ITGB1 enhances the Radioresistance of human Non-small Cell Lung Cancer Cells by modulating the DNA damage response and YAP1-induced Epithelial-mesenchymal Transition

Abstract

Objectives: Radiotherapy has played a limited role in the treatment of non-small cell lung cancer (NSCLC) due to the risk of tumour radioresistance. We previously established the radioresistant non-small cell lung cancer (NSCLC) cell line H460R. In this study, we identified differentially expressed genes between these radioresistant H460R cells and their radiosensitive parent line. We further evaluated the role of a differentially expressed gene, ITGB1, in NSCLC cell radioresistance and as a potential target for improving radiosensitivity. Materials and Methods: The radiosensitivity of NSCLC cells was evaluated by flow cytometry, colony formation assays, immunofluorescence, and Western blotting. Bioinformatics assay was used to identify the effect of ITGB1 and YAP1 expression in NSCLC tissues. Results: ITGB1 mRNA and protein expression levels were higher in H460R than in the parental H460 cells. We observed lower clonogenic survival and cell viability and a higher rate of apoptosis of ITGB1-knockdown A549 and H460R cells than of wild type cells post-irradiation. Transfection with an ITGB1 short hairpin (sh) RNA enhanced radiation-induced DNA damage and G2/M phase arrest. Moreover, ITGB1 induced epithelial-mesenchymal transition (EMT) of NSCLC cells. Silencing ITGB1 suppressed the expression and intracellular translocation of Yes-associated protein 1 (YAP1), a downstream effector of ITGB1. Conclusions: ITGB1 may induce radioresistance via affecting DNA repair and YAP1-induced EMT. Taken together, our data suggest that ITGB1 is an attractive therapeutic target to overcome NSCLC cell radioresistance.

Keywords: ITGB1; Yes-associated protein (YAP); epithelial-mesenchymal transition (EMT); non-small cell lung cancer (NSCLC); radioresistance.

Figure 1

Amongst the differentially expressed genes in H460R and H460 cells, ITGB1 is overexpressed in H460R cells. A and B. Colony formation and cell apoptosis assays were used to determine the radiosensitivity of H460R and H460 cells. C. Volcano plot of differentially expressed genes in H460R and H460 cells. D. KEGG pathway analysis of the differentially expressed genes. E. Cluster analysis of the up-regulated (29) and down-regulated (10) genes in H460R cells compared with H460 cells involved in ECM-receptor interaction, focal adhesion and cell adhesion molecule signalling pathways. F and G. qRT-PCR and western blotting were performed to detected ITGB1 expression in H460R and H460 cells.

Figure 2

ITGB1 expression is associated with radiosensitivity. A-C. Western blotting and qRT-PCR were used to detect ITGB1 expression in the indicated cell lines. D. The apoptosis rates were determined by flow cytometry. E. Representative photographs of colony formation assays and the proportions of surviving A549 and H522 cells after irradiation with 0, 2, 4, 6, and 8 Gy.

Figure 3

ITGB1 expression negatively correlates with prognosis in patients with NSCLC. A. Analysis of ITGB1 mRNA levels in healthy and NSCLC tissues from the TCGA. B and C. Overall survival and disease-free survival curves stratified by ITGB1 expression for LUAD and LUSC based on data from the GEPIA2 database. D and E. Representative immunohistochemistry images of ITGB1 (antibody clone CAB003434) in healthy lung tissues and NSCLC tissues from the Human Protein Atlas database.

Figure 4

Interfering with ITGB1 expression alter NSCLC cell proliferation and radiosensitivity. A-C. ITGB1 expression was detected by western blot assay and qRT-PCR after lentiviral transduction of A549, H522, and H460R cells. D. Representative photographs of colony formation assays and proportions of surviving A549, H460R, and H522 cells after irradiation with 0, 2, 4, 6, and 8 Gy. E. Cell Counting Kit-8 assays were used to detect the proliferation of A549, H460R, and H522 cells.

Figure 5

Effect of ITGB1 expression on irradiation-induced apoptosis and cell cycle arrest. A. Flow cytometric apoptosis assay for cells exposed to irradiation (8 Gy). B. Cell cycle analysis after irradiation (8 Gy).

Figure 6

ITGB1 is associated with DNA double-strand breaks (DNA-DSBs) induced by radiation. A and B. Representative images of γH2AX-positive nuclei in shITGB1 and ITGB1 overexpression groups at indicated times following irradiation. γH2AX signal in red, nuclear counterstaining with 4′,6-diamidino-2-phenylindole in blue. Scale bar: 50 µm. γH2AX-positive nuclei were counted in five different fields, each with at least 20 nuclei; the number is shown in relationship to the count in non-irradiated cells. Values represent the average of three independent experiments (right). C and D. Detection of γH2AX protein levels in shITGB1 and ITGB1 overexpression groups treated with or without irradiation (8 Gy).

Figure 7

ITGB1 expression correlates with the expression of proteins related to the ATM/CHK2/CDC25c pathway. A. The relationship between ITGB1 expression and that of 34 genes in the DNA-DSB response pathways. B-D. Protein levels of ATM (p-ATM), CHK2 (p-CHK2), and CDC25c (p-CDC25c) were detected by western blotting in shITGB1 and ITGB1 overexpression groups.

Figure 8

ITGB1 could promote radioresisntance of NSCLC cells by regulating EMT. A-C. Protein levels of E-cadherin, N-cadherin, Snail, vimentin, and Zeb1 were detected by western blotting of cells from shITGB1 and ITGB1 overexpression groups.

Figure 9

YAP1 is involved in the radioresistance mediated by ITGB1 overexpression in NSCLC. A and B. The correlation between ITGB1 and YAP1 expression in LUAD and LUSC based on data from the GEPIA2 database. C and D. Kaplan-Meier curves depict the probability of overall survival and disease-free survival based on the expression of ITGB1 and YAP1. Data from patients with LUAD and LUSC retrieved from the GEPIA2 database. E and F. Western blot analysis of total YAP1 protein levels. G and H. Nuclear and cytoplasmic YAP1 protein levels in shITGB1-treated and ITGB1-overexpressing cells.