The clinical significance of mesenchyme forkhead 1 (FoxC2) in gastric carcinoma

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1 Histopathology 2013, 62, DOI: /his The clinical significance of mesenchyme forkhead 1 (FoxC2) in gastric carcinoma Jin-Liang Zhu,* Yong-Xi Song,* Zhen-Ning Wang, Peng Gao, Mei-Xian Wang, 1 Yu-Lan Dong, 1 Cheng-Zhong Xing & Hui-Mian Xu Department of Surgical Oncology and General Surgery, First Hospital of China Medical University, Shenyang, China, and 1 Department of Tumor Pathology and Surgical Oncology, First Hospital of China Medical University, Shenyang, China Date of submission 6 November 2012 Accepted for publication 14 March 2013 Published online Article Accepted 18 March 2013 Zhu J-L, Song Y-X, Wang Z-N, Gao P, Wang M-X, Dong Y-L, Xing C-Z & Xu H-M (2013) Histopathology 62, The clinical significance of mesenchyme forkhead 1 (FoxC2) in gastric carcinoma Aims: Mesenchyme forkhead 1 (FoxC2) is an epithelial mesenchymal transition (EMT)-inducing factor. Previous studies have demonstrated that FoxC2 binds directly to the promoter region of p120-catenin (p120ctn). The aim of this study was to investigate the clinical significance of FoxC2 expression and the inter-relationship between FoxC2 and p120ctn, in gastric cancer. Methods and results: Immunohistochemistry was used to examine the expression of FoxC2 and p120ctn proteins in 325 gastric cancer samples. Staining for FoxC2 in cancer tissues was markedly stronger than in normal tissues. High FoxC2 expression was associated significantly with differentiation, invasion depth, lymph node metastasis and tumour stage. Patients with high FoxC2 expression or low p120ctn expression had a poor prognosis. In the high p120ctn expression group, the prognosis for patients with low FoxC2 expression was better than for the high FoxC2 group. Moreover, stepwise Cox regression showed that p120ctn was an independent prognostic factor, but FoxC2 in combination with p120ctn was not correlated significantly with survival. Conclusions: We found that FoxC2 and p120ctn play important roles in the progression and prognosis of gastric cancer. Moreover, FoxC2 and p120ctn should be evaluated further as novel biomarkers and therapeutic targets for gastric cancer treatment. Keywords: epithelial mesenchymal transition, FoxC2, gastric carcinoma, P120-catenin, prognosis Introduction Address for correspondence: Z-N Wang, Department of Surgical Oncology and General Surgery, First Hospital of China Medical University, 155 North Nanjing Street, Heping District, Shenyang City , China. josieon826@yahoo.com.cn *Jin-Liang Zhu and Yong-Xi song contributed equally to this work. Gastric cancer is the second leading cause of cancer death in both sexes worldwide; approximately people die of gastric carcinoma every year. 1,2 More than 70% of new cases occur in developing countries, and half the world total occurs in eastern Asia (mainly in China). Although early gastric cancer patients have benefited from improvements in clinical diagnosis and treatment and have a longer-term survival rate, the prognosis for patients with advanced gastric cancer is still disappointing. The most important reason for this poor prognosis is delayed diagnosis: at presentation many patients show extensive regional invasion and distant metastasis, particularly to the lymph nodes. 3 The molecular mechanisms that allow malignant tumour cells to dissociate from the primary tumor mass and invade the surrounding stroma are not yet well understood. However, accumulating evidence has indicated that epithelial mesenchymal transition (EMT) is an important mechanism during the early steps of 2013 Blackwell Publishing Limited.

2 FoxC2 in gastric carcinoma 1039 tumour progression, when neoplastic cells disseminate from the primary tumour. 4 EMT plays a crucial role in embryonic development and wound healing, but may cause organ fibrosis and promote carcinoma progression when deregulated. 5,6 Loss of expression of epithelial marker E-cadherin and gain of expression of mesenchymal markers such as N-cadherin, fibronectin or vimentin are considered important molecular markers of EMT. 5,7,8 Mesenchyme forkhead 1 (also known as Forkhead box protein C2, FoxC2) is an EMT-inducing factor and has been reported in a variety of cancers. Mani et al. 9 proposed that FoxC2 can suppress the expression of E- cadherin and act as an activator of epithelial cell dedifferentiation and metastasis in breast cancer. Yu et al. 10 also found that down-regulation of FoxC2 was critical for resveratrol-mediated suppression of tumour metastasis in in-vitro and in-vivo models. Nishida et al. 11 showed that FoxC2 is involved closely in oesophageal cancer progression and probably mediates local invasion through up-regulation of molecules such as matrix metalloproteinases MMP2 and MMP9. Jiang et al. 12 reported that overexpression of Twist, Slug and FoxC2 in stage I non-small-cell lung cancer (NSCLC) was associated with a worse survival rate. The molecular mechanisms involving these EMT markers are not well established, but down-regulation of E-cadherin has been demonstrated to have direct relevance to EMT. 7 Many EMT-inducing transcription factors, including Snail, Slug, SIP1, ZEB, Twist, Goosecoid and FoxC2, repress E-cadherin expression either directly or indirectly Conversely, loss of p120-catenin (p120ctn) expression results in destabilization of the E-cadherin complex, a critical step in invasion and metastasis Furthermore, Mortazavi et al. 23 found that the downregulation of E-cadherin level caused by FoxC2 is mediated mainly through the regulation of p120ctn. However, to our knowledge, no studies have evaluated the expression of FoxC2 and its clinicopathological significance in gastric cancer. We therefore examined the expression of FoxC2 in a large number of gastric cancer tissues and corresponding normal mucosa tissues. We also aimed to evaluate the clinical significance of FoxC2 expression and the interrelationship between FoxC2 and p120ctn in gastric cancer. Materials and methods PATIENTS AND TISSUE SAMPLES A total of 325 formalin-fixed and paraffin-embedded (FFPE) tumour samples, together with 62 corresponding normal samples, 23 intestinal metaplasia samples and 20 adjacent precursor lesion dysplasia samples, were obtained from 325 patients who underwent curative radical gastrectomy with standard lymph node dissection at The First Hospital of China Medical University between 1998 and None of the patients had received chemotherapy or radiotherapy before the surgical procedure. We obtained written informed consent from all patients, and the study was approved by the ethics committee of the China Medical University. The detailed postoperative pathology diagnosis reports were obtained and included age, gender, tumour location, size, differentiation status, growth pattern, invasion depth, lymph node metastasis, lymphatic invasion and TNM stage. The TNM classification system criteria for gastric carcinoma were utilized in accordance with the 7th 2010 American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) staging manual. Table 1. Normal-cancer, intestinal metaplasia-cancer, dysplasia-cancer paired sample comparisons (t-test) Variables No. IS Mean SD FoxC2 <0.001 Normal Cancer P120ctn Normal Cancer FoxC Intestinal metaplasia Cancer P120ctn Intestinal metaplasia Cancer FoxC Dysplasia Cancer P120ctn Dysplasia Cancer P

3 1040 J-L Zhu et al. The tumour growth pattern criteria were in accordance with the Japanese classification of gastric carcinoma, 3rd English edition. The patients comprised 235 males and 90 females, with a mean age of 57 years (range years). Of the 325 gastric cancers, 95 were T1/2 and 230 were T3/4; 225 cases (69.2%) had lymph node metastases, and 80 (24.6%) had lymphatic invasion. According to the 2000 World Health Organization histological classification of gastric carcinoma, 24 of the 325 cases, 14 were papillary adenocarcinomas, 252 tubular adenocarcinomas, 38 mucinous adenocarcinomas and 21 signet-ring cell adenocarcinomas; 47 were well differentiated, 54 moderately differentiated, 214 poorly differentiated and 10 were undifferentiated adenocarcinomas. All patients were followed-up via telephone inquiry or questionnaires, and the followup time ranged from 1 to 136 months (median 56 months). IMMUNOHISTOCHEMISTRY Four-lm-thick sections cut from the FFPE tissue blocks were deparaffinized with xylene and rehydrated using a graduated series of ethanol. The sections were incubated in boiling citric acid buffer (ph 6.0) for antigen retrieval in a steam pressure cooker. After blocking non-specific antibody binding, the sections were incubated separately overnight at 4 C with the following primary antibodies: FoxC2 monoclonal antibody, 1:100 dilution (H M02; Abnova, Taipei, Taiwan) and mouse anti-p120-catenin, 1:300 dilution (610134; BD Transduction Laboratories, San Diego, CA, USA). Immunohistochemical staining was conducted using Histostain-Plus kits (Zymed Laboratories Inc., San Francisco, CA, USA) with diaminobenzidine (DAB) as the chromogen. The sections were then counterstained with haematoxylin. Negative control staining was carried out by substituting A B C D E F Figure 1. Immunohistochemical staining for FoxC2 and p120ctn in normal and neoplastic gastric tissue. A, Normal gastric mucosa, no staining for FoxC2; B, moderate staining for FoxC2 in cytoplasm of moderately differentiated adenocarcinoma; C, strong staining for FoxC2 in cytoplasm of poorly differentiated adenocarcinoma; D, normal gastric mucosa, strong membranous staining for p120ctn; E, strong membranous staining for p120ctn in moderately differentiated adenocarcinoma; F, weak and fragmented membranous staining for p120ctn in poorly differentiated adenocarcinoma.

4 FoxC2 in gastric carcinoma 1041 Table 2. FoxC2 and p120ctn expression in relation to various clinicopathological characteristics Variable Total FoxC2 low (%) FoxC2 high (%) P value p120ctn low (%) (28.3) 233 (71.7) 121 (37.2) 204 (62.8) p120ctn high (%) P value Age at surgery (years) (33.2) 127 (66.8) 80 (42.1) 110 (57.9) > (21.5) 106 (78.5) 41 (30.4) 94 (69.6) Gender Male (27.7) 170 (72.3) 78 (33.2) 157 (66.8) Female (30.0) 63 (70.0) 43 (47.8) 47 (52.2) Pathophysiologic features Tumor size (cm) (32.5) 133 (67.5) 73 (37.1) 124 (62.9) > (21.9) 100 (78.1) 48 (37.5) 80 (62.5) Tumor location Upper (40.0) 18 (60.0) 12 (40.0) 18 (60.0) Middle (24.5) 40 (75.5) 30 (56.6) 23 (43.4) Lower (27.7) 175 (72.3) 79 (32.6) 163 (67.4) Histological grade <0.001 WD/MD (15.8) 85 (84.2) 12 (11.9) 89 (88.1) PD (33.9) 148 (66.1) 109 (48.7) 115 (51.3) Growth pattern <0.001 Expanding (22.7) 58 (77.3) 19 (25.3) 56 (74.7) Intermediate (21.2) 63 (78.8) 12 (15.0) 68 (85.0) Infiltrative (34.1) 112 (65.9) 90 (52.9) 80 (47.1) T stage < T1/ (50.5) 47 (49.5) 29 (30.5) 66 (69.5) T3/ (19.1) 186 (80.9) 92 (40.0) 138 (60.0) Lymph node metastasis < Negative (46.0) 54 (54.0) 26 (26.0) 74 (74.0) Positive (20.4) 179 (79.6) 95 (42.2) 130 (57.8) Lymphatic invasion Negative (31.0) 169 (69.0) 91 (37.1) 154 (62.9) Positive (20.0) 64 (80.0) 30 (37.5) 50 (62.5)

5 1042 J-L Zhu et al. Table 2. (Continued) Variable Total FoxC2 low (%) FoxC2 high (%) P value p120ctn low (%) p120ctn high (%) P value Tumor stage < IA, IB (61.3) 24 (38.7) 16 (25.8) 46 (74.2) IIA, IIB (30.2) 60 (69.8) 26 (30.2) 60 (69.8) IIIA, IIIB, IIIC (15.8) 149 (84.2) 79 (44.6) 98 (55.4) P120ctnexpression Low (37.2) 76 (62.8) High (23.0) 157 (77.0) WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated, undifferentiated. non-immune mouse or rabbit serum and phosphatebuffered saline (PBS) for primary antibodies. EVALUATION OF IMMUNOHISTOCHEMICAL STAINING RESULTS Immunostaining results were interpreted independently by two pathologists who were blinded to patient outcomes. A semiquantitative scoring system was used, 25 which evaluated both staining intensity (0, no stain; 1+, weak stain; 2+, moderate stain; 3+, strong stain) and the percentage of stained cells (0, <5%; 1, 5 25%; 2, 26 50%; 3, 51 75%; and 4, >75%). Scores for staining intensity and percentage positivity of cells were then multiplied to generate the immunoreactivity score (IS) for each case. With regard to staining intensity: for FoxC2, 0 = no staining, 1+ =weak cytoplasmic staining, 2+ =moderate cytoplasmic staining and 3+ =strong cytoplasmic staining; and for p120ctn, 0 = no staining, 1+ =weak or fragmented membrane staining, 2+ =moderate incomplete membrane staining, and 3+ =strong, complete membrane staining. 26 Specimens were rescored if there were IS discrepancies between the two pathologists until a consensus was reached. All cases were sorted into two groups according to the IS: for FoxC2, high expression was defined as detectable immunoreactions in cytoplasm and IS 4; and for p120ctn, high expression was defined as detectable immunoreactions in cell membranes and IS 4. STATISTICS The appropriate non-parametric tests were used to investigate FoxC2 and p120ctn expression and clinicopathological parameters (i.e. Chi-squared test, paired-samples t-test and Spearman s correlation test). Univariate survival analysis was performed using the Kaplan Meier method, and differences between the groups were analysed using the logrank test. The multivariate Cox backward stepwise regression model was used to detect independent predictors of survival. Two-tailed P-values of <0.05 were considered statistically significant. All statistical analyses were performed using SPSS software version 17.0 (SPSS for Windows, Chicago, IL, USA). Results In the epithelium of normal gastric mucosa, there was weak or negative FoxC2 immunoreactivity (mean IS, ); expression was observed primarily in the cytoplasm. Strong p120ctn immunoreactivity occurred in a predominantly membranous pattern (mean IS, ), with a small proportion of samples displaying cytoplasmic staining. Similarly, FoxC2 and p120ctn labelling of gastric cancer samples occurred predominantly in cytoplasmic and membranous patterns, respectively. However, in cancer tissues, FoxC2 staining was markedly stronger than in the corresponding normal tissues (mean IS, ; P < 0.001, Table 1, Figure 1A C); while p120ctn staining in the tumour group showed a lower IS than that of the normal group (mean IS, ; P = 0.017, Table 1, Figure 1D F). In intestinal metaplasia and precursor lesion dysplasia samples, the IS scores for FoxC2 and p120ctn were similar to the corresponding cancer samples, and no significance differences were found in these paired samples (Table 1). Furthermore, on evaluating the staining patterns for FoxC2 in all sections, no signifi-

6 FoxC2 in gastric carcinoma 1043 Table 3. Survival analysis in gastric cancer Univariate Multivariate Variable Total (%) 5 years (%) P-value RR 95% CI P-value Age at surgery (years) (58.5) 56.4 > (41.5) 50.6 Gender Male 235 (72.3) 54.6 Female 90 (27.7) 52.4 Pathophysiological features Tumour size (cm) < (Reference) 197 (60.6) 62.8 >5 128 (39.4) 41.1 Tumour location Upper 30 (9.2) 66.6 Middle 53 (16.3) 50.0 Lower 242 (74.5) 53.2 Histological grade WD/MD 101 (31.1) 54.1 PD 224 (68.9) 53.9 Growth pattern Expanding 75 (23.1) 64.5 Intermediate 80 (24.6) 54.0 Infiltrative 170 (52.3) 49.7 T stage < <0.001 T1/2 (reference) 95 (29.2) 87.4 T3/4 230 (70.8) 40.1 Lymph node metastasis < <0.001 Negative (reference) 100 (30.8) 86.9 Positive 225 (69.2) 38.8 Lymphatic invasion < Negative (reference) 245 (75.4) 60.3 Positive 80 (24.6) 34.5

7 1044 J-L Zhu et al. Table 3. (Continued) Univariate Multivariate Variable Total (%) 5 years (%) P-value RR 95% CI P-value Tumour stage <0.001 IA, IB (reference) 62 (19.0) 94.2 IIA, IIB 86 (26.5) 76.4 IIIA, IIIB, IIIC 177 (54.5) 27.0 FoxC2 expression Low (reference) 92 (28.3) 68.4 High 233 (71.7) 48.1 P120-catenin expression < Low (reference) 121 (37.2) 42.4 High 204 (62.8) 61.0 Combination of FoxC2 and P120ctn expression <0.001 FoxC2(L) P120ctn(H) (reference) 47 (14.5) 83.0 Others 278 (85.5) 49.3 WD/MD, well differentiated/moderately differentiated; PD, poorly differentiated, undifferentiated; FoxC2(L), low FoxC2 expression; P120ctn(H), high p120ctn expression; RR, relative risk; CI, confidence interval. cant differences were observed between the invasive front and other areas. Analysis of possible relationships between FoxC2 and p120ctn expression and clinicopathological parameters demonstrated that expression of FoxC2 was associated significantly with differentiation (P = 0.001), T-stage (P < 0.001), lymph node metastasis (P < 0.001) and tumour stage (P < 0.001). Expression of p120ctn was associated significantly with differentiation (P < 0.001), growth pattern (P < 0.001), lymph node metastasis (P = 0.006) and tumour stage (P = 0.009). Associations between clinicopathological features and FoxC2/p120ctn protein expression are summarized in Table 2. Our results also showed that high FoxC2 expression was associated significantly with high p120ctn expression (Table 2); 77.0% (157 of 204) of the cases with high p120ctn expression showed high FoxC2 (P = 0.007). In Kaplan Meier survival analysis (Table 3), we found that patients with high FoxC2 expression had significantly poorer cancer-specific survival rates than patients with low FoxC2 expression (P = 0.005, Figure 2A). The prognosis for high p120ctn expression tended to be better than that for low p120ctn expression patients (P < 0.001, Figure 2B). Dividing the patients into p120ctn-low and p120ctn-high groups, no significant difference was found in the p120ctn low expression group between the prognosis of patients with low FoxC2 and high FoxC2 expression (P = 0.457, Figure 3A). However, in the p120ctn high expression group, the prognosis for patients with low FoxC2 expression was better than for the high FoxC2 expression subgroup (P < 0.001, Figure 3B). In Cox multivariate analysis (Table 3), we found that T-stage, lymph node metastasis, lymphatic invasion and p120ctn expression were correlated independently with survival of gastric cancer patients (P < 0.005). Moreover, p120ctn expression was an independent prognostic factor (RR = 0.690, 95% CI , P = 0.022), but FoxC2 in combination with p120ctn was not correlated significantly with survival. Discussion Gastric cancer is understood to develop as a result of a multistep progression from chronic gastritis to atrophic gastritis, intestinal metaplasia, dysplasia and

8 FoxC2 in gastric carcinoma 1045 A 1.0 A 1.0 Survival for Low p120ctn Expression Group Accumulative survival rate B Accumulative survival rate 0.8 Low FOXC2 expression 0.6 High FOXC2 expression P = Months after surgery High p120ctn expression Low p120ctn expression 0.2 P < Accumulative survival rate Accumulative survival rate B High FOXC2 expression Months after surgery Survival for High p120ctn Expression Group High FOXC2 expression Low FOXC2 expression P = Low FOXC2 expression P < Months after surgery Figure 2. Kaplan Meier postoperative survival curves of gastric cancer patients with different levels of tumour FoxC2 (A) and p120ctn (B) expression. A, Patients with tumours showing low expression levels of FoxC2 had significantly better survival compared to those whose tumours showed high expression levels (P = 0.005). B, Patients with tumours showing high expression levels of p120ctn had significantly better prognosis compared to those with tumours showing low expression levels (P < 0.001). finally cancer. 27 The increased expression of FoxC2 in intestinal metaplasia, dysplasia precursor lesions and cancer samples suggests that FoxC2 could be one of the initial factors involved in tumour progression. EMT is a key process in normal embryonic development, but when deregulated it can also promote carcinoma progression. A variety of different Months after surgery Figure 3. Kaplan Meier postoperative survival curves of gastric cancer patients with tumours showing different levels of FoxC2 expression, according to tumour p120ctn expression. A, In the low p120ctn expression group, no significant survival difference was found between patients with low and high tumour expression of FoxC2 (P = 0.457). B, In the high-p120ctn expression group, the patients with low tumour expression of FoxC2 had a significantly better prognosis compared to those with high tumour expression of FoxC2 (P < 0.001). transcription factors, such as FoxC2, regulate the gene expression patterns that underlie EMT. 5 In recent years, accumulating evidence has indicated that deregulation of EMT factors such as Snail, Slug and Twist are involved in the progression of gastric

9 1046 J-L Zhu et al. and deregulation of FoxC2 has also been confirmed to play an important role in other cancers, such as breast, oesophageal, NSCLC and colorectal cancers. 9,11,12,32 FoxC2 is an essential regulator of vascular system development and tumour progression. 33 Hayashi et al reported that FoxC2 can regulate angiogenesis via induction of integrin beta3 and CXCR4 expression, and that expression levels of these protein increased in pathological and physiological angiogenesis, including the vasculature of ischaemic tissues such as tumours. Mani et al. 9 and Nishida et al. 11 have demonstrated previously that cells overexpressing FoxC2 express very high levels of matrix metalloproteinases (MMPs), which can facilitate tumour invasiveness and metastasis potential. Furthermore, Watanabe et al. 32 reported that FoxC2 was involved in determining the degree of lymph node metastasis in colorectal cancer. Our immunohistochemical studies revealed that increased expression of FoxC2 was associated with the histological grade, invasiveness, lymph node metastasis, tumour stage and poor prognosis, in keeping with previous reports. 9,11,12,32 These studies all suggested that FoxC2 plays an important role in gastric cancer and might be used as a diagnostic marker for gastric carcinoma precursor lesions. To investigate the mechanism by which FoxC2 is involved in the pathological regulation of EMT progression in gastric cancer we studied p120ctn, which is a member of the Armadillo (ARM)/beta-catenin gene family and is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness. 37,38 Furthermore, the findings of Mortazavi et al. 23 suggested that FoxC2 binds directly to the p120ctn promoter region and mediates the transcriptional repression of p120ctn in NSCLC. Whether this is also the case in gastric cancer is unknown, and we therefore selected p120ctn for study to explore its relationship with FoxC2. Our findings showed that reduced or absent membranous expression of p120ctn was associated with a worse histological grade of tumour, which is consistent with reported data. 39,40 We observed strong continuous expression of p120ctn along the cell membrane in normal gastric mucosa glands. However, in intestinal metaplasia, precursor lesion dysplasia samples and some gastric carcinoma tissues, especially those with an infiltrative growth pattern, p120ctn membrane expression was decreased, and was often discontinuous or absent. Also, up-regulation of cytoplasmic staining was seen, most frequently in infiltrative tumours. Furthermore, reduced membranous expression of p120ctn correlated with infiltrative growth pattern, positive lymph cancer 15,28 31 node metastasis and high TNM stage, suggesting that such decreased expression might play a critical role in gastric carcinoma progression. We also demonstrated that high FoxC2 expression was correlated significantly with high p120ctn expression. In survival analysis, we found that patients whose tumours showed increased FoxC2 expression, reduced membranous expression of p120ctn or a combination of high FoxC2 and low p120ctn expression, had a poorer prognosis (Figures 2A,B and 3B and Table 3). Our survival analysis results were in accord with earlier reported data showing that, in a variety of tumours, increased expression of FoxC2 or reduced expression of p120ctn correlated with progression and poorer prognosis. In NSCLC, Jiang et al. 12 reported that overexpression of Twist, Slug, and FoxC2 was associated with a worse survival rate. Nishida et al. 11 found that high FoxC2 expression in oesophageal cancer was associated directly with poor prognosis, while in colon carcinoma, Bellovin et al. 20 reported that cytoplasmic expression of p120ctn was associated with reduced 5- and 10-year survival rates compared with membranous expression of p120ctn. Because the increased expression of FoxC2 and reduced expression of p120ctn were correlated significantly with tumour progression, patients with tumours showing these features may require different management. Our survival analysis also found that the expression of FoxC2 was associated with poorer prognosis in the p120ctn highexpression subgroup. In multivariate analysis, using stepwise Cox regression, p120ctn was an independent prognostic factor, but FoxC2 in combination with p120ctn was not correlated significantly with survival. Thus, we suggest that FoxC2 might influence prognosis indirectly through the influence of T/N stage. The mechanism of the interaction between FoxC2 and p120ctn in cancer progression remains unclear, and further studies are warranted. In conclusion, we studied expression of FoxC2 and p120ctn in a variety of gastric tissues and found that both proteins appear to play important roles in the progression of gastric cancer. Moreover, p120ctn was an independent prognostic factor. FoxC2 in combination with p120ctn may indirectly influence the prognosis. Our findings suggest FoxC2 and p120ctn should be evaluated further as novel biomarkers and therapeutic targets in gastric cancer. Acknowledgement This work was supported by the National Science Foundation of China (no , no

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