Background Prostate adenocarcinoma (AdPC) cells can undergo lineage switching to neuroendocrine cells and develop into therapy-resistant neuroendocrine prostate cancer (NEPC). While genomic/epigenetic alterations are shown to induce neuroendocrine differentiation via an intermediate stem-like state, RNA splicing factor SRRM4 can transform AdPC cells into NEPC xenografts through a direct neuroendocrine transdifferentiation mechanism. Whether SRRM4 can also regulate a stem-cell gene network for NEPC development remains unclear. Methods Multiple AdPC cell models were transduced by lentiviral vectors encoding SRRM4. SRRM4-mediated RNA splicing and neuroendocrine differentiation of cells and xenografts were determined by qPCR, immunoblotting, and immunohistochemistry. Cell morphology, proliferation, and colony formation rates were also studied. SRRM4 transcriptome in the DU145 cell model was profiled by AmpliSeq and analyzed by gene enrichment studies. Findings SRRM4 induces an overall NEPC-specific RNA splicing program in multiple cell models but creates heterogeneous transcriptomes. SRRM4-transduced DU145 cells present the most dramatic neuronal morphological changes, accelerated cell proliferation, and enhanced resistance to apoptosis. The derived xenografts show classic phenotypes similar to clinical NEPC. Whole transcriptome analyses further reveal that SRRM4 induces a pluripotency gene network consisting of the stem-cell differentiation gene, SOX2. While SRRM4 overexpression enhances SOX2 expression in both time- and dose-dependent manners in DU145 cells, RNA depletion of SOX2 compromises SRRM4-mediated stimulation of pluripotency genes. More importantly, this SRRM4-SOX2 axis is present in a subset of NEPC patient cohorts, patient-derived xenografts, and clinically relevant transgenic mouse models. Interpretation We report a novel mechanism by which SRRM4 drives NEPC progression via a pluripotency gene network. Fund Canadian Institutes of Health Research, National Nature Science Foundation of China, and China Scholar Council.

Therapy-induced neuroendocrine prostate cancer (t-NEPC) is a highly aggressive subtype of prostate cancer with poor patient survival. Emerging evidence indicates that t-NEPC can develop when prostate adenocarcinoma cells acquire cancer stem-like cell signaling in the presence of androgen receptor inhibition, followed by redifferentiation toward neuroendocrine lineage and subsequent t-NEPC progression. Whether the stem-like signaling is controlled by the core pluripotency stem cell genes (e.g., LIN28 and SOX2) remains unknown. Here, we report that the transcription of the LIN28B isoform and SOX2 were co-upregulated in t-NEPC patient tumors, patient-derived xenografts, transgenic mice, and cell models. Immunohistochemistry validated that LIN28B and SOX2 protein expression were elevated in t-NEPC patient biopsies. Using prostate adenocarcinoma and t-NEPC cell models, we demonstrated that LIN28B induced a stem-like gene network, neuroendocrine biomarkers, and neuroendocrine cell morphology. LIN28B depletion by CRISPR inhibited t-NEPC tumorigenesis and xenograft growth. These LIN28B functions were mediated mainly through the suppression of let-7 miRNA expression, resulting in de-repression of the transcription factor HMGA2 and HMGA2-mediated SOX2 expression. This study revealed a mechanism by which t-NEPC can develop through the LIN28B/let-7/SOX2 axis that regulates a cancer cell stem-like gene network, highlighting LIN28B as a potential therapeutic target in t-NEPC.

Although potent androgen receptor pathway inhibitors (ARPI) improve overall survival of metastatic prostate cancer patients, treatment-induced neuroendocrine prostate cancer (t-NEPC) as a consequence of the selection pressures of ARPI is becoming a more common clinical issue. Improved understanding of the molecular biology of t-NEPC is essential for the development of new effective management approaches for t-NEPC. In this study, we identify a splice variant of the MYST/Esa1-associated factor 6 (MEAF6) gene, MEAF6-1, that is highly expressed in both t-NEPC tumor biopsies and neuroendocrine cell lines of prostate and lung cancers. We show that MEAF6-1 splicing is stimulated by neuronal RNA splicing factor SRRM4. Rather than inducing neuroendocrine trans-differentiation of cells in prostate adenocarcinoma, MEAF6-1 upregulation stimulates cell proliferation, anchorage-independent cell growth, invasion and xenograft tumor growth. Gene microarray identifies that these MEAF6-1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6-1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but rather facilitates t-NEPC progression by increasing the proliferation rate of cells that have acquired neuroendocrine phenotypes.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer (PCa) that becomes more prevalent when hormonal therapy, chemotherapy, or radiation therapy is applied to patients with metastatic prostate adenocarcinoma (AdPC). How AdPC cells survive these anti-cancer therapies and progress into t-NEPC remains unclear. By comparing the whole transcriptomes between AdPC and t-NEPC, we identified Bif-1, an apoptosis-associated gene, which undergoes alternative RNA splicing in t-NEPC. We found that while Bif-1a is the predominant variant of the Bif-1 gene in AdPC, two neural-specific variants, Bif-1b and Bif-1c, are highly expressed in t-NEPC patients, patient derived xenografts, and cell models. The neural-specific RNA splicing factor, SRRM4, promotes Bif-1b and Bif-1c splicing, and the expression of SRRM4 in tumors is strongly associated with Bif-1b/-1c levels. Furthermore, we showed that Bif-1a is pro-apoptotic, while Bif-1b and Bif-1c are anti-apoptotic in PCa cells under camptothecin and UV light irritation treatments. Taken together, our data indicate that SRRM4 regulates alternative RNA splicing of the Bif-1 gene that enables PCa cells resistant to apoptotic stimuli under anti-cancer therapies, and may contribute to AdPC progression into t-NEPC.

Background: Prostate adenocarcinoma (AdPC) progression to treatment-induced neuroendocrine prostate cancer (t-NEPC) is associated with poor patient survival. While AdPC and t-NEPC share similar genomes, they possess distinct transcriptomes, suggesting that RNA splicing and epigenetic mechanisms may regulate t-NEPC development. Objective: To characterize the role of alternative RNA splicing of the histone demethylase BHC80 during t-NEPC progression. Design, setting, and participants: The expression of BHC80 splice variants (BHC80-1 and BHC80-2) were compared between AdPC and t-NEPC patient tumors. Regulatory mechanisms of RNA splicing of the BHC80 gene were studied, and the signal pathways mediated by BHC80 splice variants were investigated in t-NEPC cell and xenograft models. Results: Global transcriptome analyses identified that the BHC80-2 variant is highly expressed in t-NEPC. Compared with the known histone demethylation activities of the BHC80 gene, we discovered a novel nonepigenetic action of BHC80-2, whereby BHC80-2 is localized in the cytoplasm to trigger the MyD88-p38-TTP pathway, which results in increased RNA stability of multiple tumor-promoting cytokines. While BHC80-2 does not induce neuroendocrine differentiation of cancer cells, it stimulates cell proliferation and tumor progression independent of androgen receptor signaling. Blockade of BHC80-2-regulated MyD88 signaling suppresses growth of several t-NEPC cell spheroid and xenograft models. Conclusions: Gain of function of BHC80-2 through alternative RNA splicing activates immune responses of cancer cells to promote t-NEPC development. Patient summary: The main obstacle to develop effective therapies for patients with t-NEPC is the lack of understanding on how t-NEPC is developed. Our study not only identifies a previously unknown BHC80-2-MyD88 signaling pathway that plays an important role during t-NEPC development, but also provides a proof of principle that targeting this signal pathway may offer an avenue to treat t-NEPC.