EGFR靶向抑制剂耐药新机制：GRHL2介导上皮-间质转化和抑制EGFR再循环

Acquired resistance is the main reason for failure of epidermal growth factor receptor inhibitors (EGFR-TKI) treatment. Mutations or amplification of EGFR and its downstream signal transduction could partially explain their resistance, but the resistance mechanisms of 30-40% patients is still unclear. In our previous study, we obtained gefitinib-resistant cell lines derived from Difi and HCC4006 cells, and found that: (1) transcription factor grainy head-like 2 (GRHL2) is a new key gene responsible for EGFR-TKI resistance; (2) we also found that the gefitinib-resistant cells occured epithelial - mesenchymal transition (EMT) and the EGFR molecular in cell surface decreased. We then hypothesize that: GRHL2 mediated gifitinib resistance by double regulation: activating EGFR downstream signaling pathway by promoting EMT and inhibiting EGFR recycling pathway by down-regulating the expression of Rab25. This project intends to use gene transfection, chromatin immunoprecipitation, FACS and other methods，from the molecular, cellular, animal models and clinical samples to verify our hypothesis. Our present study will clarify the role of GRHL2 played in EGFR-TKI resistance, and thus provides a new target and the theoretical basis for clinical EGFR-TKI targeted therapy and drug development.

获得性耐药是表皮生长因子受体靶向抑制剂(EGFR-TKI）治疗失败的主要原因。EGFR相关分子突变/扩增等虽能部分解释其耐药，但仍有30-40%耐药机制不明。我们通过前期研究筛选到Difi和HCC4006的吉非替尼耐药细胞株，并发现：1）转录因子粒状头样2（GRHL2）是一个新的EGFR-TKI耐药关键基因；2）耐药细胞株由上皮向间质表型转化（EMT）和细胞表面EGFR分子减少。基于E-cadherin和Rab25启动子均具有GRHL2结合位点，我们提出假设：GRHL2表达下调后通过双重调节-促进EMT激活EGFR下游信号，下调Rab25抑制EGFR再循环通路,进而介导了对吉非替尼的耐药。本课题拟利用基因转染、染色质免疫共沉淀、免疫荧光等方法，从分子、细胞、动物模型和临床样本四个层面验证上述假设，旨在阐明GRHL2介导EGFR-TKI耐药的分子机制，为临床EGFR靶向治疗提供新的干预靶点。

本项目在前期发现GRHL2表达下调能够诱导耐药的基础上，从临床样本（包括组织和血液样本）、细胞和动物三个层面，深入研究了GRHL2在结直肠癌发生发展中的作用及其下调导致耐药的机制。主要成果：1. 证实耐药细胞中，GRHL2表达下调，促进细胞发生EMT转化。另外，GRHL2表达下调同时降低EGFR受体在细胞表面的表达，从而阐明GRHL2表达降低，能够通过EMT转化和抑制EGFR受体再循环，从而产生耐药。2.发现GRHL2在肿瘤发生发展中具有双重功能：促进增殖和抑制转移。从而提出新的观点： 作为一个重要的转录因子，GRHL2在肿瘤发生的早期主要通过调节增殖相关的基因，起到促进肿瘤发生发展的作用；而在肿瘤长大到一定程度，产生缺氧，缺氧诱导GRHL2表达下调，从而通过EMT和降低细胞黏附促进肿瘤的转移。3. 发现GRHL2的一种高效调控机制：能够调控8q21-22区域多个基因的表达，从而提出GRHL2可能通过结合REX01基因改变染色质构象，产生8q22基因簇的激活效应。本课题研究为靶向药耐药提供了新的机制与干预靶点。