Summary 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.

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Ewing sarcoma breakpoint region 1 (EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5′ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5′ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5′ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to either early transcription termination, or breakpoint formation.

More than one-half of prostate tumors have a chromosomal rearrangement that results in the overexpression of an oncogenic ETS family transcription factor. The most common fusion, TMPRSS2/ERG, results in expression of ERG, a protein that is not normally expressed in prostate epithelia. When ERG is expressed in prostate cells it is thought to bind to enhancer elements and regulate gene expression by recruiting transcriptional coactivators and/or corepressors. We have recently shown that the EWS protein acts as a coactivator for ERG in prostate cells and this interaction is required for ERG-mediated xenograft tumor growth. Interestingly, the EWS interaction may be the key requirement that separates oncogenic from nononcogenic ETS factors, as EWS only interacts with four ETS family members involved in prostate cancer gene rearrangements (ERG, ETV1, ETV4, and ETV5), but not with other ETS family members. This ETS/EWS interaction also indicates a common molecular mechanism involved in prostate cancer and Ewing’s sarcoma, a cancer caused by gene fusions that express chimeric EWS/ETS proteins. ERG also interacts with corepressors such as EZH2, a subunit of PRC2. We have recently found that the interaction between ERG and EZH2/PRC2 is regulated by a series of phosphorylation events on ERG. The MAP kinase ERK can bind a high-affinity docking sequence in ERG, resulting in phosphorylation of a nearby serine, S215. This phosphorylation event leads to a conformational change in ERG that allows ERK to phosphorylate a second serine, S96. S96 phosphorylation then disrupts the interaction between ERG and EZH2/PRC2, allowing ERG to activate gene expression. Together, the interaction of ERG with EWS and the regulation of ERG function by ERK-mediated phosphorylation, represent molecular mechanisms that could serve as targets for therapeutic intervention in ERG-positive prostate cancer. Citation Format: Vivekananda Kedage, Taylor R. Nicholas, Brady G. Strittmatter, Nagarathinam Selvaraj, Justin A. Budka, Travis J. Jerde, Peter C. Hollenhorst. Regulation of ERG function in prostate cells by phosphorylation and interaction with Ewing’s sarcoma breakpoint protein EWS [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A023.

Aberrant expression of the transcription factor ERG is a key driving event in approximately one-half of all of prostate cancers. Lacking an enzymatic pocket and mainly disordered, the structure of ERG is difficult to exploit for therapeutic design. We recently identified EWS as a specific interacting partner of ERG that is required for oncogenic function. In this study, we aimed to target this specific protein-protein interaction with small molecules. A high-throughput screening (HTS) strategy was implemented to identify potential protein-protein interaction inhibitors. Secondary assays verified the function of several hit compounds, and one lead compound inhibited ERG-mediated phenotypes in prostate cells. This is the first study aimed at targeting the ERG-EWS protein-protein interaction for the development of a small molecule-based prostate cancer therapy.