Overexpression of forkhead Box C2 promotes tumor metastasis and indicates poor prognosis in colon cancer via regulating epithelial-mesenchymal transition

Abstract

Forkhead box protein C2 (FOXC2) plays a vital role in carcinogenesis; however, its significance and prognostic value in colon cancer remain unclear. In this study, FOXC2 expression was analyzed in a tissue microarray (TMA) containing 185 samples of primary colon cancer tumor samples and in human colon cancer cell lines. The effect of FOXC2 on cell proliferation, tumorigenesis, and metastasis was examined in vitro and in vivo. FOXC2 was overexpressed in human colon cancer cells and tissues, and correlated with colon cancer progression and patient survival. Functional study demonstrated that FOXC2 promoted cell growth, cell migration, and tumor formation in nude mice, whereas knockdown of FOXC2 by short hairpin RNA (shRNAs) significantly suppressed cell growth, cell migration and tumor formation. Further study found that FOXC2 enhanced AKT activity with subsequent GSK-3β phosphorylation and Snail stabilization, and then induced epithelial-mesenchymal transition (EMT) and promoted tumor invasion and metastasis. Collectively, FOXC2 promotes colon cancer metastasis by facilitating EMT and acts as a potential prognostic factor and therapeutic target in colon cancer.

Keywords: EMT; FOXC2; colon cancer; metastasis; prognostic factor.

Figure 1

Analysis of FOXC2 expression in colon cancer cell lines and tissues. (A) Western blotting (A) and Real-time PCR (B) analysis were performed to examine FOXC2 expression in eight colon cancer cell lines. (C) Western blotting analysis was performed to examine FOXC2 expression in five cases of primary colon carcinomas (T) and paired noncancerous adjacent tissues (N). GAPDH ise loading control. (D) Immunohistochemical staining of FOXC2 in TMA.FOXC2 was localized within the cytoplasm/nucleus of cancer cells. Elevated FOXC2 expression in the tumor of colon cancer tissue (D2, D4) compared to adjacent normal mucosa with negative staining (D1, D3) (orignal magnification: 400X for the inserts, 100×X for all).

Figure 2

The disease-free survival (DFS) (A) and overall survival (OS) (B) rates were stimated by the Kaplan-Meier method. Both the DFS rate and OS rate of patients with FOXC2 positive primary tumor were significantly lower than that of patients with FOXC2 negative primarytmor (log-rank test, P<0.05).

Figure 3

FOXC2 Shows Strong Oncogenicity Function. Effect of overexpression of FOXC2 in SW480 cells and knockdown of FOXC2 in RKO cell analyzed by Western blot and RT-PCR (A). Ectopic expression of FOXC2 stimulates SW480 cell proliferation, conversely, knockdown of FOXC2 inhibits RKO cell proliferation as determined by CCK8 assays (B) and colon frmation assays (C). (D) SW480-vector/SW480-FOXC2 and RKO-scramb/RKO-ShFOXC2 cells (1×10⁷) were injected in the forelimbs of nude mice. Gross tumors in the mice were showed in right. Tumor weight and tumor volumes were measuredonthe indicated days. (E) FOXC2 is associated with migration and invasive ability. Overexpression of FOXC2 promotes the migration and invasion of SW480 cells. Knockdown of FOXC2 represses migration and invasion of RKO cells. Scale bars = 100 μm. Data represent the mean ± SD and are representative of three independent experiments.

Figure 4

FOXC2 Induces Epithelial-Mesenchymal Tranitin in Colon Cancer. A. Western-blot analysis of phenotypic markers including E-cadherin, β-cadherin vimentin, N-cadherin in SW480-Vector/SW480-FOXC2 and RKO-ShFOXC2/RKO-Scramble. GAPDH expression was used s he loading control. B. Confocal microscopy analysis of phenotypic marker including E-cadherin, vimentin. The Red signal represents the staining of corresponding protein, and the green signal represents the nuclear DNA staining by DAPI.

Figure 5

FOXC2 Activates the Akt/GSK-3β/Snail Pathway to Induce EMT. (A) Western-blot analysis of the expression of the indicated proteins in the indicated cells. (B) SW480-FOXC2 cells were treated with the AKT inhibitor LY294002 (20 μM) or DMSO for 24 h, then harvested to examine the expression levels of the indicated proteins by Western blotting. SW480-FOXC2 cell invasion ability was determined by Transwell analysis (C) and proliferation ability was determined by CCK8 assays (D) after treatment with LY294002. Scale bars = 100 μm. Data represent the mean ± SD and are representative of three independent experiments.