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.

抽象的

在实体瘤中，常在低氧环境中发现癌干细胞（CSC）或肿瘤起始细胞（TIC）。然而，低氧对TIC的影响知之甚少。使用先前建立的，TIC丰富的患者来源的结直肠癌（CRC）文化，我们显示低氧增加了TICs的自我更新能力，同时诱导了分化程度更高的对应文化中的增殖停滞。基因表达数据显示自噬/自噬是TICs低氧诱导的主要途径之一。有趣的是，发现缺氧诱导的自噬可诱导Thr567残基上的EZR（ezrin）磷酸化，可通过敲低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。PRKC / PKC和EZR的自噬或药理学抑制的遗传靶向导致TICs的肿瘤启动潜力降低。此外，我们观察到稳定的ATG5敲除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，位于肿瘤缺氧和自噬区域的TIC中通常存在p-EZR。总之，我们的结果将缺氧自噬-PKC-EZR信号轴确立为TIC自我更新和CRC进展的新型调节机制。因此，自噬抑制可能代表癌症患者有希望的治疗策略。

缩写：ATG：自噬相关；BECN1：beclin 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：大肠癌；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：螯合体1；TIC：肿瘤引发细胞。