Comparing the phase separation of the androgen receptor (AR) and its splice variant (AR-V7) in prostate cancer

Synopsis

Background: The androgen receptor (AR) is the primary target for prostate cancer (PCa) therapies. However, resistance can arise through AR amplification and the expression of active splice variants like AR-V7. We recently reported that full-length AR (AR-FL) forms nuclear condensates in response to androgens to execute its oncogenic programs (PMID: 36535377). We observed that AR-V7 behaves differently from AR-FL in androgen-sensitive models. Therefore, we investigated AR-V7 condensate formation in PCa models and in vitro, compared its phase behavior to AR-FL, and identified its condensate-dependent transcriptome.

Methods: We examined condensates formed by exogenous AR-FL and AR-V7 tagged with a non-dimerizing EGFP using confocal microscopy and assessed their dynamics with Fluorescence Recovery After Photobleaching (FRAP). We used PROTAC AU15330 to degrade the chromatin remodeler SMARCA2/4 and evaluate its role in AR-V7 condensate formation, and degraded AR-FL with ARV-110 to assess the interdependency between AR-FL and AR-V7 condensates in PCa cells. We explored the role of AR-V7 condensates in transcription by examining their colocalization with RNA Pol II and MED1 using immunofluorescence and proximity ligation assays (PLA) and assessing target genes expression by qPCR. We also identified an AR-V7 mutant that disrupts condensate formation without affecting nuclear translocation or DNA binding and used it to study the condensate-dependent transcriptome by RNA-seq. Lastly, we assessed AR-V7-mEGFP droplet formation in vitro and their colocalization with red-labeled MED1 and DNA using confocal microscopy.

Results: AR-V7-mEGFP forms condensates more prominently in PCa models that endogenously express splice variants, regardless of androgens or AR-FL presence. We confirmed that AR-V7 condensates exhibit dynamic behavior and require chromatin accessibility. In both PCa cells and in vitro, AR-V7 requires 3 to 5 times higher concentrations to form condensates compared to AR-FL. AR-V7 condensates colocalize with MED1 and RNA PolII in PCa cells, and their formation correlates with the transcriptional activity. We created a phase-altering AR-V7-5YS mutant and identified KRAS signaling as dependent on AR-V7 condensates.