Abstract 2467: Interaction of CYP3A5 with AR-HSP90 complex regulating AR activation in prostate cancer cells

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Abstract Background: Previously we have demonstrated that CYP3A5 stimulates prostate cancer cell growth by promoting AR activation and downstream signaling. We further investigated the mechanism of CYP3A5's interaction with the AR-HSP90 complex known to regulate AR activation process. AR activation is tightly regulated and it involves several chaperons (HSP90, HSP70, HSP40, and HSP27), multiple cofactors and protein kinases. We hypothesize that stoichiometric CYP3A5 binding to the HSP90-AR complex can induce conformational changes in the chaperone complex, promoting AR nuclear migration and activation. Method: We used co-immunoprecipitation to identify the interaction of CYP3A5 with the components of the AR-HSP90 complex. Immunofluorescence studies were performed to identify the components of the AR-HSP90 complex that co-localize with CYP3A5. FRET analysis was used to confirm the interactions of CYP3A5 with the AR-HSP90 complex. Results: We performed co-immunoprecipitation (co-IP) experiments with extracts from flag-tagged CYP3A5 transfected LNCaP cells. Our data demonstrates that CYP3A5 co-immunoprecipitates with HSP90, AR, HSP70 and HSP40, all known components of the HSP90-AR complex. We further confirmed using reverse immunoprecipitation with AR/ HSP90 antibodies and agarose A/G beads that CYP3A5 is a part of the HSP90-AR mature complex. Empty vector transfected cell extract and IgG were used as controls for pull down and reverse IP respectively. Immunofluorescence studies demonstrated that HSP70 and HSP40 co-localized with CYP3A5 in the cytoplasm. Interestingly, the MDAPCa2b and LNCaP cells show co-localization of CYP3A5 and AR in the nucleus, in contrast to non-transformed RWPE1 cells where CYP3A5 remains in the cytoplasm. The nuclear presence of CYP3A5 is more pronounced in MDAPCa2b cells carrying the wild type CYP3A5. The FRET analysis further confirmed direct interaction between CYP3A5 and HSP40 a chaperon in the AR-HSP90 complex. Conclusions: Our data confirms that CYP3A5 directly interacts with the AR-HSP90 complex regulating AR activation process. Further understanding of this interaction can help establish CYP3A5-AR axis as a novel therapeutic target. Citation Format: Priyatham Gorjala, Oscar Goodman Jr., Ranjana Mitra. Interaction of CYP3A5 with AR-HSP90 complex regulating AR activation in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2467.