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    2020-08-12

    121 www.molecularmetabolism.com
    Original Article
    2.8. Immunoblotting
    Whole cell lysates were prepared by washing cells twice with cold PBS and lysing cells in RIPA buffer (50 mM TriseHCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM EDTA, protease inhibitors) on ice for 30 min. Lysates were cleared by centrifugation and protein concentrations were determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were diluted in Laemmli sample buffer, boiled for 5 min and resolved by SDS-polyacrylamide gel electrophoresis. The resolved proteins were then transferred onto nitrocellulose mem-branes. Membranes were blocked for 1 h in 5% milk in PBS/0.1% Tween-20 (PBS-T) at room temperature, and then probed with the following primary MG 132 in 5% milk/PBS-T overnight at 4 C: SREBP2 (1:250; BD Biosciences, 557037), SREBP1 (1:250; Santa Cruz, sc-13551), a-Tubulin (1:3000; Calbiochem, CP06), Actin (1:3000; Sigma, A2066), PARP (1:1000; Cell Signaling Technology, #9542). Primary antibodies were detected using IRDye-conjugated secondary antibodies and the Odyssey Classic Imaging System (LI-COR Biosciences). Densitometric analysis was performed using ImageJ 1.47v software.
    2.9. Quantitative RT-PCR
    3. RESULTS
    3.1. Expression of the metabolic enzyme HMGCR is elevated in primary PCa tissues and is associated with poor prognosis Given the promising epidemiological data that support an association between statin use and improved PCa patient outcome, we first evaluated whether expression of the enzymatic target of statins, HMGCR, is deregulated in primary PCa tissues. We performed immunohistochemistry (IHC) for HMGCR using a validated antibody [29] (Supplementary Fig. 1) on tissue microarrays (TMAs) comprised of matched normal and malignant prostate tissues from 149 PCa patients who underwent a radical prostatectomy (RP) (Figure 1, Supplementary Fig. 2). Staining intensity was scored as either “negative/weak” or “strong” by PCa pathologists. A greater proportion of PCa tissues scored as having high HMGCR expression compared to normal prostate tissues, suggesting that HMGCR expression is deregulated in PCa (Figure 1BeC). This observation was validated by staining an inde-pendent TMA comprised of 30 benign (normal and hyperplasia) prostate and 45 PCa tissue samples (Supplementary Fig. 3).
    We next evaluated whether high HMGCR protein expression was associated with biochemical relapse (BCR)-free survival in this cohort of patients. When considering all 149 patients, no significant associ-ation was observed between HMGCR expression and BCR-free survival (Supplementary Fig. 2A). However, 36 patients (24%) were docu-mented statin users. Given that statin-mediated HMGCR inhibition has been reported to activate a feedback response that ultimately results in the upregulation of MVA pathway enzyme expression, including HMGCR [23,30], there was the potential that statin use was a con-founding variable. Interestingly, when considering only statin non-users, a statistically significant association was observed between HMGCR expression and BCR-free survival, where patients with high 
    HMGCR expression relapsed earlier than patients with lower HMGCR expression (Figure 1D). When comparing HMGCR expression to other clinical and pathological features such as pre-treatment prostate-specific antigen (PSA) levels, Gleason score and extracapsular extension, no association was observed, even when accounting for statin use (Figure 1E, Supplementary Fig. 2B).
    3.2. Sensitivity to HMGCR inhibition is inversely associated with fluvastatin-induced SREBP2 activation in PCa cell lines
    To evaluate the effects of HMGCR inhibition on PCa viability, we treated PCa cell lines with increasing doses of fluvastatin in vitro. We chose to evaluate fluvastatin because we previously demonstrated that flu-vastatin does not interact with P-glycoprotein, a major drug efflux pump associated with drug resistance, at clinically-achievable con-centrations [31]. Fluvastatin also offers a lower potential for druge drug interactions compared to many of the other statins, as it is not metabolized by the cytochrome P450 3A4 (CYP3A4) complex, and therefore foods or the many drugs that can modulate CYP3A4 function will not affect fluvastatin activity [32]. Importantly, while hydrophilic statins (e.g. pravastatin, rosuvastatin) exhibit high hepatoselectivity, lipophilic statins have been measured in extrahepatic tissues such as the brain [33]. It is therefore hypothesized that lipophilic statins, such as fluvastatin, can reach tumors in distant organs, including the prostate. Indeed, we were able to measure fluvastatin in the prostate of NOD/SCID mice after oral delivery, albeit at a concentration approxi-mately 10-fold less than what was measured in the serum (Figure 2). Intriguingly, a range of fluvastatin sensitivities was observed among the four PCa cell lines evaluated (Figure 3A). Fluvastatin exhibited cytotoxic effects in PC-3 cells at low micromolar concentrations similar to those measurable in the mouse prostate, whereas LNCaP, DU145 and VCaP cells were less sensitive to fluvastatin exposure (Figure 3A). Treatment of statin-sensitive PC-3 cells with fluvastatin resulted in cell death, as evidenced by increased DNA fragmentation and PARP cleavage, which was fully rescued by the addition of MVA (Figure 3B). This supports that the apoptotic response in PC-3 cells is due to direct HMGCR inhibition.