Targeting 14-3-3 Zeta Overcomes Resistance To Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors In Lung Adenocarcinoma Via Bmp2/Smad/Id1 Signaling

Background:The 14-3-3 zeta protein, which acts as a putative oncoprotein, has been found to promote the proliferation, metastasis, and chemoresistance of cancer cells in several cancers including lung adenocarcinoma (LUAD); however, its significance in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance remains unknown. Methods:The Cancer Genome Atlas (TCGA) database was used to determine 14-3-3 zeta expression in pancancer and LUAD. 14-3-3 zeta and ID1 expression was then examined in clinical LUAD samples by immunohistochemistry (IHC). Lentiviral transfection with 14-3-3 zeta-specific small hairpin RNA (shRNA) was used to establish stable 14-3-3 zeta knockdown gefitinib-resistant PC9 (PC9/GR) and H1975 cell lines. The effect of 14-3-3 zeta knockdown on reversing EGFR-TKI resistance was determinedin vitroby Cell Counting Kit-8 (CCK-8), wound healing, Transwell assays, and flow cytometry. A xenograft tumor model was established to evaluate the role of 14-3-3 zeta in EGFR-TKI resistance. Microarray analysis results showed multiple pathways regulated by 14-3-3 zeta-shRNA. Results:In the present study, we demonstrated that based on the TCGA, pancancer and LUAD 14-3-3 zeta expression was elevated and predicted unfavorable prognosis. In addition, high 14-3-3 zeta expression was associated with advanced T stage, TNM stage, presence of lymph node metastasis and, importantly, poor treatment response to EGFR-TKIs in LUAD patients with EGFR-activating mutations. 14-3-3 zeta shRNA sensitized EGFR-TKI-resistant human LUAD cells to gefitinib and reversed epithelial-to-mesenchymal transition (EMT). After 14-3-3 zeta depletion, bone morphogenetic protein (BMP) signaling activation was decreased in EGFR-TKI-resistant cells in microarray analysis, which was further validated by Western blot analysis. Furthermore, the expression of 14-3-3 zeta positively correlates with ID1 expression in human EGFR-mutant LUAD patient samples.In vivo, there was a reduction in the tumor burden in mice treated with 14-3-3 zeta shRNA and gefitinib compared to mice treated with gefitinib alone. Conclusion:Our work uncovers a hitherto unappreciated role of 14-3-3 zeta in EGFR-TKI resistance. This study might provide a potential therapeutic approach for treating LUAD patients harboring EGFR mutations.