SUPPLEMENTARY DATA

14-3-3 Proteins Modulate the ETS Transcription Factor ETV1 in Prostate Cancer

Sangphil Oh, Sook Shin, Stan A. Lightfoot, Ralf Janknecht Supplementary Methods: Coimmunoprecipitation Assays and Western Blotting Transfected cells from one 6-cm dish were lysed in 650 µl of 50 mM Tris (pH 7.4), 150 mM NaCl, 50 mM NaF, 0.5% Igepal CA-630, 0.1 mM Na3VO4, 1 µg/ml pepstatin A, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 0.2 mM DTT at 4°C. Immunoprecipitations were conducted with anti-Myc 9E10 mouse monoclonal antibody or our own ETV1 antibody #959 (see below details for #959 in the ChIP section). For 14-3-3 Western blotting, we utilized rabbit polyclonal antibodies for 14-3-3τ (C-17; Santa Cruz Biotechnology sc-732) and 14-3-3β/ζ (Upstate #06-351) and mouse monoclonal antibodies for 14-3-3ε (8C3; Santa Cruz Biotechnology sc-23957) and 14-3-3σ (CS112-2A8; Upstate #05-632). To probe for ETV1 in Western blots, we employed either a commercially available rabbit polyclonal antibody (Abcam ab81086) or our own ETV1 antibody #959 or, in case of 6Myc-tagged ETV1, the 9E10 anti-Myc mouse monoclonal antibody. Supplementary Methods: RT-PCR The PCR program used was 45 min at 48°C followed by 2 min of denaturation at 96°C; 26 (MMP-1) or 18 (GAPDH) cycles of 95°C for 30 s, 55°C for 45 s, 68°C for 45 s; and a final extension at 68°C for 4 min. The DNA sequences of the primers were: MMP-1, 5’-GTTCAGGGACAGAATGTGCTA-3’ and 5’-CTGCAGTTGAACCAGCTATTAG-3’ (350 bp product) or, in Figure 1F, 5’-ACCTGAAGAATGATGGGAGGCAAGT-3’ and 5’-GTCCCGATGATCTCCCCTGACA-3’ (383 bp product and 60°C annealing temperature); GAPDH, 5’-GAGCCACATCGCTCAGACACC-3’ and 5’-TGACAAGCTTCCCGTTCTCAGC-3’ (226 bp product). RT-PCR on retrovirally infected LNCaP cells was similarly performed except that sometimes a nested PCR was added in case of MMP-1 with nested primers 5’-ACCCCAAGGACATCTACAGCTCCT-3’ and 5’-GGCCAATTCCAGGAAAGTCATGTGC-3’, resulting in a 192 bp product. Primers for ETV1 were 5’-TCCCTCCATCGCAGTCCATACCAG-3’ and 5’-GTGGCAGCTAGGCACTTCTGAGTC-3’ (314 bp product) and primers for MMP-7 were described before (Shin et al, 2013). Supplementary Methods: Chromatin Immunoprecipitation (ChIP) Assay For immunoprecipitation, we employed the following commercially available antibodies (Santa Cruz Biotechnology): sc-732 for 14-3-3τ and sc-1019 for 14-3-3ζ. In addition, an ETV1 antibody (#959) was raised in rabbits directed against the peptide GLPLKIKKEPHS(phos)PCSELGSAC and affinity-purified; this antibody was successfully

utilized before in ChIP assays (Shin et al, 2008; Shin et al, 2009; Shin et al, 2013). The following PCR program was employed to amplify the MMP-7 promoter: 2 min at 98°C followed by 6 cycles of 30 s at 98°C, 30 s at 64°C (-1°C per cycle), 25 s at 72°C, and another 20 cycles of 30 s at 98°C, 30 s at 58°C, 25 s (+1 s per cycle) at 72°C, and a final extension at 72°C for 4 min. The DNA sequences of the primers were: MMP-7-for-1, 5’-GTCCTGAATGATACCTATGAGAGC-3’; MMP7-rev-1, 5’-CCAGAGACAATTGTTCTTGGACC-3’; MMP-7-for-2, 5’- CATGGAGTCAATTTATGCAGCAGAC-3’. PCR was performed first with MMP-7-for-1/MMP-7-rev-1 primers and then with MMP-7-for-2/MMP-7-rev-1 primers. Primers for MMP-1 in the first PCR were MMP1-ChIPfor-new (5’-CCTGTAGCACCTTATGACCATCAG-3’) and MMP1-ChIPrev-new (5’-GTGGAGGAAAGCTGTGCATACTGG-3’) and in the nested PCR MMP1-ChIPfor (5’-GACCATGGAGTACTCTTTGACCTG-3’) and MMP1-ChIPrev (5’-CAGTGCAAGGTAAGTGATGGCTTCC-3’). Resulting 270 bp MMP-7 and 353 bp MMP-1 promoter fragments were visualized on agarose gels. Primers for the human MDM2 gene promoter resulting in a 338 bp product were published before (Shin and Janknecht, 2007). Supplementary Figure S1. Affinity purification of ETV1 protein complexes. ETV1 cDNA was cloned into the pNTAP-B vector (Stratagene) that contains two N-terminal tags: a streptavidin and a calmodulin binding peptide. This plasmid was then transiently transfected into human embryonic kidney 293T cells. Cells from approximately twenty 10-cm dishes were pooled, lysed and a tandem-affinity purification on streptavidin and calmodulin resin performed according to the manufacturer’s recommendations (Interplay NTAP system, Stratagene). Tandem-affinity purified ETV1-containing protein complexes were resolved by SDS-PAGE and silver-stained. Proteins identified by mass spectrometry are indicated by arrows. Supplementary Figure S2. Intracellular localization of ETV1 and 14-3-3τ in LNCaP cells. Cells were transfected with with 6Myc-ETV1 in a 6-cm dish. 20 h thereafter, cells were split and seeded onto coverslips coated with poly-L-lysine in 12-well plates. After another 20 h, cells were fixed with formaldehyde and processed for staining as described (De Haro and Janknecht, 2002). 9E10 anti-Myc mouse monoclonal antibody and C-17 14-3-3τ (Santa Cruz Biotechnology sc-732) rabbit polyclonal antibody were employed, followed by indirect staining with goat anti-mouse antibodies coupled to FITC and goat anti-rabbit antibodies coupled to rhodamine. Pictures were taken utilizing a Leica SP2 confocal microscope. Supplementary Figure S3. Quantitative real-time RT-PCR was performed utilizing the MiniOpticon PCR system and corresponding SYBR green-based kits from Bio-Rad. Levels of ETV1 mRNA were normalized to GAPDH mRNA levels, with the respective control set to 1. (A) Samples corresponding to lanes 1, 3 and 4 in Figure 3A. (B) Samples corresponding to lanes 1, 4 and 5 in Figure 3B. (C) Samples corresponding to Figure 3D. (D) Samples corresponding to

Figure 3E. (E) As for LNCaP cells shown in Figure 3E, HER2/Neu or Ras-G12V expression did not alter ETV1 transcription in human PC3 prostate cancer cells. Supplementary Figure S4. Migration and invasion of benign RWPE-1 prostate cells. (A) Representative wound closure assay showing the width of the wound at 0 h and 25 h for RWPE-1 cells infected with control, ETV1 or ETV1-A216 retrovirus. (B) Representative staining of RWPE-1 cells that invaded through a matrigel-covered membrane with 8 µm pores. Supplementary Figure S5. Specificity of rabbit polyclonal 14-3-3τ (C-17, Santa Cruz Biotechnolgy sc-732) and mouse monoclonal 14-3-3ε (8C3, Santa Cruz Biotechnology sc-23957) antibodies. Protein extracts from human LNCaP prostate cancer cells, human embryonic kidney 293T cells or from 293T cells transfected with 6Myc-tagged 14-3-3τ, ε, ζ or σ were utilized for Western blotting employing 14-3-3ε or τ antibodies. Similar expression levels of 6Myc-tagged 14-3-3 proteins were confirmed by anti-Myc Western blotting; please note that the 6Myc-tag adds ~15-20 kDa in apparent molecular weight onto 14-3-3 proteins. Anti-actin Western blotting served as a loading control. 14-3-3τ (14-3-3ε) antibodies recognized one protein band in Western blots of LNCaP and 293T cell extracts and did not cross-react with ectopically expressed 14-3-3ε (14-3-3τ), 14-3-3ζ or 14-3-3σ. Supplementary Figure S6. Overexpression of 14-3-3ε in prostate tumors. (A) Analysis of publicly available microarray data (Welsh et al, 2001; Singh et al, 2002; Wallace et al, 2008) through the Oncomine webtool. Shown are log2 median-centered ratios of 14-3-3ε mRNA levels with the median and 25-75 percentile range. N denotes normal prostate tissue, C prostate carcinoma. Statistical significance was determined with Student’s t-test. (B) Immunohistochemical analysis of 14-3-3ε expression in 31 pairs of human prostate tumors and matching normal tissue. Shown is a representative pair of tumor (top) and normal (bottom) tissue. Brown staining indicates the presence of 14-3-3ε, whereas blue color represents hematoxylin counter-staining. The specificity of the utilized mouse monoclonal 14-3-3ε antibody (8C3, Santa Cruz Biotechnology sc-23957) is documented in Supplementary Figure S5. (C) Grading of 14-3-3ε protein expression in the cytoplasm and nucleus of the 31 matching prostate tumors and normal tissues. p = 0.008 (Wilcoxon signed-rank test). Supplementary Figure S7. Upregulation of MSK2 in prostate tumors. Analysis of publicly available microarray data (Wallace et al, 2008; Grasso et al, 2012) through the Oncomine webtool. Shown are log2 median-centered ratios of MSK2 mRNA levels with the median and 25-75 percentile range. N denotes normal prostate tissue, C prostate carcinoma. Statistical significance was determined with Student’s t-test.

Supplementary Figure S8. Overexpression of 14-3-3 mRNA in prostate tumors. Analysis of publicly available microarray data through the Oncomine webtool. Shown are log2 median-centered ratios of 14-3-3 mRNA levels with the median and 25-75 percentile range. N denotes normal prostate tissue, C prostate carcinoma. Statistical significance was determined with Student’s t-test. (A) 14-3-3ζ (Tomlins et al, 2007; Wallace et al, 2008). (B) 14-3-3γ (Vanaja et al, 2003; Tomlins et al, 2007). (C) 14-3-3β (Welsh et al, 2001; Vanaja et al, 2003). References De Haro L, Janknecht R (2002) Functional analysis of the transcription factor ER71 and its

activation of the matrix metalloproteinase-1 promoter. Nucleic Acids Res 30: 2972-2979 Grasso CS, Wu YM, Robinson DR, Cao X, Dhanasekaran SM, Khan AP, Quist MJ, Jing X,

Lonigro RJ, Brenner JC, Asangani IA, Ateeq B, Chun SY, Siddiqui J, Sam L, Anstett M, Mehra R, Prensner JR, Palanisamy N, Ryslik GA, Vandin F, Raphael BJ, Kunju LP, Rhodes DR, Pienta KJ, Chinnaiyan AM, Tomlins SA (2012) The mutational landscape of lethal castration-resistant prostate cancer. Nature 487: 239-243

Shin S, Bosc DG, Ingle JN, Spelsberg TC, Janknecht R (2008) Rcl is a novel ETV1/ER81 target gene upregulated in breast tumors. J Cell Biochem 105: 866-874

Shin S, Janknecht R (2007) Concerted activation of the Mdm2 promoter by p72 RNA helicase and the coactivators p300 and P/CAF. J Cell Biochem 101: 1252-1265

Shin S, Kim TD, Jin F, van Deursen JM, Dehm SM, Tindall DJ, Grande JP, Munz JM, Vasmatzis G, Janknecht R (2009) Induction of prostatic intraepithelial neoplasia and modulation of androgen receptor by ETS variant 1/ETS-related protein 81. Cancer Res 69: 8102-8110

Shin S, Oh S, An S, Janknecht R (2013) ETS variant 1 regulates matrix metalloproteinase-7 transcription in LNCaP prostate cancer cells. Oncol Rep 29: 306-314

Singh D, Febbo PG, Ross K, Jackson DG, Manola J, Ladd C, Tamayo P, Renshaw AA, D'Amico AV, Richie JP, Lander ES, Loda M, Kantoff PW, Golub TR, Sellers WR (2002) Gene expression correlates of clinical prostate cancer behavior. Cancer Cell 1: 203-209

Tomlins SA, Mehra R, Rhodes DR, Cao X, Wang L, Dhanasekaran SM, Kalyana-Sundaram S, Wei JT, Rubin MA, Pienta KJ, Shah RB, Chinnaiyan AM (2007) Integrative molecular concept modeling of prostate cancer progression. Nat Genet 39: 41-51

Vanaja DK, Cheville JC, Iturria SJ, Young CY (2003) Transcriptional silencing of zinc finger protein 185 identified by expression profiling is associated with prostate cancer progression. Cancer Res 63: 3877-3882

Wallace TA, Prueitt RL, Yi M, Howe TM, Gillespie JW, Yfantis HG, Stephens RM, Caporaso NE, Loffredo CA, Ambs S (2008) Tumor immunobiological differences in prostate cancer between African-American and European-American men. Cancer Res 68: 927-936

Welsh JB, Sapinoso LM, Su AI, Kern SG, Wang-Rodriguez J, Moskaluk CA, Frierson HF, Jr., Hampton GM (2001) Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res 61: 5974-5978

10075

50

37

25

kDa

14-3-3ε14-3-3β, γ, η, τ, ζ

ETV1ETV1, tubulin-β1

ETV1, HSP70

Supplementary Figure S1

Supplementary Figure S2

DAPI 6Myc-ETV1 14-3-3τ Merged

A

0.0

0.4

0.8

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pQCXIP 14-3-3ε 14-3-3τ0.0

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sh-14-3-3τ #3

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DMSO U0126 SB2021900.0

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HER2/Neu Ras-G12V

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Supplementary Figure S3

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Supplementary Figure S4

14-3-3ε

kDa13072553426

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13072553426

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43Actin

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endogenousexogenous

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Supplementary Figure S5

Tumor

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0 - 1 >1 - 2 >2 - 3

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Grading of 14-3-3ε Staining

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p = 1.02E-6(N=9; C=25)

p = 7.99E-4(N=50; C=52)

p = 0.001(N=20; C=69)

Supplementary Figure S6

Wallace, MSK21.2

0.8

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C

p = 0.001(N=20; C=69)

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Supplementary Figure S7

AWallace, 14-3-3ζ4.0

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Tomlins, 14-3-3ζ1.0

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p = 4.84E-4(N=20; C=69)

BTomlins, 14-3-3γ

1.0

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-1.0

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p = 5.81E-5(N=22; C=30)

CWelsh, 14-3-3β

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2.5

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p = 6.25E-4(N=9; C=25)

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Supplementary Figure S8