LAMA3-FAK-TCF7L2正反馈通路调控腹腔转移结肠癌细胞顺铂耐药的机制研究

Intraperitoneal chemoperfusion plays important role for the management of peritoneal metastasis of colon cancer, but the efficacy is restricted by chemoresistance, which still requires intensive studies to elucidate the underlying mechanisms. Increased cell adhesion has been proved to enhance chemoresistance, which is term as cell adhesion mediated drug resistance, CAM-DR. We have previously established two cisplatin resistant colon cancer cell lines through in vivo cisplatin selection of intraperitoneal implanted colon cancer model. RNA-seq results suggested that LAMA3, a subunit of the extracellular matrix protein Laminin, was found to be elevated in the drug resistant cells. We also demonstrated that LAMA3 contributed to the chemoresistant phenotype by increasing FAK phosphorylation. More interestingly, we found that FAK inhibitor also inhibited LAMA3 expression in addition to sensitized the chemoresistant cells to cisplatin, suggesting that there might exist a positive feed mechanism. Bioinformatics analysis showed a TCF7L2 binding site at the promoter region of LAMA3. TCF7L2 belongs to the LEF/TCF transcription factor family whose activity is controlled by β-catenin, whose stability is regulated by phosphorylated FAK. Taking all the evidence together we put the hypothesis that the LAMA3, FAK, and β-catenin/TCF7L2 complex form a positive feedback pathway and contributed to the maintenance of the chemoresistant phenotype of peritoneally metastasized colon cancer cells. We will employ both molecular and functional assays to prove the existence of this feedback mechanism, which helps to develop novel therapeutics towards peritoneally metastasized colon cancer disease.

耐药是腹腔转移肠癌灌注化疗失败的主要原因，目前鲜有针对性研究。细胞黏附介导的耐药在肿瘤的耐药中发挥着举足轻重的作用。前期我们通过对腹腔种植的结肠癌细胞进行体内筛选建立了两株顺铂耐药细胞系，RNA测序结果显示黏附分子LAMA3在两耐药细胞中表达显著升高，且LAMA3通过增加FAK磷酸化调控了肿瘤细胞的耐药表型。更有意思的是我们发现FAK抑制剂可以降低耐药细胞中LAMA3表达。生物信息学预测显示LAMA3启动子区存在TCF7L2的结合位点，而文献报道FAK可以通过增加β-catenin蛋白稳定性促进TCF7L2转录活性。根据预实验结果和既往报道我们提出LAMA3-FAK-β-catenin/TCF7L2正反馈环路参与维持腹腔转移肠癌细胞顺铂耐药的假说。本项目将采用分子和功能实验证实上述反馈通路在腹腔转移肠癌细胞顺铂耐药中的作用。该假说的验证对于理解肠癌灌注化疗耐药机制具有重要的基础和转化价值。

粘附分子的异常表达在肿瘤发生、进展和恶性表型的维持中发挥着至关重要的作用。本课题组发现粘附分子LAMA3在高增殖潜能结肠癌细胞系中表达升高，结合生物信息学预测和相关前期研究基础我们提出了LAMA3-FAK-β-catenin/TCF7L2正反馈环路在其中发挥关键作用的假说。为了证实这一假说，本课题开展了一下几个方面的研究：1.通过Western blot等表达验证手段证实LAMA3在高增殖潜能结肠癌细胞中表达升高，通过功能获得和功能缺失等实验证实LAMA3促进结肠癌细胞增殖的作用；2.通过过表达及敲减实验证实LAMA3对FAK磷酸化水平的调控，并采用FAK磷酸化抑制剂从功能上验证了FAK磷酸化水平对结肠癌细胞增殖的影响；3.采用ChIP-seq及Luciferase报告基因等技术手段证实了TCF7L2对LAMA3的直接转录调控，同时确定了TCF7L2在LAMA3启动子区的结合位点；4.采用一系列过表达、敲减及挽救实验从分子和功能水平证实了LAMA3-FAK-β-catenin/TCF7L2反馈环路的存在及其在结直肠癌细胞增殖中的调控作用；5.采用免疫组化等方法检测了LAMA3-FAK-β-catenin/TCF7L2通路中关键分子在结肠癌组织中的表达，并采用统计学方法分析了其与结肠癌患者预后及增殖相关指标的相关性。同时，针对LAMA3分子量大直接过表达难度大这一问题，我们采用了CRISPRa技术，通过synergistic activation mediator系统顺利得到了解决，这一策略将为我们后续研究其他大分子量的粘附相关分子提供借鉴经验。该项目的完成一方加深了对以粘附分子为代表的微环境因素在结肠癌恶性表型维持中作用的理解，另一方面这一正反馈通路有望为结肠癌的干预提供新的靶点和思路。