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The Role of Nuclear β-Catenin Accumulation in the Twist2-Induced Ovarian Cancer EMT

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.


Introduction
Despite recent advance in the understanding of ovarian cancer development and progression, ovarian cancer remains has the highest associated mortality rate of gynecologic malignancy in mostly western countries due to the advanced stage of disease at diagnosis (stages III-IV). The vast majority of women are diagnosed with disseminated intraperitoneal carcinomatosis [1,2]. Recent literature suggests that the epithelial to mesenchymal transition (EMT) plays a critical role in the progression of ovarian carcinomas [3,4]. EMT is defined by a complex molecular and cellular program by which epithelial cells lose their differentiated characteristics such as cell-cell adhesion, apical-basal polarity, lack of cell motility and gain of mesenchymal features including motility, invasiveness and increased resistance to apoptosis [5]. The similar cellular remodeling and signaling networks appear to be active during cancer metastasis [6,7]. It has been suggested that primary EOC may undergo an EMT process during local invasion in the peritoneum and retain mesenchymal features in advanced tumors.
Transcription factors of the Twist family (Twist1, Twist2), snail family (SNAI1/snail, SNAI2/slug), as well as ZEB family (ZEB1, ZEB2), control the EMT process in cancer [5,8,9]. Twist2 has been shown to promote tumor progression through EMT in breast cancer [10]. Twist2 is a potential prognostic marker for cervical cancer [11], adenoid cystic carcinoma [12], and tongue squamous cell carcinoma [13]. Twist2 is also correlated with poor differentiation grade and short survival in head and neck squamous cell carcinomas [14]. However, whether Twist2 promotes human ovarian cancer progression remains poorly understood. Here, we investigate the role of Twist2 in ovarian cancer progression and the potential molecular mechanisms underlying the Twist2-induced cancer invasion and metastasis. We showed Twist2 is highly expressed in ovarian cancer tissues. Ectopic expression of Twist2 in ovarian cancer cells confers an EMT phenotype. Furthermore, b-catenin activation may participate in these Twist2 induced EMT properties.

Increased Twist2 expression was correlated with FIGO stage in primary ovarian cancer
IHC analyses indicated the presence of high levels of Twist2 in cancer cells of primary ovarian tumors on tissue array ( Figure 1). Twist2 expression was significantly increased in ovarian cancer (70.24%, 59 in 84 cases) relative to normal ovarian tissue (P,0.05, Figure 1, Table 1). The positive staining of Twist2 was detected both in nucleus and cytoplasm.
According to histological type classification, strong expression of Twist2 was found in 19  These data demonstrated that Twist2 protein is significantly increased in ovarian cancer tissues and the high level of Twist2 was correlated with FIGO disease stage, suggesting that Twist2 can be used as an indicator of high grade of malignancy and poor prognosis in human ovarian cancers. However there is no correlation between Twist2 expression and the tumor histological type.

Twist2 expression affected cell morphology but not the cell proliferation of SKOV-3 cells in vitro
To verify whether Twist2 is a crucial mediator of ovarian cancer, progression, we established cell lines stably overexpressing Twist2 ( Fig. 2A). We then analyzed Twist2/SKOV-3 cells and their parental cells through cell morphological observation, flow cytometry (FCM), and MTT assay.
In the initial characterization of these cells, we observed a distinct morphological change in the cells overexpressing Twist2 (Fig. 2B). The SKOV-3 cells expressing Twist2 underwent a phenotypic change and appear fibroblast-like mesenchymal morphological shape, while the control SKOV-3 cells (Vector/ SKOV-3) appear planar epithelial cell morphological shape (Fig. 2B). Immuno-fluorescent staining showed that Twist2 is localized in nuclei, which was consistent with the fact that Twist2 is a transcription factor (Fig. 2C).
We then evaluated the proliferation of SKOV-3 cells expressing Twist2through analyses of cell cycle distribution and cell growth rate. The results showed no obvious effect of Twist2 expression on cell proliferation (Fig. 3A, 3B). As Akt and Erk1/2 are important indicators involved in cell proliferation, the expressions of these proteins and their phosphorylation were examined by Western blot analysis. Consistently, we did not detect changes in Akt, Erk1/ 2, and their phosphorylation forms in Twist2/SKOV-3 cells realtive to the Vector/SKOV-3 control cells (Fig. 3C).

Twist2 Expression Enhanced Migration and Invasion of SKOV-3 Ovarian Cancer Cells
Twist2 has been shown to be an important inducer of EMT in breast and cervical cancer. EMT process promotes epithelial cells to appear fibroblast shape, characterized by increased motility and  invasiveness [15]. To determine whether Twist2/SKOV-3 cells have acquired the increased ability to migrate, we performed a wound healing assay in which the motility of cells located at the edge of the wound was evaluated on the basis of their ability to colonise the wounded area. In the presence of serum (10% FBS), wound repair was observed faster in the Twist2/SKOV-3 cells than control cells group at 24 h (Fig. 4A). As there was no increase in proliferation induced by Twist2 (Fig. 3C), the Twist2/SKOV-3 cells might have the greater cell migration potential in vitro in ''wound healing'' assay ( Fig. 4A, 4B). These results were confirmed by a transwell chamber assay containing an extracellular matrix layer (matrigel). As shown in Figure 4C, the number of Twist2/ SKOV-3 cells migrated through the pores is more than the control cells did.

Nuclear b-catenin was accumulated in ovarian carcinoma cells when Twist2 induced EMT phenotype
When Twist2 over-expresses in the ovarian cancer cells, the cancer cells morphology was transformed into fibroblast-like shape along with increased motility and invasiveness, while no change was found in cell proliferation. Our data indicated that forced expression of Twist2 might push these EOCs to undergo an epithelial-mesenchymal transition.
EMT usually involves the disruption of tight junctions, adherens junctions, which contribute to the separation into individual cells [16,17]. To further determine whether expression of Twist2 indeed promotes EMT in these ovarian cancer cells, we examined the expression of E-cadherin and N-cadherin, two well-established epithelial to mesenchymal makers, in Twist2-expressing and the vector-transfected SKOV-3 cells. We found that the expression of E-cadherin (epithelial maker) was remarkably reduced in Twist2expressing cells relative to the control cells, but the expression of N-cadherin (mesenchymal maker) increased in the cells expressing Twist2 by real-time PCR (Fig. 5A, 5B). We further confirmed the expression results of E-cadherin, N-cadherin by Western Blot (Fig. 5C).
As Twist2/SKOV-3 cells displayed the morphological changes ( Fig. 2B), enhanced potential to migrate (Fig. 4), and EMT markers expression, we concluded that ectopic expression of Twist2 promotes an EMT phenotype in epithelial ovarian carcinoma cells.
It is well recognized that the b-catenin-E-cadherin complex constitutes the structural core of the adherens contacts, and impedes b-catenin transfer to the nucleus and its transcriptional activity [18]. b-catenin was also up-regulated in the Twist2expressing cells relative to the control cells with decreased Ecadherin (Fig. 5C). Figure 5D showed b-catenin distribution by cell fractionation method. Nuclear b-catenin increased in Twist2/ SKOV-3 cells. In addition, APC mRNA decreased (Fig. 5B).

TOPflash transcriptional analysis of 293FT cells
In order to detect the effect of Twist2-induced nuclear translocation of b-catenin on gene expression, we checked downstream target genes such as c-Myc, Cyclin-D1 in Wnt signal pathway. Both c-Myc and Cyclin-D1 were increased with Twist2 overexpression ( Figure 6). We then measured TCF/LEF-dependent transcriptional activity using TOP/FOP flash reporter plasmids. After reporter genes were introduced into 293FT cells for 48 h, transcriptional activity was assessed by luciferase and renilla activity. 293FT cells showed significantly elevated LEF/ TCF dependent transcription with Twist2 co-transfection (relative to vector control; N = 3; P,0.001; Figure 7). Take together, these data demonstrate that Twist2 overexpression in SKOV-3 cells disrupts cell-cell adherens contacts and promotes cell migration. With the decreased E-cadherin, b-catenin released from E-cadherin/b-catenin complex. Meanwhile, bcatenin degradation reduced further accompanied by the decreased APC, which resulted in the accumulation of free bcatenin in the cytoplasm and caused b-catenin transfer to the nucleus to increase its transcriptional activity. These data indicated that nuclear b-catenin was involved in Twist2-induced EMT of ovarian cancer.

Discussion
Ovarian cancer is a highly metastatic disease and the identification of molecules and/or signalling pathways involved in cancer invasion and metastasis is very important for which the early-stage detection is still a barrier [4]. Ovarian cancer cells are prone to metastasis throughout the peritoneal cavity mainly by intra-abdominal dissemination and by lymphatic dissemination. Metastasis is a complex process involving changes in extracellular matrix and cell-cell junctions. Epithelial-to-mesenchymal transition (EMT) is a necessary step towards metastatic tumor progression during detachment of tumor cells from the primary tumor site and attachment to metastatic sites. Few studies have examined the factors that promote EMT of epithelial ovarian cancer (EOC) cells [4,19].
In this study, we provide the first evidence that Twist2 expression is associated with high FIGO stage in ovarian cancer. It may be expected that Twist2 plays similar EMT functions in ovarian cancer. To this end, we introduced twist2 into the SKOV-3 ovarian cancer cell line and examined cells biological behaviors including proliferation, migration and invasion. We found that over-expression of Twist2 had no effect on cell growth and Akt, Erk1/2 activation. Interesting, the Twist2/SKOV-3 cells increased the migration and invasion of ovarian cancer cells, as assessed by scratch-wound assay and invasion assay. As EMT results in enhanced cell motility and invasion, we further checked the EMT markers. A switch from E-cadherin to N-cadherin is also a key feature of EMT in ovarian cancer [4]. Overexpression of Twist2 significantly decreased E-cadherin and increased Ncadherin expression at both mRNA and protein levels respectively. E-cadherin is a transmembrane glycoprotein of the type-I cadherin superfamily. Its cytoplasmic part is linked to the actin cytoskeleton via the catenins [27]. Canonical Wnt/b-catenin signaling has a major impact on EMT during cancer progression [28,29]. Wnt signals inhibit GSK3b kinase activity to stop degradation and force a rapid increase in the levels of free, cytosolic b-catenin, and very soon thereafter, translocates into the nucleus where it binds to TCF/Lef transcription factors [30,31]. A reduction in E-cadherin levels often releases b-catenin from adheren junction, resulting in nuclear b-catenin accumulation, and the consequent transactivation of a panel of target genes, among them those specifying several EMT-inducing factors [18,28].
A recent published study on early cranial dermal development indicated a consistent role for Wnt/b-catenin signaling via Twist2 in dermal specification in different regions of the embryo [32]. Twist2 can be a direct transcription target and may mediate the functional role of Wnt signaling in dermal precursors. In our experiment, Twist2 increased nuclear b-catenin. Furthermore, soluble and nonsoluble cytosolic forms of b-catenin are strictly regulated by the proteosome/ubiquitination system that consists of GSK3b, axin, and adenomatous polyposis coli (APC) protein [25]. Due to downregulation of APC, b-catenin degradation was also repressed. Once in the nucleus, b-catenin would mediate the activation of specific mesenchymal genes. We showed an increase of c-Myc and Cyclin-D1 in Wnt/b-catenin signal pathway (Fig. 6). Because b-catenin was required for the expression of the mesenchymal genes, we checked whether the transcriptional activity of this protein was affected by ectopic Twist2. As shown in Fig. 7, the activity of the TCF4/LEF1-dependent promoter (TOP) was upregulated (two folds) in cells transfected with Twist2. Therefore, these results indicate that the expression of mesenchymal genes is under the control of b-catenin through TCF4/LEF1dependent complexes. Confirmed by the result shown in Fig. 5D, nuclear b-catenin distribution was increased in Twist2/SKOV-3 cells. Thus, Twist2 might increase TCF4/LEF1-dependent bcatenin transcriptional activity.
In this study, we provide evidence that Twist2 affects cellular behaviors in SKOV-3 cells, including changes in morphology and motility. At the molecular level, Twist2 induces significant alteration of EMT markers. Furthermore, Twist2 promoted cell migration and invasion of SKOV-3 cells. Therefore, Twist2 promoted b-catenin release from E-cadherin/b-catenin complex through inhibition of E-cadherin. With nuclear b-catenin accumulation, Wnt/b-catenin pathway was then activated to promote an EMT phenotype.  To the best of our knowledge, we show for the first time that Wnt activation in Twist2-induced EMT progress in ovarian cancer, providing new insights into their metastasis.

Conclusions
Our data suggest that upregulation of Twist2 is correlated with the FIGO stage in human ovarian cancers. Although there is no correlation between Twist2 expression and the tumor histological type, Twist2 is a potential indicator of high grade of malignancy and poor prognosis in clinical ovarian cancer. Overexpression of Twist2 induced the EMT phenotypes in human ovarian cancer cells in vitro. Downregulation of E-cadherin and APC as well as upregulation of N-cadherin and b-catenin (in cell nucleus), suggest that Twist2 promotes b-catenin release from E-cadherin/bcatenin through inhibition of E-cadherin. In addition, due to downregulation of APC, b-catenin degradation was repressed. Therefore, canonical Wnt/b-catenin pathway was then activated by nuclear b-catenin accumulation, thus evoked transcription of downstream target genes to promote tumor invasion and metastasis. Collectively, these data indicated that b-catenin is involved in Twist2-induced EMT in ovarian cancer.
Rabbit anti-phospho-Akt (Ser 473) antibody was purchased from R&D Systems Europe. Rabbit anti-Erk1/2, Rabbit anti-p-Erk1/2, Rabbit anti-Akt, Rabbit anti-p-Akt were purchased from Cell Signaling Company. ABC Kits and DAB substrate kit were purchased from Thermo Scientific and Pierce, respectively. Formalin-fixed and paraffin-embedded ovarian cancer tissue microarray chips were from Alenabio Company. The research protocol and designs were approved by the Ethics Committee of Xiamen University (ID No: 20100602). All our clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki.

Immunohistochemical Staining
Tissue array sections in formalin-fixed and paraffin-embedded human primary ovarian cancer were examined to assess the expression of Twist2, and immunohistochemical staining (IHC) was performed as described previously [25,33]. Twist2 was shown to specifically recognize the corresponding proteins (Fig. 1A). Diaminobenzidine was used to visualize the immunohistochemical reaction, followed by counterstaining with hematoxylin. To prepare the negative control, the primary antibody was replaced with normal mouse IgG. The IHC staining on tissue array was repeated for 3 times, analyzed through standard light microscopy. Positive cells showed brown granules in cytoplasm or cell nucleus.

Generation of Twist2-overexpression Stable Ovarian Cancer Cells
The human ovarian cancer cell line SKOV-3 was obtained from ATCC. The cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and penicillin/ streptomycin. The Flag-Twist2-expressing plasmid had been constructed in our previous study [25]. The Flag-Twist2-expressing  plasmid and the pBabe-puromycin vector were co-transfected into SKOV-3 ovarian cancer cells using the lipofectamine2000 TM transfection reagent (Invitrogen) according to the manufacturer's instruction. The Twist2-expressing stable clones and the vector control clones were obtained respectively through selection with puromycin. The Twist2 expression levels in the selected stable clones were then verified through immune-blot analysis with Twist2 and flag antibodies.

Cell Morphological Observation and Confocal Immunofluorescent Staining
Cell morphological shapes were observed using phase microscopy (Olympus). Immunohistochemical staining was performed on Twist2/SKOV-3 and Vector/SKOV-3 ovarian cancer cells as previously described [25]. The staining of Twist2-and vectortransfected SKOV-3 cells were observed and analyzed using the laser scanning confocal microscopy (Olympus).

Cell Growth Assay
The cells were seeded in normal medium until harvested. Cell growth was assessed via MTT assay as previously described [34]. 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 6 SD. The cell proliferation rate (%) was calculated as follows: number of cells of the experimental group/number of the control group 6100%.

Wound healing assay
Cells were seeded onto six-well plates (1610 5 cells/dish) and when they reached over 90% confluence, a scratch was made across the cell monolayer with the tip. Cells were gently washed with PBS for 3 times and maintained in the fresh medium. Cells were incubated for 24 hrs and photographed using an inverted tissue culture microscope at 6100 magnification. Assays were performed at least three times and data were presented as means 6 SD. The migration potential between Twist2-expressing cells and the control cells was compared by relative gap distance. And we analyzed the results by the one way ANNOVA test (Prism5 software). A P-value of p,0.05 was considered statistically significant.

Cell Invasion Assay
For invasion assay, 5610 3 cells were seeded on Matrigel-coated membrane inserts (BD Bioscience). The bottom chamber contained 0.75 ml McCoy's 5A supplemented with 10% fetal bovine serum as a chemoattractant. Cells were incubated for 24 hrs, the cells remaining inside the insert were removed with a cotton swab and cells that had penetrated the Matrigel to invade to the lower surface of the membrane were fixed in methanol and stained with H&E. After air drying the membrane, the cells were counted at 6100 magnification in 10 random fields of view under a microscope. Three independent experiments were performed in each case.

Cell cycling analysis via Flow Cytometry
Cell cycling was identified via flow cytometry analysis as previously described [34]. Briefly, the cells were seeded for 24 h, then were harvested, washed twice with PBS, and fixed in 70% ethanol at 4uC overnight. The cell pellets were suspended in propidium iodide staining solution (20 mg/mL propidium iodide and 0.2 mg/mL RNase in PBS) and incubated for 30 min at 37uC. The samples were then analyzed through flow cytometry.

Isolation of Subcellular Fractions, and Western Blot Analysis
Cell fractionation and Western blot analysis were performed as our previous publications [34,35]. Details of cell fractionation is as following: Whole-cell extracts were prepared by scraping cells off Petri dishes, washing cell pellets twice in PBS, and then resuspending pellets in two-packed cell volumes of RIPA buffer [150 mM NaCl/50 mM Tris?HCl, pH 7.5/0.25% (wt/ol) deoxycholate/1% Nonidet P-40/5 mM sodium orthovanadate/2 mM sodium fluoride/protease inhibitor mixture]. Nuclear and cytoplasmic extracts were isolated by washing cell pellets in buffer A (10 mM Hepes, pH 7.5/10 mM KCl/1.5 mM Mg 2 Cl/0.5 mM NaF/1 mM glycerol phosphate/protease mixture), then lysis in buffer A+B (buffer A plus 0.5% Nonidet P-40) in a 2:1 mix. After centrifugation (12,0006g for 10 min), supernatant was collected (cytoplasmic fraction). Pellets were washed in PBS, centrifuged (12,0006g for 10 min), and then flash-frozen in dry-ice-ethanol in buffer C (20 mM Hepes, pH, 7.5/420 mM NaCl/1.5 mM Mg 2 Cl/0.5 mM NaF/0.5 mM DTT/1 mM glycerol phosphate/ protease mixture), followed by a slow thaw on ice. Pellets were then centrifuged (12,0006g for 10 min), washed, and membranes lysed in RIPA buffer. The supernatant was centrifuged (12,0006g) and collected (nuclear fraction). Protein Assay Kit for protein quantity analysis was purchased from Bio-Rad company (CA,USA). The enhanced chemiluminescence (ECL) detection system was purchased from Amersham (Amersham, IL, USA). All antibodies were described as materials. All the results are the average 6 s.d. from at least three experiments.

Real-time Reverse Transcriptase-PCR
Total cellular RNA was prepared using TRIzol reagent (Invitrogen) and the expression levels of E-cadherin, N-cadherin, APC, and Twist2 mRNAs were determined by real-time reverse transcriptase-PCR using SYBR Green [36]. Triplicate 10 ng aliquots of RNA were analyzed using two-step real time RT-PCR (Applied Biosystems), and relative mRNA levels determined using the ,DELTA.,DELTA.Ct method, using the GAPDH gene as the reference gene. All primer sets employed had amplification efficiencies ranging from 98.0+/20.2% to 102.5+/21.1% (R2 values for all reactions.or = 0.995). And non-template controls were used in duplicate per analysis. Data shown are normalized to GAPDH expression and represent the average of three repeated experiments. Primer sequences were as Table 2.
Transcriptional Reporter Gene Assay TCF4/LEF1-dependent b-catenin transcriptional activity was assessed by using TOPflash and FOPflash reporter plasmid (the plasmids are provided by Professor Jianxing Ma). Briefly, the reporter genes were transfected into cells grown to semiconfluence in 12-well plates, and cultured for a further 48 h. Luciferase activity were measured by using Dual Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instructions. In order to see the effect of Twist2 on b-catenin, Twist2 plasmid was co-transfected into cells with TOPflash or FOPflash reporter genes, the luciferase and renilla activity was measured as described above.

Statistical Analysis
The results of the experimental studies are expressed as mean 6 SD. Statistical differences were analyzed through Student's t-test using SPSS 10.0 software; p,0.05 was considered to indicate statistical significance. Difference between groups was assessed by one way ANOVA tests (Prism5 software).