The role of nuclear β-catenin accumulation in the Twist2-induced ovarian cancer EMT

Abstract

Background: Twist2 has been shown to promote human tumor invasion as in breast cancer and cervical cancer. However, whether Twist2 promotes human ovarian cancer progression remains to be elucidated. Here, we investigate the role of Twist2 in ovarian cancer invasion and metastasis as well as the underlying molecular mechanisms.

Methods: Twist2 expression was detected by Immunohistochemistry (IHC) on tissue microarray of human ovarian cancers with scoring procedure according to the staining intensity and pattern. Twist2 gene was stably introduced into SKOV-3 ovarian cancer cells to examine the changes of cellular morphology, motility, invasiveness, and EMT molecular markers.

Results: Twist2 expression is significantly increased in ovarian cancers along with the FIGO disease stage, indicating that Twist2 may be associated with ovarian cancer metastasis. Overexpression of Twist2 induced the EMT phenotype including downregulation of E-cadherin, and upregulation of N-cadherin and β-catenin in human ovarian cancer cells, suggesting that Twist2 might promote β-catenin release from the E-cadherin/β-catenin complex through inhibition of E-cadherin. Thus, β-catenin degradation was inhibited due to inhibition of APC, and the Wnt/β-catenin pathway was then activated by nuclear β-catenin accumulation, which may activate transcription of downstream target genes to promote tumor invasion and metastasis. Collectively, these data indicated that β-catenin is involved in Twist2-induced EMT in ovarian cancer.

Conclusion: Our data indicates that upregulation of Twist2 is correlated with the FIGO stage in human ovarian cancers. In this report, we demonstrated that nuclear β-catenin is accumulated in Twist2-induced EMT cells to facilitates ovarian cancer invasion and metastasis.

Figure 1. Immunohistochemical (IHC) staining of Twist2 expression on paraffin-embedded ovarian carcinomas tissue array sections.

Tissue sections of ovarian carcinomas and normal ovaries were analyzed by IHC staining with a specific monoclonal antibody against human Twist2. The positive staining of Twist2 was shown in brown color. The sections were counterstained with hemotoxylin to show nuclei. Representative images of Twist2 staining in paired normal ovary and epithelial ovarian carcinomas are shown. No expression of Twist2 could been seen in normal adult ovary tissues, while positive expressions of Twist2 were mainly localized in cytoplasm or nucleus of the serous adenocarcinoma and the mucinous adenocarcinoma ovarian tumor cells. Magnitude ×200, ×400.

Figure 2. Generation of Twist2-overexpression stable ovarian cancer cells and cell morphological observation.

A. Ectopic expression of the Flag-taggedTwist2 in SKOV-3 (Twist2/SKOV-3) ovarian cancer cells were verified by Western blot. Twist2/SKOV-3 cells expressed both Twist2 and flag-tag compared with vector control cells. B. Twist2 staining was observed using the laser scanning confocal microscopy (Olympus) in SKOV-3 cells. Nuclei were counterstained with DAPI (in blue). Immunofluorescent staining of Twist2 in Twist2/SKOV-3 cells showing cells with Twist2 (in green) in cell nuclei compared with the Vector control SKOV-3 cells. The cells were from the stably transfected samples. C. Cell morphological shapes were observed using phase microscopy (Olympus). Twist2/SKOV-3 cells took on the fibroblast-like morphological shape, while the Vector/SKOV-3 control cells appeared epithelial cell shape.

Figure 3. The proliferation influence of SKOV-3 cells by Twist2.

A. Flow cytometry analysis showed no difference of the cell cycle distribution between SKOV-3 cancer cells with or without Twist2 ectopic expression. B. The proliferation rate of the transfected cells was detected and compared with that of the vector control through viable cell counts using trypan-blue staining. Triplicate assays were performed in each group of cells. Data were expressed as mean ± SD. No obvious influence of Twist2 was observed on cell growth rate. C. Expressions of Akt, Erk1/2, and their phosphoralation forms indicated Twist2 had no effects on proliferation by Western blot. Compared with Vector/SKOV-3 cells, no obvious changes of Akt, Erk1/2, and their phosphorylation forms were found in Twist2/SKOV-3 cells.

Figure 4. Overexpression of Twist2 in epithelial ovarian carcinoma cells increased cell migration, invasive potential in vitro.

A. In vitro wound healing assay of SKOV-3 cancer cells with or without Twist2 ectopic expression. Representative images of cells at “healing” sites at 12 and 24 hours after scraping are shown increased cell migration in Twist2/SKOV-3 cells. B. The analyzed migration results by the one way ANNOVA test (Graphpad Prism5 software). A P-value of 0.05 was considered statistically significant. The Twist2-expressing SKOV-3 cells migrated faster than the vector-transfected control cells. C. In vitro matrigel invasion assay of SKOV-3 cells with or without ectopic expression of Twist2. Quantification indicated that Twist2-expressing cells are 8–9 folds more invasive than the vector control cells in the in vitro matrigel invasion assay. *, p<0.005.

Figure 5. Twist2 changed the expressions of E-cadherin, N-cadherin, and distribution of β-catenin in SKOV-3 cells.

A and B. Real time PCR results of Twist2, E-cadherin, N-cadherin, and APC mRNA expressing levels in the Twist2-expressing cells and the vector-transfected control cells (*P<0.05,**P<0.01,***P<0.001). C. Western Blot results of Twist2, E-cadherin, N-cadherin, β-catenin expression in the Twist2/SKOV-3 cells compared with the Vector/SKOV-3 control cells. D. Distribution of β-catenin in cytoplasm and nucleus detected by the cell fractionation method. P-parental SKOV-3 cancer cells, V-vector-transfected control cells, T-Twist2-expressing SKOV-3 cells.

Figure 6. Downstream target proteins such as c-Myc, Cyclin-D1 in the Wnt signal pathway.

As c-Myc and Cyclin-D1 are mostly regarded as Wnt target genes, we further examined their expressions by western blot repeated for three times. Immunoblot analysis showing that Both c-Myc and cyclin-D1 increased at the Twist2/SKOV-3 group compared with the Vector/SKOV-3 group in the representative figure.

Figure 7. TCF/LEF-dependent transcriptional activity TOP/FOP flash.

In order to measure TCF4/LEF1-dependent transcriptional activity of 293FT cell lines, cells were transfected with TOP/FOP flash reporter plasmids. After genes transfection for 48 h, transcriptional activity was measured by using Dual Luciferase Reporter Assay System. Cells in FOP flash groups showed negligible levels of transcriptional activity. In contrast, cells in TOPflash groups co-transfected with Twist2 showed significant increase in signal. The results are the average ± s.d. of three experiments (N = 3; P<0.001).