Inhibition of Scavenger Receptor Class B Type 1 (SR-B1) Expression and Activity as a Potential Novel Target to Disrupt Cholesterol Availability in Castration-Resistant Prostate Cancer

There have been several studies that have linked elevated scavenger receptor class b type 1 (SR-B1) expression and activity to the development and progression of castration-resistant prostate cancer (CRPC). SR-B1 facilitates the influx of cholesterol to the cell from lipoproteins in systemic circulation. This influx of cholesterol may be important for many cellular functions, including the synthesis of androgens. Castration-resistant prostate cancer tumors can synthesize androgens de novo to supplement the loss of exogenous sources often induced by androgen deprivation therapy. Silencing of SR-B1 may impact the ability of prostate cancer cells, particularly those of the castration-resistant state, to maintain the intracellular supply of androgens by removing a supply of cholesterol. SR-B1 expression is elevated in CRPC models and has been linked to poor survival of patients. The overarching belief has been that cholesterol modulation, through either synthesis or uptake inhibition, will impact essential signaling processes, impeding the proliferation of prostate cancer. The reduction in cellular cholesterol availability can impede prostate cancer proliferation through both decreased steroid synthesis and steroid-independent mechanisms, providing a potential therapeutic target for the treatment of prostate cancer. In this article, we discuss and highlight the work on SR-B1 as a potential novel drug target for CRPC management.


Background and Focus of This Perspective
Prostate cancer (PCa) was the second most frequently diagnosed cancer and the second and fifth leading cause of cancer death among Canadian men and men worldwide, respectively, in 2020 [1]. Androgen deprivation therapy (ADT) is effective for treating PCas that fail primary curative-intent therapies; however, remission is temporary [2,3], and disease re-emergence or castration-resistant prostate cancer (CRPC) occurs in approximately 20% of patients [4][5][6]. Expertly reviewed [7] advances in the understanding of the disease landscape and the mechanisms of progression have resulted in a remarkable array of therapies that have greatly increased the survival of patients with incurable metastatic disease. The focus of this article is on the underappreciated potential of targeting cholesterol metabolism in the advanced disease state and how understanding the role of scavenger receptor class B member 1 (SR-B1) may reveal potential therapeutic opportunities related to cholesterol ester (CE) uptake for de novo steroidogenesis and cellular homeostasis. pathway and then transported through the vasculature complexed within single, phospholipid membrane structures known as lipoproteins. Apolipoproteins are proteins that bind lipids and fat-soluble vitamins to form lipoproteins [52]. Different lipids present on apolipoproteins determine the functional identity of lipoproteins and their subsequent role within the human body. Cholesterol can either be present on the hydrophilic surface of the lipoprotein as native cholesterol or within the hydrophobic core as CEs (Figure 1) [53].

Figure 1.
Lipoprotein structure-an overview. Lipoproteins are a spherical monolayer of amphipatic phospholipids (yellow) and free/unesterified cholesterol (orange). The tails of the phospholipids create a hydrophobic core of non-polar lipids, primarily cholesterol esters (green) and triglycerides (pink), surrounded by a hydrophilic membrane of phospholipids, free cholesterol and apolipoproteins (blue). Lipoproteins differ in their lipid composition, size, density, major apolipoproteins and function [53]. Figure produced using Servier medical art [54].
Lipoproteins are classified by the inverse relationship of size/density, i.e., larger and lower density and smaller and higher density [53]. Lipoproteins are large and less dense when the fat to protein ratio is increased. They are most commonly classified as highdensity lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins, very-low-density lipoproteins (VLDL), and ultra-low-density lipoproteins (ULDL), which are also known as chylomicrons ( Figure 2). Each lipoprotein type carries out a specific function and is associated with a different disease [55]. HDL collects fat molecules from cells and tissues and takes them back to the liver; LDL carries 3000-6000 fat molecules around the body; VLDL carries newly synthesized triacylglycerides from the liver to the adipose tissue; and chylomicrons carry fat from the intestines to the liver, skeletal muscle, and adipose tissue [53].
Cholesterol is also obtained from diet and bile, where it is taken up from the intestine by enterocytes. Here, it is packaged into the triacylglyceride-rich chylomicrons or VLDL that also contain cholesterol and apolipoprotein B-48 (apo B-48), a truncated form of apolipoprotein B (apo B). Chylomicrons then enter the lacteals and the bloodstream through the thoracic duct [53]. The triglyceride contents of chylomicrons are metabolized by lipoprotein, lipase and the resultant high cholesterol chylomicron remnants are then endocytosed by the liver. VLDL, secreted from the liver, contains high levels of triglyceride complexed with apolipoprotein B-100 (apo B-100). It is this full form of LDL that is recognized by the LDL receptor (LDLR). The triglycerides within these particles are similarly metabolized by lipoprotein lipase. This results in smaller particles sometimes referred to as intermediate density lipoprotein (IDL) and eventually into cholesterol containing LDL [53,56]. Oxidation of either the lipid or apolipoprotein constituents of LDL leads to conformational changes, which prevent its uptake via LDLR; instead, it is taken up by scavenger receptors [57]. The reverse role in cholesterol transport, where cholesterol is transferred from peripheral tissues to the liver, is regulated by apolipoprotein A-I (Apo A-I), which is the major component of HDL [56,58]. HDL produced by the liver receives cholesterol through apo A-I's interaction with the ATP-binding cassette transporter Figure 1. Lipoprotein structure-an overview. Lipoproteins are a spherical monolayer of amphipatic phospholipids (yellow) and free/unesterified cholesterol (orange). The tails of the phospholipids create a hydrophobic core of non-polar lipids, primarily cholesterol esters (green) and triglycerides (pink), surrounded by a hydrophilic membrane of phospholipids, free cholesterol and apolipoproteins (blue). Lipoproteins differ in their lipid composition, size, density, major apolipoproteins and function [53]. Figure produced using Servier medical art [54].
Lipoproteins are classified by the inverse relationship of size/density, i.e., larger and lower density and smaller and higher density [53]. Lipoproteins are large and less dense when the fat to protein ratio is increased. They are most commonly classified as high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins, very-low-density lipoproteins (VLDL), and ultra-low-density lipoproteins (ULDL), which are also known as chylomicrons ( Figure 2). Each lipoprotein type carries out a specific function and is associated with a different disease [55]. HDL collects fat molecules from cells and tissues and takes them back to the liver; LDL carries 3000-6000 fat molecules around the body; VLDL carries newly synthesized triacylglycerides from the liver to the adipose tissue; and chylomicrons carry fat from the intestines to the liver, skeletal muscle, and adipose tissue [53].  [59]. It is HDL whose major component is apo A-I [60][61][62].   Cholesterol is also obtained from diet and bile, where it is taken up from the intestine by enterocytes. Here, it is packaged into the triacylglyceride-rich chylomicrons or VLDL that also contain cholesterol and apolipoprotein B-48 (apo B-48), a truncated form of apolipoprotein B (apo B). Chylomicrons then enter the lacteals and the bloodstream through the thoracic duct [53]. The triglyceride contents of chylomicrons are metabolized by lipoprotein, lipase and the resultant high cholesterol chylomicron remnants are then endocytosed by the liver. VLDL, secreted from the liver, contains high levels of triglyceride complexed with apolipoprotein B-100 (apo B-100). It is this full form of LDL that is recognized by the LDL receptor (LDLR). The triglycerides within these particles are similarly metabolized by lipoprotein lipase. This results in smaller particles sometimes referred to as intermediate density lipoprotein (IDL) and eventually into cholesterol containing LDL [53,56]. Oxidation of either the lipid or apolipoprotein constituents of LDL leads to conformational changes, which prevent its uptake via LDLR; instead, it is taken up by scavenger receptors [57]. The reverse role in cholesterol transport, where cholesterol is transferred from peripheral tissues to the liver, is regulated by apolipoprotein A-I (Apo A-I), which is the major component of HDL [56,58]. HDL produced by the liver receives cholesterol through apo A-I's interaction with the ATP-binding cassette transporter ABCA1 or the cholesterol efflux regulatory protein (CERP) in peripheral tissues and releases cholesterol to tissues expressing SR-B1 Figure 3 [59]. It is HDL whose major component is apo A-I [60][61][62].
ABCA1 or the cholesterol efflux regulatory protein (CERP) in peripheral tissues and r leases cholesterol to tissues expressing SR-B1 Figure 3 [59]. It is HDL whose major com ponent is apo A-I [60][61][62].  Cholesterol-intracellular and intratumoral metabolism. Cellular uptake of cholesterol mediated by endocytosis of low density lipoproteins (LDL) via the LDL receptors (LDLRs), an partially trough HDL receptors (HDLRs), ore commonly known as scavenger receptor, Class B1 (SR B1). LDLR levels in cells is tightly regulated by proprotein convertase subtilisin/kexin type (PCSK9) and inducible degrader of LDLRs (IDOL). In endothelial cells, HDL is a chemotactic facto . Cholesterol-intracellular and intratumoral metabolism. Cellular uptake of cholesterol is mediated by endocytosis of low density lipoproteins (LDL) via the LDL receptors (LDLRs), and partially trough HDL receptors (HDLRs), ore commonly known as scavenger receptor, Class B1 (SR-B1). LDLR levels in cells is tightly regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLRs (IDOL). In endothelial cells, HDL is a chemotactic factor and signaling is mediated SR-B1 which is associated with (PDZ Domain Containing 1 (PDZK1) and Pharmaceutics 2021, 13, 1509 5 of 37 sphingosine phosphate (S1P) receptor 1 (S1PR1) a scaffolding protein, leading to phosphorylation of AKT1 (p-AKT1) which phosphorylates and activates nitric oxide synthase to produce nitric oxide (NO). Cholesterol can also be produced de novo in cells via the mevalonate pathway. Reduction of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) to mevalonate is controlled by 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the enzyme targeted by statin drugs. Cholesterol efflux is facilitated by ATP Binding Cassette Subfamily A Member 1 (ABCA1) transporter. Once in the cell, cholesterol is involved in multiple functions including cell proliferation and androgen synthesis. Excess intracellular cholesterol can get esterified to cholesterol esters by sterol O-acyltransferase 1 (SOAT1) and stored as lipid droplets. Broken and continuous arrows indicate regulation and metabolic steps respectively. Figure produced using Servier medical art [54].

Cholesterol and Prostate Cancer (PCa)
Cholesterol was first observed as crystals present within tumors in 1909 [63], and increased cholesterol levels were first reported in prostate tumors in 1942 [64]. The subsequent wealth of epidemiological evidence regarding cholesterol levels in PCa produced confounding results. Several studies reported that lowering cholesterol increased cancer incidence [65,66], while others found no association between cholesterol and cancer [67]. A meta-analysis of the relationship between circulating cholesterol and PCa was conducted in 52 population studies, of which 32 studies found an inverse association between cancer risk and cholesterol level [68]. The authors also concluded that there was no absolute level of cholesterol associated with cancer; rather, the low cholesterol cohort, relative to the cohort average in any population, had a greater prevalence of cancer.
In contrast, men with high blood-cholesterol levels have been reported to be at an increased risk of PCa and PCa mortality [69][70][71]. Furthermore, cholesterol levels have been shown to be elevated in post-ADT patient serum [72][73][74][75][76][77][78][79] and bone metastasis [80,81]. Investigations into alterations specific to cholesterol metabolism within the tumor setting have generated evidence that hypercholesterolemia was associated with an increase in tumor volume, progression, and incidence in the transgenic adenocarcinoma mouse prostate (TRAMP) [82] and increased tumor growth, AKT activation, and intratumoral androgens in xenograft models [83][84][85]. Thus, there is emerging evidence that implicates a positive association between cholesterol and PCa [86].

Intratumoral Androgen Synthesis in Castrate-Resistant Prostate Cancer
As stated earlier, AR activity persists in the majority of CRPCs, and it is presumed that progression to CRPC is driven by reactivated AR activity despite patients being on ADT and having castrate androgen levels. Steroidogenic CRPCs rely on cholesterol for androgen biosynthesis and prostate tumors have increased levels of intracellular cholesterol precursors [87,88]. This demand for cholesterol in cancer cells is met by uptake from blood and de novo cholesterol synthesis. Systemic cholesterol, predominantly found and transferred by lipoproteins, is taken up by cancer cells through the actions of LDLR [89] and scavenger receptors [90]. Upregulated expression of HMGCR, LDLR, SR-B1, sterol O-acyltransferase1 (SOAT1), the enzyme that catalyzes the reaction between long-chain fatty acids and intracellular cholesterol to form hydrophobic CE [91], and loss of the function of ATP-binding cassette transporter A1 (ABCA1), which mediates cholesterol efflux from cells, further facilitates enrichment of cellular cholesterol pools ( Figure 3) [92][93][94][95]. Many of these key nodes in cholesterol metabolism are drug targets, which might be used to modulate intratumoral cholesterol metabolism. For instance, proprotein convertase subtilisin/kexin type-9 (PCSK9), a serine protease, modulates cholesterol metabolism by binding to LDLR and targeting it for lysosomal destruction [96] (Figure 3). It is also involved in degradation of other LDLR family members, namely VLDLR, lipoprotein receptor-related protein 1 (LRP-1), and apolipoprotein E receptor 2 (apo ER2) [97]. PCSK9 inhibitors represent a new class of drugs for treatment of CRPC in patients with familial hypercholesterolemia who do not respond to statins [97,98]. Adrenal androgens have long been considered a source of ligands for AR reactivation [99]; however, it is now appreciated that elevated intratumoral androgen levels in CRPC [9] can be explained by de novo intratumoral androgen synthesis as evidenced by elevated expression of steroidogenic enzymes essential for conversion of the precursor, cholesterol, into androgens at levels sufficient to activate the AR [8,10]. These integral points in androgen biosynthesis must be explored for novel therapies in order to improve outcomes in steroidogenic CRPC. Thus, disrupting the ability of PCa to synthesize or sequester sufficient cholesterol for steroidogenic and biogenic needs is a viable therapeutic opportunity for management of CRPC. Table 1 summarizes therapies that have been developed due to their effect on cholesterol metabolism.

CYP17A1
An androgen synthesis inhibitor that works by selectively inhibiting CYP17A1, which is an enzyme required for androgen synthesis in prostatic tumor tissue, among other tissues.

Effect of a Class of Drugs That Inhibit Cholesterol Synthesis on Prostate Cancer (PCa)
The seminal discovery that a class of drugs, called "statins," are efficient inhibitors of de novo cholesterol synthesis dramatically altered cardiovascular disease mitigation [105][106][107][108]. Similar in structure to HMG-CoA, statins fit into the enzymatic active site of HMG-CoA reductase to compete with native HMG-CoA binding [109,110]. This competition reduces the rate at which mevalonate is produced, thus inhibiting production of cholesterol in the liver [111]. In addition, statins also affect systemic cholesterol availability by promoting a compensatory decrease in circulating LDL and cholesterol transport through a variety of mechanisms [93,112,113]. The broad use of statins as first-line therapeutics for prevention of cardiovascular disease by inhibition of cholesterol synthesis raised the possibility of their effect on PCa. Findings from several studies indicate that statins may also play a role as anti-cancer agents in PCa via both cholesterol and pleiotropic non-cholesterol-mediated effects [108,[114][115][116][117][118]. In preclinical settings, simvastatin and lovastatin have been found to reduce cell viability through an induction of apoptosis; to inhibit cell proliferation, colony formation, and migration; to suppress the AKT pathway; and to cause G 1 phase cell cycle arrestation in AR-positive LNCaP, AR-negative PC-3, and DU145 cells, while atorvastatin, simvastatin, and rosuvastatin have all been shown to reduce the metastatic potential of PC-3 cells [119][120][121][122][123][124][125][126][127][128].
From an epidemiological perspective, several attempts have been made to understand the effect of statins on both the probability of occurrence of PCa and the clinical outcomes of those diagnosed with PCa [116]. While PCa epidemiologists increasingly correlate statin use with decreased PCa occurrence, and improved disease prognosis , there exists a vast body of literature surrounding the total incidence of PCa with conflicting results. Several studies have reported no effect on the incidence of PCa with statin use [107,[155][156][157][158][159][160][161][162][163][164][165][166], and one study associates statin use with an increased incidence of PCa [167]. These studies have been summarized in Table 2. To investigate the association of statin use with total and advanced PCa; the latter being the most important endpoint to prevent • Analyzed data from an ongoing prospective cohort study of 34,989 US male health professionals who were cancer-free in 1990 and were followed to 2002 • In this cohort of male health professionals, use of statin drugs was not associated with a risk of PCa overall but was associated with a reduced risk of advanced (especially metastatic or fatal) PCa • No evidence of an association between statin use and total PCa • There was a protective association between statin use and advanced PCa   To determine whether the use of statins after PCa diagnosis is associated with a decreased risk of cancer-related mortality and all-cause mortality and to assess whether this association is modified by prediagnostic use of statins  [152] To assess the association between β-blockers and PCa-specific mortality in patients with high-risk or metastatic disease and to address potential confounding from the use of statins or acetylsalicylic acid (ASA)  • Study did not support any associated between statin use and PCa but concluded that a reduced risk could not be ruled out   [160] To examine the association between the long-term use of cholesterol-lowering drugs, predominantly statins, and the incidence of ten common cancers including PCa, as well as overall cancer incidence  Despite the uncertainty as to whether statins impact overall PCa incidence, evidence that statin use may reduce the incidence of advanced PCa continues to grow. In an anecdotal case, a patient with bone metastatic CRPC who started on abiraterone and prednisolone, and soon after on atorvastatin, achieved a complete PSA response with no evidence of bone metastasis in six months [169]. Reports indicating that statins improve PSA decline and overall survival of abiraterone-treated patients [170,171] corroborate observations that statin use significantly increases progression-free, and overall, survival of abirateronetreated CPRC patients [172]. These clinical results support previous conclusions that this is, in part, due to a decrease in de novo cholesterol and androgen synthesis [126,[173][174][175].
Although not without contradiction, the wealth of evidence including consistent preclinical findings suggest that all elements of cholesterol homeostasis are deregulated in PCa to promote cellular cholesterol accumulation. This demonstrates the possibility of targeting cholesterol metabolism, through statins or potentially through other cholesterol metabolism proteins, as therapeutic targets in the treatment of PCa.

SR-B1
Scavenger receptors are a diverse group of sequence-unrelated proteins united by their ability to recognize common polyionic ligands [176]. Class B is comprised of three members whose short N-and C-terminal cytosolic regions regulate signal transduction and trafficking [176]. SR-B1 is encoded by the SCARB1 gene and is a highly glycosylated, 82 kDa cell-surface-receptor protein that contains two transmembrane domains located near to its cytosolic Nand C-terminal domains [177][178][179][180].

Transcriptional Regulation
Similar to LDLR, SR-B1 is ubiquitously expressed, but shows particularly high expression in the liver as well as steroidogenic tissues including the adrenal glands, ovaries, and testes [181,182]. It is differentially regulated between the liver and steroidogenic tissues. In steroidogenic cells, SR-B1 is primarily regulated by trophic hormones such as luteinizing hormone (LH), follicle stimulating hormone (FSH), and adrenocorticotropic hormone (ACTH) [177,178,[182][183][184][185]. Its expression in liver cells, however, can be affected by both hormonal control as well as dietary fats and pharmacologic agents, notably fibrates [182,186]. Prolonged adrenal stimulation by adrenocorticotropic hormone (ACTH) has been shown to induce SR-B1 expression in adrenocortical cells and reduce hepatic expression in mice and rats, suggesting a mechanism for the preferential channeling of cholesterol to the adrenal tissue [178,187,188]. On a cellular level, SR-B1 has been shown to be regulated in a rather complex manner including generic transcription factor binding sites for sterol regulatory element-binding proteins (SREBPs), steroidogenic factor-1 (SF-1), Liver X receptors (LXRs), liver receptor homolog 1, and others [181,182,189,190]. Evidence demonstrates that SR-B1 regulation by trophic hormones may be dependent on cAMP/protein kinase A (PKA) signaling stimulating transcriptional function of steroidogenic factor 1 (SF-1) and CCAAT-enhancer-binding proteins (C/EBP) [191,192]. Cholesterol regulation of SR-B1 has also been demonstrated through SREBP and LXR functions implicating that multiple signaling pathways participate in SR-B1 expression [182]. Its allelic variants are linked to an increased risk of atherosclerosis [193], infertility [194], and/or an impaired innate immune response [195][196][197].

SR-B1 Function
SR-B1 is primarily involved in the selective uptake of CEs from circulating HDL acetylated LDL (AcLDL) and oxidized LDL (OxLDL) [198] and binding to certain viruses and bacteria by a non-endocytic process [199][200][201]. The mechanism of HDL/SR-B1-mediated cholesterol uptake is not fully understood. Unlike LDLR-mediated LDL uptake, SR-B1 does not degrade HDL, rather it mediates selective CE uptake from HDL, AcLDL, and OxLDL in the absence of holo-lipoprotein uptake: that is, without the uptake and lysosomal degradation of the HDL particle itself [202,203]. This appears to be facilitated either by a "non-aqueous" channel passing through the protein [198] or by lipoprotein internalization after binding SR-B1 in a non-clathrin-coated vesicle manner, after which the CE-depleted lipoproteins are secreted [204,205]. It has been proposed that SR-B1-mediated CE uptake is a three-step process. First, a hydrophobic tunnel is formed by the extracellular domain of the receptor between the lipoprotein particle and the cell membrane. CE diffuse through this tunnel into the cell in a concentration gradient manner [206]. The high-resolution crystal structure of the extracellular domain of LIMP-2, a homologue of SR-B1, further corroborates the validity of this mechanism [207].
SR-B1 also facilitates the efflux of free cholesterol between cells and lipoproteins [208]. This mechanism, known as reverse cholesterol transport (RCT), consists of the transport of cholesterol via HDL from peripheral tissues such as macrophages or endothelial cells to the liver where this is utilized for bile acid production, cholesterol excretion, and steroidogenic organs [180,209]. This SR-B1-mediated cholesterol uptake by steroidogenic tissues [210] is critical for androgen synthesis and for the growth and survival signaling of non-steroidogenic endothelial cells [211][212][213]. In endothelial cells, SR-B1 is critical for HDL-induced activation of endothelial nitric oxide synthase (eNOS) leading to, at least in part, the athero-protective effects associated with HDL [214][215][216][217] (Figure 3.). The activation of eNOS, in turn, leads to increased concentrations of nitric oxide (NO) known to have a number of positive effects on cardiovascular health including vasodilation, endothelial regeneration, inhibition of leukocyte chemotaxis [218,219], and prevention of SR-B1-mediated induction of apoptosis [214,220]. Several pathways have been identified as potential mechanisms for this SR-B1-dependent signaling. HDL is known to carry several lipids beyond cholesterol, including lipid-soluble vitamins and sphingolipids [214]. Although insufficient on its own, sphingosine-1-phosphate (Sp1P), when associated with HDL, has been shown to be involved in the activation of eNOS [214,[221][222][223]. The mechanism by which Sp1P or other SR-B1 inducers drive eNOS activation is believed to be through the PI3K signaling pathway [214]. HDL binding to SR-B1 has been shown to lead to the activation of the non-receptor tyrosine kinase, c-Src, via the scaffolding protein PDZ domain-containing 1 (PDZK1) that is associated with SR-B1, which, in turn, activates both the PI3K/AKT and the RAS/ERK1/2 pathways, resulting in eNOS activation through serine 1179 phosphorylation [211,212]. Although the relationship between SR-B1 and these signaling pathways has largely only been established in endothelial cells, aberrations in the PI3K pathway and the RAS pathway have been identified and implicated in advanced PCa and highlight the importance of their consideration when studying SR-B1 in this disease context [224]. In macrophages, SR-B1 is associated with a reduction in atherosclerosis [225].
SR-B1 has been associated with a reduced risk of developing atherosclerosis. In macrophages, SR-B1 functions in the uptake of modified lipoproteins as well as in secreting cholesterol to HDL. Macrophages are incapable of limiting their uptake of modified lipoproteins via SRs and depend on cholesterol efflux mechanisms via SR-B1 for maintaining cholesterol homeostasis within the cell. Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. In macrophage-deficient mice, SR-BI promotes defective efferocytosis signaling via the c-Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation [226]. Macrophage-derived foam cells, which develop as a result of excessive accumulation of lipoprotein-derived cholesterol [227][228][229][230], play an important role in all stages of atherosclerotic lesion development [231]. In early atherosclerotic lesion, it is these macrophage-derived foam cells that are the predominant constituent of the fatty streak. In advanced atherosclerotic lesions, foam cells are detected as clusters of cells surrounding a core of lipid and necrotic material, where they modulate the stability of the atherosclerotic lesion ( Figure 4).

Inhibitors of SR-B1
SR-B1 mediates the selective uptake of CE from HDL into cells and the efflux of cholesterol from cells to lipoproteins. The need to understand the mechanism behind the process, which is distinct from the endocytoic uptake of other lipoproteins, led to the characterization of small molecules that could modulate SR-B1 function.
Blocker of lipid transfer (BLT) viz. BLT-1 through 5, a family of thiosemicarbazone copper chelators, were the first inhibitors to be characterized that could inhibit both cellular selective lipid uptake of HDL CEs and efflux of cellular cholesterol to HDL [232,233]. The inhibitory effects of the BLTs were specific to SR-B1 and did not interfere with several clathrin-dependent and -independent endocytic pathways, the secretory pathway, or the actin or tubulin cytoskeletal networks [233]. Of these five compounds, BLT-1 covalently and irreversibly bound to cysteine 384 of SR-B1 [234] in nanomolar concentrations to block CE influx and cholesterol efflux, while BLT-4 was demonstrated to specifically block both SR-B1-mediated influx of CEs and ABCA1-mediated cholesterol efflux to lipid-poor apo A-I [235]. However, in spite of its specificity and nanomolar potency, BLT-1 is extremely toxic to cells, which has limited its use to short-term in vitro assays.
Glyburide, a sulfonylurea thought to be capable of binding to SR-B1, was not specific to it; rather, it bound to and inhibited sulphonylurea receptors 1 and 2 (SUR1 and SUR2), members of the ABC superfamily of proteins [235].
ITX-5061, an arylketoamide derivative and initially characterized as a type II inhibitor of p38 MAPK, is also an SR-B1 antagonist and a potent SR-B1-mediated hepatitis C virus (HCV) entry inhibitor. In animal models, ITX-5061 was shown to increase serum

Inhibitors of SR-B1
SR-B1 mediates the selective uptake of CE from HDL into cells and the efflux of cholesterol from cells to lipoproteins. The need to understand the mechanism behind the process, which is distinct from the endocytoic uptake of other lipoproteins, led to the characterization of small molecules that could modulate SR-B1 function.
Blocker of lipid transfer (BLT) viz. BLT-1 through 5, a family of thiosemicarbazone copper chelators, were the first inhibitors to be characterized that could inhibit both cellular selective lipid uptake of HDL CEs and efflux of cellular cholesterol to HDL [232,233]. The inhibitory effects of the BLTs were specific to SR-B1 and did not interfere with several clathrin-dependent and -independent endocytic pathways, the secretory pathway, or the actin or tubulin cytoskeletal networks [233]. Of these five compounds, BLT-1 covalently and irreversibly bound to cysteine 384 of SR-B1 [234] in nanomolar concentrations to block CE influx and cholesterol efflux, while BLT-4 was demonstrated to specifically block both SR-B1-mediated influx of CEs and ABCA1-mediated cholesterol efflux to lipid-poor apo A-I [235]. However, in spite of its specificity and nanomolar potency, BLT-1 is extremely toxic to cells, which has limited its use to short-term in vitro assays.
Glyburide, a sulfonylurea thought to be capable of binding to SR-B1, was not specific to it; rather, it bound to and inhibited sulphonylurea receptors 1 and 2 (SUR1 and SUR2), members of the ABC superfamily of proteins [235].
ITX-5061, an arylketoamide derivative and initially characterized as a type II inhibitor of p38 MAPK, is also an SR-B1 antagonist and a potent SR-B1-mediated hepatitis C virus (HCV) entry inhibitor. In animal models, ITX-5061 was shown to increase serum HDL and apo A-I levels without affecting LDL/VLDL levels [236]. However, phase I clinical trials with ITX-5061 in liver transplant recipients with HCV were terminated [237], [238].
R-138329 increases plasma HDL cholesterol via inhibition of SR-BI-mediated selective lipid uptake [239]. In vivo studies on mice indicate R-138329 is principally involved in the inhibition of SR-BI-mediated selective lipid uptake in the liver.
High-throughput screening of the National Institutes of Health Molecular Libraries Probe Production Centers Network compound library led to the discovery of a potent class of indolinyl-thiazole-based inhibitor of SR-B1 viz. ML278 [240,241], a bisamideheterocycle inhibitors of SR-B1 viz. ML279 [241,242] and 8-membered benzo-fused lactam viz. ML312 [241,243]. All three compounds are low nanomolar, reversible inhibitors of SR-B1 with improved potency (ML312 > ML279 > ML278), decreased toxicity liabilities, and the capability to inhibit both SR-BI-mediated lipid uptake and the efflux of free cholesterol to HDL particles [241].
Nanoparticle mimetics of HDL and apo A-II have been demonstrated to be effective targets in nasopharyngeal carcinoma [244] and in pancreatic cancers, respectively [245]. SR-B1 is up-regulated in both cancers. Thus, biomimicry may be an important role in the next generation of cancer therapies. [246]. While these approaches to generate inhibitors of SR-B1 have been successful in vitro and some in vivo models, there still exists the need to develop an inhibitor that can achieve clinical utility.

SR-B1 and PCa
Several lines of evidence link elevated SR-B1 to PCa aggressiveness. SR-B1 expression is correlated with elevated expression of the androgen synthesis enzymes, 3βhydroxysteroid dehydrogenase (3β-HSD), 17β-HSD, and the mammalian target of rapamycin (mTOR) complex 1 target, ribosomal protein S6 [247]. Pre-clinically, elevated SR-B1 expression is observed in CRPC derivatives of LNCaP, an androgen-responsive PCa cell line, [248,249], and with Western-diet-induced tumor development in the TRAMP model [81]. siRNA silencing of SR-B1 has been shown to decrease PSA secretion and cell viability in C4-2, an LNCaP-derived CRPC model [249]. We and others have demonstrated that, clinically, elevated SR-B1 expression has been found to be associated with high Gleason-grade primary PCa wherein high expression was correlated with decreased disease-specific survival of PCa patients [90,250]. Its transcript levels were elevated in PCa versus benign prostate, as well as in tumors that failed androgen receptor pathway inhibitor (ARPI) therapy and progressed and in NEPC versus CRPC. Overall, the aforementioned findings underlie the apparent potential in targeting cholesterol metabolism as a mechanism for impeding proliferation of PCa and implicating SR-B1 as an actionable target for managing CRPC. A number of studies have already investigated SR-B1 as a potential therapeutic target in CRPC-these findings are summarized in Table 3. Table 3. SR-B1 as a potential therapeutic target for management of CRPC.

Study Objectives Key Findings Conclusions
Leon et al.
(2010) [248] To assess if cholesterol and its regulation are altered in CRPC cells using a murine PCa xenograft model To silence SRB1 and assess the ability of PCa cells to maintain internal supplies of androgens without the presence of cholesterol

Stress and Autophagy
AR activity is a critical driver of metabolic reprogramming in PCa [253,254]. ARmediated enhancement of oxidative phosphorylation and anabolic activity are necessary to sustain the increased demand for the synthesis of lipids, nucleotides, and amino acids [255]. ARTAs suppress these AR-driven metabolic events and activate acute stress pathways to sustain tumor viability. Increased expression of pro-survival chaperones, such as heat shock protein (HSP) 27 and clusterin (CLU) promote pro-survival and anti-apoptotic signaling events [256][257][258][259], enhance metastatic capacity [260], and suppress mitotic activity as part of chemotherapy resistance [261]. CLU enhances PCa survival by preventing protein aggregation and inducing autophagy: an important pro-survival nutrient recycling stress response [248,262]. In this context, it is notable that SR-B1-antagonized PCa cells significantly upregulate CLU and autophagy [250].
Autophagy functions as a robust mechanism by which PCa copes with several cellular stresses including ADT, chemotherapy, and nutrient deprivation, which would otherwise be lethal [263][264][265][266]. Autophagy is generally considered to be primarily regulated by mTOR through AMP-activated protein kinase (AMPK) activation due to low glucose and ATP [267]. There appears to be a stark induction of autophagy in SR-B1-antagonized cells [250]. Although the initial results indicated the role of reduced mTOR signaling as a driver of autophagic phenotype, further characterization of the mechanism of the observed stress remain to be investigated. These include downstream mTOR signaling pathways including S6 Kinase 1 (S6K1), the eIF-4E binding proteins (4E-BP1/2), or Unc-51-like autophagy activating kinase (ULK1/2) through which mTOR mediated signaling drives its regulatory effects [268].
External to mTOR-regulated autophagy, AMPK is a critical energy/nutrient regulatory protein. It functions as a sensor for depleted ATP conditions within the cell promoting catabolic processes to generate more ATP, which have been connected to metabolic processes including autophagy [269]. Capable of activating autophagy through both ULK1 phosphorylation and mTOR, inhibition, AMPK activity has been shown to be reduced by androgen-driven reduced expression of liver kinase B1 (LKB1) and may play a role in SR-B1 driven autophagy [269,270]. Several genes involved in AMPK signaling in androgendependent PCa cell lines are involved in lipid metabolism. These include HMGCR, fatty acid synthase (FASN), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2). Inhibiting AMPK signaling using BAY-3827 resulted in downregulation of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B), and serine-threonine kinase AKT3 in these PCa cell lines. In addition, BAY-3827 inhibited expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family, which was paralleled by impaired lipid flux [271].
Autophagy is also induced independently of mTOR, through endoplasmic reticulum stress (ERS)-induced expression of chaperones such as binding immunoglobulin protein (BiP), along with inositol-requiring enzyme 1 alpha (IRE1α) through the unconventional splicing x-box protein 1 (XBP1) [272][273][274][275]. Altered cholesterol metabolism is also known to induce ERS mechanisms, and disrupted lipid equilibrium induces the unfolded protein response (UPR) [276]. The UPR, generally in response to an accumulation of misfolded proteins, activates adaptive pathways that attenuate general protein translation, increase molecular chaperone expression, and induce cell cycle arrest to alleviate the ERS [276]. SR-B1 antagonism with either siRNA knockdown or BLT-1 treatment suppresses mTOR activity and induces BiP and, to a lesser extent, IREα expression [250]. Taken as a whole, these results indicate that SR-B1 antagonism induces a strong stress response integrating autophagic and ERS pathways.

Future Perspectives
CRPC requires androgens for its development. Initial ADT on primary PCa puts selection stress on the cells to develop new methods to generate their own AR. One of the ways PCa cells do this is by initiating de novo cholesterol synthesis and enhanced expression of SR-B1. To address low blood cholesterol, PCa cells increase the expression of SR-B1 to enhance the amount of cholesterol being received by cells. This perspective provides insight on targeting cholesterol metabolism in PCa by regulating SR-B1. Analysis of the relationship between SR-B1 expression at disease onset with patient outcomes could be performed using histological methods on tissue biorepository specimens powered to identify biomarkers for recurrence-free survival [277][278][279][280]. Such repositories consist of radical prostatectomy-derived specimens and corresponding patient follow-up allowing for direct comparison of protein expression to clinical outcome. This would allow for the analysis of the role of cholesterol metabolism proteins in biochemical recurrence as well as assessment of expression across the disease state [281].
Previous studies have highlighted the results of targeting SR-B1 in well-established human and animal PCa models [250,252]. However, determining the expression of SR-B1 and the effects of targeting it in additional AR-expressing PCa models, including the AR-driven but androgen-independent cell lines LN-AI, LN95, and 22Rv1, could provide additional insight into the role of SR-B1 in further progression of the disease. Since androgen independence of these lines is attributed to increased ligand binding domain-deficient AR splice variant, AR-V7, these models would allow interrogation of disease states that are not dependent on cholesterol for de novo steroidogenesis but that are AR-driven [282]. The apparent increased expression of SR-B1 in NEPC mRNA datasets and the sensitivity of the "double negative" PCa models, PC3 and DU145, to SR-B1 antagonism [250,252] provides an interesting fundamental basis for further investigation for a role of SR-B1 in the emergence and proliferation of "androgen-indifferent" PCa. NEPC model H660 and enzalutamide-resistant LNCaP-derived 42D and 42F cells [283,284] can be used as both a direct test of the transcript predictions of increased SR-B1 expression in NEPC and as a model for analyzing SR-B1 antagonism in NEPC. A critical question is whether SR-B1 antagonism can restore sensitivity to ARTAs such as enzalutamide in treatment-resistant disease. Moreover, PCa cells that maintain steroidogenic potential and AR expression such as VCaP would provide further context to effects on steroidogenic potential [285].
Although the use of immortalized PCa cell lines provides valuable tools for evaluating potential therapeutic targets, these models are limited by selective pressures including adaptions to continued proliferation in a non-tumor environment, leading to a poor correlation with clinical outcomes [286]. The advent of patient-derived xenografts maintained in murine hosts provides a powerful approach to evaluating therapeutic potential across the heterogeneity of the disease. With the caveat that mice are HDL-dominant, xenograft tumors, directly implanted from patient to mouse, are thought to better replicate clinical PCa, avoiding the genetic drift observed within immortalized cell lines [287][288][289][290]. Examining the effects of SR-B1 antagonism in either the cell lines or patient-derived xenografts would provide insight into the role of SR-B1 in PCa and which PCa phenotype is most impacted by its antagonism. A broad survey of models would be invaluable for efforts to determine the mechanism driving increased SR-B1 expression across the phenotypic spectrum of lethal CRPC.
Beyond our comprehension of how SR-B1 antagonism may be most effective, additional investigation into the mechanism of the anti-proliferative effects is warranted with respect to both cholesterol synthesis and cholesterol uptake inhibition. The findings discussed in this perspective describe the ability to inhibit both PCa growth and progression by limiting of cholesterol availability. The inability of exogenous steroids to revert the effects of SR-B1 antagonism in AR-responsive models, and the efficacy of SR-B1 antagonism to suppress the growth of AR-null PCa models, suggests that reducing cholesterol availability, at least by SR-B1 antagonism, is not primarily responsible for anti-tumor activity [250]. This finding emphasized the impact of more general nutrient-deprived stress mechanisms driven because of impacted cholesterol accumulation. To adapt to cellular stresses such as nutrient deprivation, both cancerous and non-cancerous cells employ several mechanisms of adaptation allowing for continued survival in harsh tumor microenvironments. Specif-ically, intracellular cholesterol is generally regulated through the SREBP-1 and SREBP-2 proteins, which, under cholesterol-depleted conditions, induce the expression of proteins responsible for cholesterol uptake and synthesis [291]. The activity of the SREBP pathway should therefore be examined as the mechanism by which SR-B1-antagonized cells upregulate HMGCR expression and vice versa with SR-B1 overexpression following statin inhibition of HMGCR. For cells unable to replenish cholesterol, cellular dysfunction could impact function through several processes, including impaired membrane synthesis and lipid-raft mediated signaling leading to the manifestation of cell stress responses [292,293].
Autophagy can manifest in response to several disruptions to cellular-signaling processes. This review describes the apparent induction of ERS and the UPR in response to SR-B1 antagonism as measured through increased expression of BiP and IRE1α. ERS is generally thought to be the result of a disruption in protein homeostasis [294] but has also been demonstrated in response to disrupted lipid homeostasis [295]. Further, ERS is known to induce autophagy, which could mechanistically explain the SR-B1-antagonized phenotype [295]. There are several proposed mechanisms of ERS-mediated autophagy that could be investigated to assess the connection, including eukaryotic initiation factor 2 (eIF2α)-autophagy-related protein 12 (Atg12) expression, IRE1α-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) expression, and measuring cellular Ca 2+ and calmodulin-dependent protein kinase kinase 2 (CaMKK-β).
Although generally considered to be cholesterol-poor, increased humoral cholesterol has been demonstrated in mitochondria and may indicate a role in sustained oncogenic signaling [296]. It has been postulated that this increased cholesterol plays a role in facilitating hypoxia-inducible factor 1-alpha (HIF1α) activity and mitochondrial function in the hypoxic environment generally observed in cancer [297]. The loss of this cholesterol could result in decreased HIF1α signaling and increased hypoxic stress, leading to the observed induction of autophagy with SR-B1 antagonism [297,298]. To assess this, general measurements of mitochondrial function could be taken including ATP production and reactive oxygen species concentration or more specific assessment of HIF1α signaling.
Cholesterol constitutes a critical component of lipid rafts, which are membrane domains essential for several proliferative signaling pathways [296]. These include receptor tyrosine kinases, insulin-like growth factor 1 receptor (IGFR), and epidermal growth factor receptor (EGFR) lipid raft signaling and the small GTPase Ras family with established roles in PCa proliferation [299][300][301][302]. Both IGFR and EGFR are known to induce cancer proliferation through PI3K/AKT and Ras/MAPK signaling pathways, the disruption of which can lead to the induction of autophagy [303,304]. PI3K functions to phosphorylate phosphatidylinositol 4,5-bisphosphate (PI [4,5]P2) to PI [3][4][5]P3, leading to a conformational change inducing AKT binding and activation that leads to a proliferative phenotype through many different branches including increased growth through mTOR activation, increased nutrient metabolism through the inhibition of glycogen synthase kinase 3 (GSK3), and decreased apoptotic signaling through the activation of nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB) [305]. Ras is a small GTPase, which, in its active state, recruits and activates a kinase signaling cascade through RAF and MAPK kinases, leading to the phosphorylation of numerous targets including pro-proliferation transcription factors Fos, Jun, and Myc [306,307]. These pathways are considered some of the most critical oncogenic pathways, the disruption of which through impaired lipid raft formation could lead to reduced proliferative signaling and the induction of autophagy. Not to be overlooked, SR-B1 is also capable of inducing PI3K/AKT and Ras/MAPK signaling via Src independent of its effects on cellular cholesterol [211,212]. The examination of the activity of these pathways would provide insight into their role in the induction of autophagy in SR-B1-antagonized cells. However, determining whether the altered activity of these pathways is a result of cholesterol-mediated or independent effects would be necessary. Both fluorescent-probe and detergent-resistance assessment could be used to directly assess membrane formation in SR-B1-antagonized cells [308,309]. In general, understanding alterations in cellular signaling processes that drive the observed autophagic phenotype in response to SR-B1 antagonism provides not only valuable mechanistic insight into the role of SR-B1 in PCa but may also guide the design of novel small molecule SR-B1 inhibitors while providing rational candidates for synthetic lethal co-targeting.
Synthetic lethal co-targeting, which is already in development, can be approached by either high-throughput identification or rational design as a method to increase the cytotoxicity of individual cholesterol-metabolism-targeted agents [310,311]. In theory, highthroughput approaches would employ genome-wide knockout libraries in concert with SR-B1 antagonism to identify candidates resulting in robust cytotoxicity. Examples of the potential of rational co-targeting of SR-B1 with either HMGCR or autophagy inhibitors have previously been demonstrated by us [250]. Chloroquine and hydroxychloroquine, which are clinically used prophylactically against malaria, together inhibit the lysosomal degradation of cellular components of autophagic vacuoles and are commonly employed as an in vitro autophagy inhibitor [312]. Clinically, several trials are currently being undertaken combining chloroquine or hydroxychloroquine with standard therapy in an effort to prevent therapy-induced autophagic resistance in several cancer types [313]. Of those trials that have reported their findings, largely in limited numbers of glioblastoma multiforme patients, minimal to modest improvements in outcomes have been reported [314][315][316][317][318][319]. Although the clinical potential of chloroquine or hydroxychloroquine appears limited, their use for pre-clinical proof of principal experiments in bladder cancer and glioblastoma cells, respectively, are still valuable [320,321]. Furthermore, more potent autophagy inhibitors have been developed, which may demonstrate superior co-targeting than traditional agents [281,322].
SR-B1-antagonized cells show an enhanced upregulation of CLU, a stress-activated chaperone that regulates protein homeostasis by preventing protein aggregation, enhancing autophagosome biogenesis, inhibiting apoptosis, and promoting the survival of PCa [281,323]. To determine if CLU expression affects SR-B1-antagonism-induced cell stress, SR-B1-antagonized cells could be targeted with either CLU-targeted siRNA or OGX-011 antisense oligonucleotide. Evaluating these co-targeting approaches or those identified through high-throughput approaches in a similar manner to the experiments described in this review would likely provide therapeutic options for inducing a more robust cytotoxic response in SR-B1-antagonized cells. The research proposed here could further the understanding of the role and potential of targeting cholesterol metabolism in PCa, through expanding both the knowledge of clinical expression and the impact and effects of targeting cholesterol metabolism pre-clinically [281].
Author Contributions: All authors contributed to the writing and review of this article. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.