Abstract 1800: Inhibition of scavenger receptor class B type I suppresses androgen pathway activity and induces cytotoxic stress in C4-2 castration resistant prostate cancer cells

Abstract

Abstract Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinical benefit; however, patients continue to develop therapeutic resistance. Androgen pathway activity persists in CRPC even under maximal androgen blockade conditions. A proposed mechanism for continued androgen pathway signaling is de novo intratumoral synthesis of androgen receptor agonists from the precursor cholesterol. The high density lipoprotein(HDL)-cholesterol receptor, scavenger receptor class B type I (SR-BI), is upregulated in CRPC models in vitro and in vivo. Here, we test the hypothesis that SR-BI is a source of cholesterol for de novo steroidogenesis in CRPC cells using the castration-resistant LNCaP-derived cell line, C4-2. Cells were transfected with either non-targeted (NC) or stealth RNAi duplexes (Thermo Fisher) targeting SR-BI to silence protein expression (SR-B1 KD). Prostate specific antigen (PSA) levels in media from SR-B1 KD samples were reduced to 39% of that seen in control cell media (20.9 ± 1.4 ng/mL/μg protein SRBI-KD vs. 34.5 ± 2.4 ng/mL/μg protein NC; n = 4, p < 0.05). Intracellular testosterone concentrations measured by LC-MS showed a 2-fold reduction in SRBI-KD samples (0.20 ng/mL/mg) compared to NC (0.41 ng/mL/mg; n = 1). Additionally, SR-BI KD cells exhibited reduced cell viability as measured by MTS assay and induction of G1/S cell cycle arrest as assessed by propidium iodide staining cell cycle analysis by flow cytometry (70.9 ± 7.9% G0-G1 phase fraction SRBI-KD vs. 58.3 ± 4.1% NC; n = 4, p < 0.05). SR-BI KD cells exhibited evidence of increased cell stress relative to NC cells. SR-BI-KD cells showed elevated induction of autophagy as assessed by measuring changes in LC3-I:LC3-II ratio by immunoblotting and formation of autophagosomes by immunofluorescence, alongside induction of senescence as assessed by measuring senescence associated beta-galactosidase (SA-β-gal) activity using flow cytometry analysis and a fluorogenic substrate. Further, treating C4-2 cells with an established SR-B1 inhibitor, BLT-1, lead to decrease PSA secretion, and co-treatment with the common cholesterol synthesis inhibitor, simvastatin, synergistically decreased PSA secretion from C4-2 cells. These results suggest that under androgen-deprived conditions, SR-BI targeting can decrease androgen pathway signalling and induce cellular stress, leading to G1/S arrest and decreased proliferation in the absence of an apoptotic induction. Lastly, combined inhibition of cholesterol uptake (BLT-1) and synthesis (Simvastatin) lead to a synergistic decrease in PSA secretion indicating the possibility that a multi-faceted cholesterol blockade approach to CRPC treatment may improve response to maximal hormone blockade by disrupting intratumoral steroid production. Citation Format: Jacob A. Gordon, Ankur Midha, Mitali Pandey, Kishor Wasan, Michael E. Cox. Inhibition of scavenger receptor class B type I suppresses androgen pathway activity and induces cytotoxic stress in C4-2 castration resistant prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1800.