Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

PURPOSE: Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. EXPERIMENTAL DESIGN: Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. RESULTS: Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. CONCLUSIONS: Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.

Differential Expression of Glucose Transporters and Hexokinases in Prostate Cancer with a Neuroendocrine Gene Signature: A Mechanistic Perspective for 18F-FDG Imaging of PSMA-Suppressed Tumors.

Although the incidence of de novo neuroendocrine prostate cancer (PC) is rare, recent data suggest that low expression of prostate-specific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine hallmarks and androgen receptor (AR) suppression in PC. Previous clinical reports indicate that PCs with a phenotype similar to neuroendocrine tumors can be more amenable to imaging by 18F-FDG than by PSMA-targeting radioligands. In this study, we evaluated the association between neuroendocrine gene signature and 18F-FDG uptake-associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported 18F-FDG avidity of PSMA-suppressed tumors. Methods: Data-mining approaches, cell lines, and patient-derived xenograft models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes HK1-HK3 and GCK), and PSMA (FOLH1 gene) after AR inhibition and in correlation with neuroendocrine hallmarks. Also, we characterize a neuroendocrine-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no neuroendocrine histopathology. We measured glucose uptake in a neuroendocrine-induced in vitro model and a zebrafish model by nonradioactive imaging of glucose uptake using a fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrated that a neuroendocrine gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR inhibitors, high expression of GCK and low expression of SLC2A12 correlated with neuroendocrine histopathology and PSMA gene suppression. GLUT12 suppression and upregulation of glucokinase were observed in neuroendocrine-induced PC cell lines and patient-derived xenograft models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: A neuroendocrine gene signature in neuroendocrine PC and NELPC associates with a distinct transcriptional profile of GLUTs and hexokinases. PSMA suppression correlates with GLUT12 suppression and glucokinase upregulation. Alteration of 18F-FDG uptake-associated genes correlated positively with higher glucose uptake in AR- and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient preclinical method for monitoring nonradioactive glucose uptake.

Roles of the HOXA10 gene during castrate-resistant prostate cancer progression.

Homeobox A10 (HOXA10) is an important transcription factor that regulates the development of the prostate gland. However, it remains unknown whether it modulates prostate cancer (PCa) progression into castrate-resistant stages. In this study, we have applied RNA in situ hybridization assays to demonstrate that downregulation of HOXA10 expression is associated with castrate-resistant PCa. These findings are supported by public RNA-seq data showing that reduced HOXA10 expression is correlated with poor patient survival. We show that HOXA10 suppresses PCa cell proliferation, anchorage colony formation and xenograft growth independent to androgens. Using AmpliSeq transcriptome sequencing, we have found that gene groups associated with lipid metabolism and androgen receptor (AR) signaling are enriched in the HOXA10 transcriptome. Furthermore, we demonstrate that HOXA10 suppresses the transcription of the fatty acid synthase (FASN) gene by forming a protein complex with AR and prevents AR recruitment to the FASN gene promoter. These results lead us to conclude that downregulation of HOXA10 gene expression may enhance lipogenesis to promote PCa cell growth and tumor progression to castrate-resistant stage.

Alternative RNA splicing of the GIT1 gene is associated with neuroendocrine prostate cancer.

Potent androgen receptor pathway inhibition (ARPI) therapies have given rise to a lethal, aggressive subtype of castration-resistant prostate cancer (CRPC) called treatment-induced neuroendocrine prostate cancer (t-NEPC). Now, t-NEPC poses a major clinical problem as approximately 20% of CRPC cases bear this subtype-a rate of occurrence that is predicted to rise with the widespread use of ARPI therapies. Unfortunately, there are no targeted therapies currently available to treat t-NEPC as the origin and molecular underpinnings of t-NEPC development remain unclear. In the present study, we identify that RNA splicing of the G protein-coupled receptor kinase-interacting protein 1 (GIT1) gene is a unique event in t-NEPC patients. Specifically, upregulation of the GIT1-A splice variant and downregulation of the GIT1-C variant expressions are associated with t-NEPC patient tumors, patient-derived xenografts, and cell models. RNA-binding assays show that RNA splicing of GIT1 is directly driven by SRRM4 and is associated with the neuroendocrine phenotype in CRPC cohorts. We show that GIT1-A and GIT1-C regulate differential transcriptomes in prostate cancer cells, where GIT1-A regulates genes associated with morphogenesis, neural function, environmental sensing via cell-adhesion processes, and epigenetic regulation. Consistent with our transcriptomic analyses, we report opposing functions of GIT1-A and GIT1-C in the stability of focal adhesions, whereby GIT1-A promotes focal adhesion stability. In summary, our study is the first to report that alternative RNA splicing of the GIT1 gene is associated with t-NEPC and reprograms its function involving FA-mediated signaling and cell processes, which may contribute to t-NEPC development.

Roles of Alternative RNA Splicing of the Bif-1 Gene by SRRM4 During the Development of Treatment-induced Neuroendocrine Prostate Cancer.

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.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network.

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed "treatment-induced castration-resistant neuroendocrine prostate cancer" (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development.