for 10 minutes. In thefollowing, the samples were incubated with anti-E-cadherin anti-body (2 mg/mL; HECD1, Invitrogen) for 45 minutes at 4�C. Afterwashing steps with PBS, MECGM was added and incubated forindicated time points (0, 5, 10, and 15 minutes) at 37�C. Cellswere fixed with 4% paraformaldehyde (PFA) for 15 minutes atroom temperature. After incubation with secondary antibody(1:400) and Hoechst 33342 (5 mg/mL) for 30 minutes, cells werewashed, mounted with FluorSave Reagent (Merck) and confocalmicroscopy was performed.

E-cadherin vesicle trackingNon-targeting and V-ATPase shRNA cells were treated with

IPTG as described above. Cells were transfected with E-cad-herin-eGFP (addgene 28009) and Rab5-RFP (addgene 14437)by using Dharmafect1 (Dharmacon) according to the manufac-turer's instructions. Life-cell imagingwas performed using a Leica-SP8 confocal microscope. Cells at 24 to 48hours posttransfectionwere measured for 500 seconds. Single vesicles were detected ineach frame individually and then connected between frames with

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V-ATPase knockdown decreases mammosphereformation. A, V-ATPase downregulation is displayed.The expression of V-ATPase subunit c in nontargeting(nt) and V-ATPase shRNA (V-ATPase shRNA 1 andV-ATPase shRNA3) HMLETwist1-ER cells after inductionof shRNA by IPTG (1 mmol/L, 96 hours) is shown.One-wayANOVA, TukeyMultiple Comparison Test, SEM;� ,P<0.05,n¼ 3.B,Acidic endolysosomal compartmentsare shown by LysoTracker staining (red) in HMLE Twist1-ER nontargeting and V-ATPase shRNA HMLE cells with/without induction by IPTG (1 mmol/L). Nuclei werestained with Hoechst 33342 (5 mg/mL, blue). Scale bars,25 mm (n ¼ 3). C, Mammosphere assay of nontargeting(nt shRNA) and V-ATPase shRNA HMLE cells (V-ATPaseshRNA 1 and V-ATPase shRNA 3) induced by IPTGand before and after EMT induction by 4-OH-TX(20 nmol/L, 10 days) are shown. Scale bar, 100 mm. Thegraph depicts the number of mammospheres counted.One-wayANOVA, TukeyMultiple Comparison Test, SEM;� , P < 0.05; n ¼ 3.

Inhibition of EMT by Archazolid

www.aacrjournals.org Mol Cancer Ther; 16(11) November 2017 2331




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Archazolid A treatment during EMT decreases mammosphere formation and migration. A,Mammosphere formation of HMLE Twist1-ER cells without (co) and withEMT induction by 4-OH-TX (20 nmol/L, 10 days) and with/without treatment with Archazolid A in indicated concentrations for 24 hours before EMT induction by 4-OH-TX is shown. During EMT induction, Archazolid A (0.1 nmol/L) was added. Scale bar, 100 mm. Graphs depict mammosphere formation count (>50 mm) and sizenormalized to 4-OH-TX–treated cells. One-way ANOVA, Tukey Multiple Comparison Test, SEM, � , P < 0.05, n¼ 5).B,Mammosphere formationwith (co) andwithoutEMT induction by TGFb (5 ng/mL, 12 days) andwith/without Archazolid A treatment at 1 and 10 nmol/L concentrations for 24 hours before EMT induction are shown.During EMT induction, Archazolid A (0.1 nmol/L) was added. Scale bar, 100 mm. Graph depicts mammosphere formation count normalized to TGFb–treated cells.One-way ANOVA, Tukey Multiple Comparison Test, SEM, � , P < 0.05, ns, not significant, n¼ 3. C, Transwell migration of HMLE Twist1-ER cells with (co) and withoutEMT induction by 4-OH-TX (20 nmol/L, 10 days) and with/without Archazolid A treatment at 1 nmol/L and 10 nmol/L concentrations for 24 hours beforeEMT induction is shown. Images display migrated HMLE Twist-ER cells stained with crystal violet (purple). Scale bar, 100 mm. Graph shows the number of migratedcells after 6 hours normalized to 4-OH-TX–treated cells. One-way ANOVA, Tukey Multiple Comparison Test, SEM; � , P < 0.05; n¼ 4.D, Propidium iodide (PI, red) andHoechst 33342 (blue) staining of mammospheres of HMLE Twist1-ER cells without (co) and with EMT induction by 4-OH-TX (20 nmol/L, 10 days) and with/withouttreatment with Archazolid A in indicated concentrations for 24 hours before EMT induction by 4-OH-TX is shown. During EMT induction, Archazolid A (0.1 nmol/L)was added. Scale bar, 100 mm. E, Second round mammosphere formation assay is shown. For secondary mammosphere formation, primary mammospheres ofHMLE Twist1-ER cells without (co) and with EMT induction by 4-OH-TX (20 nmol/L, 10 days) and with/without treatment with Archazolid A in indicatedconcentrations for 24 hours before EMT induction by 4-OH-TX (as described inA)were dissociated by T/E into single cells and reseeded at 20,000 viable cells/well inpoly-HEMAcoated plates for 7 days (37�C, 5%CO2). During first and second rounds ofmammosphere formation, Archazolid A (0.1 nmol/L)was added. Scale bar, 100mm. The graphs depict mammosphere formation count (>50 mm) and size, which were normalized to 4-OH-TX–treated cells. One-way ANOVA, Tukey MultipleComparison Test, SEM; �, P < 0.05; n ¼ 3.

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Archazolid A inhibitsmigration andmammosphere formation ofmesenchymal HMLE cells.A, Transwellmigration of fully transitionedmesenchymal HMLE Twist1-ERcells prestimulatedwith/without Archazolid A (1 or 10 nmol/L) for 24 hours determined by Boyden chamber experiments is shown. Scale bar, 100 mm. Graph displaysthe number ofmigrated cells after 6 hours related to positive control (PC). For negative control (NC) onlymediumwithout FCSwas added. One-wayANOVA, Tukey'sMultiple Comparison Test, SEM; �, P < 0.05, n¼ 3). B, Mammosphere formation of fully transitioned mesenchymal HMLE Twist1-ER cells with/without 1 nmol/LArchazolid A treatment for 24 hours is shown. Scale bar, 100 mm. The graph displays the mammosphere count related to untreated control (Student t test, SEM;� ,P <0.05, n¼ 3).C,Mammosphere formation ofMDA-MB-231 cellswith/without Archazolid A (1 nmol/L or 10 nmol/L) treatment for 24 hours is shown. Scale bar, 100mm. The graph displays the mammosphere count and size related to untreated control (n¼ 3). D, Mammosphere formation of MDA-MB-231 cells with/withoutConcanamycin (5 nmol/L or 10 nmol/L) treatment for 24 hours is shown. Scale bar, 100 mm. The graph displays themammosphere count and size related to untreatedcontrol (n¼ 3).






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V-ATPase inhibition abrogates EMT. A, Immunoblots of HMLE Twist1-ER cells before and after EMT induction by 4-OH-TX (20 nmol/L, 10 days) and with/withoutArchazolid A treatment at 1 nmol/L and 10 nmol/L concentrations for 24 hours before EMT induction for E-cadherin, claudin, b-catenin, fibronectin, vimentin,and b-tubulin (loading control) are shown (n¼ 3). During EMT induction, Archazolid A (0.1 nmol/L) was added. B, Immunoblots of HMLE Twist1-ER cellsbefore and after EMT induction by TGFb (5 ng/mL, 12 days) and with/without Archazolid A treatment at the indicated concentrations for 24 hours before EMTinduction for E-cadherin and vimentin are shown. A prestained gel served as a loading control (n¼ 3). (Continued on the following page.)

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a minimum pair-wise distance algorithm. Mean squared displa-cements (MSD)were calculated for each track,fitted, and averagedwith an active transport model (Eq. A). For the fit, the first half ofthe data points were used.

r2̂ >ð Þ� � ¼ 4D> þ V � >ð Þ½ �2̂ ðAÞ

Statistical analysisAll experiments were performed at least three times unless

otherwise indicated in the figure legend. Statistical analysis wasperformed using GraphPad Prism software version 5.04. Graphdata represent means � SEM. One-way ANOVA/Tukey multiplecomparison test and individual Student t tests were conducted. Pvalues less than 0.05 were considered statistically significant.

ResultsInhibition of V-ATPase abrogates mammosphere formation

HMLEs overexpressing Twist1 fused to the ligand bindingdomain of a mutated estrogen receptor (ER) served as a modelto study EMT (14, 32). Tamoxifen-mediated Twist activation inHMLE cells induced a mesenchymal phenotype and EMT (Sup-plementary Fig. S1A and S1B) and resulted in the formation ofmammospheres (Supplementary Fig. S1C; ref. 32). Besides that,as other way to induce EMT, TGFb treatment of HMLE cells wasapplied.

To investigate the function of V-ATPase in mammosphereformation, we generated inducible V-ATPase knockdownHMLE cells by stable transduction of HMLE cells with an IPTGinducible V-ATPase V0 subunit c shRNA vector. IPTG treatmentof V-ATPase shRNA HMLE cells induced V-ATPase downregula-tion as shown by RT-PCR (Fig. 1A). In addition, LysoTrackerstaining showed decreased acidification of the lysosomes of V-ATPase shRNA HMLE cells upon IPTG treatment (Fig. 1B). Ofnote, the mammosphere forming potential which was inducedby Tamoxifen-mediated Twist activation was inhibited in theIPTG-treated V-ATPase shRNA knockdown HMLE cells com-pared with IPTG-treated HMLE cells transduced with non-targeting shRNA (Fig. 1C).

To judge the V-ATPase as a pharmacologically accessible targetin mammosphere formation, epithelial HMLE cells were pre-treated with Archazolid A (1 nmol/L and 10 nmol/L, 24 hours)before EMT was induced by Tamoxifen-driven Twist activation orTGFb. During EMT induction (10 days), Archazolid Awas presentat a very low concentration (0.1 nmol/L) which did not affect cellviability or proliferation (25, 33, 34) and Supplementary Fig. S2).During the assays subsequent to EMT induction, Archazolid wasremoved, in order to ensure that specifically its influence on EMTwas analyzed. Diminished acidification of lysosomes confirmedV-ATPase inhibition by Archazolid A (Supplementary Fig. S3). Of

note, Archazolid A treatment ofHMLE cells in their epithelial statedecreased the formation mammospheres induced by tamoxifen-driven Twist activation as well as TGFb (Fig. 2A and B). Moreover,Archazolid A treatment of epithelial cells before EMT inductionreduced cellmigration (Fig. 2C). This was not due to increased celldeath as shown by Nicoletti assays (Supplementary Fig. S2A) aswell as by staining ofmammospheres with propidium iodide (PI)that indicates dead single cells but living mammosphere-formingcells regardless fromArchazolid treatment (Fig. 2D). Importantly,as shown by repeated mammosphere assays, the effect of Arch-azolid A was sustained (Fig. 2E). This set of data shows thatArchazolid A inhibits two crucial functional characteristics oftumor-initiating cells, namely migration and self-renewal. AsArchazolid A was applied on epithelial cells before EMT induc-tion, a function of V-ATPase in the EMT process is suggested.

So far, our data showed that mammosphere formation andmigration are reduced when inhibition of V-ATPase is applied inepithelial cells before EMT. Besides interfering with EMT, it is ofutmost clinical importance, to target already existing, poorlyaccessible mesenchymal cells. Therefore, Archazolid A wasapplied to fully transitioned mesenchymal HMLE cells that hadalready undergone EMT. In fact, Archazolid A inhibited bothmigration (Fig. 3A) and mammosphere formation (Fig. 3B) offully transitioned mesenchymal HMLE cells. An apoptotic effectcould be excluded (Supplementary Fig. S2B). Moreover, in orderto verify that the effect of V-ATPase inhibition is not cell typespecific, we used basal triple-negative MDA-MB-231 breast cancercells. Archazolid was shown previously to reduce MDA-MB-231lysosome acidification (24). In fact, V-ATPase inhibition both byArchazolid A (Fig. 3C) as well as by Concanamycin (Fig. 3D)reduced mammosphere formation of MDA-MB-231 cells, verify-ing that V-ATPase inhibition even is effective in cells with a highEMT/CSC phenotype.

V-ATPase inhibition abrogates EMTTo verify our hypothesis that V-ATPase inhibition interferes

with EMT, expression of classical EMT markers was analyzed.Changes of self-renewal markers Sox2 and OCT-4—that onlymight be detectable after cell sorting—could not be observed inwhole-cell populations (Supplementary Fig. S4). In fact, EMTinduction by Tamoxifen-driven Twist activation or TGFbdecreased the expression of epithelial markers like E-cadherin,b-catenin, or claudin-1 and increased the expression of mesen-chymalmarkers like vimentin,fibronectin, andN-cadherin (Fig. 4;Supplementary Fig. S4). Whereas mRNA expression was notmodified by V-ATPase inhibition as shown by RT-PCR (Supple-mentary Fig. S5), Archazolid A strongly influenced the proteinlevel of EMT markers: levels of the epithelial markers E-cadherin,b-catenin and claudin-1 were increased by Archazolid A whereasthe expression of the mesenchymal markers fibronectin andvimentin was reduced as shown by Western blot analysis

(Continued.) During EMT induction, Archazolid A (0.1 nmol/L) was added. C, Immunostainings of HMLE Twist1-ER cells before and after EMT induction by 4-OH-TX(20 nmol/L, 10 days) and with/without Archazolid A treatment at 1 nmol/L and 10 nmol/L concentrations for 24 hours before EMT induction for E-cadherin,b-catenin, vimentin, N-cadherin (green), andHoechst 33342 (nuclei, blue, 5mg/mL) are shown. DuringEMT induction, ArchazolidA (0.1 nmol/L)was added. Scale bar,10 mm (n¼ 3).D, Immunostainings of HMLE Twist1-ER cells before and after EMT induction by TGFb (5 ng/mL, 12 days) and with/without Archazolid A treatment at 1nmol/L and 10 nmol/L concentrations for 24 hours before EMT induction for E-cadherin, b-catenin, vimentin, N-cadherin (green), and Hoechst 33342 (nuclei,blue, 5mg/mL) are shown. During EMT induction, Archazolid A (0.1 nmol/L)was added. Scale bar, 10mm(n¼ 2).E, Immunostainings of IPTG-induced nontargeting (ntshRNA) and V-ATPase knockdown (V-ATPase shRNA_1 and V-ATPase shRNA_3) HMLE cells before and after EMT induction by 4-OH-TX (20 nmol/L,10 days) probed for b-catenin, vimentin (green), and Hoechst 33342 (nuclei, blue, 5 mg/mL) are shown. Scale bars, 10 mm (n¼ 3). F, Immunostainings of MDA-MB-231cells treated with/without Archazolid (0.1 nmol/L or 10 nmol/L as indicated) probed for E-cadherin (green), and Hoechst 33342 (nuclei, blue, 5 mg/mL) is shown.Scale bars, 10 mm (n¼ 3).






(Fig. 4A and B). These results were confirmed by immunostain-ings: the EMT-driven loss of the epithelialmarkers E-cadherin andb-catenin and the increase of the mesenchymal markers vimentinandN-cadherinwere prevented both byArchazolid A (Fig. 4C andD) and V-ATPase knockdown (Fig. 4E).

As V-ATPase inhibition interfered with mammosphere forma-tion of fully transitioned mesenchymal cells with EMT/CSCphenotype as well, we checked its effect on E-cadherin as a centralEMTmarker inMDA-MB-231 cells. Cytoplasmic E-cadherin local-ization was not changed by Archazolid in these cells (Fig. 4F),suggesting that different targets of V-ATPase are addressed byArchazolid in these cells.

In summary, this set of data suggests that V-ATPase inhibitionpreserved an epithelial phenotype and interfered with EMT.

V-ATPase inhibition disturbs E-cadherin recyclingIn order to elucidate the underlying mechanism of V-ATPase in

EMT, we focused on E-cadherin as the endosomal trafficking andrecycling of E-cadherin represents a crucial process during EMT(35).We investigatedwhether V-ATPase inhibition influenced thelocalization of E-cadherin to endosomes by using Rab5 as anendosomal marker. In epithelial cells, E-cadherin was localizedmainly to the membrane (Fig. 5A, left). In cells that had under-gone EMT due to Tamoxifen-driven Twist activation, E-cadherinwas diminished at cell–cell contacts and localized intracellularly(Fig. 5A, middle). Prevention of EMT by IPTG-induced V-ATPaseknockdown resulted in localization of E-cadherin at the mem-brane and in enlarged Rab5-positive endosomes (Fig. 5A, right),suggesting that Archazolid A inhibits E-cadherin internalizationand degradation. To get a more detailed insight into the impactof V-ATPase in E-cadherin recycling during EMT, which representsa highly dynamic process, surface-E-cadherin was analyzedby internalization assays. In epithelial HMLE Twist1-ER cells,E-cadherin was internalized and recycled back to the membrane(Fig. 5B, left). In mesenchymal cells that had undergone EMT,surface E-cadherin was internalized and the total concentrationof the protein was decreased (Fig. 5B, middle). Archazolid Atreatment before EMT induction inhibited E-cadherin internali-zation and resulted in accumulation of E-cadherin at the cellsurface (Fig. 5B, right). Thus, cells treated with Archazolid Amaintained epithelial characteristics. This was confirmed bylive-cell–imaging experiments and subsequent single-particletracking of E-cadherin containing vesicles. The average meansquared displacement (MSD) analysis of tracked E-Cadherincontaining vesicles shows active endosomal transport processes.The velocities of E-cadherin–containing Rab5-positive vesicleswere highly increased in cells that had undergone EMT (Fig. 6Band D; Supplementary Fig. S6) compared with epithelial cellswithout EMT induction (Fig. 6A and D; Supplementary Fig. S6).The velocities of E-cadherin containing vesicles were decreasedin V-ATPase knockdown cells (Fig. 6C and D; SupplementaryFig. S6). In summary, our data suggest that Archazolid A-mediatedinhibition of EMT is linked with a disturbed internalization andincreased accumulation of E-cadherin at the cell surface.

DiscussionThis study introduces V-ATPase inhibition as a promising new

strategy to interfere with EMT in cancer. During recent years,important functions of V-ATPase in cancer have been elucidated,like an implication of V-ATPase in tumor cell death, invasion, and

metastasis (23–25, 34, 36). As a consequence, V-ATPase hasemerged as a promising target for cancer therapy. However, onlyfew reports point to a function of V-ATPase in TICs and the modeof action of V-ATPase to contribute to TIC regulation has not been

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V-ATPase inhibition disturbs E-cadherin recycling. A, Immunostainings of E-cadherin (green) and Rab5 (red) in HMLE Twist1-ER cells before and after EMTinduction by 4-OH-TX (20 nmol/L, 10 days) in IPTG-induced nontargetingshRNA (nt shRNA) and V-ATPase knockdown cells (V-ATPase shRNA_1) areshown. Colocalization is indicated in orange. Nuclei were stained with Hoechst33342 (blue). Scale bars, 25 mm (n¼ 2). B, Surface E-cadherin (green) recyclingof HMLE Twist1-ER cells with/without Archazolid A (Archa A) pretreatment(1 nmol/L) for 24 hours and without (epithelial, epi) and with subsequent EMTinduction by 4-OH-TX (20 nmol/L, 10 days) is shown. During EMT induction by4-OH-TX, Archazolid A (0.1 nmol/L) was added. Subsequent to surface E-cadherin staining, cells were fixed after 0, 5, 10, and 20 minutes of recycling.Nuclei were stained with Hoechst 33342 (blue, 5 mg/mL; n¼ 3).

Merk et al.





elucidated until now. Inhibition of V-ATPase has been shown toeradicate TICs in rhabdomyosarcoma (37). Furthermore, knock-down as well as pharmacologic V-ATPase inhibition by bafilo-mycin A1, a first-generation V-ATPase inhibitor, diminished theneurosphere-forming ability of glioblastoma and suppressed theexpression of stem cell markers (38). In line with this study, ourresults revealed a requirement of V-ATPase for mammosphereformation of mesenchymal HMLE cells, which was blocked byArchazolid A-mediated pharmacologic inhibition of V-ATPase.Moreover, investigating the underlying mechanism of V-ATPaseinmammosphere formation, our study reveals that the inhibitionof V-ATPase blocks the EMT process. So far, little is known aboutthe function of V-ATPase in EMT: V-ATPase has been shown topromote EMT in kidney proximal tubular cells, suggesting animplication of V-ATPase in the pathogenesis of kidney tubuloin-terstitial fibrosis (39). In breast cancer, V-ATPase has been shownto contribute to DMXL-2–driven EMT via Notch signaling acti-vation (40).However, the detailed function of V-ATPase, itsmodeof action, and the therapeutic effects of V-ATPase inhibition inEMT in cancer have not been investigated so far.

According to an increasing number of studies, EMT can lead tothe generationof TICs andplays a central role in cancermetastasis.The induction of EMT in nontumorigenic mammary epithelialcells promotes a mesenchymal phenotype and induces TIC for-mation (6). EMT is associated with a poor clinical outcome incancers such as bladder cancer, oral squamous cell carcinoma,ovarian cancer, cervical cancer, as well as breast cancer (41–45).Moreover, EMT is associated with therapy resistance in somecancers, including lung, pancreatic, and breast cancer (46–48).

Thus, pharmacologic targeting of EMT represents an effectivestrategy to inhibit metastasis and TIC formation and mightincrease the vulnerability of cancer cells to chemotherapeutics.As a consequence, an increasing number of EMT- and TIC-target-ing therapies have been developed during recent years but, unfor-tunately, only very few showed promising results in clinic. Indetail, several Notch pathway inhibitors were clinically tested, butrevealed cytotoxic effects. Only compounds which block TGFb–induced EMT such as galunisertib and fresolimumab revealedsuccessful anti-tumor activity in clinical trials (49, 50). Accordingto our present study, inhibition of V-ATPase by Archazolid Amight be judged as a possible new option to target EMT and theformation of tumor-initiating cells.

In search for themechanism of howV-ATPase is involved in theEMT process, we focused on the cell-adhesion protein E-cadherin.Reduced cell–cell adhesion and changes in the cytoskeleton areessential features in EMT. Particularly the loss of E-cadherinrepresents a hallmark of EMT as it results in dissociation ofcell–cell contacts in mesenchymal cells and accounts for metas-tasis (51). Besides genetic and epigenetic downregulation, endo-cytosis and recycling represent crucial posttranscriptional process-es in the regulation of E-cadherin dynamics. Because of its abilityto acidify endolysosomal compartments, V-ATPase has beenassociated with trafficking and recycling of receptors like theepidermal growth factor receptor (EGFR), or the transferrin recep-tor (23, 25). Furthermore, V-ATPase has been associated withendosomal trafficking of cell-adhesion proteins, including E-cadherin (39, 52, 53). In our study, we showed that V-ATPaseinhibition could prevent the EMT-induced loss of E-cadherin,

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V-ATPase knockdown hindersE-cadherin vesicle trafficking.A–C, Average mean squareddisplacement (MSD) of trackedE-cadherin containing vesicles. Theconcave upwards form of the MSDplot is characteristic for activetransport processes. A, The averageMSD from 1,862 trajectories fromcontrol cells (nontargeting shRNAcells that were not induced by IPTGand not treated with 4-OH-TX, ntshRNA). B, The average MSD for1,418 trajectories from nontargeting(nt) IPTG-induced shRNA cells afterEMT induction by 4-OH-TX (ntshRNA þ IPTG/4-OH-TX), whichshow faster endosomal transportthan (C) the averageMSD from 1,879trajectories of IPTG-induced V-ATPase knockout cells (V-ATPaseshRNA 3 þ IPTG/4-OH-TX, C)and control cells (A). D, Averageendosomal vesicle velocities derivedfrom fitting MSD with a diffusionmodel with active transport(Eq. A). Error bars, SD. Although thevelocities of individual trajectoriesshow a broad distribution(Supplementary Fig. S7), thereis a significant change in themean velocity between the differenttreatments (one-way ANOVA;� , P < 0.05).






b-catenin, and claudin-1 and diminished the upregulation ofmesenchymal markers like vimentin, fibronectin, and N-cad-herin. In detail, V-ATPase inhibition interfered with E-cadherininternalization, recycling, and lysosomal degradation, whichresulted inmaintenance of E-cadherin at the cell surface, opposinga mesenchymal and preventing an epithelial phenotype.

Importantly, in linewithprevious studies, we show that besidesinterfering with the EMT process by disturbing E-cadherin, V-ATPase inhibition as well interferes with cells that already have anestablished EMT/CSC phenotype. Interestingly, this was notbased on E-cadherin. By disturbing the endolysosomal system,V-ATPase inhibition addresses various targets including the Rho-GTPase Rac1 (25), integrin activation (24), ironmetabolism (23),Notch (54), amongst others. Most probably, these targets con-tribute to the effects of Archazolid on cells with EMT/CSCphenotype.

Taken together, our finding that V-ATPase inhibition preventsE-cadherin loss upon EMT induction and thereby inhibits malig-nant TIC formation evokes new options for interfering with EMTin cancer therapy. To conclude, our study provides evidence for V-ATPase inhibition by Archazolid A as a promising new strategy toblock EMT and suggests investigating V-ATPase inhibition as apotential option to interfere with TICs in cancer therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H. Merk, A.M. Vollmar, J. PachmayrDevelopment of methodology: H. Merk, S. Zahler, A.M. Vollmar, J. PachmayrAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.K. Messer, M.A. Ardelt, S. ZahlerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H.Merk, P.K.Messer,M.A. Ardelt, D. Lamb, S. Zahler,J. PachmayrWriting, review, and/or revision of the manuscript: H. Merk, P.K. Messer,R. M€uller, A.M. Vollmar, J. PachmayrAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. M€uller, J. PachmayrStudy supervision: J. Pachmayr

AcknowledgmentsWe thank Dr. Christina Scheel (Helmholtz-Center Munich, Institute of Stem

Cell Research) for providing the HMLE cells. We thank Silvia Schnegg and JuliaBlenninger for their kind help with the experiments.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 8, 2017; revised June 21, 2017; accepted June 26, 2017;published OnlineFirst August 3, 2017.

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2017;16:2329-2339. Published OnlineFirst August 3, 2017.Mol Cancer Ther Henriette Merk, Philipp Messer, Maximilian A. Ardelt, et al. Inhibit EMTInhibition of the V-ATPase by Archazolid A: A New Strategy to

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