超过40%的肺腺癌患者拥有EGFR突变并对EGFR抑制剂类药物敏感，但大部分患者会产生获得性耐药，限制了靶向EGFR的长期疗效，约5-22%的耐药患者由MET通路异常所致。我们前期对MET依赖性的耐药组织进行转录组学分析发现其中磷酸酶PTPRO水平下调，而过表达PTPRO可使耐药细胞复敏。利用底物陷阱技术结合蛋白质谱发现肺腺癌中c-Met为PTPRO的底物，并通过磷酸蛋白组学、磷酸酶活性检测等手段首次证实PTPRO可去磷酸化c-Met-Y1234/1235位点，抑制c-Met活化并逆转MET依赖性耐药。此外，预实验结果表明转录因子FOXO3可与PTPRO启动子区结合，并促进PTPRO转录。另一方面，PTPRO上调FOXO3蛋白稳定性及其胞核转运，从而形成FOXO3-PTPRO正反馈环路。本课题拟深入探索PTPRO-FOXO3环路在逆转肺腺癌MET依赖性EGFR-TKI耐药中的作用及机制。

Up to 40% of lung adenocarcinoma patients possess EGFR mutation and can be targeted by EGFR-TKI (Tyrosine Kinase Inhibitor) such as gefitinib, representing the majority of lung adenocarcinoma nonoperative therapy. However, almost all those patients end up with acquired resistance towards EGFR-TKI drugs. Multiple mechanisms can result in EGFR-TKI resistance, including EGFR-T790M mutation and MET amplification. Aberrant c-Met (encoded by MET gene) signaling accounts for approximately 20% cases of EGFR-TKI resistance. Here we aimed to investigate the underlying mechanisms and potential targets to help overcome MET-dependent EGFR-TKI resistance in lung adenocarcinoma. Firstly, we collected gefitinib resistant lung adenocarcinoma tissues with MET amplification, and conducted next-generation sequencing which revealed that transcription of PTPRO (Receptor Protein Tyrosine Phosphatase Type O) was significantly decreased in gefitinib resistant tissues compared to the gefitinib sensitive tissues. Secondly, we screened potential PTPRO substrates in lung adenocarcinoma by combining substrate trapping strategy with SILAC (Stable Isotope Labeling by Amino acids in Cell culture) mass spectrometry. Accordingly, we identified a novel interaction between PTPRO and c-Met, which was further verified by coimmunoprecipitation and GST pull-down. Thirdly, phosphoproteomic analysis and phosphatase activity test towards different designated peptides demonstrated that PTPRO can directly dephosphorylate the p-Y1234/1235 site of c-Met protein. Fourthly, preliminary cellular and molecular data confirmed the role of PTPRO on reversing c-Met-related EGFR-TKI resistance through dephosphorylating c-Met and blocking its downstream signaling. Finally, we found that transcription factor FOXO3 (Forkhead Box Protein O3) can bind to the PTPRO promoter region and enhance PTPRO transcription. Interestingly, nucleus transportation and transcription activity of FOXO3 can be upregulated by PTPRO-c-Met signaling pathway, therefore forming a positive feedback loop. Taken together, this project aims to investigate the role and mechanism of PTPRO-FOXO3 feedback loop on modulating c-Met-related EGFR-TKI resistance in lung adenocarcinoma. Our study will not only illuminate the underlying molecular mechanisms above, but also help direct clinical trials on dealing with EGFR-TKI resistance.