Proteomic and Phosphoproteomic Analyses Reveal the Oncogenic Role of PTK7-NDRG1 Axis in Non-small-cell Lung Cancer Cell Resistance to AZD9291

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the most important chemotherapeutics for non-small-cell lung cancer (NSCLC) therapy. The resistance to EGFR-TKIs is one of the biggest obstacles to NSCLC outcome. In this study, taking advantage of phospho- and proximal proteomic techniques, we analyzed the network rearrangement in cell lines responding to AZD9291 treatment and found that cell-cell adhesion was dramatically enhanced in AZD9291-resistant cells. Further analysis revealed that protein tyrosine kinase 7 (PTK7) expression was significantly elevated. Knockdown or overexpression assays showed that PTK7 played a critical role in improving cell adhesion, which enhanced drug resistance. Because PTK7 is a membrane-localized pseudokinase, the proximal labeling probe BirA* was fused to reveal PTK7-interacting proteins. We found that PTK7 interacted with and stabilized NDRG1, which is located predominantly adjacent to adherens junctions. Downregulation of PTK7 or NDRG1 eliminated the resistance of H1975-resistant (H1975-R) and PC9-resistant (PC9-R) cells to AZD9291, suggesting that the PTK7-NDRG1 axis might be a potential target to eliminate the EGFR-TKI resistance during NSCLC therapy.表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）是非小细胞肺癌（NSCLC）治疗中最重要的化疗药物之一。EGFR-TKI耐药是非小细胞肺癌临床治疗结局的最大阻碍之一。本研究借助磷酸化蛋白质组学与邻近蛋白质组学技术，分析了经AZD9291处理的细胞系中的信号网络重排现象，发现AZD9291耐药细胞的细胞间黏附能力显著增强。进一步分析显示，蛋白酪氨酸激酶7（PTK7）的表达水平显著升高。敲低与过表达实验证实，PTK7可通过增强细胞黏附能力进而提升细胞的药物耐药性，在该过程中发挥关键调控作用。鉴于PTK7属于膜定位假激酶，本研究通过将其与邻近标记探针BirA*融合以筛选其互作蛋白，结果发现PTK7可与主要定位于黏着连接附近的NDRG1结合并稳定其表达。下调PTK7或NDRG1的表达，可消除H1975耐药株（H1975-R）与PC9耐药株（PC9-R）对AZD9291的耐药性，提示PTK7-NDRG1轴或可作为消除非小细胞肺癌EGFR-TKI治疗耐药性的潜在治疗靶点。