Targeting 14-3-3ζ overcomes resistance to EGFR-TKI in lung adenocarcinoma via BMP2/Smad/ID-1 signaling

Abstract Background: 14-3-3ζ protein which acts as a putative oncoprotein has been found to promote 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 14-3-3ζ expression in pan-cancer and LUAD. 14-3-3ζ and ID1 expression was then examined in clinical LUAD samples by immunohistochemistry (IHC). Lentiviral transfection with 14-3-3ζ-specific shRNA was used to establish stable 14-3-3ζ knockdown gefitinib resistant PC9 (PC9/GR) and H1975 cell lines. The effect of 14-3-3ζ knockdown on reversing EGFR-TKI resistance was determined in vitro by CCK-8, wound healing, transwell assays and flow cytometry. A xenograft tumor model was established to evaluate the role of 14-3-3ζ in EGFR-TKI resistance. Microarray analysis results showed the multiple pathways regulated by 14-3-3ζ-shRNA. Results: In the present study, we firstly demonstrated that 14-3-3ζ expression was elevated and predicted unfavourable prognosis in pan-cancer including LUAD based on TCGA. In addition, high 14-3-3ζ expression was significantly associated with advanced T stage, TNM stage, present of lymph node metastasis and, importantly, poor treatment response to EGFR-TKI in LUAD patients with EGFR-activating mutations. 14-3-3ζ shRNA significantly sensitized EGFR-TKI-resistant human LUAD cells to gefitinib and, notably, reversed epithelial-to-mesenchymal transition (EMT). BMP signaling activation was decreased in EGFR-TKI resistant cells followed by 14-3-3ζ depletion in microarray analysis, which was further validated by Western blot analysis. Furthermore, the expression of 14-3-3ζ positively correlates with ID1 expression in human EGFR-mutant LUAD patient samples. In vivo , 14-3-3ζ shRNA and gefitinib treatment resulted in a significant reduction in the tumor burden compared to that treated with gefitinib alone. Conclusion: Our work uncovers a hitherto unappreciated role of 14-3-3ζ in EGFR-TKI resistance. This study might provide a potential therapeutic approach for treating LUAD patients harboring EGFR mutations.