Drug repurposing through fasting could pinpoint new cancer cell liabilities and define new treatment options. By screening over 800 approved drugs in PK9 pancreatic ductal adenocarcinoma (PDAC) cells, we identified azoles, inhibitors of cholesterol biosynthesis, as agents whose cytotoxic activity against cancer cells was synergistically enhanced by starvation conditions. We hypothesized that starvation and azoles would cooperate by blunting cholesterol production in PDAC cells. Interestingly, we found other cholesterol inhibitors had their antitumor effects strongly enhanced by starvation. In addition, combined starvation and cholesterol inhibitors reduced tumor growth in gastrointestinal mouse models and intracellular cholesterol levels both in vitro and in vivo. Furthermore, methyl-beta-cyclodextrin, which depletes intracellular cholesterol, and starvation showed a synergistic cytotoxic effect against Capan-1 and MIA PaCa-2 cells (another PDAC cell line). Adding back cholesterol or LDL in gastrointestinal tumor cells/xenografts prevented the synergistic interaction between starvation and cholesterol inhibitors. Combined therapy inhibited pAKT (partially restored with LDL add-back) whereas, its antitumor activity was abolished by the simultaneous supplementation with circulating growth-promoting factors (IGF1, insulin, and leptin). In addition, combined therapy reduced mitochondrial oxidative phosphorylation (OXPHOS) and energy status in gastrointestinal tumor xenografts, whereas cholesterol restoration abolishes this effect. Taken together, these findings support the rationale for conducting clinical studies to assess the safety, feasibility, and activity of combining periodic cycles of fasting with inhibitors of cholesterol biosynthesis in cancer patients
