Supplemental Information

Loss of Estrogen Regulated MIR135A1 at 3p21.1 Promotes Tamoxifen Resistance in Breast Cancer

Weijie Zhang, Mingming Wu, Qing-Yun Chong, Min Zhang, Xiao Zhang, Lan Hu,

Yanghao Zhong, Pengxu Qian, Xiangjun Kong, Sheng Tan, Gaopeng Li, Keshuo Ding,

Peter E. Lobie, and Tao Zhu.

Supplemental data include 5 supplemental tables, and 7 supplemental figures and legends. Supplemental Tables

Supplementary Table S1. Sequences of the miRNA mimics/siRNAs, shRNAs, and primers for cloning, ChIP assays and qRT-PCR analysis. miRNAs Sense Strand (5'-3') Antisense Strand (5'-3') hsa-miR-135a UAUGGCUUUUUAUUCCUAUGU ACAUAGGAAUAAAAAGCCAUA mimics GA UU siERK1 GAAACUACCUACAGUCUCUTT AGAGACUGUAGGUAGUUUCTT siERK2 GUGCUCUGCUUAUGAUAAUTT AUUAUCAUAAGCAGAGCACTT siAKT GCACCUUCAUUGGCUACAATT UUGUAGCCAAUGAAGGUGCTT Negative Control (NC) UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT shRNAs Sense Strand (5'-3') CCGGCTACAGGCCAAATTCAGATAACTCGAGTTATCTGAATTTGGC shESR1 CTGTAGTTTTT CCGGGTGAATGCACTGGTGTCTCATCTCGAGATGAGACACCAGTGC shESRRA ATTCACTTTTT CCGGCGGAAGTCAGATTGTAGCCAACTCGAGTTGGCTACAATCTGA shNCOA1 CTTCCGTTTTT CCGGGCTTGACTGGTTTGAGACACACTCGAGTGTGTCTCAAACCAG shPIM2 TCAAGCTTTTT CCGGCCACCTCTCAATGTCGACAAACTCGAGTTTGTCGACATTGAG shMRAS AGGTGGTTTTTG CCGGGTCATCAAGATTGGGTTGTTTCTCGAGAAACAACCCAATCTTG shLCP1 ATGACTTTTTG CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTA shScr GG Primers for plasmid construction Sense Strand (5'-3') Antisense Strand (5'-3') pri-miR-135a-1 CGAATTCAGGAACTCCATGCCA AATGTCGACCCTGAAGTCAGCC cloning primer GCACAG CTTCAGTG TCACATAGGAATTTAAGCCATACGATTCACATAGGAATTTAAGCCA TAACCGGTTCACATAGGAATTTAAGCCATATCACTCACATAGGAAT TTAAGCCATACCCGGTGCATGACTAAGCTAGATAATCACATAGGAA TTTAAGCCATACGATTCACATAGGAATTTAAGCCATAACCGGTTCA CATAGGAATTTAAGCCATATCACTCACATAGGAATTTAAGCCATAA miR-135a Sponge CGCGTCGACCTAAGTATCCTCCTCTTGGA ESR1 3’UTR TACTCGAGAGCCCTGGGGCATG GTGCGGCCGCGCTATGGCTTGG cloning primer GAGCTGAAC TTAAACATATC ESR1 3’UTR Mut GCAGATATGTTTAACCGCCCAT CTCAAAAGGCATGGGCGGTTAA primer GCCTTTTGAG ACATATCTGC ESRRA 3’UTR ATCTCGAGAGGGGTGGGACTG CCGCGGCCGCTTTCTCTTTGCAG cloning primer GTGGGGG CAAATATAC ESRRA 3’UTR CAGTGCTGCCCCTTGCACGTGC CACTCTGGGGGCACGTGCAAGG MutA primer CCCCAGAGTG GGCAGCACTG GTAGGGGGCCTTGCGGGGGGG CATCCCGTGCAGCCCCCCCGCA ESRRA 3’UTR CTGCACGGGATG AGGCCCCCTAC MutB primer

NCOA1 3’UTR CGCGCTCGAGCCACTTTTAAAG ATGCGGCCGCCTTCAGGGATGC cloning primer GAATGTGAAA TTTATTATCC NCOA1 3’UTR CAGACTTCAAGGAAGTCCCCA AGGAAGGACCTGGGGACTTCCT MutA primer GGTCCTTCCTTCTGCATCTC TGAAGTCTGAGTCCATGC NCOA1 3’UTR CATTTCCAGCTTGAAAGCCTAC GGTAGGGACTAGAGTAGGCTTT MutB primer TCTAGTCCCTACC CAAGCTGGAAATG PIM2 3’UTR ATCTCGAGAATGGTCAGAAGA GCGCGGCCGCCTATTACTTTTAT cloning primer GCCATCCCA TAAATAGTGGG PIM2 3’UTR Mut GCCTGGATTATTTAAATGTGGA ATAGTGGGTTTCCACATTTAAA primer AACCCACTAT TAATCCAGGC MRAS 3’UTR ATCTCGAGCAGGCCTGAGGCCC GCGCGGCCGCCACTCGTTTCAC cloning primer TGGGCACA GACAATGGCC MRAS 3’UTR CATACAAAGTACAAAACAACA GGAAAGAAGCTTGTTGTTTTGT MutA primer AGCTTCTTTCC ACTTTGTATG MRAS 3’UTR CAGCACCAAGACCTGTTCCCTT CAGTTGACTCCAAGGGAACAGG MutB primer GGAGTCAACTG TCTTGGTGCTG LCP1 3’UTR ATCTCGAGGGCCCAATGGGGCT GCGCGGCCGCGGCTACAGTAAG cloning primer GGGTGGGA TTCAATCTGAAG LCP1 3’UTR Mut GCTCCAGGGATGATTCCCAAAG CCAAGTTGAACTTTGGGAATCA primer TTCAACTTGG TCCCTGGAGC Promoter L GCGCTCGAGAGCAGGGAAGTG TATAAGCTTTCAGGGAGCTCTT cloning primer GCTCATTATTTC GGCTCAGGGAG Promoter S ATACTCGAGTGAGAGAGACCC TATAAGCTTCCCTGCTGTTTGCA cloning primer TCAGGGGGACCT ACTCTGGGCA Promoter L Mut CGGCACCGTCCAGGGGCACAA CACAGGCACTTGTGCCCCTGGA primer GTGCCTGTG CGGTGCCG Primers for ChIP assay Sense Strand (5'-3') Antisense Strand (5'-3')

Site 1 Primer ACTTCGCGGCCACTCATC CCTCACTGCCCATAGTCCTG

Site 2 Primer GGAACAGTGTATGCCCAGTCCT GGAGTAGCCCCAGCACATTCCA

Site 3 Primer AGGCTCACAGGTTTTACTCCA ACTGCAGGAATATCTAGGCTCT

Site 4 Primer TCGCACATGACCCAGACCC TGGCACTAGGCAGGCACCA

Site 5 Primer ACCCAGTGCCAATACTCATGCT CTGTGCTGGCATGGAGTTCCT

Site 6 Primer CAGAGCCCGCTTACCACGTT GGCTGGCAGGACTTTCCCC ACGGCTCCAATCCCTATATGAG Site 7 Primer GGCCCCTCCACACCCTCAG T

TFF1 site Primer CCGGCCATCTCTCACTATGAA CCTCTTTCCCATGGGAGTCTC

NC site Primer ATGGTTGCCACTGGGGATCT TGCCAAAGCCTAGGGGAAGA Primers for RNA Pull down assay Sense Strand (5'-3') Antisense Strand (5'-3')

ESR1 site GCCGTAATGATTCTATAATGCC AAATCAAAGACTGCACTTCC

ESRRA site CCATAACGTGCCCCCAGAGTGT TAGGTTTCTGCCTCCCACGCATC NCOA1 site TGAAAGCCAGCCATATTACTCT TCTGCGCGTCTAAATATCACA

PIM2 site GACCCTACTCTGTTATCCCAA CTTATTTCCCCTAGCCCAT

MRAS site 1 CAAAGTACAAAACAAGCCAT CTATAAAAGACATCCAAGCTCA

MRAS site 2 TTCATGCTGGATCTTAACCAA TCTGCTCATTGACTAGGCTT

LCP1 site GGATGATTCAAGCCATTCCAA CTCCTGCAAAGAATGAAGCAC

GAPDH site TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG Primers for qRT-PCR Sense Strand (5'-3') Antisense Strand (5'-3') GTCGTATCCAGTGCAGGGTCCG miR-135a RT AGGTATTCGCACTGGATACGAC Primer TCACAT miR-135a qRT-PCR Primer CGGCTATGGCTTTTTATTCC GTGCAGGGTCCGAGGT TGGAACGATACAGAGAAGATT snRNA U6 AGCA AACGCTTCACGAATTTGCGT pri-miR-135a-1 TATAGGGATTGGAGCCG GCAGCCTGGGTTGAGA pri-miR-135a-2 TAAAGTCTCATGTAGGGATG GCTCCACAGGCTAACG TFF1 ATACCATCGACGTCCCTCCA AAGCGTGTCTGAGGTGTCCG

NR3C2 GGCCAGCTTAATATTGTCCAG GTGCCTGAACATGAATGCTT

APC GAGGTCAGCCTAAACCCAT ACCGGAGTATTTTCAATAGCAA

MTSS1 AGGCAGATCAGCAGGAGT ACAGAAGGCTTGGTGGAA

JAK2 GAATAGCCAAAGAAAACGAT ATCTGTACCTTATTCGCTTC

SIAH1 CAGCAGTTCTTCGCAATCGT TCGCCTATGACCATTTAGCTC

HOXA10 CTTCCGAGAGCAGCAAAGCCT CTTCCGACCACTCTTTGCCGTGA

ESR1 GCATTCTACAGGCCAAATT CACAGGACCAGACTCCATAA

ESRRA CGTGGCTACCCTCTGTGACCT GCACCTCCATCCACACGCTCT

NCOA1 GAGCCAGGGACCGAGAA GCCGTGCAATACAAATCAG

PIM2 CACTGCCATTCCCGTGGAGT AAGCAGGGCACCAGAACCAA

MRAS AAAGCCTTCCATGACCTCGTT GTCTCCCCGCCATTTGGTT TCCCAATGAACCCAAACACGA LCP1 A TCATCAATTGTGTCTGGCACT

GAPDH TGCACCACCAACTGCTTAGC GGCATGGACTGTGGTCATGAG

Supplementary Table S2. List of antibodies used in western blot and chromatin immunoprecipitation assay.

Incubation Assay Antibody Origin Dilution period ERα WB mmab #sc-8005, Santa Cruz 1:500 Over night ERα IHC mmab #sc-8005, Santa Cruz 1:50 Over night pERα (S118) WB mmab #2511, Cell Signaling 1:1000 Over night pERα #64508, Cell (S167) WB rmab Signaling 1:1000 Over night ERα ChIP mmab #sc-8005, Santa Cruz 5 ug Over night ESRRA WB mmab #sc-65718, Santa Cruz 1:500 Over night ESRRA IHC mmab #sc-65718, Santa Cruz 1:50 Over night NCOA1 WB rpab #sc-8995, Santa Cruz 1:500 Over night NCOA1 IHC rpab #sc-8995, Santa Cruz 1:50 Over night PIM2 WB mmab #sc-13514, Santa Cruz 1:500 Over night PIM2 IHC mmab #sc-13514, Santa Cruz 1:50 Over night #sc-292844, Santa MRAS WB rpab Cruz 1:500 Over night #sc-292844, Santa MRAS IHC rpab Cruz 1:50 Over night #sc-133218, Santa LCP1 WB mmab Cruz 1:500 Over night #sc-133218, Santa LCP1 IHC mmab Cruz 1:50 Over night pAKT (S473) WB rpab #9271, Cell Signaling 1:1000 Over night pAKT #sc-7985-R, Santa (S473) WB rpab Cruz 1:1000 Over night pAKT (S473) IHC rpab #9271, Cell Signaling 1:100 Over night AKT1 WB mmab #sc-5298, Santa Cruz 1:1000 Over night pERK1/2 (T202/Y204) WB mmab #9106, Cell Signaling 1:1000 Over night pERK1/2 (T202/Y204) IHC mmab #9106, Cell Signaling 1:100 Over night tERK1/2 WB mmab #13-6200, Zymed 1:1000 Over night TWIST1 WB rpab #sc-15393, Santa Cruz 1:1000 Over night EZH2 WB rpab #5246, Cell Signaling 1:1000 Over night FN1 WB mmab # 610077, BD 1:5000 Over night #13409-1-AP, Occludin WB rpab Proteintech 1:1000 Over night γ-catenin WB mmab #sc-8415, Santa Cruz 1:1000 Over night #60008-1-Ig, β- WB mmab Proteintech 1:5000 Over night

Abbreviations: WB, Western blot; ChIP, chromatin immunoprecipitation; mmab, mouse monoclonal antibody; rmab, rabbit monoclonal antibody; rpab, rabbit polyclonal antibody. Supplementary Table S3. The correlation of MIR135A1 deletion with the clinicopathologic features of breast cancer in the TCGA breast tumor dataset Parameter n MIR135A1 loss, n (%) p value Menopause pre 213 78 (37) 0.072 peri 35 14 (40) post 649 190 (29) Age <=35 29 15 (52) 0.006 35-55 401 139 (35) >55 573 162 (28) Vital status alive 906 277 (31) 0.052 dead 97 39 (40) Recurrence free 746 225 (30) 0.294 recurred 63 23 (37) Histological type IDC 730 265 (36) 0.000 ILC 177 33 (19) Other types 95 18 (19) Pathologic T T1 262 57 (22) 0.000 T2 582 213 (37) T3* 156 45 (29) Pathologic stage I 169 40 (24) 0.052 II 570 191 (34) III* 246 79 (32) Estrogen receptor － 215 112 (52) 0.000 ＋ 744 192(26) Progesterone receptor － 308 151 (49) 0.000 ＋ 648 153 (24) HER2 low 306 104 (34) 0.447 high 525 165 (31) * includes higher categories. Supplementary Table S4. The correlation of pri-miR-135a-1 expression with the clinicopathologic features of breast cancer in the TCGA breast tumor dataset Parameter n Lower pri-miR-135a-1, n (%) p value Menopause pre 213 78 (37) 0.000 period 35 22 (63) post 649 358 (55) Age <=35 29 13 (45) 0.041 35-55 401 182 (45) >55 573 306 (53) Vital status alive 906 453 (50) 0.989 dead 97 48 (50) Recurrence free 746 369 (50) 0.353 recurred 63 35 (56) Histological type IDC 730 366 (50) 0.989 ILC 177 88 (50) Other types 95 47 (50) Pathologic T T1 262 132 (50) 0.746 T2 582 294 (51) T3* 156 75 (48) Pathologic stage I 169 79 (47) 0.148 II 570 285 (50) III* 230 129 (56) Estrogen receptor － 215 132 (61) 0.000 ＋ 744 339 (46) Progesterone receptor － 308 193(63) 0.000 ＋ 648 277 (43) HER2 low 306 174 (57) 0.004 high 525 244 (47) * includes higher categories. Supplementary Table S5. List EMT markers examined in the Oxford breast tumor set CDH1, CDH2, VIM, FN1, SNAI1, SNAI2, TCF3, TWIST1, ZEB1, JUP, CTNNB1, ZEB2, TJP1, OCLN, GJA1, GJB1, CTNNA1, FOXC2, MMP2, MMP9, EZH2 ERα cofactors and interacting AHR, BCAR1, BRCA1, CAV1, CCND1, EFNA5, ESR1, ESR2, ESRRA, ESRRB, ESRRG, IGF1, MTA1, NCOA1, NCOA2, NCOA3, NCOR1, NCOR2, NR0B1, NR0B2, NRIP1, PELP1, PHB2, SAFB, TFF1, VDR Predicted miR-135a targets using TargetScan Version 6.2, which mRNA levels are negatively correlated with miR-135a expression in breast cancer tissues (GSE22220, p<0.05) CXCL10, ADAM8, LRP8, SLC1A3, POLQ, IQGAP3, N4BP1, MKLN1, SOX11, CXCL9, NFE2L3, BIRC5, KIF23, CCR1, CHST11, MYD88, PVR, CTPS, TREM1, CCL7, RASAL1, CD86, UHRF1, STK17B, ADCY7, RIPK2, GOPC, LOC221710, SF3B3, C21orf91, MRPS12, AQP9, KCNE3, NFKBIE, GCH1, COTL1, MKI67, PMM2, CALU, NIPA2, OAS3, NINJ2, FUT4, NAPG, UBE2L6, ALDH1B1, MAPK1, ODC1, IL10RA, DOCK2, RORA, BCAT1, PIM2, SLA, YKT6, C7orf29, FMNL2, GBP4, NLN, KCNJ10, EIF2C3, CAMK2D, CTSS, BID, ACPP, C9orf25, NPR3, CTSC, LIMK2, TOMM34, EOMES, GPR65, ATP6V1C2, TOM1L2, DIAPH3, KCNG1, P2RX7, PRDM1, LCP1, TIMELESS, TRIM16, EMILIN2, GJB5, STX6, CCNF, TGM2, SLC31A1, STAT2, EIF5A2, DHODH, SQSTM1, OAS2, CEBPG, ITK, EHD4, ANGPT1, PCNX, B4GALT5, SEC14L4, MMP13, DHDDS, POP1, YBX2, XK, ATP6V1E1, GPR3, PGAM1, FBXW2, ZBED4, ARFGEF2, FKBP11, SH2D1A, TES, GM2A, KHDRBS3, KIAA1609, INHBA, CORO1C, PFKFB4, SSR1, SNTB1, PRKAA1, INPP4A, CD68, SYNGR1, GABPB2, DDN, HMGB2, VPS4A, RTKN2, RRBP1, SLC5A6, VAPB, EYA3, PRKX, SCNM1, NHLRC1, RRAGD, TRIM11, PLXNA2, SLC5A3, DGKH, SPN, NR2C2, MEF2D, PIK3CD, ARIH1, GMEB1, MXD3, GMEB2, ESRRA, PAQR3, MFHAS1, HPS5, HRH1, PHTF2, COL4A1, ACTR1A, AKR1A1, RNF24, MRAS, LOC401097, RASSF2, EFS, PLCG1, APOBEC2, F11R, TIMM8B, RASAL2, ZFP30, NSMAF, NIN, HPCAL4, KCNS1, AKAP2, DISC1, UCHL5, MMP11, KIAA0226, DDHD1, LYNX1, APOL3, ETV6, CDX2, PTPRJ, ATP6V1C1, ZFP91, IPMK, ANKRD1, GNAI3, COX5A, MGAT4A, PRPF4B, SLC13A4, TLL2, MID1, TGFA, GABRA5, UBE2V1, TLR8, MLANA, ZNF365, CBLB, TOMM22, NPC1, PCDHGB4, SLC31A2, SMURF2, ATP11C, NDRG4, OSBPL6, ITGAV, KIAA1033, MOV10, SHB, SNTA1, SIX4, MB, SHC3 Supplementary Figure Legends

Fig. S1 MIR135A1 gene copy number and miR-135a-1 expression are decreased in breast cancer. A, The copy number of human 3p miRNA was determined by genomic qPCR in 4 benign breast tissues and 20 breast cancer specimens. ACTB, GAPDH and SSTR2 were used as control. B, The reads per million (RPM) of pri-miR-135a-1 in TCGA breast cancer samples with deleted or non-deleted MIR135A1 locus. C, The RPM of pri-miR-135a-1 and pri-miR-135a-2 in

TCGA breast cancer samples. D-F, The expression level of miR-135a in Oxford breast tumors (GSE22216, n=210) with different status of distant relapse (DR), tumor size and histological grade. B-E, Mann-Whitney test, F, Kruskal-Wallis test.

Fig. S2 miR-135a inhibits cell proliferation, survival, epithelial-mesenchymal transition (EMT), migration and invasion of breast cancer cells. A, The expression levels of miR-135a in breast cancer cells stably transfected with plasmids containing pri-miR-135a-1 or miR-135a-1 sponge, or the empty vector, were determined by qRT-PCR. snRNA U6 was used as internal control. B, The cell viabilities of MCF-7-sponge cells transfected with miR-135a mimics (left), or T47D

(middle) and MDA-MB-231 (right) cells with forced expression of miR-135a or sponge were determined by MTT assay over a period of 5 days. C, The cell cycle progression and D, early apoptotic population in T47D and MDA-MB-231 cells with forced expression of miR-135a or sponge were determined by flow cytometric analyses. E, The 3D growth in matrigel and soft agar colony formation of T47D (left) and MDA-MB-231 (right) cells were quantified. F-G, Transwell migration and invasion assays of MCF-7 (left) and T47D (right) cells. (F) Representative pictures were taken at 200X magnification. (G) The number of crystal violet stained colonies was counted in 5 different fields in each well and triplicates were determined for each sample. H, The correlation between the levels of miR-135a and the EMT markers in

Oxford breast tumor set was analyzed using Pearson’s correlation coefficient (R). The heatmap diagram represents the log2 transformed value of median centered gene levels in samples. I, The quantification of Fig. 2K data. N indicates the number of independent repeats. Results are presented as mean ± S.D. *P<0.05; **P<0.01;

***P<0.001; ns, not significant (ANOVA test). Fig. S3 miR-135a impairs the in vivo tumor growth. A, The image of tumors from

MCF-7 stably transfected with miR-135a or sponge, or empty plasmid was shown. B,

The expression level of miR-135a in tumors (A) was assessed by ISH. Fig. S4 miR-135a-1 transcription is positively regulated by ERα. A, The expression of miR-135a was compared in ERα negative and positive breast tumors

(GSE22216, n=210). B, Top panel, The expression of miR-135a in MCF-7 cells upon forced expression of ERα or treatment with 1µM fulvestrant (ICI 182,780) as determined by qRT-PCR. Bottom panel, The ERα protein levels in the respective

MCF-7 cells were determined by western blot. snRNA U6 was used as miRNA input control in qRT-PCR and β-actin was used as input control in western blot. C, The quantification of Fig. S2B data. D-E, The expression levels of pri-miR-135a-1 and pri-miR-135a-2 in MCF-7 cells at the indicated periods after (D) hormone starvation

(E) and subsequent treatment with 10nM E2 were determined by qRT-PCR. GAPDH was used as an input control. F, The schematic representation of ERα binding site

(solid black bar) upstream of the MIR135A1 gene based on the ChIP-Seq data in

U2OS-ERα cells. Red arrows show the direction of transcription of pri-miR-135a-1 and pri-miR-135a-2. G, Top panel, Schematic representations of the TFF1 promoter region with an ERα binding site (positive control) and a DNA region at chromosome

12p13.3 with no predicted ERα binding sites (negative control). Red bars represent amplicons in the post-ChIP qPCR analysis. Bottom panel, MCF-7 cells were treated with 10nM E2 for the indicated durations and the cross-linked whole cell extracts were subjected to ChIP. The amount of enrichment of both the negative (left) and positive (right) control DNA regions in the ERα- and RNA polymerase

II-immunoprecipitated samples were plotted. H-I, The western blot analysis (H) and quantified result (I) of ERα protein levels in HEK-293T cells with forced expression of ERα (top) and in MCF-7 cells. J, MCF-7 cells were transfected with either the luciferase reporter plasmid containing the MIR135A1 promoter (Pro L) or the empty vector. The luciferase reporter activities in the transfected cells treated with 10nM estrogen and/or 1µM tamoxifen/fulvestrant were determined. Renilla luciferase activity was used as input control. N indicates the number of independent repeats.

Results are presented as mean ± S.D.. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (A, Mann-Whitney test; others, ANOVA test).

Fig. S5 miR-135a regulates estrogen-independent growth and tamoxifen resistance in ERα+ breast cancer cells. A-B, (A) Cell viability and (B) foci formation of T47D cells stably transfected with either the empty vector or the miR-135a sponge. The cells were hormone starved for 6 days and then cultured with or without E2 for 6 days in the MTT assay and for 12 days in the foci formation assay.

C-D, (C) Cell viability and (D) foci formation of MCF-7 parental and tamoxifen resistant (TamR) cells treated with 1µM tamoxifen or EtOH vehicle for 6 days in the

MTT assay and for 12 days in the foci formation assay. E-F, (E) Cell viability and (F) foci formation of T47D parental and TamR cells treated with 1µM tamoxifen or EtOH vehicle for 6 days in the MTT assay and for 12 days in the foci formation assay. G,

The expression level of miR-135a in tamoxifen resistant cells and parental cells was shown. Data were derived from GSE56411. H, The expression level of miR-135a in primary lesion or tamoxifen resistant, recurrent lesion was shown. Data were derived from GSE83292. I, miR-135a expression in MCF-7 parental and TamR cells treated with 1µM tamoxifen for the indicated time points were analyzed by qRT-PCR. snRNA U6 was used as miRNA input control. J, Cell viabilities of MCF-7-vector and

–miR-135a cells treated with 1µM tamoxifen or EtOH vehicle for 6 days were determined by MTT assay. K, Cell viabilities of T47D-vector, –miR-135a and -sponge cells treated with 1µM tamoxifen or EtOH vehicle for 6 days were determined by

MTT assay. L, The expression level of miR-135a in tumors (Fig.5K) was assessed by

ISH. The percentages of tamoxifen-mediated decrease in cell viabilities were shown.

Results are presented as mean ± S.D. in A, C, E, G-K. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (ANOVA test).

Fig. S6 Validation of miR-135a targets in breast cancer cells. A-B, The expression of previously published miR-135a targets in (A)MCF-7 and T47D cells, or (B)

MDA-MB-231 cells transfected with miR-135a mimics or control scrambled oligonucleotides was analyzed by qRT-PCR. GAPDH was used as input control. C,

The correlation between the levels of miR-135a and verified miR-135a targets in

Oxford breast tumor set was analyzed using Pearson’s correlation coefficient (r). The

X axis represents log2 transformation of miR-135a level; the Y axis represents log2 transformation of the miR-135a target mRNA levels. D, The quantification of western blotting data from Fig. 6F. E-F, The western blot analysis (E) and quantified result (F) of protein levels of ESR1, ESRRA, NCOA1, PIM2, MRAS and LCP1 in T47D cells stably transfected with plasmids containing miR-135a or sponge, or the empty vector.

β-actin was used as input control. G, The protein levels of the miR-135a targets in tumors (Fig. 3A) were assessed by IHC. H, TFF1 mRNA levels (positive control) in

MCF-7 and T47D cells transfected with miR-135a mimics or control scrambled oligonucleotides were determined by qRT-PCR. GAPDH was used as input control.

I-J, The western blot analysis (I) and quantified result (J) of protein levels of the miR-135a targets in MCF-7 cells transfected with respective shRNA plasmids. β-actin was used as input control. K, Cell viabilities of MCF-7 cells transfected with shRNA plasmids targeting the respective miR-135a targets over a period of 6 days were determined by MTT assay. shEEN represents shRNA that targets ESR1, ESRRA and

NCOA1, while shPML represents shRNA that targets PIM2, MRAS and LCP1. L,

Migration and invasion of MCF-7 cells transfected with the indicated shRNA plasmids were determined by the transwell assay. M-N, The western blot analysis (M) and quantified result (N) of protein levels of miR-135a targets in MCF-7 and T47D parental and tamoxifen resistant (TamR) cells. β-actin was used as input control. O,

MCF-7 TamR cells transfected with the indicated shRNA plasmids and treated with

1µM tamoxifen or EtOH vehicle for 6 days. The resulting cell viabilities were determined by MTT assay. N indicates the number of independent repeats. Results are presented as mean ± S.D. . *P<0.05; **P<0.01; ***P<0.001; ns, not significant

(ANOVA test). Fig. S7 miR-135a modulates ERα-ERK1/2-AKT crosstalk in breast cancer cells.

A-B, The quantification of (A) Fig. 7A and (B) Fig. 7B. C, The protein levels of phosphorylated ERK1/2 and AKT in tumors (Fig. 3A) were assessed by IHC. D-E,

The western blot analysis (D) and quantified result (E) of protein levels of phosphorylated ERK1/2 and AKT in MCF-7 and T47D tamoxifen resistant cells, and respective parental cells. F-G, The western blot analysis (F) and quantified result (G) of protein levels of phosphorylated ERK1/2 and AKT in MCF-7 cells treated with

10µM MEK1/2 inhibitor (U0126) and/or 100nM AKT inhibitor IV (AI IV). H,

MCF-7 TamR cells were treated with 10µM U0126 and/or 100nM AI IV in the presence of either 1µM tamoxifen or ethanol vehicle. Cell viability was determined by

MTT assay. I-J, The western blot analysis (I) and quantified result (J) of protein levels of phosphorylated ERα, ERK1/2 and AKT in MCF-7 cells transfected with

ERK1/2 siRNA and/or AKT siRNA. K-L, The western blot analysis (K) and quantified result (L) of protein levels of phosphorylated ERα in MCF-7 cells treated with 10µM U0126 and/or 100nM AI IV in the presence of 10nM estrogen. M, The quantification of Fig. 7E. N-Q, The western blot analysis (N, P) and quantified result

(O, Q) of (N, O) MCF-7 TamR cells, and (P, Q) MCF-7 Sponge cells treated with

10µM U0126 and/or 100nM AI IV, and respective control cells. N indicates the number of independent repeats. Results are presented as mean ± S.D.. **P<0.01;

***P<0.001; ns, not significant (ANOVA test). Fig S1

A Normal Breast cancer B TCGA BRCA C TCGA BRCA

hsa-miR-3135a Gain P<0.001 hsa-miR-466 P<0.001 hsa-miR-128-2 hsa-miR-1226 hsa-miR-3134 hsa-miR-4793 Normal hsa-miR-4791 hsa-miR-5688 hsa-miR-885 hsa-miR-3714 Loss hsa-miR-563 hsa-miR-26a-1 hsa-miR-2115 1 RPM

hsa-let-7g - hsa-miR-135a-1 hsa-miR-138-1 hsa-miR-711 hsa-miR-3938 hsa-miR-4270 135a - hsa-miR-4787 hsa-miR-378b

hsa-miR-1324 eads Per Million

hsa-miR-4273 miR R hsa-miR-4443 hsa-miR-3923 hsa-miR-4792 hsa-miR-564 hsa-miR-4271 hsa-miR-5193 hsa-miR-4442 hsa-miR-191 hsa-miR-425 Not Deleted Deleted miR-135a-1miR-135a-2 hsa-miR-4444-2 hsa-miR-566 N=654 N=308 N=1206 N=1206 E F D GSE22216 GSE22216 GSE22216 16 P=0.075 P=0.019 DR Free Patients(N=131) vel 14 DR Patients(N=79) P<0.001 135a le 135a level - 135a level - - 12 R

10 elative miR R Relative mi Relative miR

8 0% 20% 40% 60% 80% 100% <=2cm >2cm Grade I Grade II Grade III Percent of patients N=86 N=124 N=42 N=81 N=63 Fig S2 A B *** Vector 64 miR-135a MCF-7 T47D MDA-MB-231 *** 1.5 Sponge Vec Vec

n of *** 1.5 16 miR-135a miR-135a *** *** *** *** 1 Sponge Sponge 4 1 *** 135a/U6 ** *** - 0.5 miR 1 0.5 elative expressio Relative cell viability

R * * ** ** 0.25 0 0 MCF-7 TamR T47D MDA-MB-231 d. 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 C G2/M S G0/G1 D E T47D MDA-MB-231 100 15 ** 1.5 Vec 3D culture 3D culture *** ls miR-135a 1.5 75 Soft Agar Soft Agar *** ** Sponge ns *** ** 10 1 ** ***

** *** 1 50 *** *** *** *** 5 0.5 135a % of gated % of cel 135a

0.5 Fold changes - 25 - * *** Fold changes Apoptosis cell (%) Vec miR Sponge Vec miR Sponge 0 0 0 0 T47D MDA-MB-231 T47D MDA-MB-231 miR-135a Sponge miR-135a Sponge

F MCF-7 T47D G Vec miR-135a Sponge Vec miR-135a Sponge MCF-7 n miR-135a 2 Sponge ***

Migratio **

1 ** Fold Fold changes ion ***

Invas 0 Migration Invasion H GSE22220 -0.5 -0.3 -0.2 0 0.2 0.3 0.5 miR-135a Pearson R P-value T47D GJA1 0.289 2.43E-05 CTNNA1 0.134 0.054 OCLN 0.194 0.0050 miR-135a *** JUP 0.124 0.074 CDH1 0.042 0.55 3 Sponge CTNNB1 0.099 0.16 TCF8 0.082 0.24 *** TJP1 0.054 0.44 SNAI2 0.011 0.88 VIM 0.031 0.65 2 ZFHX1B -0.072 0.31 MMP2 0.033 0.64 TWIST1 -0.132 0.058 MMP9 -0.269 8.61E-05 1

GJB1 -0.033 0.63 Fold changes FOXC2 0.023 0.74 *** FN1 -0.218 0.0016 ** SNAI1 -0.179 0.0097 CDH2 -0.087 0.21 0 TCF3 -0.076 0.28 EZH2 -0.328 1.41E-06 Migration Invasion I MDA-MB-231 N=4~5 MCF-7 N=4~5

* * ** ** * *** ** * * * *** malized intensity *** * ** Fold Fold changes of ** ** ** *** ** ** nor

Twist1 Ezh2 FN1 Occludin γ-catenin Twist1 Ezh2 FN1 Occludin γ-catenin Fig S3

A B Vec miR-135a Sponge

Vector

miR-135a

Sponge Fig S4 A D MCF-7 –E2 E MCF-7 +E2 GSE22220 1.5 3 miR-135a-1 ns P=0.007 miR-135a-1 ** miR-135a-2 miR-135a-2

ns 1 ns 2 * * * ns * 135a level ns

- ns ns *** 0.5 *** 1 ns ns ns tive miR Relative Expression Relative Expression Rela

0 0 ERα Negative Positive d. 0 2 4 6 h. 0 1 4 12 24 48 N=80 N=127 U2OS-ERα binding Sequence B MCF-7 F Chr 3 hsa-miR-135a-1 ERα-6291 H HEK293T 2 ** 52328587 52329259 52329931 52330603 52331275 Chr 12 hsa-miR-135a-2 EV ERα 1.5 97954423 97955256 97956089 97956922 97957755 pression of ERα 135a/U6 - 1 ns G 1k bps miR * N.C. 12p13.3 0.5 β-Actin

Relative ex ERα site TFF1. TFF1 0 MCF-7 ICI: ﹣ ﹣ + + 25 ERα 10nM E2 0h *** *** ERα: ﹣ + ﹣ + 10nM E2 6h shScr shERα 15 10nM E2 24h ERα 5 ERα

β-Actin N.C. TFF1 5 β-Actin * 15

Fold Fold change % Input of *** 25 Pol II

C I J MCF-7 5 MCF-7 N=3 HEK-293T N=3 MCF-7 N=3 EV Pro L * 4 *** ** * * 3 ** *** RLuc / ns ns tensity normalizedof 2

* intensity in fLuc ns α α Relative activity of

ER ** ER 1 d changes normalizedof Fol Fold Fold changes ** 0 ICI: ﹣﹣ + + EV ERα shScrshERα 10nM E2: - - - ERα: ﹣ + ﹣ + + + + 1µM Tam: - + - - + - 1µM ICI: - - + - - + Fig S5

A B C DMCF-7 T47D T47D MCF-7 1.5 E2 + - 1.5 Tam ﹣ + +E2 -E2 EtOH Tam ns ** *** 4%

*** 11% 7 - 1 25% 1 37% Vec MCF

lative cell viability 0.5 0.5 Re Relative cell viability TamR Sponge

0 0 Vec Sponge MCF-7 TamR

E T47D F T47D I 1.5 Tam ﹣ + 1.5 MCF-7 TamR EtOH Tam *** 1uM Tam 1 27% 1 iability T47D ns * ** 13% *** 135a/U6 -

0.5 miR 0.5 *** Relative cell v Relative expression of

TamR ns

0 0 T47D TamR d. - 1 2 3 4 - 4 G J K MCF-7 T47D GSE56411 1.5 1.5 EtOH Tam * 12% EtOH Tam *** *** 1 1 47% *** 36% *** 135a - 63%

e expression of 65% miR 0.5 0.5 Relative cell viability Relative cell viability

Relativ *** *** *** 0 0 MCF-7 TamR-1 TamR-4 TamR-8 Vec miR-135a Vec miR-135a Sponge

H Par Vec Par Sponge TamR Vec TamR miR-135a GSE83292 L No No Tam n level of 135a - miR Relative expressio With Tam With

Primary lesion Relapse lesion Fig S6 A ns B D MCF-7 N=4~5 2 MCF7 Control 3 Control MCF7 miR-135a * T47D Control miR-135a ns * T47D miR-135a ns **

ssion * ns ns 2 ns ** ns ns ** *** 1 ns ** 1 *** ** HOXA10/GAPDH Fold Fold changes of ** ** ** ** Relative expre normalized intensity Relative expression of 0 0 NR3C2 APC MTSS1 JAK2 SIAH1 MDA-MB-231 ERα ERRα NCOA1 PIM2 MRAS LCP1 C H -0.5 -0.3 -0.2 0 0.5 GSE22220 0.2 0.3 Control miR-135a miR-135a Pearson R p-value 1 ESR1 0.381 1.51E-08 NCOA1 -0.023 0.75 ESRRA -0.175 0.012 *** 0.5 MRAS -0.166 0.017 *** LCP1 -0.230 8.63E-04 TFF1/GAPDH

PIM2 -0.264 1.20E-04 Relative expression of 0 MCF-7 T47D

E I M MCF-7 T47D T47D shScr shLCP1 shScrshNCOA1 Parental TamR Parental TamR LCP1 LCP1 NCOA1 LCP1 MRAS β-Actin β-Actin MRAS shScr shMRAS shScr shESRRA PIM2 PIM2 MRAS ERRα NCOA1 NCOA1 β-Actin β-Actin ERRα ERRα shScr shPIM2 shScr shESR1

ERα PIM2 ERα ERα

β-Actin β-Actin β-Actin β-Actin

MCF-7 N=3 N=3 F T47D N=3 J N * * * ** * * * * * d intensity ** * * * * ** * ** * * * ** ns

** Fold changes of

Fold Fold changes of * * * * ** Fold changes of * normalized intensity normalize *** normalized intensity

ERα ERRα NCOA1 PIM2 MRAS LCP1 ERα ERRα NCOA1 PIM2 MRAS LCP1 ERα ERRα NCOA1 PIM2 MRAS LCP1 G ERα ERRα NCOA1 PIM2 MRAS LCP1 Mouse IgG c Ve 135a - miR Rabbit IgG onge Sp

K MCF-7 L O MCF-7 TamR MCF-7 shScr ns ns EtOH Tam shESR1 shScr 2% 7% ns 1 shESRRA shE-E-N ***14% *** 1 shP-M-L 12% *** shNCOA1 ns 16% *** shE-E-N 1 ** 7% 29% ges shPIM2 ll viability 18% shMRAS *** *** *** shLCP1 *** *** 33% 0.5 shP-M-L 0.5 0.5 *** Fold Fold chan Relative ce Relative cell viability

0 0 0 d. 1 2 3 4 5 Migration Invasion shRNA: Scr ESR1 ESRRA NCOA1 E-E-N PIM2 MRAS LCP1 P-M-L Fig S7

A MCF-7 N=3 B MCF-7 N=3 C Vec miR-135a Sponge

ns S473 ns pAKT ns ns ns * * ** ns ns ns ns d intensity * * * ns ns * * ns ns * * * * *** Fold Fold changes of *** **** Fold changes of normalize ** normalized intensity ** * pERK1/2 T202/Y204

pERK tERK pAKT tAKT pERK tERK pAKT tAKT MCF-7 N=3 G 2 D MCF-7 T47D F MCF-7 Veh U0126: U0126 Parental TamR Parental TamR ﹣ + ﹣ + AI IV: ﹣ ﹣ + + AI IV pAKT U0126+AI IV ns pAKT ns S473 ns ns S473 ns 1 tAKT * tAKT * *** pERK1/2 Fold changes of ** **

normalized intensity ** T202/Y204 pERK1/2 ** T202/Y204 tERK1/2 0 tERK1/2 pERK tERK pAKT tAKT

β-Actin K MCF-7 β-Actin E2: - + + + + E U0126: - - + - + N=3 AI IV: - - - + + * H MCF-7 TamR pERα * ns EtOH S167 0% * 1 Tam ** ** pERα 21% S118 ns ns ns * ns β-Actin

Fold Fold changes of 0.5 normalized intensity ***

Relative cell viability 64%*** MCF-7 N=3 71% L pERK tERK pAKT tAKT 0 U0126: - + - + AI IV: - - + + I MCF-7 siERK1/2: - + - + siAKT1: - - + + Fold Fold changes of * pERα normalized intensity ** ** ** *** S167 *** *** *** pERα J S118 S167 S118 MCF-7 N=3 pERα pAKT M MCF-7 N=3 S473 ns ns tAKT * * * ns ** * * pERK1/2 * * * *** ** T202/Y204 Fold changes of ** ** ** ** ** normalized intensity *** ** ** tERK1/2 Fold Fold changes of

normalized intensity ** ** pERK tERK pAKT tAKT pERα S118 S167 β-Actin P pERα S118 S167 N=3 MCF-7 MCF-7 N=3 N O Vector Sponge Q 2 MCF-7 Vector+Veh U0126: - - + - + Sponge+Veh Parental TamR AI IV: Sponge+U0126 * - - - + + Sponge+AI IV pERα pERα Sponge+U0126+AI IV S167 ** S167 1 pERα pERα S118 S118 rmalized intensity

Fold Fold changes of * Fold Fold changes of ** ** no normalized intensity ** ** ** ** β-Actin β-Actin *** 0 pERα S118 S167 pERα S118 S167