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Open Full Page Published OnlineFirst May 7, 2019; DOI: 10.1158/0008-5472.CAN-18-2529 Cancer Molecular Cell Biology Research Upregulation of Scavenger Receptor B1 Is Required for Steroidogenic and Nonsteroidogenic Cholesterol Metabolism in Prostate Cancer Jacob A. Gordon1,2, Jake W. Noble1, Ankur Midha3, Fatemeh Derakhshan4, Gang Wang4, Hans H. Adomat1, Emma S. Tomlinson Guns1,2, Yen-Yi Lin1, Shancheng Ren5, Collin C. Collins1, Peter S. Nelson6, Colm Morrissey7, Kishor M. Wasan2,8, and Michael E. Cox1,2,9 Abstract Aberrant cholesterol metabolism is increasingly appre- viability and induced endoplasmic reticulum stress and ciated to be essential for prostate cancer initiation and autophagy. The inability of exogenous steroids to reverse progression. Transcript expression of the high-density lipo- these effects indicates that AR pathway activation is insuffi- protein-cholesterol receptor scavenger receptor B1 (SR-B1) cient to overcome cytotoxic stress caused by a decrease in the is elevated in primary prostate cancer. Hypothesizing that availability of cholesterol. Furthermore, SR-B1 antagonism SR-B1 expression may help facilitate malignant transforma- decreased cholesterol uptake, growth, and viability of the tion, we document increased SR-B1 protein and transcript AR-null CRPC cell model PC-3, and the small-molecule SR- expression in prostate cancer relative to normal prostate B1 antagonist block lipid transport-1 decreased xenograft epithelium that persists in lethal castration-resistant prostate growth rate despite poor pharmacologic properties. Overall, cancer (CRPC) metastasis. As intratumoral steroid synthesis our findings show that SR-B1 is upregulated in primary and from the precursor cholesterol can drive androgen receptor castration-resistant disease and is essential for cholesterol (AR) pathway activity in CRPC, we screened androgenic uptake needed to drive both steroidogenic and nonsteroi- benign and cancer cell lines for sensitivity to SR-B1 antag- dogenic biogenic pathways, thus implicating SR-B1 as a onism. Benign cells were insensitive to SR-B1 antagonism, novel and potentially actionable target in CRPC. and cancer line sensitivity inversely correlated with expres- sion levels of full-length and splice variant AR. In androgen- Significance: These findings highlight SR-B1 as a potential responsive CRPC cell model C4-2, SR-B1 antagonism sup- target in primary and castration-resistant prostate cancer that is pressed cholesterol uptake, de novo steroidogenesis, and AR essential for cholesterol uptake needed to drive steroidogenic activity. SR-B1 antagonism also suppressed growth and and nonsteroidogenic biogenic pathways. Introduction tion-resistant disease (CRPC; refs. 2, 3). Its levels are elevated in patient serum and bone metastasis after androgen deprivation Cholesterol is essential for rapid cancer growth (1), and has therapy (ADT), and hypercholesterolemia correlates with been specifically linked to prostate cancer progression to castra- increased prostate cancer–specific mortality (4–6). In addition, association of elevated squalene monooxygenase (SQLE) expression 1 Vancouver Prostate Centre, Vancouver Coastal Health Research Institute, with higher Gleason grade and disease-specific mortality indicates Vancouver, Canada. 2Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada. 3Institute of Immunology, Freie Universitat€ a role for de novo intratumoral cholesterol synthesis in lethal Berlin, Berlin, Germany. 4Department of Pathology, British Columbia Cancer prostate cancer (7). The increased appreciation that statin use is Agency, Vancouver, Canada. 5Department of Urology, Second Military Medical correlated with decreased prostate cancer occurrence and University, Shanghai, China. 6Human Biology Division, Fred Hutchinson Cancer improved disease prognosis (8–10), together with evidence link- 7 Research Center, Seattle, Washington. Department of Urology, University of ing statin use to improved PSA declines and overall survival in 8 Washington, Seattle, Washington. College of Pharmacy and Nutrition, Univer- abiraterone-treated patients (11, 12), highlight the benefitof sity of Saskatchewan, Saskatoon, Canada. 9Department of Urologic Sciences, University of British Columbia, Canada. reducing de novo cholesterol and androgen synthesis to achieve maximal suppression of androgen receptor (AR) pathway activa- Note: Supplementary data for this article are available at Cancer Research tion, and management of advanced prostate cancer (13–16). Online (http://cancerres.aacrjournals.org/). Cholesterol needs can also be met by elevating systemic uptake Corresponding Author: Michael E. Cox, University of British Columbia, JBRC, via the actions of low-density lipoprotein receptor (LDLr) and Rm. 240, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, Canada. scavenger receptors (SR), particularly the Class B1 allele, SR-B1 Phone: 604-875-4111, ext. 68369; Fax: 604-875-5654; E-mail: [email protected] (SCARB1; ref. 17). LDLr transcript levels are lower in more aggressive tumors (7, 18). Although elevated SCARB1 transcript Cancer Res 2019;79:3320–31 levels have been suggested to correlate with decreased disease-free doi: 10.1158/0008-5472.CAN-18-2529 survival (18), analyses of the well-annotated Health Professional Ó2019 American Association for Cancer Research. Follow-up, Physicians' Health Study, and Swedish Watchful 3320 Cancer Res; 79(13) July 1, 2019 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 7, 2019; DOI: 10.1158/0008-5472.CAN-18-2529 SR-B1: A Novel Prostate Cancer Target Waiting cohorts demonstrated unchanged expression relative to low-bicarbonate DMEM (ATCC) supplemented with 10% FBS. tumor Grade or disease outcome (7). Whether SR-B1 expression Unless otherwise noted, all other reagents were from VWR or persists in CRPC, and how it might promote mechanisms of Thermo Fisher Scientific. malignant transformation, remain to be determined. SR-B1 internalizes high-density lipoprotein (HDL) cholesterol, Block lipid transport-1 and acetylated or oxidized LDL, and has allelic variants linked to Block lipid transport-1 (BLT-1; ChemBridge), a selective inhib- increased risk of atherosclerosis and an impaired innate immune itor of cholesteryl ester transfer through SR-B1 (29), or dimethyl response (19). It is also critical for cholesterol uptake as a sulfoxide (DMSO, vehicle), was added to cells cultured in phenol precursor for androgen synthesis in steroidogenic tissues (20). red-free media supplemented with 5% charcoal-dextran–stripped Experimentally, linkage of SR-B1 expression to prostate cancer FBS (CSS, Invitrogen) at the indicated final concentrations. Unless aggressiveness includes elevated expression in de novo androgenic otherwise specified, all assays were conducted 3 days posttreat- CRPC derivatives of LNCaP (13, 16), and increased tumor growth ment initiation. in TRAMP (21). SR-B1 also signals growth and survival of non- steroidogenic endothelial (22), and breast cancer cells (23), and RNA interference association of elevated expression with aggressive characteristics One day after transfection with either Stealth RNAi duplexes and poor prognosis of breast, and clear cell renal carcinomas, targeting SR-B1 (SRB1-KD: Oligo ID HSS101571: AUAAUCC- indicates roles for SR-B1 in multiple malignancies (24–26). GAACUUGUCCUUGAAGGG, catalog. no. 1299001) or Lo GC Hypothesizing that SR-B1 expression may help facilitate malig- Negative Control duplexes (NC: catalog. no. 12935-110; Invitro- nant transformation by increasing levels of metabolically avail- gen), cells were cultured in phenol red-free RPMI1640 with 5% able cholesterol, we demonstrate increased SR-B1 expression in CSS for C4-2 cells, or DMEM with 10% FBS for PC3 cells (30). the transition from normal prostatic tissue to cancerous tissue, Unless otherwise specified, all assays were conducted 4 days and persistent high expression in metastases. We go on to show posttransfection. sensitivity of androgenic prostate cancer cell lines to SR-B1 antag- onism, and how targeting SR-B1 suppresses cancer growth AR activation reagents through induction of endoplasmic reticulum (ER) stress and Metribolone (R1881, Perkin Elmer), dehydroepiandrosterone autophagy via both steroid and nonsteroid-based mechanisms. (DHEA, Steraloids), and progesterone (Sigma-Aldrich) were These results implicate systemic cholesterol uptake mechanisms, added to culture medium simultaneously with BLT-1. DHEA was particularly SR-B1, as potentially actionable targets for managing added to culture media 1 day post-SRB1-KD/NC transfection. CRPC. Immunoblotting Cellular protein levels were determined by immunoblot anal- Materials and Methods ysis as described in Supplementary Methods. Samples were nor- IHC and mRNA expression analysis of clinical prostate cancer malized using primary antibodies targeting GAPDH (sc-32233) samples from Santa Cruz Biotechnology, and b-actin (A2228), or vinculin IHC staining of the prostate cancer Donor Rapid Autopsy (V4505) from Sigma-Aldrich. Antibodies targeting the AR (sc- Program at the University of Washington (UWRA, Seattle, WA) 7305) was from Santa Cruz Biotechnology, SR-B1 (NB400-104) metastatic CRPC tissue microarray was performed using SR-B1 was from Novus Biologicals, and clusterin (CLU: 4214S and sc- primary antibody: AB52629 (Abcam; ref. 27). Metastatic speci- 6419) were from Cell Signaling Technology and Santa Cruz mens were obtained from patients who
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