Probes for INS

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castrate-resistant prostate cancer characterized by poor patient outcome. Whole transcriptome sequencing analyses identified a NEPC-specific RNA splicing program that is predominantly controlled by the SRRM4 gene, suggesting that SRRM4 drives NEPC development. However, whether SRRM4 expression in patients may aid pathologists in diagnosing NEPC and predicting patient survival remains to be determined. In this study, we have applied RNA in situ hybridization and immunohistochemistry assays to measure the expressions of SRRM4, NEPC markers (SYP, CD56, and CHGA), and adenocarcinoma (AdPC) markers (AR, PSA) in a series of tissue microarrays constructed from castrate-resistant prostate tumors, treatment-naïve tumors collected from radical prostatectomy, and tumors treated with neoadjuvant hormonal therapy (NHT) for 0-12 months. Three pathologists also independently evaluated tumor histology and NEPC marker status. Here, we report that SRRM4 in castrate-resistant tumors is highly expressed in NEPC, strongly correlated with SYP, CD56, and CHGA expressions (Pearson correlation r = 0.883, 0.675, and 0.881; P < 0.0001) and negatively correlated with AR and PSA expressions (Pearson correlation r = -0.544 and -0.310; P < 0.05). Overall survival is 12.3 months for patients with SRRM4 positive tumors, comparing to 23 months for patients with SRRM4 negative tumors. In treatment-naïve AdPC, low SRRM4 expression is detected in ∼16% tumor cores. It correlates with SYP and CHGA expressions, but not Gleason scores. AdPC treated with >7 month NHT has significantly higher SRRM4 expression. Based on these findings, we conclude that SRRM4 expression in castrate-resistant tumors is highly correlated with NEPC and poor patient survival. It may serve as a diagnosis and prognosis biomarker of NEPC.

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) 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. Cellmorphology, 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.