BACH1 promotes lung adenocarcinoma cell metastasis through transcriptional activation of ITGA2

Abstract

BACH1 plays an important role in promoting cancer. This study aims to further verify the relationship between the expression level of BACH1 in lung adenocarcinoma prognosis, as well as the influence of BACH1 expression on lung adenocarcinoma and the potential mechanism. The expression level of BACH1 in lung adenocarcinoma and its relationship with prognosis was evaluated by lung adenocarcinoma tissue microarray analysis combined with bioinformatics approaches. Gene knockdown and overexpression were used to investigate the functions and molecular mechanisms of BACH1 in lung adenocarcinoma cells. The regulatory downstream pathways and target genes of BACH1 in lung adenocarcinoma cells were explored by bioinformatics and RNA sequencing data analysis, real-time PCR, western blot analysis, and cell immunofluorescence and cell adhesion assays. Chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to verify the target gene binding site. In the present study, BACH1 is abnormally highly expressed in lung adenocarcinoma tissues, and high BACH1 expression is negatively correlated with patient prognosis. BACH1 promotes the migration and invasion of lung adenocarcinoma cells. Mechanistically, BACH1 directly binds to the upstream sequence of the ITGA2 promoter to promote ITGA2 expression, and the BACH1-ITGA2 axis is involved in cytoskeletal regulation in lung adenocarcinoma cells by activating the FAK-RAC1-PAK signaling pathway. Our results indicated that BACH1 positively regulates the expression of ITGA2 through a transcriptional mechanism, thereby activating the FAK-RAC1-PAK signaling pathway to participate in the formation of the cytoskeleton in tumor cells and then promoting the migration and invasion of tumor cells.

Keywords: BACH1; ITGA2; cytoskeleton; lung adenocarcinoma; metastasis.

FIGURE 1

BACH1 expression is significantly upregulated in lung adenocarcinoma (LUAD) and negatively correlated with the overall survival (OS) of LUAD patients. (A) Tissue microarray for BACH1 Ab staining and representative scanned images with low or high BACH1 by immunohistochemistry (IHC). (B) BACH1 protein levels in adjacent (N) and tumor (T) tissue in tissue microarray (TMA) as examined by IHC (C) Kaplan–Meier survival curves showing the relationship between BACH1 protein level and OS in TMA from Shanghai Outdo Biotech. ***p < 0.001.

FIGURE 2

BACH1 promotes lung adenocarcinoma (LUAD) cell migration and invasion. (A, C) Western blot analysis validation of the knockdown or overexpression (OE) effect of BACH1 in H1975 and PC9 cells. (B, D) Quantification of wound healing assays in BACH1 knockdown or overexpression H1975 and PC9 cells. (E, F) Representative images and quantification of the Transwell invasion assay in BACH1 knockdown or overexpression H1975 and PC9 cells. (G, H) Representative images and quantification of the Transwell migration assay in BACH1 knockdown or overexpression H1975 and PC9 cells. *p < 0.05; **p < 0.01.

FIGURE 3

BACH1 promotes metastasis of lung adenocarcinoma (LUAD) cell through cytoskeleton regulation. (A) Gene Set Enrichment Analysis identified BACH1‐related signaling pathways based on The Cancer Genome Atlas LUAD dataset. (B) BACH1‐knockdown PC9 and sh‐Mock PC9 cells’ differentially expressed genes were considered for Gene Ontology analysis. (C) F‐actin staining with TRITC phalloidin in the BACH1 knockdown H1975 and PC9 cells (red). DAPI was used to stain nuclei (blue). (D, E) Adhesion of the BACH1 knockdown H1975 and PC9 cells on fibronectin (10 μg/mL) was analyzed at 0, 30, 60, and 120 min time points. **p < 0.01; ***p < 0.001. OD, optical density.

FIGURE 4

BACH1 directly targets ITGA2 and activates the focal adhesion kinase (FAK) signaling pathway. (A) Venn diagram depicts the overlap between BACH1‐binding genes and differentially expressed genes (DEGs) following BACH1 silencing. (B) Cluster analysis of potential target genes on BACH1 silencing in PC9 cells. (C) Quantitative RT‐PCR analysis of potential BACH1‐binding genes in H1975 and PC9 cells with BACH1 knockdown or overexpression. (D) Western blot (WB) analysis of potential BACH1‐binding genes in H1975 and PC9 cells with BACH1 knockdown or overexpression (OE). (E) Agarose gel electrophoresis showed ChIP assays determining BACH1 binding to the ITGA2 promoter in PC9 cells. B‐BS, BACH1 binding site. (F) Sequence of predicted BACH1‐binding site 2 on ITGA2 promoter (B‐BS2‐wt) and the corresponding mutant sequence (B‐BS2‐mut) constructed in the pGL3‐basic luciferase reporter vector. (G) 293 T were cotransfected with either control (p‐ctrl) or p‐BACH1 in conjunction with pGL3‐ITGA2‐B‐BSs‐wt or pGL3‐ITGA2‐BBS2‐mut. Results were expressed as fold change to negative control (NC) after normalizing firefly luciferase activity to Renilla luciferase activity. (H) WB analysis of FAK, pFAK, RAC1, P21‐activated kinase (PAK), and pPAK protein level in BACH1 knockdown H1975 and PC9 cells. (I) WB analysis of FAK, pFAK, RAC1, PAK, and pPAK protein level in BACH1 overexpression H1975 and PC9 cells. **p < 0.01; ***p < 0.001. ns, not significant.

FIGURE 5

ITGA2 promotes metastasis of lung adenocarcinoma cell through cytoskeleton regulation. (A) Western blot analysis validation of the knockdown effect of ITGA2 in H1975 and PC9 cells. (B) Quantification of wound healing assays in ITGA2 knockdown H1975 and PC9 cells. (C) Representative images and quantification of the Transwell invasion assay in ITGA2 knockdown H1975 and PC9 cells. (D) Representative images and quantification of the Transwell migration assay in ITGA2 knockdown H1975 and PC9 cells. (E) F‐actin staining with TRITC phalloidin in the ITGA2 knockdown H1975 and PC9 cells (red). DAPI was used to stain nuclei (blue). (F, G) Adhesion of the ITGA2 knockdown H1975 and PC9 cells on fibronectin (10 μg/mL) was analyzed at 0, 30, 60, and 120 min time points. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

BACH1 depends on ITGA2 expression to promote metastasis of lung adenocarcinoma cell. (A) Quantification of the wound healing assays using transfected H1975/PC9 cells. (B) Representative images and quantification of the Transwell invasion assays using transfected H1975/PC9 cells. (C) Representative images and quantification of the Transwell assays using transfected H1975/PC9 cells. (D, E) Western blot analysis of focal adhesion kinase (FAK), pFAK, RAC1, P21‐activated kinase (PAK), and pPAK protein level using transfected H1975/PC9 cells. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7

BACH1 depends on ITGA2 expression to regulate lung adenocarcinoma cell cytoskeleton. (A) F‐actin staining with TRITC phalloidin using transfected H1975/PC9 cells (red). DAPI was used to stain nuclei (blue). (B, C) Adhesion of the transfected H1975/PC9 cells on fibronectin (10 μg/mL) was analyzed at 0, 30, 60, and 120 min time points. *p < 0.05; **p < 0.01; ***p < 0.001. OD, optical density; OE, overexpression.

FIGURE 8

ITGA2 downregulation alleviates BACH1 induced lung adenocarcinoma metastatic ability in vivo. (A) Representative luciferase images and quantification of average luciferase intensity of lungs in the in vivo metastasis assay. (B, C) Representative photographs and quantification of metastatic tumor nodes in mouse lungs from the in vivo metastasis assay. The blue arrows indicate tumor nodules. **p < 0.01; ***p < 0.001. OE, overexpression.