comparisons test where each group was compared to the
comparisons test, where each group was compared to the solvent controls group).
pools of sterols and other non-sterol isoprenoids are depleted . The depletion of sterols typically leads to the cleavage and activation of the SREBP transcription factors, which subsequently activate the tran-scription of MVA pathway genes, thus restoring MVA pathway activity [30,34]. In this study, we demonstrated that relatively statin-insensitive PCa cell lines cleave SREBP2 and activate this restorative feedback loop in response to fluvastatin treatment. Inhibiting this sterol-regulated feedback loop with dipyridamole potentiated fluvastatin-induced apoptosis in these cell lines (Figure 5). This was also ach-ieved by knockdown of SREBP2 (Figure 4CeF), which is consistent with the results of our recent genome-wide shRNA dropout screen that
identified SREBP2 knockdown as a potentiator of statin-induced tumor cell death . This suggests that statin-induced SREBP2 activation is a tumor vulnerability across multiple cancer types.
Intriguingly, while we demonstrated that dipyridamole can inhibit both SREBP1 and SREBP2 activation in response to statin treatment (Figure 5CeD), knockdown of SREBP2 alone was sufficient to phe-nocopy the effects of dipyridamole and potentiate statin-induced apoptosis in LNCaP cells (Figure 4CeF). While SREBP1 primarily regulates the expression of genes involved in fatty ATPγS tetralithium salt metabolism, it shares a subset of target genes with SREBP2 , suggesting some functional redundancies between these transcription factors. Indeed, it
126 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/).
Figure 6: The combination of fluvastatin and dipyridamole delays prostate tumor growth. (A) Male NOD/SCID mice were injected subcutaneously with 5 million LNCaP cells. Once tumors reached a volume of 200 mm3, the mice were randomized to receive 50 mg/kg/day fluvastatin (oral), 120 mg/kg/day dipyridamole (i.p. injection), the combination or vehicle controls. The drug combination resulted in significantly reduced tumor volumes. Error bars represent the mean SD, n ¼ 4e5 mice per treatment group, *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (B) After 12 days of treatment, tumors were excised, fixed and assayed for TUNEL staining by IHC. TUNEL-positive cells were quantified and representative images are shown (scale bar ¼ 100 mm). Box plot with whiskers representing minimum and maximum values, n ¼ 4e5 mice per treatment group, *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). (C) Male NOD-SCID mice were engrafted subcutaneously with LTL-484 patient-derived xenograft tissue. Once tumors reached a volume of 200 mm3, the mice were randomized to receive fluvastatin and dipyridamole (as above) or vehicle controls. The drug combination resulted in significantly reduced tumor volumes. Error bars represent the mean SD, n ¼ 6e9 mice per treatment group, *p < 0.05 (Student t test, unpaired, two-tailed). (D) After 24 days of treatment, the mice were euthanized, and the tumors were excised and weighed. Tumors from the mice treated with the drug combination weighed significantly less than those from the mice treated with the vehicle controls. Box plot with whiskers representing minimum and maximum values, n ¼ 6e9 mice per treatment group, *p < 0.05 (Student t test, unpaired, two-tailed).
has previously been reported that SREBP1 can compensate for reduced SREBP2 expression in a tissue-specific manner . Hence, it is possible that inhibition of SREBP2 alone in some tumors may be insufficient to potentiate statin-induced apoptosis due to compensation by SREBP1, and therefore an inhibitor against both SREBP1 and SREBP2, such as dipyridamole, would have broader utility and greater anti-cancer efficacy in combination with a statin.
In contrast to the other PCa cell lines that were evaluated, the statin-sensitive PC-3 cell line failed to induce SREBP2 target gene expression in response to fluvastatin treatment (Figure 3DeE). In line with this observation, co-treatment with dipyridamole did not potentiate cell death in this cell line (Figure 5A, Supplementary Fig. 5). Impaired feedback regulation of sterol metabolism has been documented in a number of different cancer types [23,35,36]; however, the mecha-
nisms of deregulation in these cancer cells remain to be elucidated. In the context of PC-3 cells, the failure to upregulate HMGCR and
HMGCS1 expression in response to fluvastatin was not due to a lack of SREBP2 expression (Figure 3D). Rather, these cells expressed high levels of both full-length and cleaved SREBP2, suggesting that addi-tional post-translational or epigenetic mechanisms may be contrib-uting to their inability to mount this feedback response. Further investigation into the mechanisms of impaired feedback regulation of the MVA pathway in cancer is warranted, as this could potentially reveal predictive biomarkers of statin sensitivity.