Epithelial to mesenchymal transition rewires the molecular path to PI3-Kinase-dependent
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1 Supplemental material for Epithelial to mesenchymal transition rewires the molecular path to PI3-Kinase-dependent proliferation Megan B. Salt 1,2, Sourav Bandyopadhyay 1,3 and Frank McCormick 1 Authors Affiliations: 1-Helen Diller Family Comprehensive Cancer Center; 2-Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, California; 3-California Institute for Quantitative Biosciences, San Francisco, California.
2 Supplemental Figure Legends Table S1. EMT associated changes in gene expression. Significantly (A) up- and (B) downregulated genes in comparing H358-TwistER cells to control H358-GFP expressing cells treated with 100nM 4OHT for 12 days. The four columns on the right are associated with significantly upregulated genes, while the four columns furthest left described significantly downregulated genes. The first column for the up- and downregulated genes shows the Probe ID associated with each gene from the Affymetric Human Gene 1.0 ST microarrays. The gene name are shown in the next column, followed by the log 2 (fold change) in expression for each gene after 4OHT treatment in the H358-TwistER cells. The final column shows the p-value for each gene adjusted for the false discovery rate. Figure S1. Akt inhibition reduces serum-independent proliferation. (A) Proliferation of H358- TwistER cells with increasing concentrations (um) of GSK (top) or MK-2206 (bottom). (B) Lysates from H358-TwistER cells treated for 48hrs in serum free media with indicated concentrations of GSK (top), or MK-2206 (bottom). Figure S2. The effects of TGFβ1 are specific to epithelial cells and lead to reduced serumindependent proliferation. (A) Proliferation of untreated (top) or TGFβ1 pre-treated (bottom) H358 and H441 cells in the presence (10%) or absence (SS) of serum. (B) Lysates from indicated cell lines treated with 2ng/ml TGFβ1 for 14d. Figure S3. sirnas targeting ErbB3 and NRG1 are efficient and on target and knockdown of autocrine TGFα/EREG does not affect ErbB3-PI3K signaling. (A) Knockdown efficiencies at the protein level of indicated oligos 72hrs post-transfection. (B) Fold change in RNA level 48hrs posttransfection with indicated oligos. (C) Proliferation of H35-TwistER cells transfected with the indicated
3 sinrg1 oligos in the absence (-) or presence (+) of exogenous NRG1 ligand (100ng/ml). (D) Lysates from H358-TwistER cells (NT) collected 72hrs after transfection with indicated sirna oligos, serum starved overnight. (E) qpcr quantification of fold change RNA level 72hrs post-transfection with indicated sirnas. (F) Immunoprecipitation of ErbB3 and p85 from H358-TwistER lysates 72hrs after transfection with indicated oligos, serum starved overnight. Figure S4. Proliferation of epithelial cells after knockdown of TGFα/EREG and ErbB3 in serum and in mesenchymal cells after EGFR expression in H358-TwistER cells. (A) Proliferation of H358- TwistER cells in the presence of indicated sirna oligos and serum conditions. (B) Proliferation of H358-TwistER cells before EMT in the presence of 10% serum after transfection with indicated sirnas. (C) Proliferation of H358-TwistER cells after 14d 4OHT pre-treatment, after transfection with indicated expression constructs. Figure S5. ErbB3 and NRG1 promote Akt activity in H441 and H2122 cells and ErbB3 knockdown reduces serum-independent proliferation. (A) Lysates from H441 and H2122 cell lines collected 72hrs after transfection with indicated sirnas, serum starved overnight. (B) Immunoprecipitation of p85 from H441 and H2122 lysates 72hrs after transfection with NRG1 targeting sirna, serum starved overnight. (C) Proliferation of H441 and H2122 cells in the absence of serum after transfection with indicated sirnas. SS: serum starved; IP: Immunoprecipitation; WCL: whole cell lysates. Figure S6. GAB1 mediates growth factor stimulated signaling from EGFR to PI3K-Akt, and is required for proliferation in H358-TwistER cells after EMT. (A) Lysates from H358-GFP expressing control cells 72hrs after transfection with indicated sirnas, serum starved overnight, and treated -/+ EGF for 10 minutes. (B) H358-TwistER cells pre- (NT) and post- (4OHT) EMT 72 hrs after transfection with control of GAB1 targeting sirnas. Lysates collected from cells asynchronously growing in serum,
4 or starved overnight and treated 10min -/+ EGF as indicated. (C) Proliferation of H358-TwistER cells with and without 4OHT pre-treatment after transfection with indicated sirnas in 10% serum. Figure S7. KRas is required for proliferation before and after the induction of EMT. Proliferation of H358-TwistER cells (A) before and (B) after 4OHT induced EMT in the presence of indicated sirnas. (C) Lysates from H358-TwistER cells 72hrs after transfection with indicated sirnas, serum starved overnight. (D) Relative cell numbers of H358-TwistER with and without 4OHT pre-treatment after 72 hours growth in the presence of indicated doses of PD (uM). Figure S8. EMT alters sensitivity to inhibition of EGFR but does not affect sensitivity to PI3K inhibitors. (A) Relative cell number of H358-TwistER with and without 4OHT pre-treatment after 72 hours growth in the presence of indicated doses of Erlotinib (top) or BKM-120 (bottom). (B) Relative number of H441 cells with and without 2 weeks TGFβ1 pre-treatment (2ng/ml) after 72 hours treatment with Erlotinib (top) or BKM-120 (bottom). (C) Relative number of H358 and H441 cells after transfection with ErbB3 sirnas followed by treatment the next day with indicated doses of Erlotinib for 72 hours. Sensitivity to Erlotinib and Lapatinib compared to EMT Score (see methods) across all cell lines in the CCLE (D) and NSCLC lines (E). Sensitivity to GDC-0941 across NSCLC cell lines (F). Drug sensitivities are based on AUC from dose-responses analysis in the Cancer Cell Line Encyclopedia, with AUC<1 (area under the curve) used to define drug sensitive lines. *** indicates p<0.0001, n.s.=not significant.
5 Supplemental Methods Gene expression analysis RNA was extracted from three biological replicates of each condition using Qiagen RNeasy mini kit according to manufacturer s instructions. Quality was analyzed on a 2100 BioAnalyzer (Agilent). GeneChip Human Gene 1.0 ST Arrays (Affymetrix) were used to quantitate gene expression in each sample. RNA preparation and data collection was performed at the Gladstone Institute Genomics Core Facility. Additional data analysis was performed by the Bioinformatics Core at the Helen Diller Family Comprehensive Cancer Center. Raw data and results can be found in the GEO database, accession number GSE Phospho-Receptor Tyrosine Kinase Arrays Phospho-RTK arrays were purchased from R&D Systems. Equal quantities of protein lysates from cells with and without 4OHT pre-treatment, were collected in supplied lysis buffer supplemented with protease inhibitor cocktail (Roche) and incubated on membranes and analyzed according to manufacturer s protocol. sirna oligonucleotides The following sirna oligonucleotides were used for this study. sirna Name Company Catalogue Number Target Sequence Control Qiagen Proprietary ErbB3 pool Dharmacon M See below (pool of 08/22/23/24) ErbB3 08 Dharmacon D GCAGUGGAUUCGAGAAGUG ErbB3 22 Dharmacon D AGAUUGUGCUCACGGGACA ErbB3 23 Dharmacon D GUGGAUUCGAGAAGUGACA
6 ErbB3 24 Dharmacon D GCGAUGCUGAGAACCAAUA NRG1 pool Dharmacon M See below (pool of 21/22/23/24) NRG1 21 Dharmacon D UUUCAAACCCCUCGAGAUA NRG1 22 Dharmacon D GGGGAGUGCUUCAUGGUGA NRG1 23 Dharmacon D UUGUAAAAUGUGCGGAGAA NRG1 24 Dharmacon D ACAUCCACCACUGGGACAA TGFα pool Dharmacon M Pool: CAGCAGUGGUGUCCCAUUU GCAUGUGUCUGCCAUUCUG GUACGUUGGUGCACGCUGU ACUGCUGCCAGGUCCGAAA Ereg pool Dharmacon M Pool: GCUCUGACAUGAAUGGCUA GUACAACUGUGAUUCCAUC GAAGUGGGUUAUACUGGUG GCUUUGACCGUGAUUCUUA GAB1 pool Dharmacon M Pool: AUACUUAGAUCUCGACUUA CAUCAAAGCUAGACACUAU GAUGCUGGAUUGACAUUUA GAGAGUGGAUUAUGUUGUU ErbB2 14 Qiagen S AACAAAGAAATCTTAGACGAA ErbB2 15 Qiagen S CACGTTTGAGTCCATGCCCAA EGFR 10 Qiagen S TACGAATATTAAACACTTCAA EGFR 12 Qiagen S CAGGAACTGGATATTCTGAAA KRas 234 Mirimus N/A Proprietary KRas 355 Mirimus N/A Proprietary
7 SYBR Green qpcr RNA was isolated from cells using the RNEasy Mini kit (Qiagen). cdna was synthesized from total RNA using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). 10ng cdna template was used in qpcr reaction along with 1X SYBR green master mix (Applied Biosystems), 0.2uM indicated forward and 0.2uM reverse primers. Data was collected on the Stratagene Mx 3000P machine and analyzed using Δ(Δct) method to calculate fold change. Primers sequences are shown below. The following qpcr primers were used for this study. Primers Targets ErbB3 forward ErbB3 reverse NRG1 forward NRG1 reverse EGFR forward EGFR reverse EREG forward EREG reverse TGFα forward TGFα reverse Sequence GGTGATGGGGAACCTTGAGAT CTGTCACTTCTCGAATCCACTG GCCAGGAATCGGCTGCAGGT AGCCAGTGATGCTTTGTTAATGCGA AGGCACGAGTAACAAGCTCAC ATGAGGACATAACCAGCCACC ATACTGGTGTCCGATGTGAACA CCGACGACTGTGATAAGAAACA AGGTCCGAAAACACTGTGAGT AGCAAGCGGTTCTTCCCTTC
8 Table S1. Up- and downregulated genes after EMT in H358-TwistER cells. Upregulated Genes Downregulated Genes Probe Id Gene logfc adj.p.val Probe Id Gene logfc adj.p.val CDR E CDH E RUNX1T E MACC E LOX E GRHL E DPP E AGR E IGF E EPCAM E COL12A E RBM35A E TMEM E RAB E PTGS E GPR E KAL E TSPAN E TNFAIP E FGFBP E NRK E MAL E NA MYO5C E PMP ANKRD E INHBA E KRT E GGTA E ITGB E DAB E CD E CADM E KLK HMCN E EREG E COL14A E CCDC88C C4orf E GPR E LRRC E TC2N E SEMA5A E DSC E CPA E IL BBOX E TMEM E LOH3CR2A E TMEM RBPMS E DAPP E COL3A E C3orf E EPHA E MALL E BNC E LAMC C7orf E HOOK E RNF E AP1M FAM133A MPZL E TCP11L E GPR LIX E SPINT NEXN E KRT6A FBN E TP CCDC E ERP E-05
9 NA KLRC FRMD SCNN1A HHIP E PPP1R9A E CALCRL NP LMCD E ZNF FN CKB CLIC S100A E YPEL E RAB27B E ID E CDS E FILIP1L E ABCA ANTXR E EHF KDELR SCIN E LOC E AP1S SMAD CYP1B E OR7E19P STEAP SEPP GALNT TNFSF SORT E ROBO E LAMA E IGFBP E NAALADL IL7R E CXADR PLXNC E RBM E GPR137B E FLJ GPRC5B E TACSTD KDR E NA E RSPO E FGD EBF E CLIP E REPS E CLDN E PROS PRRG HEG E FXYD LIMCH E ARAP KIAA ME GBP LUM TLE CNTN NPNT RASEF DEPDC E TPD GJA PKP FOXN ACSL UCP ZNF NA TSPAN LOC MYO5B TSPAN ARHGEF
10 BACE RTKN MRAS E MMP CHN ANXA COL8A ELOVL MAGED GATM AXL ERBB FBLN DSP PCDH GPRC5A CCDC102B TMEM30B PNMA FAM160A SESN E TMPRSS WIPI KLHL GSN C12orf GPR TJP TLR ATP8A SERPINF FUT RNF MPZL CREB3L CLDN SPARC LYN RECK LAMB CDH ALDH1A PGCP BSPRY MAN1A MUC IL1R SPAG ID MYO KIAA TNIK KIFAP ZDHHC TFPI AGPAT DEPDC CGN SMAD MAP SDC CYB5A DOCK LIPH GPNMB FGF WIF SH3BGRL SRPX SVIP ATXN7L C12orf LHFP CMTM C9orf TNFRSF10A CRISPLD GJB SEC24D C1orf TRAM FAM84B
11 TMEM ZBED NA SERPINB ADAMTS OSBPL ADAMTS LOC FAM114A WDR RHOBTB JUP RARB SMPDL3B RALYL MYO3B SERPINE ITGA GPX SAT PID PHLDA ZMAT HIST1H2AA NA GLRB TTC SPINK GPC BMP DKK RNF PTGES FZD CRIM C6orf GLIPR PLCH GSTA CELSR SNAI MBOAT SYTL OSTF CCL MYEOV CHRNA MPP MRC MAP7D PRR AFAP1L LOC ASAH ZEB ERMP PTCH DCLK ANXA PKIB PLSCR CTH PAM FRK SGK RP6-213H TMEM PROM PRDM TNS TRPC FAM47E LOC MFSD NCAM SPP C1orf SLC39A AKT SEC11C EHD EGLN
12 SCUBE NA F2RL SFN SCG TEX FAT GCLC EGR TGFA SEMA6D SQRDL TFPI GBP APOL PLEKHG ST3GAL GRPR RGS SYTL EPB41L SLC9A3R MGP KRT19P CXCL CYP4X CLEC2B KIAA MYL TMC CERCAM TRIM SHC PLS FRMPD FRRS CCBE MAPKAPK PELI B3GNT NA CCNYL HECW CAST PLCB PIK3R UNQ C6orf USP GCNT CAPN HIST1H2BA ST6GALNAC EZR TCEAL C6orf MAGI C6orf LRIG TMC CNTN PTPLB PCDH TM4SF PCOLCE OLFML2B PAPSS CHAC ZFHX MNS ACTA ACPP TMEM63C B3GNT SLC40A UACA PLAC ZCCHC CPS EPB41L4B TMEM VAV
13 PLXDC MKNK SNX TRMT2B SAMSN DSC ISL ACYP PBX UBASH3B NA TMTC ALDH1L UTX CYBRD CASP PTRF LOC C6orf TMEM RGL PDK PDGFRA PLA2G4A SLC30A MATN LOC PRMT RCAN MYB KLHDC NEBL CTSO CST LXN SLC41A GABRE FOXA IFITM PDCD2L TMEM CRYBG GPR RALB GLT8D FLJ AMIGO CDGAP ANKRD TP53INP GCOM SLC16A DTWD GHR TM7SF LBH PARD6B PDCD PFKFB EPHA GMDS SLC44A ENDOD PCDHB UCHL RHOU AKAP PRTG IFRD RELL EXPH FKBP GRAMD COBLL DUSP JMY CLDN CPE PGM2L MAML CMTM
14 EPHA KLRK CTGF B3GALT TGFB PDIK1L KIAA TUBD TUFT C21orf CALD CTSL IL31RA TXNRD C8orf PMEPA IFI44L RASA SPOCK TOM1L MFAP GGCT FABP INADL APBB DLEU GPM6A MRPS LPHN ABHD RUNDC3B RASSF PDE5A MBOAT ZEB FAM49B KLHL ALCAM ARHGAP ACP RBM PDCL RGS SLC25A NEO ASB PPAP2A MYH CABLES LRBA LPHN LRRFIP ARMCX TDRKH HAPLN MAP3K7IP SCYL1BP AIM SPG UTP KIAA EPB TLN NDUFB RIMS BIRC MSRB MRTO C12orf TNFAIP SLCO5A PPA AS3MT IDH3A F2R HIST1H1B ATXN7L TRIM SLC9A RAB3IP TLL SP
15 GPAM IKZF PSAP BRCC STAT RBBP THRB HSDL LEPRE C12orf CPNE ARRDC GLI HSPA4L KCNMA CHMP4A NA STRBP KIAA ARPC5L SLC16A JUNB CLEC7A CCDC TUBB2B FLJ TGFBR FAM83B ZNF PPM2C ZNF ZNF ARHGAP MLLT NA MRPS LHFPL ROD SCN3A FUCA NAP1L KRT FAM122C PPAPDC ANK HPGD CNTNAP SYK CRISPLD AIFM LGR SPTBN DNM LIPG ZNF SELI AOX ANXA GPR GFM ZBTB FERMT CXCR LRRFIP SLITRK NA C7orf ARMCX FOXP COL6A ASAP APCDD TPM
16 RAI RNF SEC23A AASS ATXN7L IFNAR LPAR EFNA RCOR ATXN7L MTMR DENND5A GOLSYN Sep ICK MGC CDH SORBS SSFA C1orf IGSF DKK ARHGAP IFNGR INPP CGNL DDAH CDRT SAMD4A LECT GRAMD1C GAB ATP8B HHIPL KCNH C3orf NUPR PPARG GBP NFIB CD99L
17 ENG MAP3K CDH PRRX LGR TSPYL SMARCA NA GNAI PCDH NAV RAB8B LDB MX DAPK CARD RBMS PLCL VWDE ARID5B NCRNA BHLHB TMEM SATB DPT DOCK PHLDB SSPN RGMB MAGEH SOX MAMDC CYR TMEM39A PHF21A NES BMPR DSEL ADC FCHSD KIAA
18 GLRX IFI SGEF TP53TG CYP7B TLR ARNT TBC1D2B FKBP MGC SLC5A PRPS ST STXBP PXK VLDLR ZNF ATP10D RCAN TTC DST DPYD TM4SF EFNB STAT5B SPRED MAP7D ZFPM PDE3A KLF ATP8B FAM169A PCDHB PCLO C9orf MOXD PTPN SEC31A LOXL CD PRKD
19 LOC CNTLN NA LY6K
SUPPLEMENTARY INFORMATION
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NES: normalized enrichment score (analyzed using KEGG pathway gene sets in the GSEA software); FDR:
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