Targeting 14-3-3ζ Overcomes Resistance to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Lung Adenocarcinoma via BMP2/Smad/ID1 Signaling.

Jinfang Cui,Xuesong Chen,Xuejiao Han,Hailing Lu,Yanbo Chen,Li Cai,Xiaomin Xu,Jing Hu,Ying Xing,Yang Song

doi:10.3389/fonc.2020.542007

Abstract

Background: The 14-3-3ζ 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 14-3-3ζ expression in pancancer 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 small hairpin RNA (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 Cell Counting Kit-8 (CCK-8), wound healing, Transwell assays, and flow cytometry. A xenograft tumor model was established to evaluate the role of 14-3-3ζ in EGFR-TKI resistance. Microarray analysis results showed multiple pathways regulated by 14-3-3ζ-shRNA.Results: In the present study, we demonstrated that based on the TCGA, pancancer and LUAD 14-3-3ζ expression was elevated and predicted unfavorable prognosis. In addition, high 14-3-3ζ 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. 14-3-3ζ shRNA sensitized EGFR-TKI-resistant human LUAD cells to gefitinib and reversed epithelial-to-mesenchymal transition (EMT). After 14-3-3ζ 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 14-3-3ζ 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 14-3-3ζ shRNA and gefitinib compared to mice 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.

Highlights

Lung cancer remains the leading cause of cancer death worldwide, and pathologically, most lung cancer is non-small cell lung cancer (NSCLC) [1, 2]
We provided evidence that 14-3-3ζ inhibition significantly attenuated lung adenocarcinoma (LUAD) cells refractory to EGFR-TKIs in vitro and in vivo, and this was accompanied by epithelial–mesenchymal transition (EMT) reversal
Our IHC analysis results revealed that high 14-3-3ζ expression was associated with advanced T stage, advanced TNM stage, and the presence of lymph node metastasis (Figures 2B–D; Supplementary Table 2)

Summary

Introduction

Lung cancer remains the leading cause of cancer death worldwide, and pathologically, most lung cancer is non-small cell lung cancer (NSCLC) [1, 2]. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, lead to unprecedented clinical benefits and provide a new weapon against LUAD with EGFR-activating mutations [6]. Most LUAD patients eventually develop acquired resistance to EGFR-TKIs [6]. The identified acquired resistance mechanisms have been mainly categorized as secondary mutations in the EGFR gene (T790M and other rare mutations), the activation of alternative signaling pathways, and phenotypic changes such as epithelial–mesenchymal transition (EMT) [7, 8]. The 14-3-3ζ 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); its significance in epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) resistance remains unknown

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Results

Discussion

Conclusion