br We also demonstrated that in addition to impaired feedbac
We also demonstrated that, in addition to impaired feedback regula-tion, the MVA pathway was deregulated at the level of HMGCR protein expression in primary PCa tissues. Analysis of matched normal and malignant prostate tissues from PCa patients revealed that a greater proportion of prostate tumors express high levels of HMGCR compared to normal prostate controls (Figure 1B, C, Supplementary Fig. 3). Moreover, high HMGCR expression in prostate tumors was significantly associated with earlier BCR (Figure 1D), which is consistent with
MOLECULAR METABOLISM 25 (2019) 119e1302019 University Health Network. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 127 www.molecularmetabolism.com
evidence that HMGCR overexpression and MVA pathway activation contribute to tumorigenesis . Deregulation of the MVA pathway at the level of HMGCR expression in PCa suggests that prostate tumors may be particularly sensitive to statin-induced apoptosis; however, data in the literature are conflicting as to whether or not HMGCR expression alone can accurately predict statin sensitivity [20,22,23,42]. One possible explanation for these conflicting data is the poor specificity of many commercially-available HMGCR Jasplakinolide [22,29]. In this study, we used a monoclonal antibody that we previ-ously showed can detect human HMGCR at the predicted 97 kDa molecular weight by immunoblotting, and which reliably detects the upregulation of HMGCR expression following statin treatment in cell lines  and in the mouse liver (Supplementary Fig. 1). Further comprehensive studies using validated HMGCR reagents are needed to accurately evaluate the utility of HMGCR expression as a predictive biomarker of statin sensitivity.
Interestingly, the association between HMGCR expression and BCR was only observed among statin non-users. While statins decrease serum cholesterol by primarily inhibiting cholesterol biosynthesis in the liver, it has been suggested that statin use may result in the compensatory activation of the MVA pathway in extrahepatic tissues [43,44]. In line with this hypothesis, data from a window-of-opportunity clinical trial in breast cancer revealed that women treated with high cholesterol-lowering doses of atorvastatin had higher HMGCR expression in their tumor tissue after two weeks of treatment . In our cohort of PCa patients, no significant difference in HMGCR expression was observed between statin users and non-users; how-ever, the dose, type and duration of statin use at the time of RP varied among patients. As a result, HMGCR expression at the time of RP is a poor marker of statin-regulated MVA pathway activity in this cohort of patients. Analysis of tissue samples pre- and post-statin treatment from recently completed window-of-opportunity clinical trials in PCa (e.g. Murtola et al.  or NCT01992042) will be useful in determining how statin use affects intratumoral expression of MVA pathway genes and in identifying potential biomarkers of statin sensitivity.
A number of mechanisms have been proposed to explain why PCa cells are dependent on MVA metabolism [44,45]. Cholesterol serves as the precursor for steroid hormones, including androgens, which are important for PCa cell survival and disease progression . Choles-terol is also an important component of lipid rafts, which facilitate cell signaling events initiated at the cell membrane that are important for cell survival . However, with the exception of a few studies , attempts to rescue the anti-cancer effects of statins with sterols have failed in a variety of different cancer cell types, including PCa [48e50]. Rather, in these studies, addition of the non-sterol isoprenoid ger-anylgeranyl pyrophosphate (GGPP), and sometimes farnesyl pyro-phosphate (FPP), more consistently rescued statin-induced cell death. Our observations, as well as those of others , that inhibition of the sterol-regulated feedback loop of the MVA pathway potentiates statin-induced apoptosis offers a possible explanation for these results. Rather than compensate for the inhibition of HMGCR, increasing sterol levels retains SREBP2 in its inactive form at the ER, thus abrogating the ability of cells to compensate for MVA pathway inhibition, which in turn potentiates cell death. This indicates that both the sterol and non-sterol isoprenoid branches of the MVA pathway are important for PCa cell survival. While depletion of non-sterol isoprenoid pools results in cancer cell death, the depletion of sterols triggers a homeostatic feedback response through which the cell attempts to compensate for the depletion of these crucial MVA-derived metabolites.