BACH1 promotes the progression of esophageal squamous cell carcinoma by inducing the epithelial-mesenchymal transition and angiogenesis

Abstract

Metastasis to regional lymph nodes or distal organs predicts the progression of the disease and poor prognosis in esophageal squamous cell carcinoma (ESCC). Previous studies demonstrated that BTB and CNC homology 1 (BACH1) participates in various types of tumor metastasis. However, the function of BACH1 in ESCC was rarely reported. The present study demonstrated that BACH1 protein was overexpressed in ESCC tissues compared with paired esophageal epithelial tissues according to immunohistochemical staining (IHC). Higher levels of BACH1 mRNA were associated with decreased overall survival (OS) and shorter disease-free survival (DFS) of ESCC patients based on an analysis of The Cancer Genome Atlas (TCGA) datasets. BACH1 significantly enhanced the migration and invasion of ESCC in vitro. Mechanistically, BACH1 promoted the epithelial-mesenchymal transition (EMT) by directly activating the transcription of CDH2, SNAI2, and VIM, as determined by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR). BACH1 overexpression significantly enhanced CDH2 promoter activity according to the results of a luciferase assay. The results of subsequent experiments indicated that BACH1 enhanced the growth of tumor xenografts. The density of CD31⁺ blood vessels and the expression of vascular endothelial growth factor C (VEGFC) in tumor xenografts were significantly associated with BACH1 levels according to the results of IHC and immunofluorescence (IF) analyses performed in vivo. Moreover, ChIP-qPCR analysis demonstrated that the transcriptional activity of VEGFC was also upregulated by BACH1. Thus, BACH1 contributes to ESCC metastasis and tumorigenesis by partially facilitating the EMT and angiogenesis, and BACH1 may be a promising therapeutic target or molecular marker in ESCC.

Keywords: BACH1; ESCC; angiogenesis; metastasis; the EMT.

FIGURE 1

BACH1 is expressed at a high level in ESCC. (A) Representative images of BACH1 expression in paired esophageal epithelial tissues (upper row) and ESCC tissues (lower row) obtained by IHC staining of ESCC tissue arrays. (B) Comparison of the IHC scores of BACH1 in paired esophageal epithelial tissues and ESCC tissues by Mann‐Whitney U test. (C) Comparison of BACH1 mRNA levels in ESCC tissues and noncancerous tissues in the TCGA database by the Mann‐Whitney U test. (D, E) Kaplan‐Meier plot of the TCGA data showing overall survival (OS) and disease‐free survival (DFS) of ESCC patients with high and low levels of BACH1 assigned based on the cut‐off values obtained by the ROC curve analysis.

FIGURE 2

The expression of BACH1 in ESCC cells. (A) Western blot analysis and (B) qPCR analysis of the levels of BACH1 in various ESCC cell lines. (C) Immunofluorescence analysis of BACH1 (green) and DAPI (blue, cell nuclei) in ESCC cell lines. Scale bars, 30 μm. (D) Western blot analysis and (E) qPCR analysis of the expression of BACH1 in the transient or stable knockdown of BACH1 in KYSE30 and KYSE170 cells and the corresponding control cells and the cells with stable overexpression of BACH1 using KYSE150‐BACH1 and the corresponding KYSE150‐vector control cells. The protein levels of BACH1 were normalized to those of β‐actin, and fold changes are shown relative to the control group. *P < 0.05, **P < 0.01 and ***P < 0.001 by Student's t test.

FIGURE 3

Effects of BACH1 expression on the metastasis‐related properties and proliferation of ESCC cells in vitro. (A) Wound healing assays were performed in KYSE30‐siBACH1‐1/‐2, KYSE170‐siBACH1‐1/‐2, and KYSE150‐BACH1 cells and corresponding control cells cultured for 24 h. (B) Transwell assays were performed in KYSE30‐siBACH1‐1/‐2, KYSE170‐siBACH1‐1/‐2, and KYSE150‐BACH1 cells and corresponding control cells cultured for 24 h. (C) CCK‐8 proliferation assays and (D) colony formation assays in KYSE170‐si/shBACH1 and KYSE150‐BACH1 cells and corresponding control cells. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student's t test or two‐way ANOVA combined with multiple comparisons test.

FIGURE 4

BACH1 promoted metastasis by activating EMT‐related genes. (A) Representative images showing that BACH1 overexpression in KYSE150 cells and BACH1 knockdown in KYSE170 and KYSE30 cells using two siRNA oligos (siBACH1‐1 and siBACH1‐2) resulted in a morphological transition compared with the control cells. Scale bar, 20 μm. (B‐D) The mRNA levels of CDH1, CDH2, SNAI2, and VIM in BACH1‐overexpressing cells (B) and BACH1‐knockdown cells (C, D) were quantified by qPCR. (E) Western blot analysis of the EMT markers E‐cadherin, N‐cadherin, vimentin, and Slug in BACH1‐overexpressing cells and BACH1‐knockdown cells. The values of the ratios under each Western blot image indicate the levels of the expression of EMT markers, which were normalized to the expression of β‐actin and to the corresponding levels in the control group. (F) Representative images of BACH1 expression in ESCC tissues with various expression levels of N‐cad, Slug, and vimentin. (G) Correlation of the expression of N‐cadherin, Slug, and vimentin with BACH1 levels in the ESCC tissues (n = 77); Chi‐squared test was used for comparison. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student's t test.

FIGURE 5

BACH1 transcriptionally activated CDH2, SNAI2, and VEGFC to promote the EMT and angiogenesis. (A) Upper panel, schematic diagram showing the BACH1/antioxidant response element (ARE) consensus motif and predicted BACH1‐binding sequence in the CDH2 promoter region ~3400 bp upstream of the transcriptional start sites (TSSs). Lower panel, schematic diagram showing 350 bp promoter sequences containing the BACH1‐binding sequence or deletion subcloned into the pGL3 luciferase reporter vector. (B, C) ChIP‐qPCR analysis of BACH1 binding to the promoter regions of HMOX1, SNAI2, VIM, and CDH2 in (B) KYSE170 and (C) KYSE150 cells (comparison vs. control IgG binding; HMOX1 was used as a positive control). (D) Luciferase assays of the CDH2 promoter and the mutant promoter in KYSE170 cells with exogenous overexpression of BACH1 or cells transfected with an empty vector. (E) The mRNA levels of VEGFC in BACH1‐knockdown (KYSE170‐siBACH1‐1/‐2 and KYSE30‐siBACH1‐1/‐2) and BACH1‐overexpressing (KYSE150‐BACH1) cells were quantified by qPCR. (F) Analysis of VEGFC mRNA levels in ESCC tissues and paracancerous tissues based on the GSE23400 dataset. (G) The mRNA expression of VEGFC was positively correlated with BACH1 expression according to the analysis of the data of the TCGA database. (H) ChIP‐qPCR analysis of BACH1 binding to the promoter regions of VEGFC compared with that for the control IgG binding. **P < 0.01, ***P < 0.001 by Student's t test.

FIGURE 6

BACH1 promoted in vivo cell growth and angiogenesis in ESCC. (A‐C) KYSE170‐shBACH1 and KYSE170‐shCtrl cells were subcutaneously injected into the flanks of nude mice (n = 6). The xenograft tumors were obtained after 4 weeks, and (B) the tumor volume and (C) weight were measured. (D‐F) KYSE150‐BACH1 and empty vector control cells were subcutaneously injected into the flanks of nude mice (n = 6). The xenograft tumors were obtained after 4 weeks, and (E) the tumor volume and (F) weight were measured. (G‐H) Immunohistochemical staining for N‐cadherin, vimentin, and Slug in xenograft tumors derived from KYSE170‐shBACH1 and KYSE170‐shCtrl cells. Scale bar (40×), 50 μm. (I, J) Immunohistochemical staining for (I) CD31 and (J) VEGFC in xenograft tumors derived from KYSE170‐shBACH1 and KYSE170‐shCtrl cells. Scale bars (20×), 200 μm, scale bars (40×), 50 μm. (K) HE and immunofluorescence staining for CD31 in xenograft tumors derived from KYSE150‐BACH1 and KYSE150‐vector cells. Scale bars (20×), 100 μm, scale bars (40×), 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student's t test.