Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA

Significance Pluripotency factors, such as NANOG, play a critical role in the maintenance and specification of cancer stem cells, which are required for primary tumor formation and metastasis. In this study, we report that exposure of breast cancer cells to hypoxia (i.e., reduced O2 availability), which is a critical feature of the tumor microenvironment, induces N6-methyladenosine (m6A) demethylation and stabilization of NANOG mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype. We show that inhibiting the expression of AlkB homolog 5 (ALKBH5), which demethylates m6A, or the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α, which activate ALKBH5 gene transcription in hypoxic breast cancer cells, is an effective strategy to decrease NANOG expression and target BCSCs in vivo. N6-methyladenosine (m6A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α–dependent expression of AlkB homolog 5 (ALKBH5), an m6A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m6A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.

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