ABSTRACT

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enrichedpatient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L, or BECN1. Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5. Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients.

Abbreviations

ATG: autophagy related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CQ: chloroquine; CSC: cancer stem cells; CRC: colorectal cancer; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PRKC/PKC: protein kinase C; SQSTM1/p62: sequestosome 1; TICs: tumor-initiating cells.

中文翻译：

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缺氧诱导的自噬通过激活 PRKC/PKC-EZR (ezrin) 通路来驱动结直肠癌的发生和进展。

 抽象的

在实体瘤中，癌症干细胞（CSC）或肿瘤起始细胞（TIC）通常存在于缺氧环境中。然而，缺氧对 TIC 的影响知之甚少。使用先前建立的富含 TIC 的患者来源的结直肠癌 (CRC) 培养物，我们发现缺氧增加了 TIC 的自我更新能力，同时在其分化程度更高的对应培养物中诱导增殖停滞。基因表达数据显示巨自噬/自噬是 TIC 缺氧诱导的主要途径之一。有趣的是，缺氧诱导的自噬被发现会诱导 EZR (ezrin) 在 Thr567 残基处的磷酸化，这可以通过敲除ATG5、BNIP3、BNIP3L或BECN1来逆转。此外，我们确定 PRKCA/PKCα 是参与缺氧诱导的自噬介导的 TIC 自我更新的潜在激酶。自噬的基因靶向或 PRKC/PKC 和 EZR 的药理学抑制导致 TIC 的肿瘤启动潜力降低。此外，我们观察到稳定敲低ATG5后体内肿瘤的发生和生长显着减少。对人类 CRC 样本的分析表明，p-EZR 经常存在于位于肿瘤缺氧和自噬区域的 TIC 中。总而言之，我们的结果确立了缺氧-自噬-PKC-EZR 信号轴作为 TIC 自我更新和 CRC 进展的新型调节机制。因此，自噬抑制可能代表了癌症患者的一种有前途的治疗策略。

 缩写

ATG：自噬相关； BECN1: 贝克林 1; BNIP3：BCL2 相互作用蛋白 3； BNIP3L：BCL2 相互作用蛋白 3 样； CQ：氯喹； CSC：癌症干细胞； CRC：结直肠癌； HIF1A/HIF-1α：缺氧诱导因子1亚基α； MAP1LC3/LC3：微管相关蛋白1轻链3； PRKC/PKC：蛋白激酶C； SQSTM1/p62: 隔离体 1; TIC：肿瘤起始细胞。