MZT2A promotes NSCLC viability and invasion by increasing Akt phosphorylation via the MOZART2 domain

Abstract

Mitotic spindle organizing protein 2A (MZT2A) is localized at the centrosome and regulates microtubule nucleation activity in cells. This study assessed the role of MZT2A in non-small-cell lung cancer (NSCLC). Differential MZT2A expression was bioinformatically assessed using TCGA database, the GEPIA database, and Kaplan-Meier survival data to determine the association between MZT2A expression and NSCLC prognosis. Furthermore, NSCLC tissue specimens were evaluated by immunohistochemistry. MZT2A was overexpressed or knocked down in NSCLC cells using cDNA and siRNA, respectively. The cells were subjected to various assays and treated with the selective Akt inhibitor LY294002 or co-transfected with galectin-3-binding protein (LGALS3BP) siRNA. MZT2A mRNA and protein levels were upregulated in NSCLC lesions and MTZ2A expression was associated with poor NSCLC prognosis. MZT2A protein was also highly expressed in NSCLC cells compared with the expression in normal bronchial cells. MZT2A expression promoted NSCLC cell viability and invasion, whereas MTZ2A siRNA had the opposite effect on NSCLC cells in vitro. At the protein level, MZT2A induced Akt phosphorylation, promoting NSCLC proliferation and invasion (but the selective Akt inhibitor blocked these effects) through upregulation of LGALS3BP via the MTZ2A MOZART2 domain, whereas LGALS3BP siRNA suppressed MTZ2A activity in NSCLC cells. The limited in vivo experiments confirmed the in vitro data. In conclusion, MZT2A exhibits oncogenic activity by activating LGALS3BP and Akt in NSCLC. Future studies will assess MTZ2A as a biomarker to predict NSCLC prognosis or as a target in the control of NSCLC progression.

Keywords: Akt; LGALS3BP; MZT2A; NSCLC; prognosis.

FIGURE 1

MZT2A overexpression and association with poor NSCLC prognosis. A, Differential MZT2A expression in NSCLCs using TCGA data (i) and GEPIA data (ii). LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma. B, Kaplan‐Meier survival analysis. High MZT2A expression is associated with poor NSCLC prognosis. C, Immunohistochemistry. MZT2A expression in our cohort of 132 NSCLC cases was analyzed. i, Weak MZT2A expression in normal bronchial epithelium. ii, Strong staining of MZT2A in mitosis‐stage cells. iii, iv, MZT2A expression in LUSC and adenocarcinoma cells. D, Western blot. NSCLC cell lines and HBE cells were grown and harvested for western blot analysis. The data showed that MZT2A expression was highest in H292 cells and lowest expressed in H460 cells. The graph is the quantified data for the western blots. E, Immunofluorescence. NSCLC A549 and H1299 cells were grown and immunostained with an MZT2A antibody. The data show that MZT2A was mainly localized in the cytoplasm with some staining in the nuclei

FIGURE 2

MZT2A promotion of NSCLC cell viability and invasion, as well as regulation of protein expression in A549 and H1299 cells. A, Western blot. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to western blot analysis. The data showed that MZT2A cDNA or siRNA transfection, respectively, upregulated and downregulated MZT2A expression. Graphs show the quantified data. B, RT‐qPCR. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to RT‐qPCR. C, Cell viability MTT assay. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to cell viability assays. Data show that the upregulation or downregulation of MZT2A expression promoted or reduced NSCLC cell viability. D, Transwell invasion assay. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to Transwell assays. Graphs show the quantified data. E, Western blot. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to western blot analysis of the proteins associated with cell proliferation and invasion. Graphs show the quantified data. F, RT‐qPCR. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to RT‐qPCR analysis of the gene associated with cell proliferation and invasion. Graphs show the quantified data

FIGURE 3

MZT2A regulation of Akt phosphorylation in NSCLC. A, Western blot. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to western blot analysis. Graphs show the quantified data. B, RT‐qPCR. NSCLC A549 and H1299 cells were grown, transfected with MZT2A cDNA or siRNA, and subjected to RT‐qPCR. C, Western blot. A549 and H1299 cells were grown, transfected with MZT2A cDNA, treated with or without the selective Akt inhibitor LY294002, and subjected to western blot analysis. Graphs show the quantified data. D, Cell viability MTT assay: A549 and H1299 cells were grown, transfected with MZT2A cDNA, treated with or without the selective Akt inhibitor LY294002, and subjected to MTT assays. E, Transwell assay. A549 and H1299 cells were grown, transfected with MZT2A cDNA, treated with or without the selective Akt inhibitor LY294002, and subjected to Transwell assays. Graphs show the quantified data

FIGURE 4

MZT2A‐activated Akt signal transduction via LGALS3BP. A, Co‐IP‐western blot. A549 and H1299 cells were grown and subjected to Co‐IP‐western blot analysis to detect the intact interaction between MZT2A and LGALS3BP proteins B, Immunohistochemistry and Pearson correlation analysis. NSCLC tissue sections were immunostained with an anti‐MZT2A or LGALS3BP antibody and the expression of MZT2A and LGALS3BP protein was scored as high or low. Tissue sections were subjected to Pearson correlation analysis (shown in the graph; P < .05). C, Western blot. A549 and H1299 cells were grown, transfected with MZT2A cDNA and/or LGALS3BP siRNA, and subjected to western blot analysis. D, Cell viability MTT assay. A549 and H1299 cells were grown, transfected with MZT2A cDNA and/or LGALS3BP siRNA, and subjected to MTT assays. E, Transwell assay. A549 and H1299 cells were grown, transfected with MZT2A cDNA and/or LGALS3BP siRNA, and subjected to Transwell assays. Graph shows the quantified data

FIGURE 5

The MZT2A functional domain MOZART2 mediates MZT2A functions in the regulation of Akt phosphorylation. A, Illustration of our creation of 2 mutated MZT2A MOZART2 domain constructs. ARS: antibody recognition sequences B, Western blot. A549 and H1299 cells were grown and transfected with the 2 mutated MZT2A cDNA constructs, and then were subjected to western blot analysis. C, D, Co‐IP‐western blot. A549 and H1299 cells were grown, transfected with the 2 mutated MZT2A cDNA, and subjected to Co‐IP‐western blot. E, Western blot. A549 and H1299 cells were grown, transfected with the 2 mutated MZT2A cDNAs, and subjected to Co‐IP‐western blot analysis. F, Transwell assay. A549 and H1299 cells were grown, transfected with the 2 mutated MZT2A cDNAs, and subjected to Transwell assays. Graphs show the quantified data. G, Cell viability MTT assay. A549 and H1299 cells were grown, transfected with the 2 mutated MZT2A cDNAs, and subjected to MTT assays

FIGURE 6

MZT2A activation of Akt phosphorylation in vivo. A, Nude mice bearing tumors (shown in the graph; P < .05). B, Tumor growth curve. Tumor size were measured at 7, 13, 18, 22, 25, and 27 d after injection. C, Western blot. Tumor proteins subjected to western blot analysis