Abstract 4392: Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer

Abstract

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with poorly understood drivers of disease progression. We recently characterized five mCRPC phenotypes, including an amphicrine phenotype that co-expresses androgen receptor (AR) and neuroendocrine prostate cancer (NEPC) biomarkers. We determined that loss of RE1-silencing transcription factor (REST), a master regulator of neuronal differentiation, drives the amphicrine phenotype, but is not sufficient for prostate cancer (PC) adenocarcinoma to NEPC conversion. Furthermore, loss of REST through SRRM4-mediated splicing of REST pre-mRNA to REST4 has been suggested to drive adenocarcinoma to NEPC conversion. However, the roles of pre-mRNA splicing factors and REST activity in lineage switching require further investigation. Methods: Transcriptomic (RNASeq) and immunohistochemical/immunofluorescent analysis (IHC and IF) were conducted on amphicrine and NEPC patient metastases, LuCaP patient-derived xenograft (PDX) models and modified CRPC cell lines. The roles of SRRM3 and SRRM4 were examined using overexpression studies in AR-expressing and AR-null CRPC cell lines. Results: RNASeq, IHC and IF of metastatic specimens, LuCaP PDX models and VCaP cells confirmed the existence of the amphicrine phenotype in vitro and in vivo. Interestingly, transcriptome analysis of amphicrine patient specimens and LuCaP 77CR revealed that loss of REST repressor activity occurred without SRRM4 expression in a subset of tumor specimens. Indeed, BaseScope analysis using primers specific to REST4 and SRRM4 verified that amphicrine 77CR tumors were positive for REST4 expression but negative for SRRM4 expression, suggesting an alternative mechanism of REST splicing. Notably, overexpression of SRRM4 in AR-expressing C4-2B and AR-null PC-3 cells did not induce REST splicing. Moreover, RNASeq of SRRM4-overexpressing cells displayed heterogeneous transcriptome profiles inconsistent with canonical amphicrine or NEPC gene expression profiles. Interestingly, SRRM3 transcript was expressed at high levels in amphicrine and NEPC patient and LuCaP PDX biospecimens that lacked SRRM4 expression, suggesting an SRRM3-mediated mechanism of REST splicing. Studies interrogating the roles of SRRM3 in REST splicing and CRPC cellular plasticity are ongoing. Conclusions: Our data highlights an unrecognized mechanism of adenocarcinoma to amphicrine or NEPC conversion that hinges on a SRRM3-REST regulatory axis rather than REST-loss or SRRM4-mediated REST splicing. Identifying the mechanisms that may convert adenocarcinoma to treatment-resistant amphicrine or NEPC phenotypes in mCRPC patients will inform treatment and identify potential molecular pathways for therapeutic intervention. Citation Format: Mark P. Labrecque, Ilsa M. Coleman, Lisha G. Brown, Bryce Lakely, Lori Kollath, Daniel W. Lin, Lawrence D. True, Eva Corey, Peter S. Nelson, Colm Morrissey. Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4392.