Open Collections

Determining the role of scavenger receptor class B type I as a source of cholesterol for de novo androgen synthesis in castration-resistant prostate cancer

University of British Columbia

Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinical benefit; however, patients develop therapeutic resistance. Persistent androgen signaling in CRPC is maintained in part by intratumoral steroidogenesis from the precursor cholesterol. The high density lipoprotein-cholesterol (HDL) receptor, scavenger receptor class B type I (SR-BI), is upregulated in CRPC models in vitro and in vivo. This thesis tests the hypothesis that depriving CRPC cells of HDL as a cholesterol source by silencing SR-BI will diminish de novo steroidogenesis and resultant androgen receptor-mediated signaling necessary for CRPC viability. The effects of SR-BI silencing were studied using CRPC C4-2 cells transfected with either Stealth RNAi duplexes targeting SR-BI (SRBI-KD) or Lo GC Negative Control (NC) duplexes. Cells cultured in androgen-depleted conditions for varying times post-transfection were assessed for SR-BI expression, prostate specific antigen (PSA) expression by chemiluminescence, cholesterol levels by fluorometry, and steroid levels by liquid-chromatography-mass spectrometry. HDL-uptake was measured by flow cytometry of the fluorescent cholesterol mimetic 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-HDL). Cell cycle state was assessed by flow cytometry of propidium iodide DNA staining, while cell cycle markers were assessed by immunoblotting. Adaptive stress responses were assessed by immunoblotting for autophagy markers, as well by flow cytometry for senescence associated beta-galactosidase (SA-β-gal) using a fluorogenic substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside. SRBI-KD treatment reduced SR-BI levels by ~57% by 2 days and ~86% by 4 days post-transfection. This correlated with reduced DiI-HDL-uptake by 22%, reduced cellular testosterone levels two-fold, and reduced PSA secretion by 39% compared to NC cells. These changes were accompanied by reduced proliferation, G₁S cell cycle arrest, and a small, but measurable increase in cell death at 4 to 6 days post-transfection. Cell stress was evidenced by enhanced autophagy activity and induced stress marker expression while senescence was evidenced by increased SA-β-gal activity. These studies indicate that SR-BI silencing reduced DiI-HDL uptake which reduced cellular androgen content and androgen signaling, resulting in induction of an adaptive stress response characterized by cell cycle arrest, autophagy, senescence, and eventually death of these CRPC cells. The data presented herein provide support for SR-BI as a cholesterol source for androgen synthesis in CRPC cells.

Text

2015-08-12

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/54366

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/