Upregulated forkhead-box A3 elevates the expression of forkhead-box A1 and forkhead-box A2 to promote metastasis in esophageal cancer

Abstract

Esophageal cancer (EC) is one of the most lethal cancers currently known. Members of the forkhead-box A (FOXA) family, including FOXA1 and FOXA2, have been reported to regulate EC progression. However, the role of FOXA3, which is another FOXA member, has not yet been investigated. In the present study, public dataset analyses and immunohistochemistry of 96 samples from patients with EC were performed to determine the potential roles of FOXA3 in EC. The results revealed that FOXA3 was significantly upregulated in EC tumor tissues and Barrett's esophagus tissues. In addition, FOXA3 upregulation was positively associated with tumor invasion, distant metastasis, tumor-node-metastasis stage and shorter overall survival in patients with EC, and multivariate analysis identified FOXA3 as an independent prognostic marker. In vitro experiments demonstrated that the migratory and invasive abilities of EC109 and EC9706 cell lines were inhibited following FOXA3 knockdown. Notably, FOXA3 expression levels were positively correlated with FOXA1 and FOXA2 expression levels according to The Cancer Genome Atlas dataset analysis. Furthermore, FOXA3 knockdown decreased the expression levels of FOXA1 and FOXA2 in EC109 and EC9706 cell lines. Conversely, FOXA1 or FOXA2 overexpression compensated for the effects of FOXA3 knockdown on the migratory and invasive capacities of EC cells. In conclusion, the present study demonstrated that FOXA3 upregulation in EC cells promoted metastasis through regulation of other FOXA members.

Keywords: compensation; esophageal cancer; forkhead-box A2; forkhead-box A3; metastasis.

Figure 1.

FOXA3 expression patterns in EC tissues. (A-C) Relative mRNA expression levels of FOXA3 in EC, BE and normal esophageal tissues from different public datasets. (D) Representative IHC staining images of FOXA3 and regional magnification in EC and normal tissues. Scale bar, 200 µm. (E) IHC score of FOXA3 expression in EC and normal tissues. BE, Barrett's esophagus; EC, esophageal cancer; FOXA3, forkhead-box A3; IHC, immunohistochemistry.

Figure 2.

Predictive value of FOXA3 expression in patients with EC. (A and B) Association analysis of FOXA3 expression levels with various clinicopathological features. (C-E) Kaplan-Meier survival analysis demonstrated the association between FOXA3 expression and OS in patients at (C) TNM I–IV, (D) TNM I and (E) TNM II–IV. (F) Cox multivariate analysis for identification of the independent prognostic factors for OS of patients with EC. EC, esophageal cancer; FOXA1, forkhead-box A1; FOXA2, forkhead-box A2; FOXA3, forkhead-box A3; HR, hazard ratio; IHC, immunohistochemistry; OS, overall survival; TNM, tumor-node metastasis.

Figure 3.

FOXA3 promotes migration and invasion of EC cells. FOXA3 knockdown efficiency in EC109 and EC9706 cells determined by (A) RT-qPCR and (B) western blotting. (C) Migratory ability of EC cells following FOXA3 knockdown determined by wound healing assay. Representative images (10× magnification) and statistical data are presented. (D and E) Migratory and invasive abilities of EC cells following FOXA3 knockdown determined by Transwell assay. Representative (20× magnification) and statistical data are presented. (F) mRNA (left) and protein (right) expression levels of the EMT-associated markers E-cad, N-cad, Snail, Slug, and Twist in EC9706 cells following FOXA3 knockdown. (G) Metastatic ability of EC cells following FOXA3 knockdown in vivo. Representative and statistical data are presented. All experiments were repeated at least three times. *P<0.05, ***P<0.001. E-cad, E-cadherin; FOXA3, forkhead-box A3; N-cad, N-cadherin; NS, not significant; si, small interfering; Slug, snail family transcriptional repressor 2; Twist, twist family bHLH transcription factor.

Figure 4.

FOXA3 elevates the expression levels of other FOXAs in EC. (A) FOXA1 and FOXA2 mRNA expression levels in EC109 and EC9706 cells following FOXA3 knockdown determined by RT-qPCR. (B) FOXA1 and FOXA2 protein expression in EC109 and EC9706 cells following FOXA3 knockdown determined by western blotting. (C) Correlation analysis of FOXA1, FOXA2 and FOXA3 expression levels from in TCGA dataset. (D) FOXA2 overexpression efficiency on EC cells with FOXA3 knockdown determined by western blotting. Relative expression ratios of FOXA1/GAPDH or FOXA2/GAPDH are shown. (E) Effect of FOXA2 overexpression in EC cells with FOXA3 knockdown determined by Transwell invasion assays. Representative (20× magnification) and statistical data are presented. All experiments were repeated at least three times. *P<0.05; **P<0.01; ***P<0.001. FOXA1, forkhead-box A1; FOXA2, forkhead-box A2; FOXA3, forkhead-box A3; si, small interfering; TCGA, The Cancer Genome Atlas.