title: Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m⁶A-demethylation of NANOG mRNA.
abstract: N(6)-methyladenosine (m(6)A) 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 m(6)A 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 m(6)A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m(6)A 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.
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Organ Transplant Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China;
authors.forename: Chuanzhao
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
authors.forename: Debangshu
authors.initials: D
authors.lastname: Samanta
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
authors.forename: Haiquan
authors.initials: H
authors.lastname: Lu
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
authors.forename: John W
authors.initials: JW
authors.lastname: Bullen
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
authors.forename: Ivan
authors.initials: I
authors.lastname: Chen
authors.affiliation: Organ Transplant Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China;
authors.forename: Xiaoshun
authors.initials: X
authors.lastname: He
authors.affiliation: Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 gsemenza@jhmi.edu.
authors.forename: Gregg L
authors.initials: GL
authors.lastname: Semenza
mesh_terms: D000071502:AlkB Homolog 5, RNA Demethylase / Q000235:genetics / Q000502:physiology*; D051792:Basic Helix-Loop-Helix Proteins / Q000502:physiology*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002384:Catalysis; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D008745:Methylation; D000071317:Nanog Homeobox Protein / Q000235:genetics*; D014411:Neoplastic Stem Cells / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000071502:AlkB Homolog 5, RNA Demethylase; D051792:Basic Helix-Loop-Helix Proteins; D000071317:Nanog Homeobox Protein; D012333:RNA, Messenger; D000099162:Endothelial PAS Domain-Containing Protein 1; C486780:NANOG protein, human; C559920:ALKBH5 protein, human
keywords: hypoxia-inducible factors; metastasis; pluripotency factors; self-renewal; tumorigenesis