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• W2532275275 abstract "•EWS interacts specifically with ETS proteins that promote prostate cancer•EWS is required for oncogenic ETS functions in prostate cells•EWS acts as a co-activator for oncogenic ETS transcription factors•Oncogenic ETS function through similar mechanisms in prostate and Ewing’s sarcoma More than 50% of prostate tumors have a chromosomal rearrangement resulting in aberrant expression of an oncogenic ETS family transcription factor. However, mechanisms that differentiate the function of oncogenic ETS factors expressed in prostate tumors from non-oncogenic ETS factors expressed in normal prostate are unknown. Here, we find that four oncogenic ETS (ERG, ETV1, ETV4, and ETV5), and no other ETS, interact with the Ewing’s sarcoma breakpoint protein, EWS. This EWS interaction was necessary and sufficient for oncogenic ETS functions including gene activation, cell migration, clonogenic survival, and transformation. Significantly, the EWS interacting region of ERG has no homology with that of ETV1, ETV4, and ETV5. Therefore, this finding may explain how divergent ETS factors have a common oncogenic function. Strikingly, EWS is fused to various ETS factors by the chromosome translocations that cause Ewing’s sarcoma. Therefore, these findings link oncogenic ETS function in both prostate cancer and Ewing’s sarcoma. More than 50% of prostate tumors have a chromosomal rearrangement resulting in aberrant expression of an oncogenic ETS family transcription factor. However, mechanisms that differentiate the function of oncogenic ETS factors expressed in prostate tumors from non-oncogenic ETS factors expressed in normal prostate are unknown. Here, we find that four oncogenic ETS (ERG, ETV1, ETV4, and ETV5), and no other ETS, interact with the Ewing’s sarcoma breakpoint protein, EWS. This EWS interaction was necessary and sufficient for oncogenic ETS functions including gene activation, cell migration, clonogenic survival, and transformation. Significantly, the EWS interacting region of ERG has no homology with that of ETV1, ETV4, and ETV5. Therefore, this finding may explain how divergent ETS factors have a common oncogenic function. Strikingly, EWS is fused to various ETS factors by the chromosome translocations that cause Ewing’s sarcoma. Therefore, these findings link oncogenic ETS function in both prostate cancer and Ewing’s sarcoma. In 50%–70% of prostate tumors, a chromosomal rearrangement results in fusion of a transcriptionally active promoter and 5′ UTR to the open reading frame of an ETS family transcription factor, resulting in aberrant expression in prostate epithelia (Tomlins et al., 2005Tomlins S.A. Rhodes D.R. Perner S. Dhanasekaran S.M. Mehra R. Sun X.W. Varambally S. Cao X. Tchinda J. Kuefer R. et al.Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer.Science. 2005; 310: 644-648Crossref PubMed Scopus (3105) Google Scholar, Tomlins et al., 2007Tomlins S.A. Laxman B. Dhanasekaran S.M. Helgeson B.E. Cao X. Morris D.S. Menon A. Jing X. Cao Q. Han B. et al.Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer.Nature. 2007; 448: 595-599Crossref PubMed Scopus (652) Google Scholar). The most common fusion is TMPRSS2:ERG, occurring in approximately one-half of prostate tumors. ETV1 and ETV4 rearrangements occur in an additional 5%–10% of tumors. Expression of ERG, ETV1, and ETV4 in prostate cells is oncogenic, particularly when coupled with a second mutation activating the PI3K/AKT or androgen receptor pathways (Aytes et al., 2013Aytes A. Mitrofanova A. Kinkade C.W. Lefebvre C. Lei M. Phelan V. Lekaye H.C. Koutcher J.A. Cardiff R.D. Califano A. et al.ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer.Proc. Natl. Acad. Sci. U S A. 2013; 110: E3506-E3515Crossref PubMed Scopus (89) Google Scholar, Baena et al., 2013Baena E. Shao Z. Linn D.E. Glass K. Hamblen M.J. Fujiwara Y. Kim J. Nguyen M. Zhang X. Godinho F.J. et al.ETV1 directs androgen metabolism and confers aggressive prostate cancer in targeted mice and patients.Genes Dev. 2013; 27: 683-698Crossref PubMed Scopus (120) Google Scholar, Carver et al., 2009Carver B.S. Tran J. Gopalan A. Chen Z. Shaikh S. Carracedo A. Alimonti A. Nardella C. Varmeh S. Scardino P.T. et al.Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate.Nat. Genet. 2009; 41: 619-624Crossref PubMed Scopus (517) Google Scholar, King et al., 2009King J.C. Xu J. Wongvipat J. Hieronymus H. Carver B.S. Leung D.H. Taylor B.S. Sander C. Cardiff R.D. Couto S.S. et al.Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis.Nat. Genet. 2009; 41: 524-526Crossref PubMed Scopus (376) Google Scholar, Zong et al., 2009Zong Y. Xin L. Goldstein A.S. Lawson D.A. Teitell M.A. Witte O.N. ETS family transcription factors collaborate with alternative signaling pathways to induce carcinoma from adult murine prostate cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 12465-12470Crossref PubMed Scopus (169) Google Scholar). Fusion transcripts involving other ETS genes (ETV5, FLI1, EHF, ELF2, ELK4, and ETV6) have been identified at low frequency (<2%), but it is not clear if these are oncogenic or passenger mutations (Rickman et al., 2009Rickman D.S. Pflueger D. Moss B. VanDoren V.E. Chen C.X. de la Taille A. Kuefer R. Tewari A.K. Setlur S.R. Demichelis F. Rubin M.A. SLC45A3-ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer.Cancer Res. 2009; 69: 2734-2738Crossref PubMed Scopus (158) Google Scholar, Robinson et al., 2015Robinson D. Van Allen E.M. Wu Y.M. Schultz N. Lonigro R.J. Mosquera J.M. Montgomery B. Taplin M.E. Pritchard C.C. Attard G. et al.Integrative clinical genomics of advanced prostate cancer.Cell. 2015; 161: 1215-1228Abstract Full Text Full Text PDF PubMed Scopus (1990) Google Scholar). Although significant progress has been made in understanding the oncogenic gene targets of ERG, ETV1, and ETV4, the molecular mechanisms used by these three ETS transcription factors to activate transcription of these target genes are largely unknown. Furthermore, although these rearrangements tend to be mutually exclusive (Svensson et al., 2011Svensson M.A. Lafargue C.J. Macdonald T.Y. Pflueger D. Kitabayashi N. Santa-Cruz A.M. Garsha K.E. Sathyanarayana U.G. Riley J.P. Yun C.S. et al.Testing mutual exclusivity of ETS rearranged prostate cancer.Lab. Invest. 2011; 91: 404-412Crossref PubMed Scopus (64) Google Scholar), it is not clear if ERG, ETV1, and ETV4 utilize a common molecular mechanism to promote prostate cancer. The human genome encodes 28 ETS factors, and these are extensively co-expressed, with at least 15 present in any individual cell type (Hollenhorst et al., 2004Hollenhorst P.C. Jones D.A. Graves B.J. Expression profiles frame the promoter specificity dilemma of the ETS family of transcription factors.Nucleic Acids Res. 2004; 32: 5693-5702Crossref PubMed Scopus (171) Google Scholar). ETS proteins share a highly conserved ETS DNA binding domain and can be divided based on similarities in this domain into subfamilies of no more than three members each (Hollenhorst et al., 2011bHollenhorst P.C. McIntosh L.P. Graves B.J. Genomic and biochemical insights into the specificity of ETS transcription factors.Annu. Rev. Biochem. 2011; 80: 437-471Crossref PubMed Scopus (330) Google Scholar). Within a subfamily, amino acid homology extends across the entire protein, but between subfamilies, the only homology is in the ETS DNA binding domain. ETV1, ETV4 (PEA3), and ETV5 comprise the PEA3 subfamily, but ERG is in a distinct subfamily, and therefore, outside of the DNA binding domain, ERG has no amino acid sequence similarity with ETV1, ETV4, and ETV5. Instead, ERG is homologous with FLI1 and FEV, which have no clear roles in prostate cancer. These sequence comparisons make it difficult to predict a conserved functional mechanism through which ERG, ETV1, and ETV4 promote prostate cancer that would not also extend to the non-oncogenic ETS factors normally expressed in prostate cells. Oncogenic ETS proteins are known to promote cell migration, invasion, and epithelial-mesenchymal transition (EMT) when overexpressed in prostate epithelial cells (Tomlins et al., 2008Tomlins S.A. Laxman B. Varambally S. Cao X. Yu J. Helgeson B.E. Cao Q. Prensner J.R. Rubin M.A. Shah R.B. et al.Role of the TMPRSS2-ERG gene fusion in prostate cancer.Neoplasia. 2008; 10: 177-188Abstract Full Text PDF PubMed Scopus (543) Google Scholar, Wu et al., 2013Wu L. Zhao J.C. Kim J. Jin H.J. Wang C.Y. Yu J. ERG is a critical regulator of Wnt/LEF1 signaling in prostate cancer.Cancer Res. 2013; 73: 6068-6079Crossref PubMed Scopus (63) Google Scholar). By directly comparing overexpression of multiple ETS factors in RWPE1 immortalized-normal prostate epithelial cells, we previously demonstrated that only ERG, ETV1, ETV4, and ETV5, but not FLI1 or other ETS proteins, promote cell migration (Hollenhorst et al., 2011aHollenhorst P.C. Ferris M.W. Hull M.A. Chae H. Kim S. Graves B.J. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells.Genes Dev. 2011; 25: 2147-2157Crossref PubMed Scopus (123) Google Scholar), indicating that these four ETS proteins share a common biological function that is unique in the ETS family. These ETS proteins activated transcription of cell migration genes by binding cis-regulatory sequences that have neighboring ETS and AP-1 transcription factor binding sites. However, this ETS/AP-1 binding is not specific to ERG, ETV1, ETV4, and ETV5, because ETS1 can also bind ETS/AP-1 sequences and activate transcription of cell migration genes in KRAS mutant cancer cells (Plotnik et al., 2014Plotnik J.P. Budka J.A. Ferris M.W. Hollenhorst P.C. ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells.Nucleic Acids Res. 2014; 42: 11928-11940Crossref PubMed Scopus (74) Google Scholar). Therefore the molecular mechanism behind the specific biological function of ERG, ETV1, ETV4, and ETV5 in prostate cells is unknown. Prostate cancer is not the only malignancy caused by ETS gene rearrangements. Ewing’s sarcoma is caused by chromosomal translocations involving one of five different ETS genes. Prostate cancer chromosomal rearrangements generally promote expression of full-length or N-terminally truncated ETS proteins (Clark et al., 2007Clark J. Merson S. Jhavar S. Flohr P. Edwards S. Foster C.S. Eeles R. Martin F.L. Phillips D.H. Crundwell M. et al.Diversity of TMPRSS2-ERG fusion transcripts in the human prostate.Oncogene. 2007; 26: 2667-2673Crossref PubMed Scopus (206) Google Scholar). In contrast, the oncogenic product of an Ewing’s sarcoma translocation is a fusion protein consisting of an N-terminal domain of the RNA binding protein EWS fused to a C-terminal region of an ETS protein (Delattre et al., 1992Delattre O. Zucman J. Plougastel B. Desmaze C. Melot T. Peter M. Kovar H. Joubert I. de Jong P. Rouleau G. et al.Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours.Nature. 1992; 359: 162-165Crossref PubMed Scopus (1536) Google Scholar, Patel et al., 2012Patel M. Simon J.M. Iglesia M.D. Wu S.B. McFadden A.W. Lieb J.D. Davis I.J. Tumor-specific retargeting of an oncogenic transcription factor chimera results in dysregulation of chromatin and transcription.Genome Res. 2012; 22: 259-270Crossref PubMed Scopus (78) Google Scholar). In this fusion oncoprotein, the N terminus of EWS contributes a strong transcriptional activation domain, and the C terminus of the ETS protein contributes the ETS DNA binding domain, both of which are necessary for transformation of Ewing’s sarcoma cells (May et al., 1993May W.A. Lessnick S.L. Braun B.S. Klemsz M. Lewis B.C. Lunsford L.B. Hromas R. Denny C.T. The Ewing’s sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1.Mol. Cell. Biol. 1993; 13: 7393-7398Crossref PubMed Scopus (443) Google Scholar). Interestingly, the five ETS genes involved in Ewing’s sarcoma fusions (FLI1, ERG, FEV, ETV1, and ETV4) partially overlap with those rearranged in prostate cancer. However, the most common Ewing’s fusion is EWS:FLI1 (85%), and FLI1 has not been shown to drive prostate tumorigenesis and does not promote prostate cell migration (Hollenhorst et al., 2011aHollenhorst P.C. Ferris M.W. Hull M.A. Chae H. Kim S. Graves B.J. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells.Genes Dev. 2011; 25: 2147-2157Crossref PubMed Scopus (123) Google Scholar). Furthermore, there is no known connection between the molecular mechanism of prostate cancer ETS rearrangements and Ewing’s sarcoma fusion proteins. This study investigated the role of the wild-type EWS protein in the oncogenic mechanism of ETS genes rearranged in prostate cancer. EWS was found to interact specifically with the ETS proteins ERG, ETV1, ETV4, and ETV5. This interaction occurred both in cell lines and between purified proteins, indicating it is direct. Fusion of the EWS N terminus with any ETS protein promoted prostate cell migration, indicating that this interaction is sufficient for an oncogenic phenotype. Furthermore, using both a knockdown of EWS and a point mutation in ERG that fails to interact with EWS, we demonstrate that the EWS-ETS interaction is critical for oncogenic ETS proteins to activate gene expression and drive cell migration and transformation in prostate cells. Activation of gene expression via ETS and EWS occurred through both ETS/AP-1 and GGAA-repeat cis-regulatory sequences. The necessity of EWS was specific to cell lines that express oncogenic ETS proteins, indicating that therapeutic strategies to inhibit this interaction may result in few side effects. These data suggest that a protein-protein interaction with EWS defines the specific oncogenic function of the ETS family transcription factors that drive prostate tumorigenesis and sets the stage for future work to further define this interaction in vivo and in human tumors. The EWS protein is fused to ETS transcription factors in Ewing’s sarcoma, but the oncogenic role for wild-type EWS is less clear. To test if EWS naturally interacts (absent fusion) with any ETS protein, we immobilized 21 purified full-length ETS proteins, representing every ETS subfamily, on beads, and we used these for affinity purification of interacting partners from PC3 prostate cancer cell nuclear extract. An immunoblot indicated that four of these 21 ETS proteins strongly and specifically interact with EWS (Figure 1A). Surprisingly, these four ETS proteins (ERG, ETV1, ETV4, and ETV5) cannot be grouped by common sequence but are linked by a common biological function: the ability to promote prostate cancer. This indicates that an interaction with EWS could define a common molecular mechanism for prostate cancer ETS oncoproteins. To test if this interaction occurs in cancer cells, two prostate cancer cell lines that overexpress an oncogenic ETS factor were examined. VCaP cells have a TMPRSS2:ERG fusion (Tomlins et al., 2005Tomlins S.A. Rhodes D.R. Perner S. Dhanasekaran S.M. Mehra R. Sun X.W. Varambally S. Cao X. Tchinda J. Kuefer R. et al.Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer.Science. 2005; 310: 644-648Crossref PubMed Scopus (3105) Google Scholar), and immunoprecipitation with an anti-ERG antibody enriched for EWS (Figure 1B). PC3 cells overexpress ETV4 (Hollenhorst et al., 2011cHollenhorst P.C. Paul L. Ferris M.W. Graves B.J. The ETS gene ETV4 is required for anchorage-independent growth and a cell proliferation gene expression program in PC3 prostate cells.Genes Cancer. 2011; 1: 1044-1052Crossref PubMed Scopus (49) Google Scholar), and immunoprecipitation with an anti-ETV4 antibody also enriched for EWS (Figure 1C). Reverse co-immunoprecipitation with anti-EWS antibody enriched for ERG (Figure 1B) and ETV4 (Figure 1C). EWS also co-immunoprecipitated with ERG in an ERG-positive primary prostate tumor (Figure 1D). To further validate the in vivo interaction, both ERG and EWS genomic occupancy was mapped in VCaP cells by chromatin immunoprecipitation sequencing (ChIP-seq). ERG-bound regions showed significant overlap with those identified in previous studies (Chng et al., 2012Chng K.R. Chang C.W. Tan S.K. Yang C. Hong S.Z. Sng N.Y. Cheung E. A transcriptional repressor co-regulatory network governing androgen response in prostate cancers.EMBO J. 2012; 31: 2810-2823Crossref PubMed Scopus (128) Google Scholar, Yu et al., 2010Yu J. Yu J. Mani R.S. Cao Q. Brenner C.J. Cao X. Wang X. Wu L. Li J. Hu M. et al.An integrated network of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression.Cancer Cell. 2010; 17: 443-454Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar) and were enriched for ETS binding sites and histone H3 acetylation (Figures S1A and S1B). EWS co-occupied 1,242 of these ERG-bound genomic regions (Figure 1E; Table S1), a greater than 3-fold enrichment over the random expectation (p < 0.0001). Together, these data indicate that oncogenic ETS proteins interact with EWS in prostate cancer cells. To test if the interaction between oncogenic ETS proteins and EWS is direct, glutathione S-transferase (GST)-tagged full-length EWS was purified from bacteria (Figure 1F) and mixed with beads bound to purified full-length ETS proteins. ERG, ETV1, ETV4, and ETV5 interacted with purified EWS, while the close ERG homolog, FLI1, did not (Figure 1G). The interaction of ETV1 with EWS was dramatically weaker than ERG, ETV4, or ETV5, indicating that the ETV1-EWS interaction may require additional partner proteins or posttranslational modifications. Together, these data suggest a direct and specific interaction between EWS and oncogenic ETS proteins. Overexpression of ERG, ETV1, ETV4, and ETV5, but no other ETS proteins, promotes migration of the normal prostate cell line RWPE1 (Hollenhorst et al., 2011aHollenhorst P.C. Ferris M.W. Hull M.A. Chae H. Kim S. Graves B.J. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells.Genes Dev. 2011; 25: 2147-2157Crossref PubMed Scopus (123) Google Scholar). Strikingly, even the close ERG homolog FLI1 lacks this function. However, in Ewing’s sarcoma, both FLI1 and ERG are oncogenic when fused to the N terminus of EWS due to a chromosomal rearrangement. To test if fusion of EWS could impart an oncogenic function on ETS proteins expressed in prostate cells, we compared full-length wild-type versions of seven ETS proteins to the same proteins N-terminally fused to amino acids 1–264 of EWS. To avoid super-physiological expression levels, the cytomegalovirus (CMV) promoter in the viral expression vector was replaced with the weaker HNRNPA2B1 promoter, a promoter that drives ETS expression in some prostate tumor rearrangements (Tomlins et al., 2007Tomlins S.A. Laxman B. Dhanasekaran S.M. Helgeson B.E. Cao X. Morris D.S. Menon A. Jing X. Cao Q. Han B. et al.Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer.Nature. 2007; 448: 595-599Crossref PubMed Scopus (652) Google Scholar). ERG expression from this vector was lower than ERG expression in VCaP cells or from a CMV vector (Figure S2A) but still caused a more than 3-fold increase in RWPE1 cell migration (Figure 2A). In contrast, expression of six non-oncogenic ETS proteins, including FLI1, did not significantly change cell migration (Figures 2A and S2B). Expression of the EWS N terminus by itself had no effect on cell migration, but every EWS-ETS fusion protein tested caused significant increases in cell migration. None of the EWS-ETS fusion proteins accumulated to appreciably higher levels in the cell than the ETS protein alone (Figure 2B). In Ewing’s sarcoma fusions, the N terminus of ERG or FLI1 is removed and replaced by the N terminus of EWS. To test if this truncation of the ETS N terminus affected cell migration, we also tested EWS-NtermΔERG and EWS-NtermΔFLI1 constructs analogous to the fusion proteins found in Ewing’s sarcoma. These EWS-NtermΔETS constructs drove cell migration to the same extent as EWS-ETS constructs (Figures 2A and 2B). Therefore, an obligate interaction with the EWS N terminus is sufficient to allow an ETS protein to act like an oncogenic ETS factor. To test if this role of EWS was limited to cell migration, a second phenotype, clonogenic survival, was assayed (Figure 2C). ERG expression alone caused little increase in clonogenic survival of RWPE1 cells. Previous reports indicate that ERG and activated AKT are both required for tumor formation (Carver et al., 2009Carver B.S. Tran J. Gopalan A. Chen Z. Shaikh S. Carracedo A. Alimonti A. Nardella C. Varmeh S. Scardino P.T. et al.Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate.Nat. Genet. 2009; 41: 619-624Crossref PubMed Scopus (517) Google Scholar, King et al., 2009King J.C. Xu J. Wongvipat J. Hieronymus H. Carver B.S. Leung D.H. Taylor B.S. Sander C. Cardiff R.D. Couto S.S. et al.Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis.Nat. Genet. 2009; 41: 524-526Crossref PubMed Scopus (376) Google Scholar). Expression of myristoylated AKT increased AKT activation but alone had little affect on clonogenic survival. However, expression of both ERG and myristoylated AKT together resulted in a dramatic increase in clonogenic survival (Figure 2C). Similar to the cell migration phenotype, FLI1 expression with activated AKT caused significantly lower increases in clonogenic growth than ERG with activated AKT, but EWS fused to FLI1 was similar to ERG (Figure 2C). Both cell migration and clonogenic growth assays indicate that the EWS interaction is sufficient to allow non-oncogenic ETS proteins to function like ERG. To identify the region of ERG that interacts with EWS, various ERG truncations and deletions were purified and tested for the EWS interaction in PC3 nuclear extract (Figure 3A). A deletion of the C terminus of ERG after amino acid 391 resulted in a loss of EWS binding. However, a truncated ERG containing amino acids 275–455 retained EWS binding. Therefore, the region of ERG, C-terminal to the ETS domain, between amino acids 391 and 455, was necessary for the EWS interaction. A deletion analysis of ETV5 identified a region necessary for EWS interaction that was N-terminal to the ETS domain spanning amino acids 357–368 (Figure 3B). This region of ETV5 has high homology to PEA3 subfamily proteins ETV1 and ETV4 but no homology to any region of ERG (Figures S3A and S3B). Similarly, the EWS interacting region of ERG had no homology with any region of ETV1, ETV4, or ETV5, indicating that ERG and PEA3 proteins interact with EWS through distinct interaction domains. The EWS interaction region of ERG has 75% identity with FLI1 (Figure S3A), which did not physically interact with EWS (Figure 1A). To identify the difference that allows ERG, but not FLI1 to bind EWS, point mutations were created in ERG in amino acids that differ from FLI1 between 391 and 455. Two of these ERG point mutants were purified and tested for the interaction with EWS from PC3 cell nuclear extract (Figure 3C). One mutant, ERG P436A, eliminated the EWS interaction. Purified EWS protein was also used to show that ERG 1–391 and ERG P436A fail to directly interact with EWS (Figure 3D). To test if the P436A mutation disrupted the ERG-EWS interaction in cells, FLAG-ERG and FLAG-ERG P436A were expressed in RWPE1 cells and immunoprecipitated with anti-ERG antibody. Only ERG, and not ERG P436A, interacted with EWS (Figure 3E). Therefore ERG P436 is required for the EWS interaction both in cells and in vitro. Four oncogenic ETS proteins uniquely promote prostate cell migration (Hollenhorst et al., 2011aHollenhorst P.C. Ferris M.W. Hull M.A. Chae H. Kim S. Graves B.J. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells.Genes Dev. 2011; 25: 2147-2157Crossref PubMed Scopus (123) Google Scholar) and uniquely interact with EWS (Figure 1A). To test if the EWS interaction is required for the cell migration function, the ability of ERG and ERG P436A to promote migration of RWPE1 cells was compared. Despite being expressed at the same level, ERG promoted cell migration, and ERG-P436A inhibited cell migration (Figures 4A and S4A). This indicated not only that the EWS interaction was necessary for this ERG function but also that loss of the EWS interaction reversed ERG’s function. A similar result was observed in the clonogenic growth assay, where the P436A mutation abrogated the ability of ERG to promote colony formation (Figure 4B). Neither ERG nor ERG P436A expression altered cell proliferation when plated at higher density (Figure S4B). To verify the necessity of EWS for an oncogenic ETS to promote cell migration, EWS was depleted from two prostate cancer cell lines, PC3 and DU145, using two independent small hairpin RNAs (shRNAs) (Figure 4C). PC3 prostate cancer cells migrate due to overexpression of the oncogenic ETS ETV4 (Hollenhorst et al., 2011cHollenhorst P.C. Paul L. Ferris M.W. Graves B.J. The ETS gene ETV4 is required for anchorage-independent growth and a cell proliferation gene expression program in PC3 prostate cells.Genes Cancer. 2011; 1: 1044-1052Crossref PubMed Scopus (49) Google Scholar), while DU145 prostate cancer cells migrate due to a KRAS gene rearrangement and do not express any of the oncogenic ETS (Selvaraj et al., 2014Selvaraj N. Budka J.A. Ferris M.W. Jerde T.J. Hollenhorst P.C. Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation.Mol. Cancer. 2014; 13: 61Crossref PubMed Scopus (29) Google Scholar, Wang et al., 2011Wang X.S. Shankar S. Dhanasekaran S.M. Ateeq B. Sasaki A.T. Jing X. Robinson D. Cao Q. Prensner J.R. Yocum A.K. et al.Characterization of KRAS rearrangements in metastatic prostate cancer.Cancer Discov. 2011; 1: 35-43Crossref PubMed Scopus (82) Google Scholar). EWS knockdown significantly decreased migration of PC3 cells, but not DU145 cells (Figures 4C and S4C). This indicates that EWS is required for cell migration in a cell line that expresses an oncogenic ETS, but not for cell migration in general. EWS knockdown had no effect on PC3 or DU145 cell proliferation (Figure S4D). To further confirm the specificity of EWS for oncogenic ETS-induced cell migration, we used the RWPE1 system. We have previously shown that either ERG or KRAS overexpression causes RWPE1 cells to migrate by activating a similar gene expression program (Hollenhorst et al., 2011aHollenhorst P.C. Ferris M.W. Hull M.A. Chae H. Kim S. Graves B.J. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells.Genes Dev. 2011; 25: 2147-2157Crossref PubMed Scopus (123) Google Scholar, Selvaraj et al., 2014Selvaraj N. Budka J.A. Ferris M.W. Jerde T.J. Hollenhorst P.C. Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation.Mol. Cancer. 2014; 13: 61Crossref PubMed Scopus (29) Google Scholar). The necessity of EWS for this migration was compared by EWS knockdown in RWPE1-ERG and RWPE1-KRAS cells. EWS knockdown significantly reduced migration in ERG-overexpressing cells, but not the KRAS-overexpressing cells (Figures 4D and S4C). EWS knockdown had no effect on RWPE1-ERG or RWPE1-KRAS cell proliferation (Figure S4E). To show specificity, the EWS knockdown phenotype was rescued by EWS overexpression (Figures 4E and S4F). To extend these findings to another oncogenic ETS function, roles in anchorage-independent growth were tested. The oncogenic ETS protein ETV4 is required for anchorage-independent growth of PC3 prostate cancer cells (Hollenhorst et al., 2011cHollenhorst P.C. Paul L. Ferris M.W. Graves B.J. The ETS gene ETV4 is required for anchorage-independent growth and a cell proliferation gene expression program in PC3 prostate cells.Genes Cancer. 2011; 1: 1044-1052Crossref PubMed Scopus (49) Google Scholar), and loss of EWS significantly inhibited growth of PC3 cells in soft agar (Figures 4F and S4G). ETV1 and ERG are overexpressed in MDA-PCa-2B and VCaP cell lines, respectively, due to chromosomal rearrangements like those observed in patient tumors (Tomlins et al., 2007Tomlins S.A. Laxman B. Dhanasekaran S.M. Helgeson B.E. Cao X. Morris D.S. Menon A. Jing X. Cao Q. Han B. et al.Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer.Nature. 2007; 448: 595-599Crossref PubMed Scopus (652) Google Scholar). EWS knockdown reduced soft-agar growth of both of these cell lines (Figures 4F and S4G). Together, these data show that the EWS interaction is responsible for common biological functions of oncogenic ETS proteins. To directly test the role of the EWS-ERG interaction in tumor growth, we employed the established subcutaneous xenograft tumor growth model (Shaw et al., 2010Shaw A. Gipp J. Bushman W. Exploration of Shh and BMP paracrine signaling in a prostate cancer xenograft.Differentiation. 2010; 79: 41-47Crossref PubMed Scopus (19) Google Scholar). Since ERG accelerates tumor formation when expressed in prostate cells with activated Akt signaling, we combined RWPE1 prostate cells stably expressing ERG, myristoylated AKT (myr-AKT), or both with cancer-associated fibroblasts subcutaneously into the flank of immunocompromised mice. ERG expression and AKT activation were confirmed by immunoblot (Figure 5A). An RWPE1 vector-only control formed no tumors, while RWPE1 cells expressing ERG or myr-AKT alone formed small masses that became stagnant after an initial growth phase between weeks 1 and 4 (Figure 5B). RWPE1 cells expressing both ERG" @default.
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• W2532275275 title "An Interaction with Ewing’s Sarcoma Breakpoint Protein EWS Defines a Specific Oncogenic Mechanism of ETS Factors Rearranged in Prostate Cancer" @default.
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