利用CRISPR-Cas9全基因组文库筛查技术研究胃癌细胞对整合素激酶抑制剂耐受的机制

ILK-pAKT signalling is required for cell proliferation in various human malignancies. We screened over 2000 small molecule compounds in order to identify a specific and selective ILK kinase inhibitor. We found that QLT0267 is a potent and selective ILK inhibitor. Studies demonstrate that this small molecule inhibitor significantly suppresses proliferation of many type of cancer cells by the blocking ILK-pAKT signalling without affecting normal cell cycle progression. Our previous work demonstrates that ILK is overexpressed in human gastric cancer and can be an independent prognostic factor. However, some gastric cancer lines with high ILK-pAKT signalling have innate resistance to ILK inhibition. Treatment with QLT0267 in these lines did not influence cell proliferation in vitro and in vivo. The underlying mechanism needs to be determined. ..Based on preliminary data, this project will use a genome-scale lentiviral sgRNA library as a loss of function genetic screening for drug resistance to ILK inhibitor in gastric cancer cells. A negative selection screen for essential genes will be performed corresponding to fundamental processes to test this screen system. Candidate genes contributing to ILK inhibitor resistance will be identified by a positive selection screen in vitro and in vivo. Lastly, a comprehensive bioinformatics study will be conducted by interrogating TCGA and GSEA datasets to investigate and demonstrate clinical relevance and the underlying complex ILK-pAKT compensatory signalling network. Genome-wide investigation of gastric cancer drug resistance will be conducted using this new technology. Our study will provide new insights and data for the translational application of CRISPR-Cas9 in the high throughput screenings of human cancer cells. This type of screen could be used as a model for investigating drug sensitivity in not just gastric cancer cells, but many human malignancies.

恶性肿瘤细胞的增殖依赖ILK-pAKT信号的异常激活。我们通过对2000多种小分子化合物筛选获得特异性ILK激酶抑制剂，可高选择性地阻断ILK对AKT的磷酸化激活作用，并对多种恶性肿瘤细胞的增殖产生明显抑制作用。该药物目前处于II期临床试验。然而ILK信号具有细胞特异性。我们的前期工作显示ILK在人胃癌组织中呈高表达，且可作为独立的预后因素。但部分高表达ILK-pAKT信号的胃癌细胞对ILK抑制剂具有自然耐药的能力，该机制目前还不清楚。本课题拟利用CRISPR-Cas9技术构建的全基因组文库筛查药物敏感基因，全面深入地研究胃癌细胞对该抑制剂的耐药机制及ILK-pAKT信号网络，为定位该药物的受益人群、疗效判断及生存预后寻找有效的生物学标志物，为临床治疗提供新的靶点。本项目中对CRISPR文库筛查系统的检测和优化也为该技术用于临床药物筛选、高通量基因功能筛查等实际应用奠定基础。

胃癌是一个严重影响人类健康的疾病，已经成为第四常见的恶性肿瘤并且死亡率位居第二，全世界范围内每年有超过950,000新增胃癌病例。胃癌由多方面因素，主要包括环境因素和遗传因素等共同作用下形成。而环境因素主要有吸烟、幽门螺杆菌感染以及不良的饮食习惯等。胃癌患者早期症状不明显，大多数患者被确诊时已进展至中晚期，失去了手术机会，对放化疗等治疗敏感性也因人而异，预后较差。尽管我国的胃癌发病率及死亡率均有一定程度上的下降，在流行病学、病理学、分子机制、治疗手段及策略方面也有许多新的认识和进步，但目前依然有很多问题需要解决。. 本研究利用CRISPR/Cas9技术利用ILK sgRNA构建了胃癌ILK敲除细胞株，并以此为基础揭示了胃癌细胞通过ILK抗衰老的机制调控细胞周期从而促进细胞增殖，发现ILK敲除的胃癌细胞激活p21/p53信号，能够同时增强胃癌细胞氧化磷酸化和糖酵解能力，并且增加细胞因子分泌水平。深入研究发现ILK是通过炎症通路尤其是NF-κB通路共同参与抑制大量炎症相关基因表达如S100A8/A9来抵抗细胞衰老。并且我们发现ILK衰老与其激酶功能及激酶结构域相关，最后验证ILK在临床胃癌患者的胃癌组织中高表达预后更差，并与肿瘤分期以及分化程度相关。该课题明确了ILK在胃癌进展的重要作用，阐明了其具体分子机制，为胃癌治疗提供新的肿瘤标志物选择以及靶向治疗提供新的作用靶点和方向。