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.

Western blot (protein immunoblot) is a widely used analytical technique in molecular biology. Utilizing the specific recognizing primary antibody, proteins immobilized on various matrix are investigated by subsequent visualization steps, for example, by the horse radish peroxidase conjugated secondary antibody incubation. Methods to improve the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a new strategy to amplify Western blot signals by constructing a probe with proximal labeling and IgG targeting abilities. The R118G mutation attenuated the biotin‐AMP binding affinity of the bacterial biotin ligase BirA*, offering a proximity‐dependent labeling ability, which could be used as a signal amplifier. We built a BirA*‐protein A fusion protein (BioEnhancer) that specifically binds to IgG and adds biotin tags to its proximal amine groups, enhancing the immunosignal of target proteins. In our experiments, the BioEnhancer system amplified the immunosignal by tenfold compared to the standard western blot. Additionally, our strategy could couple with other signal enhancement methods to further increase the western blot sensitivity.