Abstract B001: CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance

Abstract

Abstract Purpose: Androgen receptor (AR) plays a major role in promoting growth and progression of prostate cancer and thus has been successfully targeted therapeutically. Therapeutic resistance to androgen deprivation therapy (ADT) accounts for much of the morbidity and mortality attributable to prostate cancer. Therapeutic resistance is typically accompanied by insufficient blockade of AR activation, whose initial step involves translocation of AR to the nucleus. In recent studies we demonstrated that CYP3A5 facilitates the nuclear translocation of AR, promoting prostate cancer growth. This work focuses on understanding the role of CYP3A5 in regulating AR nuclear translocation and therapeutic resistance. Method: To understand how CYP3A5 effects AR downstream signaling, we used RT2 profiler PCR arrays to help identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy and cell fractionation experiments were used to further confirm the effect of CYP3A5 modulation of AR signaling. As concomitant CYP3A5 inducer/inhibitor medications can affect intratumoral CYP3A5 activity and thus may modify AR downstream signaling, we utilized cell fractionation and confocal microscopy to analyze the effect of these medications on AR nuclear translocation. Further, to decipher the role of CYP3A5 in the AR translocation process we performed a series of co-immunoprecipitation (co-IP) experiments with CYP3A5 and components of HSP90-AR complex. We used extracts of LNCaP transfected with a CYP3A5-Flag-tagged construct for IP, with either Flag-agarose or HSP90 and AR antibodies with A/G beads. Finally, to decipher CYP3A5’s role in therapeutic resistance we analyzed cDNA after CYP3A5 siRNA treatment using drug resistance RT2 profiler PCR array. Results: The q-PCR based profiler assay revealed a set of nine AR regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005. MYC and PIK3R3 are known to be involved in regulating cell growth by either affecting cell cycle progression and/or apoptosis. PSA, known to aid in the cell growth, is regulated by SPDEF, a transcription factor. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. Concomitantly prescribed CYP3A inhibitors amiodarone and ritonavir blocked AR nuclear migration and decreased PSA expression whereas inducers phenytoin and rifampicin promoted increased AR nuclear migration and increased PSA production, supporting our hypothesis. Co-immunoprecipitation experiments revealed that CYP3A5 is a part of the HSP90-AR complex. Both Flag-immunoprecipitation and indirect immunoprecipitation revealed that CYP3A5 associates with the HSP90-AR complex. The CYP3A5 siRNA also downregulated 11 genes (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB, and HPRT1) known to confer drug resistance, with a p value of ≤0.005. The role of TOP2A, MYC, and BRCA is well known in prostate cancer drug resistance; thus, their downregulation with CYP3A5 siRNA treatment may predict better outcome. Conclusion: Our results indicate that CYP3A5 is a part of the HSP90-AR complex and positively regulates AR nuclear translocation promoting AR downstream signaling and therapeutic resistance. Therefore, targeting CYP3A5 to block AR nuclear translocation may be a therapeutic adjunctive approach in advanced prostate cancer. As CYP3A5 siRNA also downregulates drug-resistance genes, targeting CYP3A5 may also help overcome therapeutic resistance. As many concomitant medications can alter CYP3A5 activity, thus they can modify AR signaling. We hypothesize that AR directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors. These observations may play a significant role in African Americans who express high level of wild-type CYP3A5 and often have aggressive prostate cancer. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B001.