Statins: protectors or pretenders in prostate cancer?

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Review

Statins: protectors or pretenders in prostate cancer? Hyeongsun Moon1, Michelle M. Hill1, Matthew J. Roberts2,3,4, Robert A. Gardiner2,3, and Andrew J. Brown5 1

The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, QLD 4102, Australia 2 The University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, QLD 4006, Australia 3 Department of Urology, Royal Brisbane and Women’s Hospital, Brisbane, QLD 4006, Australia 4 School of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia 5 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia

The role of statin therapy in prostate cancer (PCa) prevention and treatment is plagued by controversy. This critical review of published clinical series reveals several caveats in earlier studies, which reported no benefit. Recent studies that adjust for confounding factors have demonstrated statin therapy to be associated with PCa prevention and favorable clinical outcomes. Developed as inhibitors of cholesterol synthesis, the expected mechanism of statin action is systemic cholesterol reduction. By lowering circulating cholesterol, statins indirectly reduce cellular cholesterol levels in multiple cell types, impacting on membrane microdomains and steroidogenesis. Although non-cholesterol mechanisms of statin action have been proposed, they are limited by the uncertainties surrounding in vivo tissue statin concentrations. Are statins a useful therapy in the fight against PCa? PCa is the most commonly diagnosed cancer in men [1]. Although androgen-deprivation therapy may slow and temporarily reverse tumor progression, the clinical response to this therapy inevitably changes, a state termed castration-resistant PCa, for which there is currently no cure [2]. Over the past 15 years a beneficial link between the use of the ‘statin’ class of FDA-approved cholesterollowering drugs and PCa has been suggested. In this article we critically review the current evidence from clinical studies and the range of possible molecular mechanisms of statins in PCa prevention and therapy. Clinical studies of statins in PCa Important considerations Following FDA approval for treating hyperlipidemia in 1987, statins have become among the most commonly Corresponding author: Brown, A.J. ([email protected]). Keywords: statin; HMG-CoA reductase; prostate cancer; cholesterol; androgen synthesis; prenylation. 1043-2760/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2013.12.007

prescribed medications worldwide, and are ingested regularly by 25% of adults aged 45 years and over in the USA [3]. Importantly, use of these drugs provides significant benefits in primary and secondary prevention of major occlusive vascular events [4]. These medications have well-studied side-effect profiles and are generally tolerated without concerns (discussed in Box 1), to such an extent Glossary Abiraterone acetate: an inhibitor of cytochrome P450 17a-hydroxylase/17,20lyase (CYP17), a key enzyme that catalyzes the biosynthesis of androgens from pregnane precursors. FDA-approved for metastatic castration-resistant PCa patients. Adrenal gland: an endocrine organ and extra-testicular source of androgens. Production of these androgens may be considered a therapeutic target in castration-resistant PCa patients. Biochemical recurrence (BCR): also called biochemical failure, is defined as an increase in serum PSA following treatment for PCa with curative intent. Ezetimibe: a drug that inhibits intestinal cholesterol absorption by binding to Niemann–Pick C1-like 1 (NPC1L1) protein. Often considered for combination therapy with statin in hyperlipidemic patients. Fibrates: agonists of peroxisome proliferator-activated receptor-a (PPAR-a); lipid-lowering medications that reduce triglyceride-rich lipoproteins by inhibiting the synthesis of very low density lipoprotein (VLDL). Liver X receptor (LXR): a sterol-activated nuclear transcription factor which participates in cellular cholesterol efflux. Myositis: muscular pain or discomfort from infection or other causes such as autoimmune diseases. Niacin: a type of vitamin B (vitamin B3 or nicotinic acid). Can be used as a lipidlowering drug. Prostate cancer (PCa): an adenocarcinoma arising from the prostatic epithelium. Prostate-specific antigen (PSA): a serine protease of the kallikrein-related peptidase family that is released by prostate epithelial cells. Often observed to be elevated in the serum of PCa patients, hence its use as a biomarker. Resins: bile acid resins used as lipid-lowering agents; they bind to cholesterolderived bile acids, reducing their absorption and thus their enterohepatic recirculation from the small intestine. Rhabdomyolysis: the destruction or degeneration of skeletal tissues with massive release of proteins (creatine kinase) into the bloodstream, potentially causing end-organ failure. Sterol-regulatory element-binding protein (SREBP): a transcription factor that enables cholesterol synthesis and uptake. Three isoforms are found in mammals: SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1 participates in fatty acid synthesis whereas SREBP-2 is involved in cholesterol metabolism. Statin: a class of drugs that competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the mevalonate pathway which is involved in cholesterol synthesis and the formation of isoprenoid lipid anchors (prenylation). Statins inhibit the mevalonate pathway but also increase the expression of low-density lipoprotein (LDL) receptors in hepatocytes, thereby reducing circulating blood cholesterol levels. Hydrophobic (lipophilic) to hydrophilic (lipophobic) statins (in order of hydrophobicity): lovastatin > simvastatin > atorvastatin >> fluvastatin > rosuvastatin > pravastatin.

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Review Box 1. Side-effects of statins The most common side-effects of statins include muscle-related symptoms (myalgia in 0.1–15% of patients) and liver enzyme elevations, which may result in patients not taking their medication [65–67]. True incidence estimates of adverse events of statins may be higher, as described in observational studies and post-marketing surveillance data, owing to reporting and patient selection in initial studies as well as a lack of consensus definitions [66,67]. However, severe muscle conditions such as myopathy (0.0002%), myositis (0.03%), and rhabdomyolysis (0.002%) are rare in people taking commercially available statins [65]. Further meta-analyses have also shown these risks are not significantly increased when compared with placebo [68]. Other rare potential side-effects include gastrointestinal and urinary tract dysfunctions, autoimmune disorders, and cardiac dysfunctions such as atrial fibrillation and bradycardia [65]. When combined with other drugs (i.e., warfarin) that are metabolized by similar liver enzymes, adverse reactions may be more frequent because of enzyme competition and the resulting higher concentrations of circulating statin [65]. The etiology appears to be multifactorial, being statin-specific (type, dose, metabolism, drug interactions) and relating to patient factors (age, other medical conditions, genetics) [66–68]. However, various algorithms may predict risk of statin-associated adverse effects [66], and strategies exist for the management of adverse effects [68].

that they have been suggested as an important part of a smart drug combination advocated for the populace at large [5]. However, contradictory findings have been reported for statins in PCa. Older studies are mostly observations of large populations or evaluations of databases from other trials in case–control or cohort formats, which were often underpowered and failed to adjust for confounding factors such as serum prostate-specific antigen (PSA; see Glossary) [6]. Clinical use of serum PSA as a surrogate marker for PCa is common, resulting in a higher incidence of PCa diagnosis and treatment [7]. To compound complexities, serum PSA is also reduced with obesity, a condition that contributes to higher rates of advanced and aggressive PCa [8,9]. Furthermore, even statins are known to reduce serum PSA [10–16], and this may complicate the utility of serum PSA in this population further because it may reduce the index of suspicion for PCa and the need for prostate biopsy-based diagnosis. Overall, it is clear that reports on statins and PCa should be assessed in an informed manner, especially with respect to numbers of PCa cases and adjustment for serum PSA and other clinical variables, together with informed assessment of data collection and reporting methods. There are many reports of no beneficial or harmful effects of statins on PCa-specific endpoints. Of note, Chan and colleagues studied data collected as part of a prospective study on male osteoporotic fractures and found no association between statin use and PCa endpoints (total, low/high stage, low/high grade PCa), with adjustment for standard clinical parameters but not PSA, presumably because they did not possess these data [17]. Similarly, Vinogradova et al. used a series of nested community-based case–control series of statin users with primary cancers (n = 88 125) and matched controls (n = 362 254), but did not find any beneficial association with PCa [18]. When adjustments for serum PSA are made, an association between statin use and reduced risk of diagnosis of PCa (overall), aggressive, and fatal disease has been 2

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reported [10,12,15,19,20]. Geybels and colleagues reported that, in a retrospective evaluation of 1001 patients who were interviewed prior to PCa diagnosis, and clinical outcome determined using the Surveillance, Epidemiology, and End Results program (SEER) registry, PCa specific mortality was lower (1 vs 5% at 10 years), with cancer stage determined to be favorable in statin users [10]. Breau and colleagues examined a longitudinally based cohort with biennial examinations and observed that statin users demonstrated a reduced risk of undergoing prostate biopsy or high-grade PCa diagnosis [19]. In addition, a longer duration of statin use was associated with a lower risk for adverse PCa outcomes and a reduced likelihood of returning an abnormal PSA result when compared to a given agespecific PSA reference range. This relationship of duration of statin use with PCa outcomes may be dose-dependent, with Lustman and colleagues observing this relationship in 1813 PCa cases in a population of 66 741 patients, supporting a longer duration of statin use being associated with decreased PCa incidence, whereas a stronger association was observed for increasing total dose, for both hydrophilic and hydrophobic statins [20]. These observations were further confirmed when associations between statin use and PCa were examined by Murtola and colleagues in the Finnish PCa screening trial of 23 320 men with 9 years of follow-up [12]. They found that statin use was associated with reduced PCa incidence, and reported a dose-dependent (cumulative amount used) relationship for users of commonly prescribed statins, including simvastatin, atorvastatin, and fluvastatin. Statins outperformed other hypolipidemic agents [fibrates, acipimox (niacin), and resins], although this group was substantially smaller (n = 437) than the statin (n = 6692) or non-user (n = 16 516) groups. Users of statins and other hypolipidemic agents demonstrated lower serum PSA levels and higher free (unbound) to total PSA ratios, with a lower ratio often associated with PCa [21]. In addition, relative risk of PCa was reduced in patients undergoing prostate biopsy. A recent meta-analysis of statin use and risk of PCa based on 27 observational studies (15 cohort, 12 case– control), with a pooled population of 1 893 571 men and 56 847 cases of PCa, reported a significant 7% reduction in risk of overall PCa [6]. A more striking and clinically valuable finding was the 20% reduction in advanced PCa. An association with long-term statin use was not established, but a cumulative trend of change in risk reporting from positive to negative was observed for the period 1993–2011. There was a significant inverse association between risk of total PCa and statin use in studies published after 2007, without publication bias, presumably with more consistent adjustment for PSA. The followup periods for most included studies were extensive (median 8.5 years) given that PCa is known to be a slowly growing disease and a life-expectancy of >10 years is required to provide evidence for a survival benefit from screening [22]. However, risk adjustment for possible geographical influence on PCa risk and statin use was not performed, and remains undetermined to our knowledge. This meta-analysis provides a timely summary, highlighting the shift in recent trends in evidence in favor

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Review of statin use to improve PCa outcomes, unlike previous meta-analyses on this topic [6]. Studies in PCa populations Given the evolving evidence regarding the benefits of statins to PCa outcomes, study designs have also evolved to include only cohorts of patients within PCa studies, such as patients undergoing radical prostatectomy (RP), radioor chemoprevention (e.g., dutasteride) therapy, [11,13,14,23–32]. Overall, however, the results of these studies using targeted populations are conflicting with respect to the effects of statins on PCa outcomes. Favoring statin therapy, Loeb et al. observed in 1351 men undergoing RP that statin users had many favorable clinical outcomes related to RP compared with non-users, including significantly lower serum PSA levels, smaller tumor volumes, lower percentage of cancer in RP specimens and fewer positive surgical margins, but were significantly older and had higher body-mass indices [13]. Kollmeier and colleagues observed that of 1681 patients receiving radiotherapy for high-risk localized PCa without post-radiation adjuvant chemotherapy, statin use was associated with improved biochemical recurrence rates (BCR) and increased BCR-free survival at 5 and 8 years follow-up. However, statin use did not correspond with improved distant metastasis-free survival [23]. This effect may be more pronounced when patients receive androgendeprivation therapy after radiotherapy, and Gutt and colleagues described a highly significant reduction in BCR and improved relapse-free survival with statin use in this population (n = 691), which was also associated with lower pretreatment PSA levels, tumor stage and Gleason score [24]. These studies suggest that statins may provide some benefit in specific patients and may work better in different clinical situations or in combination with particular therapies such as metformin [33]. A recently published meta-analysis by Park and colleagues compared the effect of statins in PCa populations stratified by primary treatment [25]. They found that, in 13 studies, statins demonstrated no overall effect on recurrence-free survival. However, statin use was associated with a 32% improvement in recurrence-free survival in radiotherapy patients, despite significant overall heterogeneity. Furthermore, they suggested that statins may influence the efficacy of the primary treatment modality in PCa recurrence owing to a radio-sensitizing effect of these drugs, a contention supported by evidence from both in vitro and in vivo studies [24,26]. Other studies have suggested that pre-intervention statin use does not influence BCR, although it is unclear whether appropriate adjustments were made in all studies. Rieken et al. observed in 6842 patients undergoing RP that statin users did not differ from non-users in terms of BCR over a median follow-up of 25 months, but noted that the patients were older and had a higher rate of positive surgical margins [27]. They also reported an 11% total BCR rate, which is higher than those cited in other series over the same follow-up period [28]. Chao and colleagues observed outcomes and no dose–response relationships between treatment with RP and radiotherapy [29,30], as did Ku et al. for patients who underwent RP, in the context

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of a lower pre-operative PSA in statin users [14]. One small study demonstrated an association of statin use with an increased likelihood of harboring aggressive PCa and a reduced 5 year BCR-free survival in the cohort of patients who underwent RP [31]. Statin use in men with PCa was also reported as an independent predictor of BCR after treatment, considered to be the result of delayed diagnosis secondary to the PSA-lowering effects of statins [31]. By contrast, Freedland and colleagues compared statin use to PCa outcomes utilizing data from 6729 men in the REDUCE trial (in which dutasteride was compared with placebo in men with serum PSA levels between 2.5 and 10 ng/ml for chemoprevention of PCa) [11]. The authors observed that statin users had a lower average serum PSA and were older, but statin use was not associated with overall, low-grade, or high-grade PCa. However, men with aggressive tumors or reduced PSA levels (<2.5 ng/ml) were excluded from the study. Furthermore, the primary outcome of the REDUCE trial demonstrated that dutasteride reduced low-risk PCa diagnosis, which may have further affected PCa outcomes related to statin use [32]. Ongoing clinical trials Several randomized trials are being performed to assess prospectively the effects of statins on various PCa parameters. These include simvastatin (see www.ClinicalTrials.gov, Identifier: NCT00572468) and atorvastatin (NCT01821404, NCT01759836) versus placebo, with circulating biomarker levels and specific histology parameters (such as apoptosis) in RP specimens being examined. Further prospective, non-randomized studies are underway to assess the ability of statins and other medications to reduce BCR following RP or radiotherapy in at-risk patients, employing specifically atorvastatin and the non-steroidal anti-inflammatory drug, celecoxib (NCT01220973), and simvastatin and the anti-diabetic drug, metformin (NCT01561482). The latter is of particular interest because a combination of statins and metformin has been observed to reduce significantly PCa incidence by 68% [33]. The results of these prospective studies are eagerly anticipated, as is the initiation of further prospective studies using combinations of the above approaches, together with complementary medications such as anti-inflammatory drugs [15] and chemopreventive agents such as dutasteride (NCT01428869). Molecular mechanism of statin action Can statins directly affect PCa cells? Along with clinical outcomes in PCa patients, preclinical studies have also demonstrated suppressed tumor growth in PCa xenograft mouse models with statin therapy [34,35]. However, unlike in humans, statin use is not associated with reduced blood cholesterol levels in mice [36]. Therefore, statins may have cholesterol-independent and direct actions on PCa cells. Indeed, many publications have shown that statins display direct actions on PCa cells (reviewed in [15]). Most of these studies found that micromolar but not nanomolar concentrations of various statins were active in vitro, causing PCa cell apoptosis, G1 cell cycle arrest, autophagy, and degradation of androgen receptor as examples [15,37]. 3

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Figure 1. Potential mechanisms of statin use against prostate tumor progression. The major physiological effect of statin action is via reduction of hypercholesterolemia. Hypercholesterolemia may promote PCa progression by modulating cholesterol-enriched lipid raft function and intratumoral de novo androgen synthesis (castrationresistant PCa). Statins may also directly act on prostate tumors (cancer cells as well as tumor microenvironment) through the inhibition of the mevalonate pathway. (A) Inhibition of prenylation is one potential mechanism of statins (*prenylation may be affected only at sufficient statin concentrations, possibly not achieved in prostate tissue with clinical doses of statins). (B) PCa cells may synthesize de novo androgen from cholesterol (i.e., circulating blood cholesterol and from cellular cholesterol synthesis via the mevalonate pathway). Abbreviations: Akt, v-Akt murine thymoma viral oncogene/protein kinase B; AR, androgen receptor; Cav1, caveolin-1; EGFR, epidermal growth factor receptor; farnesyl-PP, farnesyl-pyrophosphate; HMGCR, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase; VEGFR, vascular endothelial growth factor receptor.

To the best of our knowledge, penetration and accumulation of statins in the prostate, among other tissues, is largely unknown because concentrations have not been established. With clinically recommended doses, the mean serum concentration of statins (simvastatin, atorvastatin, and lovastatin) range from 1–15 nM to peak at 19–50 nM [38]. Given that the liver, the main organ of drug metabolism, only accumulates twice the statin concentration of serum [38], it can be speculated that passive tissue penetration into prostate epithelial cells across multiple cell layers would most likely be at low nanomolar levels. Intriguingly, Murtola et al. reported that low doses of statins (10 nM and 100 nM) cause cell cycle arrest and growth inhibition of normal prostate epithelial cells, but not PCa cells [39], suggesting that direct effects of statins on PCa cells may be clinically limited.

tumor growth, mobility, and metastasis, the inhibition of prenylation has been suggested as a plausible non-cholesterol-dependent anti-cancer mechanism of statins (reviewed in [40]). However, the relative sensitivities of isoprenylation and cholesterol synthesis to mevalonate inhibition require consideration. The in vitro concentration of statin required to inhibit protein prenylation has been demonstrated to be more than 100-fold higher than the concentration required to inhibit cholesterol synthesis [41,42]. Thus, when mevalonate is limited, cells seem to maintain the prenylation pathway at the expense of cholesterol synthesis. Hence, the differential in vitro sensitivity of prenylation and cholesterol synthesis to inhibition of mevalonate synthesis [41,42] suggests that the main therapeutic mechanism of statin action, in the context of PCa prevention, is likely to be through reduction of cholesterol.

Inhibition of prenylation Statins were developed for their inhibition of cholesterol synthesis through the mevalonate pathway. In addition to cholesterol, this pathway also plays a key role in synthesis of isoprenoid precursors for modification and physiological targeting of the Ras superfamily of oncogenes (Figure 1A). Because Ras, Rac, and Rho are important mediators of

Modulation of membrane microdomains Statins predominantly act on hepatocytes in the liver, the major organ for cholesterol production [43]. Therefore, the first benefit of statin use in cancer patients is to lower circulating blood cholesterol levels and attenuate cholesterol-mediated pathways in prostate tumors, including the tumor microenvironment. Cholesterol-dependent effects of

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Review statins are supported by epidemiological correlations between hypercholesterolemia and risk of aggressive PCa [12]. Post hoc analysis of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study cohort (29 093 smokers, after more than 10 years follow-up) also reported that a higher serum total cholesterol was associated with greater risks of overall and advanced PCa [44]. Furthermore, similarly to statin use and PCa risk, low blood cholesterol levels have been inversely correlated with PCa risk (reviewed in [45]), and reduced total and lowdensity lipoprotein (LDL)–cholesterol are associated with reduced BCR in patients receiving androgen-deprivation therapy after radiotherapy [24]. Therefore, epidemiological reports on the association between hypercholesterolemia and PCa indicate that the major beneficial mechanisms of statins against PCa are likely to be cholesterol-mediated. The major molecular mechanisms that affect the pathways in promoting prostate tumor progression are illustrated in Figure 1. Cholesterol is an essential component of mammalian cell membranes, and plays a regulatory role by organizing cholesterol-enriched membrane microdomains known as lipid rafts (Box 2) [46]. Hypercholesterolemia leads to altered membrane signaling and trafficking through the accumulation of lipid raft microdomains [36,47]. Preclinical studies using LNCaP (lymph node carcinoma of the prostate) xenograft mice and transgenic PCa mice (TRAMP, transgenic adenocarcinoma of the mouse prostate) have shown increased tumor growth and angiogenesis as a result of diet-induced hypercholesterolemia [36,48]. The xenograft tumor models showed elevated cholesterol content in lipid rafts with increased phosphorylation of Akt (v-Akt murine thymoma viral oncogene/protein kinase B) [36], whereas disruption of lipid rafts by depleting membrane cholesterol is known to inhibit the epidermal growth factor receptor/PI3K (phosphoinositide Box 2. Lipid rafts and caveolin-1 Lipid rafts are specialized membrane microdomains, enriched in cholesterol and sphingolipids, which act as signaling platforms [46]. Morphologically, there are two major types of lipid rafts: planar lipid rafts and a flask-shaped subtype of lipid rafts known as caveolae. The cholesterol-binding protein caveolin-1 is an integral membrane protein for caveola formation, but also acts as a tumor promoter in advanced PCa [69,70], likely due to formation of an aberrant noncaveolar lipid raft microdomain [71]. Caveolin-1 enhances PI3K/Akt signaling while inhibiting serine/threonine protein phosphatases, PP1 and PP2A, leading to elevated proliferation and pro-survival pathways [70]. Caveolin-1 also upregulates androgen receptor and IL-6 expression [71], and this contributes to the generation of reactive stroma in the tumor microenvironment [72]. Furthermore, in addition to cellular caveolin-1 in endothelial cells, secreted caveolin-1 from PCa cells has pro-angiogenic effects through the PI3K–Akt–endothelial nitric oxide synthase signaling module [73] and VEGF-stimulated angiogenic activities [50]. In vascular endothelium, statin use reduces caveolin-1 expression, whereas hypercholesterolemia upregulates caveolin-1 expression [51]. Although endothelial cells have abundant caveolae, caveolin-1 in prostate carcinoma presumably does not form caveolae owing to lack of PTRF/cavin-1 expression [71]. Cholesterol induces caveolin-1 transcription via SREBP in human skin fibroblast cells [74]; however, this remains to be demonstrated in PCa cells. Nevertheless, statin may suppress prostate tumor progression by reducing cholesterolenriched membrane microdomains, including caveolin-1.

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3-kinase)/Akt pathway in PCa cells [47]. This can be influenced by the cholesterol-lowering effects of statins. In endothelial cells, cholesterol accumulation enhances angiogenesis through increased lipid raft colocalization of caveolin-1 and vascular endothelial growth factor (VEGF) receptor 2, whereas reduced VEGF-mediated signaling has been seen with apolipoprotein AI-mediated cholesterol efflux or depleting membrane cholesterol by cyclodextrin [49]. Endothelial caveolin-1 is known to activate a proangiogenic signaling pathway [50], and the expression of caveolin-1 is downregulated by statins [51]. The cholesterol absorption inhibitor, ezetimibe, also reduces endothelial caveolin-1 expression and decreases angiogenesis, as well as reducing serum cholesterol, in LNCaP xenograft mice [52]. Given that statins do not reduce serum cholesterol levels in mice [36], these experiments strongly support the proposition that reduced tumor growth and angiogenesis through disruption of lipid rafts are likely to be the main cholesterol-dependent actions of statins in PCa. Modulation of de novo androgen synthesis Although the link between androgen levels and PCa risk is contentious (discussed in Box 3), it is clear that androgens are essential for the survival of normal and malignant prostate tissues. Inhibition of adrenal and intratumoral de novo androgen synthesis through inhibition of the steroidogenic enzyme CYP17 is achieved by the drug abiraterone acetate, which has been reported to delay progression, improve symptoms [53], and increase overall survival in patients with castration-resistant PCa [54]. Recent studies have also demonstrated that PCa cells have the ability to produce dihydrotestosterone, a more potent androgen in vivo, through a backdoor steroidogenic pathway [55]. Furthermore, a mutation of HSD3B1 (hydroxy-D5-steroid dehydrogenase, 3b- and steroid D-isomerase) in castrationresistant PCa accelerates the conversion of adrenalderived steroid dehydroepiandrosterone to dihydrotestosterone [56]. These alternative steroidogenic pathways may develop in vivo in castration-resistant PCa cells as an adaptation by the tumor to androgen-deprivation therapy. By reducing cellular cholesterol, a precursor of steroidogenesis, statins may also limit adrenal as well as prostate de novo androgen synthesis (Figure 1B). In support of the contention that circulating cholesterol Box 3. Statins, androgens, and PCa risk Cholesterol is the starting substrate for steroidogenesis, and statindependent reduction of circulating cholesterol may decrease circulating testosterone levels [75]. However, the relationship between high testosterone level and PCa risk is unclear, with many studies reporting contradictory results. Some studies have reported that patients with advanced prostate tumors may demonstrate low testosterone levels [76], whereas other studies have found no relationship between high testosterone or other androgens and PCa risk [77]. Furthermore, obesity is associated with PCa risk [78]; nevertheless, obesity in men is associated with low serum testosterone and hypercholesterolemia, and hence with a higher risk of cardiovascular disease [79]. Therefore, the benefit of statin against PCa risk may include mechanisms other than a potential reduction of steroidogenesis. Further studies are necessary to understand why PCa occurs more frequently in patients with metabolic perturbations, such as hypercholesterolemia and hyperglycemia, concomitant with hormonal imbalances. 5

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contributes to ‘intratumoral’ de novo androgen synthesis, diet-induced hypercholesterolemia was associated with increased tumor growth and intratumoral levels of dihydrotestosterone in LNCaP xenograft mice, without a change in circulating testosterone levels [57]. However, two other studies in mice disagree with this finding. Martinez-Martos et al. reported that diet-induced hypercholesterolemia significantly reduced circulating testosterone levels, potentially as a consequence of impaired testicular function [58]. Seo et al. found that administration of lovastatin in conjunction with ezetimibe increased intraprostatic expression levels of steroid 5a-reductase type 2, an enzyme that catalyzes the conversion of testosterone to dihydrotestosterone [59], and recently targeted for PCa prevention with the drug dutasteride with promising results [32]. Thus, dihydrotestosterone levels in the prostate may not be efficiently reduced by statins and ezetimibe. Further research is necessary to determine the effects of hypercholesterolemia or statin use on local and systemic androgen changes in the prostate and prostatic tumors.

plasma levels of LDL–cholesterol and C-reactive protein, a commonly used clinical biomarker for inflammation [61]. Statins have been reported to reduce the production of proinflammatory cytokines such as interleukin 6 (IL-6), IL8, and tumor necrosis factor a (TNF-a), which enhance prostate tumor progression through cytokine signaling pathways [15]. By contrast, statins activate CD4+ regulatory T cells, which suppress immune responses [15]. However, most of the earlier in vitro studies were performed using micromolar concentrations of statins, and should be re-evaluated with the therapeutic nanomolar concentration in mind. Recent studies highlighted the role of the cholesterol regulatory pathway, including sterol regulatory element-binding protein (SREBP) and liver X receptor (LXR), on immune regulation [62]. Therefore, statins may modulate systemic and intratumoral inflammation via both cholesterol-dependent and -independent mechanisms. Considering the emerging key roles of immune suppression and tumor microenvironment in cancer progression, the multi-faceted anti-tumor actions of statins could be employed to reduce drug resistance.

Anti-inflammation The systemic anti-inflammatory action of statins has been associated with its beneficial effect in cardiovascular and other disease states. Chronic inflammation in the tumor microenvironment has been associated with PCa progression, and preoperative statin use was associated with reduced intratumoral inflammation in a cohort of 295 men, as judged by immunohistochemical analysis of immune infiltration into the malignant glands and lymphoid nodules [60]. Cholesterol-independent mechanisms have been proposed because of the low correlation between

Concluding remarks and future perspectives Critical evaluation of current clinical evidence mostly supports a beneficial association of statin therapy with patients at risk for PCa or undergoing cancer treatments. Clinically, statins are invaluable in improving cardiovascular disease outcomes, demonstrating the most convincing results compared with other cholesterol-lowering medications [4,63]. Because statins are generally accepted as safe drugs with relatively few side-effects (Box 1), they will continue to be used for this indication. With mounting evidence for a beneficial role in PCa, statins may be an

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Figure 2. Statin use and cholesterol homeostasis. (A) When cellular cholesterol level is low (or when patients receive statin), sterol-regulatory element binding protein (SREBP) cleavage activating protein (SCAP) escorts SREBP from the endoplasmic reticulum (ER) to the Golgi. Through the proteolytic activation of SREBP at the Golgi membrane, the mature form of SREBP translocates into the nucleus, and binds to the sterol regulatory elements (SREs) of target genes, thereby upregulating genes involved in cholesterol synthesis (e.g., HMG-CoA reductase; HMGCR), and cholesterol uptake (i.e., low-density lipoprotein receptors; LDLR). (B) By contrast, sufficient or high cholesterol (hypercholesterolemia) causes the tethering of SCAP–SREBP in the ER, through SCAP binding to insulin-induced gene (INSIG). An oxysterol, generally a cholesterol derivative, binds to liver X receptor (LXR) which dissociates the corepressor complex that has been repressing the transcription of cholesterol efflux genes such as ABCA1 (a member of the ABCA subfamily of ATP-binding cassette transporters). Cellular cholesterol efflux is increased with the transcription of ABCA1, reversing cellular cholesterol accumulation. Influenced by [62,80].

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Review Box 4. Physiological complexity of statins and cholesterol homeostasis Cellular cholesterol levels are regulated through cholesterol uptake, synthesis, and efflux, with the main mode of regulation being via the master transcription factors, SREBP and LXR. The roles of SREBP and LXR in cholesterol accumulation in the setting of PCa have recently been reviewed [64]. These transcription factors have opposing effects, and in turn are exquisitely sensitive to cell cholesterol status, being reciprocally regulated like children on a seesaw (Figure 2). Activated by low cholesterol status, SREBP increases cholesterol uptake and synthesis, whereas LXR is activated by high cholesterol status and stimulates cholesterol efflux. Accordingly, SREBP and LXR have been suggested as proand anti-tumor transcription factors, respectively. Conceptually, reducing cellular cholesterol levels can be achieved via synthetic inhibition by statins, reducing cholesterol uptake/synthesis by inhibiting SREBP, or increasing cholesterol efflux by activating LXR. However, cholesterol homeostatic responses can blur the distinctions between absolute cause and effect. Hence, hypercholesterolemia inhibits SREBP, but also activates LXR. By contrast, statin enhances SREBP activities as a rebound effect by reducing blood and cellular cholesterol levels [80], but statin also inhibits the production of LXR ligands, in turn attenuating LXR activities [81]. Therefore, the therapeutic benefit of statin or the pro-tumor effects of hypercholesterolemia are complicated by these homeostatic responses, creating uncertainty regarding the potential roles of SREBP and LXR in PCa. Use of statin and targeting cholesterol homeostasis-related transcription factors might be similarly related to modulation of PCa progression through cellular cholesterol levels, but the precise causal mechanisms involved need to be clarified.

Box 5. Outstanding questions  What is the concentration range of statins in prostate tissues in patients receiving statin therapy? Is this different between normal and malignant cells?  Does hypercholesterolemia promote PCa development?  Is there a causal relationship between androgen levels and PCa risk and progression?  Are there specific genes/proteins regulated by cellular cholesterol levels involved in PCa progression? Are they androgen-independent?  Androgen-deprivation therapy causes metabolic disorders, including hypercholesterolemia [82]. Could statins effectively inhibit adrenal and/or intratumoral de novo androgen synthesis in castration-resistant PCa? Would hypercholesterolemia directly promote castration-resistant PCa progression through androgenindependent mechanisms?  Do cholesterol homeostasis-related transcription factors have pleiotropic effects on PCa progression?  Are the paradoxical actions of statins on SREBP versus LXR a disadvantage or limitation of their actions against PCa?

adjunctive therapy to consider in the treatment and prevention of aggressive PCa, but administration should be tempered by an awareness of possible unwanted sideeffects. The potential mechanisms of statins on prostate tumor progression include both cholesterol-dependent and -independent effects. However, the strongest evidence points to intratumoral effects of reduced circulating and cellular cholesterol levels. Related to cholesterol-sensitive mechanisms in PCa progression, recent studies have also suggested that the cholesterol homeostasis-related master transcription factors SREBP and LXR (see Box 4) could be targeted. Potentially, SREBP inhibitors such as betulin

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and d-tocotrienol attenuate mevalonate pathways, including cholesterol synthesis and cholesterol-independent (i.e., prenylation), mechanisms to reduce tumor progression in a manner similar to statins [64]. However, SREBP inhibitors have different in vitro effects when compared with statins (Box 4), and therefore the entire spectrum of SREBP target genes needs to be determined to evaluate fully SREBP as a potential therapeutic target. Furthermore, although the aim of LXR agonists in reducing cellular cholesterol levels is similar to that of SREBP antagonists or mevalonate pathway inhibitors, the specific effects in PCa remain to be evaluated fully. Before expanding therapeutic recommendations, further investigation and understanding of cholesterol homeostatic relationships with statin use in PCa, as well as studying other molecular therapeutic targets in these pathways, are required (Box 5). Acknowledgments M.M.H. is supported by an Australian Research Council Future Fellowship (FT120100251). M.J.R. is supported by a Doctor in Training Research Scholarship from Avant Mutual Group Ltd. The laboratory of A.J.B. is supported by grants from the National Health and Medical Research Council (1008081) and the National Heart Foundation of Australia (G11S5757).

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