Cancer Cell, Volume 24

Supplemental Information

DNp73 Exerts Function in Metastasis Initiation

by Disconnecting the Inhibitory Role of EPLIN

on IGF1R-AKT/STAT3 Signaling

Marc Steder, Vijay Alla, Claudia Meier, Alf Spitschak, Jens Pahnke, Katharina Fürst, Bhavani S. Kowtharapu, David Engelmann, Janine Petigk, Friederike Egberts, Susanne G. Schäd-Trcka, Gerd Gross, Dirk M. Nettelbeck, Annett Niemetz, and Brigitte M. Pützer

Inventory of Supplemental Information

Supplemental data (figures and figure legends)

Figure S1 is related to Figure 1

Figure S2 is related to Figure 4

Figure S3 is related to Figure 5

Figure S4 is related to Figure 7

Supplemental experimental procedures

Supplemental references

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2

Figure S1, related to Figure 1. DNp73 expression promotes invasion but does

not affect proliferation of melanoma cells.

(A) Invasive capacity of SK-Mel-29 cells infected with Ad vector encoding DNp73 was

analyzed by Boyden chamber assay. Values of invasion were obtained by counting

five fields per membrane. The data are means ± SD of three separate experiments.

Mean invasion of control virus infected cells was set as 1.0. * indicates p < 0.01 as

determined by Student’s t-test.

(B) Proliferation kinetics of melanoma cells in the absence and presence of DNp73

was measured by XTT viability assay at indicated times (upper panel). DNp73

transduced NIH3T3 cells were used as positive control (lower panel). Each data point

represents the mean number of viable cells in triplicate dishes. Error bars = SD.

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4

Figure S2, related to Figure 4. Regulation of EPLIN by antagonistic p53 family

members.

Reporter assays of the pGL3_LIMA1-intronic fragment (0.5 µg) cotransfected with 0.5

µg of plasmids expressing empty vector, p53 or p73β, and increasing amounts (0.5

µg, 1 µg, 1.5 µg) of DNp73 expression construct in H1299 cells (A). SK-Mel-29 cells

with endogenously high EPLIN levels were cotransfected with the pGL3_LIMA1-

intronic fragment and DNp73 expression plasmid or control (B). Luciferase activities

are measured 24 or 48 hrs after transfection relative to pcDNA (set as 1) and

normalized to total protein concentration. Each bar graph shows means ± S.D. of

three different experiments, each performed in duplicate.

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6

Figure S3, related to Figure 5. Role of EPLIN proteins in melanoma cell

invasion and cytoskeletal reorganization.

(A) Lentiviral-mediated expression of EPLINα or EPLINβ on melanoma cell invasion

was analyzed by Boyden chamber assay. Cells with empty control virus were

normalized to 1. Values of invasion are shown as relative fold change, * p < 0.01.

Values are means ± SD. Protein levels of both EPLIN isoforms were verified by

Western blot using actin as control.

(B) Confocal fluorescence microscopy of phallacidin stained F-actin in SK-Mel-29

cells after knockdown of EPLINβ with sh817 and EPLINα/β with sh941 compared to

scr control. Arrows indicate stress fibers aligned along the major cell axis. Nuclei

were visualized by propidiumiodid (blue). Scale bar: 10 µm. Percentages of SK-Mel-

29 with stress fibers aligned along the major cell axis after EPLIN depletion:

shEPLIN(814) 47 ± 23; shEPLIN(941) 34 ± 11. Parental SK-Mel-29 expressing

scrambled shRNA show no stress fibers.

(C) Box plot of EPLINβ quantification in human melanoma samples shown in Fig. 5E

(right) by qPCR. The horizontal line represents the median. Relative mRNA

expression was normalized to 18S rRNA. Relative expression was calculated using

the comparative Ct method. Statistical significance was determined by Student’s t-

test (twotailed).

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8

Figure S4, related to Figure 7. Expression pattern of different DNp73 or p63

isoforms, p63/DNp63 function, and status of TP53, BRAF and NRAS mutations

in melanoma cell lines.

(A) Detection of p63 protein expression in non-metastatic and metastatic melanoma

cell lines. Actin was used as loading control.

(B) Effect of p63 on transcriptional regulation of LIMA1 and melanoma cell

invasiveness. Reporter assay of SK-Mel-103 cells cotransfected with 0.5 µg of

pGL3_LIMA1-intronic fragment and 0.5 µg of indicated expression plasmids.

Luciferase activities (RLU) are shown 24 hrs after transfection relative to pcDNA set

as 1 and normalized to total protein concentration. Error bars indicate one SD of the

mean from three separate experiments (left). Immunoblot of EPLIN isoforms in

different melanoma cell lines after overexpression of p63 or DNp63. p63/DNp63 and

actin levels are as indicated (middle). Migration/invasion was determined by Boyden

chamber assay of SK-Mel-103 cells after overexpression of p63 or p73, and both

(right). Fold changes are relative to cells transfected with GFP plasmid. Values are

means ± SD. * indicates p < .01.

(C) Quantitative RT-PCR analysis of different DNp73 isoforms in the cell models

used. Data are represented as mean ± SD of duplicates from three independent

experiments. The status of TP53, BRAF and NRAS is as indicated (mt, mutant; wt,

wild-type).

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Supplemental Experimental Procedures

Cell Culture, ASO, and Proliferation Assay

All melanoma cell lines other than Mel888, C8161, and A375M (D. Nettelbeck) were

provided by M. Soengas and maintained as HEK293, HEK293T, H1299 (obtained

from ATCC), NIH3T3 (Cell Line Service), and Madin Darby canine kidney (MDCK)

cells (Cell Line Service) in Dulbecco's Modified Eagle´s Medium (DMEM, PAA)

supplemented with 10% fetal bovine serum (FBS, Biochrom) using standard

conditions and procedures. Stable SK-Mel-29 cell lines were generated by

transfection with empty vector or pIRESpuro2-ΔEx2/3βp73 expressing the DNp73

isoform ΔEx2/3β. The NIH3T3-∆Ex2/3βp73 cell line has been described previously

(Stiewe et al., 2002). Transfections were performed using Effectene reagent

(QIAGEN). For inhibitor studies cells were exposed to the JAK-specific inhibitor

AG490 (Merck), which inhibits p-STAT3, and Akt Inhibitor VIII (Merck) that selectively

inhibits Akt1/Akt2, both dissolved in DMSO and used at a concentration of 20 µM or 1

µM respectively for 48 hr. Cells were treated with the IGF1R Inhibitor II (Merck) for 1

hr (30 µM) and lysed after 24h of cultivating. Locked nucleic acid (LNA) antisense

oligonucleotide ASO116 gapmer specifically directed against the ∆Ex2/3p73 splice

junction and scrambled control oligo (ASOscr) described by Emmrich et al. (2009)

were introduced into cells by nucleofection (Amaxa). Cell growth rates were

measured by TACS™ XTT Cell Proliferation Assay (Trevigen) according to the

manufacturer´s instructions.

Plasmids and cDNA Synthesis

Expression plasmids for TAp73α, TAp73β, and ΔEx2/3βp73 (DNp73) were described

previously (Stiewe et al., 2003). cDNAs encoding EPLINα and EPLINβ were

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amplified by PCR from SK-Mel-29 cells using primers fw 5´-

ATGGAAAATTGTCTAGGAGA-3´, rev 5´-TCACTCTTCATCCTCATCCTC-3´

(EPLINα), and fw 5´-ATGGAATCATCTCCATTTAATA-3´, rev 5´-

TCACTCTTCATCCTCATCCTC-3´ (EPLINβ), cloned into pcDNA3.1/V5-HisTOPO

(Invitrogen) and sequence verified. Both isoforms were inserted into the lentiviral

vector pWPXL (Addgene). The LIMA1 reporter fragments were amplified from

chromosomal DNA isolated from SK-Mel-29 and subcloned into TA-cloning vector

(Invitrogen). DNA fragments were inserted into pGL3-Basic luciferase reporter vector

(Promega) using KpnI and XhoI restriction sites. Primers used are: LIMA1-intronic

fragment: fw 5´-AAAACATGTTTGTATTTATGCA-3´, rev 5´-

ACATCCTTCCCATCTCCACACA-3´; EPLINα promoter: fw 5´-

TGCCTGGCCCTGATAAGTGAGAGAAT-3´, rev 5´-

CTGTGAACAGTTACAGCCTTGCCA-3´, EPLINβ promoter: fw 5´-

ACATCCAAGAGAAGCCCTGACCT-3´, rev 5´-TCACGTTCTCGGGTAGCCAGAAG-

3´.

Generation of Viral Vectors and shRNAs

The adenoviral (Ad) vectors Ad.DNp73 (encoding ΔEx2/3βp73), Ad.GFP, Ad.p73,

and Ad.p53 have been previously described (Emmrich et al., 2009; Stiewe et al.,

2002; Buhlmann et al., 2008). Adenoviruses were generated and amplified in 293

cells as described (He et al., 1998). Infection was carried out at a multiplicity of

infection (MOI) that allows 100% transduction of target cells. For production of

lentiviruses expressing shRNA against E-Cadherin (shCDH1, TRCN0000039666),

Slug (shSlug, TRCN0000015390) EPLINα and -β isoforms (941; TRCN0000149941),

EPLINβ (817, TRCN0000146817), or scrambled shRNA (shscr), Mission shRNA

plasmids (Sigma) were used. EPLIN (941)-shRNA sequence is

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CCGGGTCTCTGAATTGGTCGAGTTTCTCGAGAAACTCGACCAATTCAGAGACTT

TTTTG and EPLIN (817)-shRNA sequence is

CCGGCATGGAGAAGAAGAGAAGTAACTCGAGTTACTTCTCTTCTTCTCCATGTTT

TTTG. VSV-G enveloped pseudotyped lentiviral vectors were generated by

cotransfection of HEK293T cells with plasmids pMD2.G and psPAX2 (Addgene)

using calcium phosphate as described (Salmon and Trono, 2007).

RNA Isolation and qRT-PCR

Total RNA was extracted with NucleoSpin® RNA II (Machery-Nagel) and reverse

transcribed with Omniscripr RT (QIAGEN). cDNA samples were mixed with iQTM

SYBR® Green Supermix and analyzed on iQTM5 Multicolor Real-Time PCR Detection

System (Bio-Rad). Relative gene expression was calculated using iQTM5 Optical

System Software. The following primer pairs were used: DNp73: fw 5´-

GGCTGCGACGGCTGCAGGCC-3´, rev 5´- CAGGCGCCGGCGACATGG-3´; EPLIN:

fw 5´-ACGAAATCAGCAAGCCCGAAGT-3´, rev 5´-

TTTCCTGATAGGTGGGGACACA-3´; EPLINβ: fw 5´ -

CTCCAAGTACCAGAAAGCAG-3´, rev 5´-GTCTGCTCTGTGCCTAATCT-3´; DPP4:

fw 5´-CGCAGGAGCTGTGAATCCAAC-3´, rev 5´- TCCCGATGACTTCCCAGGTG-3´;

CHL1: fw 5´-AGCTTTGGCTGAACCCTA-3´, rev 5´-TAGATGCACCCGTGTTTG-3´;

CDH1: fw 5´-GCTTTGACGCCGAGAGCTACA-3´, rev 5´-

TCCCAGGCGTAGACCAAGAAA-3´; SEMA6A: fw 5´-

TTCCCAAGAGTGGCTCAGGTTT-3´, rev 5´-AACATCACGCCCGTTGATACGA-3´;

MOAP1: fw 5´-AGAGCTGCAGTGTGGCAGAAAT-3´, rev 5´-

GCGTGACTAGTCTCCGCAGTAAG-3´; CSPG2: fw 5´-

ACTGATGGCAGCACACTGCAA-3´, rev 5´-CGGCTCCAACGATGATCATG-3´;

CCL2: fw 5´-AGATGCAATCAATGCCCCAGTCAC-3´, rev 5´-

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TAGCTGCAGATTCTTGGGTTGTGG-3´; EPHA2: fw 5´-

TCGGGAGAAGGATGGCGAGTT-3´, rev 5´-TTGCAGACCAGGTTGCTGTTGA-3´;

S100A2: fw 5´-TGGTCTGCCACAGATCCA-3´, rev 5´-CCATGGCAGGAAGTCAAG-

3´; RHOB: fw 5´-GTGCTCTGCCAAGACCAAGGAA-3´, rev 5´-

AGCAGTTGATGCAGCCGTTCT-3´; ZEB1: fw 5´-TTTTCCTGAGGCACCTGAAGAG-

3´, rev 5´-GTGTTCTTCAGAGAGGTAAAGCG-3´; S100A4: fw 5´-

CGGGCAAAGAGGGTGACAAGTT-3´, rev 5´-TCCCTGTTGCTGTCCAAGTTGC-3´;

B2M was used as reference: fw 5´-TGCCGTGTGAACCATGTGACTT-3´, rev 5´-

CCAATCCAAATGCGGCATCTTC-3´.

Microarray Experiments

Equal amounts of total RNA were analyzed using Affymetrix GeneChip Human

Genome U133 Plus 2.0 Arrays. Each analysis was done in triplicate. Background

corrected signal intensities were determined and processed using MAS5 function of

the R/Bioconductor affy package. Gene transcripts not detected in any sample were

excluded from statistical analysis. Normalization of expression data, statistical tests,

and clustering was accomplished by GeneSpring GX 9.0 (Agilent Technologies).

Expression profiles of mock and SK-Mel-29.DNp73 cells were statistically analyzed

for differential gene expression using t-test and multiple testing correction (Benjamini

and Hochberg False Discovery Rate). A list of 1376 genes displaying a minimum 2-

fold induction or reduction (P < .05) were included for clustering compared to

expression profiles of SK-Mel-103 and SK-Mel-147. DNp73-regulated genes were

analyzed using database for annotation, visualization and integrated discovery

(DAVID) based on Gene Ontology molecular function, biological process, and cellular

component (Huang Da et al., 2009). Oncomine™ (Compendia Bioscience, Ann

Arbor, MI) was used for extraction of microarray data from tumor profiling studies.

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Immunoblotting, Immunofluorescence, and Actin Staining

Cells were lysed in RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM NaF, 2 mM

Na3VO4 (SOV), 0.1% SDS, 0.1% Triton X-100, 1% deoxycholate, 5 mM EDTA)

supplemented with cOmplete ULTRA Protease- and PhosSTOP Phosphatase

inhibitors (Roche) and total protein concentration was quantified by Bradford assay

(Bio-Rad). Equal amounts of protein were separated by SDS-PAGE, transferred to

nitrocellulose membranes (Amersham) and probed with primary antibodies against

p73 (ER15), p53, Fibronectin, E-cadherin, and N-cadherin from BD Bioscience,

EPLIN (20), MITF (N-15), Vimentin (V9), Slug (H-140), p63 (4A4) (Santa Cruz

Biotechnology), IGF1R, phospho-IGF1R, AKT, phospho-AKT(Ser473), p44/42 MAPK

(Erk1/2), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-

MEK1/2(Ser217/221), STAT3, phospho-STAT3(Tyr705) from Cell Signaling

Technology, TBP (Abcam), and Actin (Sigma). For optimized protein detection

SuperSignal West Femto Chemiluminescent Substrate (Pierce) was used. For

immunofluorescence, cells grown on glass slides were fixed in 4%

paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with

10% FCS and 1% BSA in PBS. Cells were incubated with the primary antibodies

diluted in 3% BSA in PBS/0.02% Triton X-100, washed in PBS, and incubated for 45

min with fluorescence-labelled secondary antibody. Staining of F-actin was

performed with BODIPYFL phallacidin (Invitrogen). Images were obtained using an

inverted confocal laser scanning microscope (Zeiss). Cells displaying cytoskeletal

changes (stress fibers aligned along the major cell axis) were quantified by counting

the number of positive cells from five randomly chosen confocal images per

condition.

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Wound Healing, Matrigel Invasion, and Cell Adhesion Assay

Cells were grown in monolyers and wounded with a pipette tip. Detached cells were

washed off with PBS and new medium was added. Migration of cells was measured

as reduction of the wound area in each photographed field. At least four fields were

photographed for each condition each time and the gap was calculated using ImageJ

software (http://rsbweb.nih.gov/ij). Images were obtained at 24, 48, and 72 hr after

scratching. Invasion assays were performed as a modified Boyden chamber assay.

Polyethylene terephtalate membranes with 8 µm sized pores (cell culture inserts, BD

Falcon) were coated with 3.1 mg/ml diluted Matrigel Basement Membrane Matrix (BD

Biosciences). Cells were seeded onto the top of the membrane in serum-free

medium. Medium containing 25% FBS was added in the lower chamber as

chemoattractant. After 72 hr cells that had not penetrated the filters were removed by

scrubbing with cotton swabs. Chambers were fixed in 100% methanol for 2 min and

stained with DAPI. Five sections per membrane were counted under the microscope.

For adhesion assays, cells were seeded onto 24-well dishes coated with laminin (5

µg/ml, Sigma) or fibronectin (25 µg/ml, BD Biosciences) and grown for 4 to 8 hr. After

washing with PBS, adherent cells were fixed with 10% formaldehyde and stained

with 0.1 % crystal violet. Cells were lysed with 2% SDS and intensity of released dye

was determined in a microplate reader at 595 nm. For apoptosis inhibition, the

caspase inhibitor Z-VAD-FMK (Tocris Bioscience) was added with Ad.TAp73 at a

final concentration of 50 µM. All experiments were conducted in the presence of 5

μg/mL of mitomycin-C to inhibit cell proliferation.

Luciferase Reporter Assay and Chromatin Immunoprecipitation

Luciferase activity was measured 24 hours after transfection using the Luciferase

Reporter Assay System (Promega) and normalized to total protein concentration in

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cell extracts. ChIP assays were performed essentially as described. Protein-DNA

complexes were immunoprecipitated using anti-p73 (BD Bioscience) or control IgG

antibody. Primer sequences: EPLINα: 5́ -CTCATATGCATGTAAATTATGTCAG-3´,

5´-CTTTTAAATAAGACAAAGATGCAGG-3´; EPLINβ: 5´ -

TTGCAGTGAGGGAAGTGG-3´, 5´-TGCTGTCGCCATCTTACGC-3´; LIMA1-intronic

fragment: 5´- ACCTACCTCATGACTTGTTGC-3´, 5´-

GCCTGCTCCATATAAACAATG-3´; CDKN1A: 5´- GCACTCTTGTCCCCCAG-3´, 5´-

TCTATGCCAGAGCTCAACAT-3´.

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Supplemental References

Buhlmann, S., Racek, T., Schwarz, A., Schaefer, S., and Pützer, B.M. (2008). Molecular mechanism of p73-mediated regulation of Hepatitis B virus core promoter/enhancer II: Implications for hepatocarcinogenesis. J. Mol. Biol. 378, 20-30. He, T.C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B. (1998). A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA. 95, 2509-2514. Huang Da, W., Sherman, B.T., and Lempicki, R.A. (2009). Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1-13. Huang Da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57. Salmon, P., and Trono, D. (2007). Production and titration of lentiviral vectors. Curr. Protoc. Hum. Genet. 12, 12.10.