Mebendazole Treatment Disrupts the Transcriptional Activity of Hypoxia-Inducible Factors 1 and 2 in Breast Cancer Cells

SIMPLE SUMMARY: Mebendazole (MBZ), an orally available, FDA-approved anthelmintic, has demonstrated efficacy in reducing solid tumor growth and preventing or treating metastasis in multiple preclinical models of cancer. MBZ was also well tolerated in a recently completed phase I clinical trial. Given the success of MBZ, we aimed to identify additional mechanisms of action for MBZ beyond those that have been previously reported, which include tubulin disruption, inhibiting angiogenesis, promoting apoptosis, and maintaining cell stemness. We demonstrate that MBZ can inhibit the transcriptional activity of HIFs in breast cancer cell lines and in preclinical models of breast cancer by preventing the induction of HIF-1α, HIF-2α, and HIF-1β at the protein level under hypoxic conditions. We show that MBZ treatment has dual use as a chemotherapeutic agent as well as blocking the hypoxia-induced phenotype that promotes chemoresistance. ABSTRACT: Breast cancer is the most diagnosed cancer in women in the world. Mebendazole (MBZ) has been demonstrated to have preclinical efficacy across multiple cancers, including glioblastoma multiforme, medulloblastoma, colon, breast, pancreatic, and thyroid cancers. MBZ was also well tolerated in a recent phase I clinical trial of adults diagnosed with glioma. The mechanisms of action reported so far for MBZ include tubulin disruption, inhibiting angiogenesis, promoting apoptosis, and maintaining stemness. To elucidate additional mechanisms of action for mebendazole (MBZ), we performed RNA sequencing of three different breast cancer cell lines treated with either MBZ or vehicle control. We compared the top genes downregulated upon MBZ treatment with expression profiles of cells treated with over 15,000 perturbagens using the clue.io online analysis tool. In addition to tubulin inhibitors, the gene expression profile that correlated most with MBZ treatment matched the profile of cells treated with known hypoxia-inducible factor (HIF-1α and -2α) inhibitors. The HIF pathway is the main driver of the cellular response to hypoxia, which occurs in solid tumors. Preclinical data support using HIF inhibitors in combination with standard of care to treat solid tumors. Therefore, we tested the hypothesis that MBZ could inhibit the hypoxia response. Using RNA sequencing and HIF-reporter assays, we demonstrate that MBZ inhibits the transcriptional activity of HIFs in breast cancer cell lines and in mouse models of breast cancer by preventing the induction of HIF-1α, HIF-2α, and HIF-1β protein under hypoxia. Taken together, our results suggest that MBZ treatment has additional therapeutic efficacy in the setting of hypoxia and warrants further consideration as a cancer therapy.