PFTK1激活Wnt/β-catenin信号促进肺癌靶向治疗耐受的作用和机制

We previously established an EGFR-TKIs resistant lung cancer cell line 827/AR and identified that PFTK1 expression was upregulated in 827/AR cells and lung cancer tissues. Our previous experimental results suggested that PFTK1 would interact with Wnt co-receptor LRP6 to enhance the activation of Wnt/β-Catenin singling, which subsequently would regulated PFTK1 expression to form positive regulatory loop. To confirm the roles and mechanisms of cross-interaction between PFTK1 and Wnt/β-Catenin singling in EGFR-TKIs resistance, in the present program, we will first confirm the roles of PFTK1 in EGFR-TKIs resistance using various in vitro and in vivo models. Then, we will explore the mechanisms of PFTK1 mediated activation enhancement of Wnt/β-Catenin singling, the mechanisms of Wnt/β-Catenin singling in PFTK1 induced EGFR-TKIs resistance and mechanisms of Wnt/β-Catenin singling mediated activation of PFTK1 in lung cancer, through experimental methods such as Co-IP, ChIP, Wnt/β-Catenin signaling activation detection, site directed mutation and promoter reporter assay. Third, we will analyze retrospectively and prospectively the correlations between PFTK1 and EGFR-TKIs therapy sensitivity, between PFTK1 and Wnt/β-Catenin singling activation, between PFTK1 and lung cancer recurrence and metastasis, and between PFTK1 and prognosis of patients based on the detection on lung cancer clinical tissue samples. These studies will provide a valuable theoretical basis for elucidating the new mechanisms of targeted therapy resistance, identifying new EGFR-TKIs resistance markers, exploring new methods for prediction of targeted therapy sensitivity, and screening new drugs for reversing targeted therapy resistance in lung cancer.

项目组前期通过自建的体外EGFR-TKIs治疗耐药模型筛查到一个在耐药细胞和肺癌组织中高表达的耐药候选新基因PFTK1,发现该基因能作用于Wnt共受体LRP6而活化Wnt/β-Catenin信号，而后者能激活下游分子正反馈调节PFTK1。为了证实二者的互动在耐药中的作用和机制，本项目拟首先采用多对EGFR-TKIs耐药和敏感细胞确定PFTK1在体内外耐药中的作用；其次通过定点突变、免疫共沉淀、Chip、激光共聚焦、Wnt活性检测、报告基因等技术探讨PFTK1活化Wnt/β-Catenin信号的机制，以及β-Catenin活化PFTK1参与治疗耐药的机制；第三通过回顾性和前瞻性分析探讨PFTK1在肺癌组织样本中的表达与EGFR-TKIs治疗敏感性、复发转移、生存预后的关系。这些研究将为阐述肺癌靶向治疗耐药新机制，识别和鉴定新的耐药标志物，探讨靶向治疗敏感性预测新方法提供有价值的理论依据。

肺癌靶向治疗耐受与常规化疗耐受一样，已成为临床治疗的瓶颈。识别EGFR-TKIs治疗耐药发生机制，探寻阻止和逆转靶向治疗耐药的新方法则成为肺癌研究的热点课题。项目组旨在探讨PFTK1介导肺腺癌对吉非替尼耐受的作用及相关分子机制，为丰富EGFR-TKIs治疗耐受网络调控机制和探寻临床治疗的新靶点具有非常重要的意义。项目总体完成情况良好，申请书中大部分内容均基本完成，且在原有基础上进行了新的延伸，主要成果如下：.（1）明确了PFTK1在肺腺癌吉非替尼治疗耐受中的作用.在肺腺癌吉非替尼耐药细胞（HCC827/GR和PC9/GR）以及各自的亲本细胞（HCC827和PC9）中证实PFTK1介导肺腺癌细胞对吉非替尼的耐受，体内CDX模型亦得到相似的结果。在肺腺癌组织中证实PFTK1在吉非替尼耐受组的表达量远高于其在敏感组的表达量，且其高表达与肺腺癌患者无病生存期负相关。.（2）证实了PFTK1通过与p-LRP6结合活化Wnt/β-Catenin信号通路的作用机制.在肺腺癌细胞中通过生信预测、转录组测序、免疫共沉淀以及双荧光素酶报告基因等多种实验方法证实PFTK1能够与p-LRP6结合，进而活化Wnt/β-Catenin信号通路。.（3）发现DNMT3A通过启动子甲基化调控PFTK1的表达.生信预测结合甲基化抑制剂、染色质沉淀实验以及双荧光素酶报告基因实验证实DNMT3A上调PFTK1启动子甲基化水平以沉默其表达。在吉非替尼耐药细胞中DNMT3A表达量下调，PFTK1启动子的甲基化水平也显著降低，进而促进PFTK1的表达。.（4）探究PFTK1在临床治疗中的潜在意义.在肺腺癌吉非替尼耐药细胞（HCC827/GR和PC9/GR）中使用新型抑制剂FMF-14-159-2，可显著降低PFTK1的表达，增加上述细胞对吉非替尼的敏感性。在吉非替尼耐受的CDX模型中，使用FMF-14-159-2处理，可显著抑制瘤体生长。.上述研究内容阐述了PFTK1通过Wnt/β-Cantenin信号通路介导肺腺癌对吉非替尼耐受的作用及相关分子机制，并初步探索了以PFTK1作为临床治疗靶点的可行性。目前已培养研究生5名，发表文章3篇，其中SCI收录文章1篇。