Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA
Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA
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DOI：
10.1073/pnas.1602883113
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发表时间：
2016-04-05
影响因子：
11.1
通讯作者：
Semenza, Gregg L.
中科院分区：
文献类型：
--
作者：
Zhang, Chuanzhao;Samanta, Debangshu;Semenza, Gregg L.
mRNA的n -6-甲基腺苷（m(6)A）修饰在胚胎干细胞多能性调控中起作用。然而，决定甲基化和去甲基化之间平衡的生理信号尚未被描述，也没有研究解决m(6)A在癌症干细胞中的作用。我们报道，乳腺癌细胞暴露于缺氧刺激缺氧诱导因子(HIF)-1 α和HIF-2 α依赖于AlkB同源物5 （ALKBH5）和m(6)A去甲基化酶的表达，该酶在3'- utr中m(6)A残基处去甲基化编码多能因子的NANOG mRNA。缺氧以HIF-和alkbh5依赖的方式诱导NANOG mRNA和蛋白表达增加，以及乳腺癌干细胞（BCSC）表型。将NANOG 3'-UTR插入到荧光素酶报告基因中，导致O-2、hif和ALKBH5对荧光素酶活性的调控，在甲基化残基突变时失去了调控。ALKBH5过表达降低NANOG mRNA甲基化，增加NANOG水平，增加BCSCs百分比，表型复制缺氧的作用。在MDA-MB-231人乳腺癌细胞中，敲低ALKBH5的表达会导致bscs数量减少，从而显著降低其肿瘤启动能力。因此，hif依赖性ALKBH5表达介导了缺氧肿瘤微环境中BCSCs的富集。
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)-1 alpha- and HIF-2 alpha-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 O-2, 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.