SYMPK regulates AR alternative polyadenylation and AR variant expression in advanced prostate cancer

Localized prostate cancer can be cured by radiation or surgery but advanced prostate cancer continues to be a clinical challenge. Advanced prostate cancer can initially be controlled by endocrine therapies that target the androgen receptor (AR), however, these tumors will inevitably develop resistance. This stage of the disease, termed castration-resistant prostate cancer (CRPC), is responsible for practically all prostate cancer-specific deaths. Truncated AR variant (AR-V) proteins are expressed in CRPC cells, and can function as ligand-independent, constitutively active transcription factors that promote resistance to endocrine therapies. Several well-characterized AR-Vs, such as AR-V7 and AR-V9, arise from splicing of AR exon 3 to different cryptic exons (CEs) located within AR intron 3. Splicing of these CEs is coordinated by a consensus AAUAAA poly(A) site located at the end of AR exon CE3. To define sequence elements in AR pre-mRNA required for usage of the CE3 poly(A) site, we designed inhibitory antisense oligomers to target regions upstream and downstream of the core AAUAAA motif. We found that sequences directly flanking the AAUAAA motif positively regulated AR-V expression, whereas sequences ~50 nucleotides downstream of the CE3 poly(A) site negatively regulated AR-V expression. To identify trans-acting factors that recognize and drive usage of the CE3 poly(A) site, we performed RNA-immunoprecipitation (RIP) using an RNA bait containing the CE3 poly(A) site or an RNA bait with the AAUAAA motif mutated. Proteins that preferentially bound the CE3 poly(A) site in RIP experiments were identified using mass spectrometry. We identified the scaffold protein SYMPK as a factor that bound specifically to RNA bait containing the CE3 poly(A) site. Consistent with a functional role for this binding event, knockdown of SYMPK reduced AR-V expression and inhibited growth of CRPC cell lines. We have also found SYMPK regulates the growth of AR negative prostate cancer cell lines, demonstrating AR-independent functions for SYMPK in regulating the growth of prostate cancer cells. This work highlights SYMPK as a novel regulator of AR-V expression and a therapeutic target for the treatment of multiple subtypes of advanced prostate cancer.