Endometrial cancer stem cells (ECSCs) are stem-like cells endowed with self-renewal and differentiation abilities, and these cells are essential for cancer progression in endometrial cancer (EC). As hallmarks of the tumour microenvironment (TME), hypoxia and hypoxia-inducing factors (HIFs) give rise to the dysregulation of tumour stemness genes, such as SOX2. Against this backdrop, we investigated the regulatory mechanisms regulated by HIFs and SOX2 in ECSCs during EC development. Here, ECSCs isolated from EC cell lines and tissues were found to express stemness genes (CD133 and aldehyde dehydrogenase, ALDH1) following the induction of their ECSC expansion. Notably, m⁶A methylation of RNA and HIF-1α/2α-dependent AlkB homologue 5 (ALKBH5) participate in the regulation of HIFs and SOX2 in EC, as confirmed by the observations that mRNA levels of m⁶A demethylases and ALKBH5 significantly increase under hypoxic conditions in ECSCs. Moreover, hypoxia and high ALKBH5 levels restore the stem-like state of differentiated ECSCs and increase the ECSC-like phenotype, whereas the knockdown of HIFs or ALKBH5 significantly reduces their tumour initiation capacity. In addition, our findings validate the role of ALKBH5 in promoting SOX2 transcription via mRNA demethylation, thereby maintaining the stem-like state and tumorigenicity potential of ECSCs. In conclusion, these observations demonstrate a critical role for m⁶A methylation-mediated regulation of the HIF-ALKBH5-SOX2 axis during ECSC expansion in hypoxic TMEs.

子宫内膜癌干细胞 (ECSC) 是具有自我更新和分化能力的干细胞样细胞，这些细胞对子宫内膜癌 (EC) 的癌症进展至关重要。作为肿瘤微环境 (TME) 的标志，缺氧和缺氧诱导因子 (HIF) 会导致肿瘤干细胞基因（如 SOX2）的失调。在此背景下，我们研究了 EC 发育过程中 ECSC 中 HIF 和 SOX2 调控的调控机制。在这里，发现从 EC 细胞系和组织中分离的 ECSC 在诱导其 ECSC 扩增后表达干细胞基因（CD133 和醛脱氢酶，ALDH1）。值得注意的是，m ⁶RNA 和 HIF-1α/2α 依赖的 AlkB 同源物 5 (ALKBH5) 的甲基化参与了 EC 中 HIF 和 SOX2 的调节，正如观察到的 m ⁶ A 去甲基化酶和 ALKBH5 的mRNA 水平在缺氧条件下显着增加所证实的那样ECSC。此外，缺氧和高 ALKBH5 水平恢复分化的 ECSCs 的茎样状态并增加 ECSC 样表型，而 HIFs 或 ALKBH5 的敲低显着降低了它们的肿瘤起始能力。此外，我们的研究结果证实了 ALKBH5 在通过 mRNA 去甲基化促进 SOX2 转录中的作用，从而保持了干细胞样状态和致瘤潜力。总之，这些观察结果表明 m ⁶在缺氧 TME 中 ECSC 扩增期间甲基化介导的 HIF-ALKBH5-SOX2 轴调节。