Oncogene FOXK1 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition - PubMed (original) (raw)

. 2016 Aug 9;7(32):51150-51162.

doi: 10.18632/oncotarget.9457.

Ying Peng 1, Meiyan Wu 1, Wenjing Zhang 3, Mengnan Zhang 1, Ruyi Xie 1, Pei Zhang 1, Yang Bai 1, Jinjun Zhao 4, Aimin Li 1, Qingzhen Nan 1, Ye Chen 1, Yuexin Ren 1, Side Liu 1, Jide Wang 1

Affiliations

Oncogene FOXK1 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition

Yao Wu et al. Oncotarget. 2016.

Abstract

Transcriptional factor FOXK1 is a member of the FOX family, involved in the cell growth and metabolism. The higher expression of FOXK1 leads to a variety of diseases and may play an important role in the development of various tumors. However, the role of FOXK1 in the progression of colorectal cancer (CRC) remains unknown. We demonstrated that FOXK1 was overexpressed in 16 types of solid tumor tissues via tissue multi-array (TMA). We found that FOXK1 induced elevated expressions and transactivities of five major oncogenes in CRC. Moreover, the elevated expression of FOXK1 was showed to be correlated with tumor progression and was a significant predictor of overall survival in CRC patients. Furthermore, it was showed that the depletion of FOXK1 expression could inhibit the migratory and invasive abilities of CRC cells. In contrast, ectopic expression of FOXK1 elicited the opposite effects on these phenotypes in vitro. FOXK1 promoted tumor metastasis through EMT program induction. In addition, TGF-β1 induced FOXK1 expression in a time-dependent pattern and the knockdown of FOXK1 inhibited TGF-β1-induced EMT. In vivo, higher expression of FOXK1 promotes CRC cell invasion and metastasis, and induces EMT in CRC as well. Alltogether, it was concluded that the higher expression of FOXK1 could indicate a poor prognosis in CRC patients since that FOXK1 induces EMT and promotes CRC cell invasion in vitro and in vivo.

Keywords: FOXK1; colorectal cancer; epithelial-mesenchymal transition; invasion; metastasis.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1

Figure 1. FOXK1 expression in CRC were higher than normal cells and increased multiple oncogenes expression

A, B. FOXK1 expression in normal and malignant human colorectal tissues was detected by TMAs and IHC. C. Whole lysates of FHC, HT29, SW480, LoVo, SW1116, SW620, Colo205 and DLD1 were collected, and FOXK1 was detected by Western blot. GAPDH was used as the internal control (GAPDH: glyceraldehyde-3- phosphate dehydrogenase). D. Proteins isolated from resected tumors and adjacent non-tumorous tissue specimens were subjected to Western blotting analysis. T, CRC tissues: N, normal tissues. E. Expression of multiple oncogenes in stable transfectants of SW480/Vector, SW480/FOXK1 as detected by Western blot and qRT-PCR in SW480 cells. *, P < 0.05; **, P < 0.01. F. Luciferase (Luc) reporter constructs contain the Survivin, cyclin D1, AP-1, and ZEB1, TERT promoter of a luciferase gene in FOXK1 transfection experiments. *, P < 0.05. Scale bars, 100 μm in A; 50 μm in B.

Figure 2

Figure 2. Correlation between FOXK1 protein expression and the clinicopathological parameters of colorectal carcinoma

Figure 3

Figure 3. FOXK1 expression in CRC was associated with poor prognosis

A. Expression analysis of FOXK1 protein in CRC by immunohistochemistry. Immunoreactivity in FOXK1 staining was localised in the nucleus. (a) negative expression of FOXK1 in CRC; (b) weak expression of FOXK1 in CRC; (c) moderate expression of FOXK1 in CRC; (d) strong expression of FOXK1 in CRC. B. Kaplan -Meier survival analysis of overall survival in all patients, C. patients at the early stage of CRC and D. patients at the late stage of CRC according to FOXK1 expression. The log-rank test was used to calculate P values. Scale bars, 100 μm in A.

Figure 4

Figure 4. FOXK1 was associated with the invasive and metastatic potential of CRC

A, B. The representative results of IHC staining for FOXK1 of CRC tissues with the primary and lymph node metastatic sites. Arrow, stroma cells. C. FOXK1 expression was detected by Western blot. D. Images of the wound closure of monolayer SW480 and SW1116 transfected with siRNA. *, P < 0.05; **, P < 0.01, ***, P < 0.001. E. Invasive potential of SW480 and SW1116 transfected with the Scr siRNA or FOXK1 siRNA; **, P < 0.01. The experiments were repeated at least three times. Scale bars, 100 μm in A and B.

Figure 5

Figure 5. FOXK1 regulated epithelial-to-mesenchymal transition (EMT) in vitro

A. Morphology of stable transfectants of SW480/Vector, SW480/FOXK1 as visualized under phase-contrast microscope. B. Stable transfectants stained with rhodamine-phallotoxin, with F-actin filaments visualized under fluorescent microscopy. C. E-cadherin expression correlates with increased in immunofluorescence analysis after SW480 FOXK1-siRNA. D. The EMT biomarkers, including E-cadherin, N-cadherin, vimentin, and fibronectin, were detected by Western blot. These experiments were repeated three times with identical findings. Scale bars represent 50 μm in A and B; 20μm in C.

Figure 6

Figure 6. FOXK1 participates in TGF-β1-induced EMT

A. Western blot of FOXK1, vimentin and E-cadherin in the indicated cells in response to treatment with 10 ng/mL TGF-β1 for 0, 24, and 48 hours. B. Twenty-four hours post-transfection of Scr siRNA or FOXK1 siRNA, the cells were treated with TGF-β1 (2 ng/ml) for an additional 48 h. FOXK1 expression was detected by Western blot. The expression of E-cadherin and vimentin was detected by Western blot, with GAPDH as the internal control. C. The morphology of SW480 and SW1116 cells was observed under an inverted microscope. D. Representative images and data of a transwell assay for SW480 and SW1116 cells. Each bar represents the mean ± SD. ***, P < 0.001 compared with that in the absence of TGF-β1. Compared with those transfected with FOXK1-siRNA and treated with TGF-β1: ***, P < 0.001 in SW480 and SW1116. All images are representative of three independent experiments with similar findings. Scale bars, 20 μm in C.

Figure 7

Figure 7. FOXK1 promoted tumour metastasis in vivo

A. External whole-body fluorescence images of liver by injection of SW480/pEGFP-FOXK1 and SW480/pEGFP-N1 (Vector), or B. SW480/pEGFP-FOXK1 shRNA and SW480/pEGFP-src shRNA were obtained 30 days after spleen injection. The mice were sacrificed. C. Metastatic cancer tissues (arbitrary polygonal) were stained with H&E. D. E-cadherin expressions of the liver metastasis of colon carcinoma detected by IHC. E. Expression of E-cadherin-positive tumors derived from SW480 cells was determined by qRT-PCR; **, P < 0.01. Scale bars, 100 μm in C and D.

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