C9orf86介导的上皮间质转化在非小细胞肺癌ALK抑制剂获得性耐药中的机制研究

Acquired drug resistance is the major barrier for the long-term survival of advanced non-small-cell lung cancer (NSCLC) patients harboring ALK fusion gene, the mechanisms of which remain not fully understood. In our previous study, we have shown that C9orf86 could activate p-ERK/Sox2 pathway. Sox2 could directly bind to the promoter region of Snail gene and down-regulate the expression of epithelial markers (β-Catenin and E-Cadherin) while up-regulate the mesenchymal markers (Vimentin and Fibronectin). Furthermore, we found that C9orf86 was over-expressed both in crizotinib-resistant clinical samples and cell lines. The established acquired ALK inhibitor-resistant cells exhibited typical EMT markers. Based upon this, we hypothesized that C9orf86 could modulate EMT-associated markers and enhance steemness in NSCLC vis p-ERK/Sox2 pathway, thus resulting in the resistance to ALK inhibitors. This project will go further. We will use genetic modification, luciferase activity assay, ChIP and other molecular tools to reveal the mechanisms of ALK inhibitor resistance based on cell lines, mice and human tissue level. The results of our study could shed light into the understanding of how to overcome the resistance to ALK inhibitors.

获得性耐药是限制ALK基因融合晚期非小细胞肺癌患者长期生存的难题，其机制未完全清楚。我们前期发现C9orf86可激活p-ERK/Sox2通路，Sox2可直接结合Snail基因启动子并激活其转录活性，进而下调上皮标志物β-Catenin和E-cadherin，上调间质标志物Vimentin和Fibronectin。进一步，我们在克唑替尼获得性耐药标本及细胞株H3122/CR中发现C9orf86表达升高，高表达C9orf86与克唑替尼疗效不佳相关。并且，H3122/CR细胞株表现出典型的EMT标志。据此我们假设：C9orf86通过调控p-ERK/Sox2信号通路促进EMT的发生及干细胞特性的增强，从而诱发ALK抑制剂耐药。本课题将运用基因调控、荧光素酶活性实验、ChIP等生物学技术，从细胞、动物及临床标本水平深入研究C9orf86与ALK抑制剂耐药的关系，并为克服ALK抑制剂耐药提供新的思路。

肺癌，特别是非小细胞肺癌的发生率和死亡率正逐年提高，近年来EGFR、ALK、RET和ROS1等肺癌驱动基因的发现及相应靶向药物治疗大大延长了患者的生存。但几乎都不可避免的发生获得性耐药，其发生机制仍需深入研究。我们前期预实验中发现C9orf86可诱导肺癌细胞EMT的发生，并初步探索了其分子机制。本课题进一步更全面的研究C9orf86的表达水平及EMT的产生。进而我们探索了C9orf86与细胞周期的关系，结果显示C9orf86高表达可促进肿瘤细胞增殖，干扰C9orf86可将肿瘤细胞阻断在G2期，并诱导其凋亡。同时发现C9orf86高表达可增强肺癌干细胞特性。另一方面探讨了高表达C9orf86介导EMT的分子机制，研究显示在ALK阳性细胞株中，C9orf86可通过激活p-ERK/Sox2通路，调控下游snail诱导EMT产生。体内实验应用临床患者标本，深入研究C9orf86与ALK抑制剂耐药相关，在ALK阳性晚期肺癌患者中，C9orf86高表达患者的预后更差。本课题组研究发现C9orf86与ALK抑制剂耐药相关，特异性阻断C9orf86，可能给应用ALK抑制剂的晚期非小细胞肺癌患者带来更大的获益，也为克服ALK抑制剂耐药提供新的思路。

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