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  • br Inhibition of HMGCR activity results in the


    Inhibition of HMGCR activity results in the depletion of intracellular sterol levels, which in turn results in the activation of SREBP2 [30,34]. SREBP2 resides in the endoplasmic reticulum (ER) in its precursor, full-length form. In response to sterol depletion, SREBP2 is escorted to the Golgi apparatus, where it is cleaved. Cleavage of SREBP2 liberates the N-terminal transcription factor, which then translocates to the nucleus to activate the transcription of sterol metabolism genes, including those that encode enzymes of the MVA pathway (Figure 3C). Most normal and cancer MG-132 demonstrate robust SREBP2 activation in response to sterol depletion; however, impair-ment of this sterol-regulated feedback response has been docu-mented in a subset of cancer cells [23,35,36]. We next evaluated SREBP2 activation in PCa cell lines in response to fluvastatin treat-ment. Intriguingly, while increased SREBP2 cleavage was evident after fluvastatin treatment in LNCaP, DU145 and VCaP cells, no fluvastatin-induced SREBP2 cleavage was observed in statin-sensitive PC-3 cells (Figure 3D). In line with this observation, treatment of PC-3
    cells with fluvastatin failed to induce the expression of the SREBP2 target genes HMGCR and HMG-CoA synthase 1 (HMGCS1) after 16 h of treatment (Figure 3E). In contrast, treatment of LNCaP cells with fluvastatin resulted in the upregulation of both HMGCR and HMGCS1 mRNA expression (Figure 3F). This response was completely abro-gated by the addition of 25-hydroxycholesterol (25-HC), supporting that this restorative feedback mechanism is sterol-regulated in LNCaP cells (Figure 3F).
    122 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 (
    Figure 2: Fluvastatin can be measured in the mouse prostate. Male NOD/SCID mice were treated with PBS or 50 mg/kg/day fluvastatin by oral gavage for 4 consecutive days. 2 h after the last treatment, serum samples were collected, the mice were euthanized and prostate and liver tissues were harvested. Fluvastatin concentrations were quantified by HPLC-MS/MS. Error bars represent the mean SD, n ΒΌ 5 mice per group.
    3.3. Inhibition of the sterol-regulated feedback loop of the MVA pathway potentiates fluvastatin-induced cell death in PCa cell lines Given that fluvastatin sensitivity seemed to be inversely associated with the ability of PCa cells to activate SREBP2 and upregulate the expression of sterol metabolism genes in response to fluvastatin treatment, we next evaluated whether inhibition of the sterol-regulated feedback loop of the MVA pathway potentiated the cytotoxic effects of fluvastatin. Given that the addition of 25-HC prevented the upregulation of HMGCR and HMGCS1 mRNA expression in response to fluvastatin treatment (Figure 3F), we tested whether 25-HC could sensitize PCa cells to flu-vastatin. Treatment of LNCaP and DU145 cells with a sub-toxic con-centration of 25-HC significantly decreased the IC50 value of fluvastatin, suggesting that inhibition of SREBP2 activation can potentiate the cytotoxic effects of fluvastatin (Figure 4AeB). As a complementary approach, we knocked down SREBP2 in LNCaP cells using two inde-pendent doxycycline-inducible shRNAs (Figure 4C). SREBP2 knockdown abrogated fluvastatin-induced HMGCS1 expression and significantly decreased the IC50 value of fluvastatin (Figure 4DeE). Moreover, treatment of LNCaP cells with fluvastatin in the presence of SREBP2 knockdown resulted in increased apoptosis, as evidenced by increased PARP cleavage (Figure 4F). Collectively, these data suggest that inhib-iting the sterol-regulated feedback loop of the MVA pathway is a viable approach to potentiate statin-induced PCa cell death.
    3.4. Dipyridamole inhibits fluvastatin-induced SREBP activation and potentiates fluvastatin-induced apoptosis in PCa cell lines There is significant interest in targeting the SREBP family of tran-scription factors in PCa, as reactivation of lipogenesis has been shown to promote disease progression [17]. In addition to SREBP2, the master transcriptional regulator of fatty acid metabolism (SREBP1) has also been implicated as a viable therapeutic target in PCa [37]. Small molecule inhibitors, such as fatostatin, have been identified to inhibit both SREBP1 and SREBP2 and exhibit anti-cancer activity in vivo [37]; however, fatostatin has yet to be evaluated in clinical trials. More recently, our lab identified that the drug dipyridamole, which is currently approved as an anti-platelet agent, can also inhibit statin-induced SREBP2 activation [38]. Given that inhibiting SREBP2 poten-tiated statin-induced cell death in PCa cells (Figure 4), dipyridamole could potentially offer an immediately-available option to increase the therapeutic window of statins as anti-PCa agents.