br response to intracellular sterol depletion Aberrant SREBP
response to intracellular sterol depletion. Aberrant SREBP2 expression and activity has been associated with PCa progression [15e17], suggesting that prostate tumors may be particularly dependent on cholesterol and other isoprenoid metabolites, and therefore vulnerable to HMGCR inhibition by statins. Statins have been shown to induce cancer cell-specific apoptosis in a number of different cancer cell types via the direct inhibition of HMGCR ; however, heterogeneous responses to statin exposure have been reported [18e21]. A number of mechanisms have been proposed to explain why some cancer Fer-1 are more sensitive to statins than others. For example, in breast cancer, high basal expression of sterol biosynthesis genes has been associated with resistance to atorvastatin . Moreover, we previously demonstrated in multiple myeloma that lovastatin sensitivity was inversely associated with the ability of cells to induce the expression of MVA metabolism genes in response to statin exposure . In PCa, however, the determinants of statin sensitivity remain to be defined.
Before statins can be repurposed for the treatment of PCa, data from prospective clinical trials are necessary. While such data in PCa have been limited thus far , statins have been evaluated in clinical trials in a number of other cancer types. In line with the epidemiological data, mixed efficacies have been reported by these studies and the need for patient stratification and/or combi-nation therapies has been proposed [25e27]. Hence, a better
A Acetyl-CoA B C Normal Tumor
HMG-CoA Statin HMGCR
(e.g. GGPP) Androgens
(Statin non-users only)
Figure 1: Expression of the metabolic enzyme HMGCR is elevated in primary PCa tissues and is associated with poor prognosis. (A) Schematic representation of the MVA pathway. Statins inhibit the rate-limiting enzyme of the pathway, HMGCR. GGPP ¼ geranylgeranyl pyrophosphate. (B) Representative images of a patient-matched normal and malignant prostate tissue pair stained for HMGCR expression. Scale bars ¼ 300 mm (top row) and 100 mm (bottom row). (C) Prostate tumor tissues stained more intensely for HMGCR expression compared to adjacent normal prostate tissue controls. N ¼ 149 matched normal and tumor tissues. p < 0.0001 (McNemar’s test). (D) HMGCR expression in prostate tumors was associated with early biochemical relapse (BCR) in patients who were statin non-users. Hazard Ratio (95% confidence interval) ¼ 0.43 (0.24e0.97); p ¼ 0.04 (Log-rank test). (E) HMGCR expression among statin non-users by clinical and pathological features. IQR ¼ interquartile range.
120 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/).
understanding of the mechanisms of MVA pathway deregulation and statin sensitivity in PCa will be crucial for the successful design of future clinical trials. Here, we provide evidence that deregulation of the MVA pathway at the level of HMGCR expression is associated with poor prognosis in PCa patients; however, inhibition of HMGCR activity in vitro was insufficient to induce apoptosis in the majority of PCa cell lines evaluated. Sensitivity to fluvastatin was inversely associated with SREBP2 activation following statin treatment. Importantly, inhibition of SREBP2 with the clinically-approved agent dipyridamole potentiated fluvastatin-induced apoptosis in PCa cells that were relatively insensitive to fluvastatin as a single agent. Taken together, these findings provide strong rationale for the combined inhibition of HMGCR and SREBP2 to induce PCa cell death and warrant the clinical evaluation of fluvastatin and dipyridamole for the treatment of PCa.
2. MATERIALS AND METHODS
2.1. Tissue microarrays and immunohistochemistry
Tissue microarrays (TMAs) comprised of radical prostatectomy (RP) tissue samples from 149 PCa patients treated at the Princess Margaret Cancer Centre between 2003 and 2013 were obtained with Research Ethics Board approval. Each patient was represented by 3 malignant and 2 benign cores. TMAs were probed with a monoclonal antibody against HMGCR (A9, prepared in-house) by the Pathology Research Program (PRP) Laboratory (University Health Network, Toronto, Canada), and staining was scored by practicing PCa pathologists. Clinical and pathological data, including statin use information, were obtained through a comprehensive chart review. As validation, an independent TMA (US Biomax, PR807b) was stained and scored. To evaluate apoptosis in our xenograft experi-ments, excised tumor tissues were fixed in 10% buffered formalin for at least 24 h, paraffin-embedded, sectioned and stained with an anti-TUNEL antibody (prepared in-house) by the PRP Laboratory. TUNEL positivity was quantified using Aperio ImageScope v22.214.171.1240 software.