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Upregulated N‐cadherin expression is associated with poor prognosis in epithelial‐derived solid tumours: A meta‐analysis

Abstract Background N‐cadherin is an important molecular in epithelial‐mesenchymal transition (EMT) and has been reported to be associated with aggressive behaviours of tumours. However, prognostic value of N‐cadherin in solid malignancies remains controversially. Materials and Methods The Pubmed/MELINE and EMBASE databases were used for a comprehensive literature searching. Pooled risk ratio (RR) and hazard ratio (HR) with their corresponding 95% confidence intervals (CIs) were employed to quantify the prognostic role. Results Involving 36 studies with 5705 patients were performed to investigate relationships between N‐cadherin upregulation and clinicopathological features, survival. Results suggested upregulated N‐cadherin was associated with lymph node metastasis (RR = 1.16, 95% CI [1.00, 1.35]), higher histological grade (RR = 1.36, 95%CI [1.14, 1.62]), angiolymphatic invasion (RR = 1.19, 95% CI [1.06, 1.34]) and advanced clinical stage (RR = 1.32, 95% CI [1.06, 1.64]), while upregulated N‐cadherin was apt to be associated with distant metastasis (RR = 1.43, 95% CI [0.99, 2.05]). Moreover, N‐cadherin was correlated with poor prognosis of 3‐year survival (HR = 1.78, 95% CI [1.51, 2.10]), 5‐year survival (HR = 1.57, 95% CI [1.17, 2.10]) and overall survival (OS) (HR = 1.32, 95% CI [1.20, 1.44]). Subgroup analyses according to cancer types were also conducted for applying these conclusions to some tumours more properly. No publication bias was found except subgroup analysis of distant metastasis (P = .652 for Begg's test and 0.023 for Egger's test). Conclusions Taken together, upregulation of N‐cadherin is associated with more aggressive behaviours of epithelial‐derived solid malignancies and can be regarded as a predictor of poor survival.


| INTRODUCTION
As a life-threatening disease, cancer has increasingly been a heavy burden of healthcare system. 1,2 The failure of cancer therapy and poor outcomes are mostly owing to the development of local invasion and distant metastasis. Activating of invasion and metastasis has been demonstrated as one of the most important hallmark capabilities of cancer. 1 To date, researchers have paid tremendous attention to the biological behaviour of cancer cells, aiming at finding potential therapeutic targets.
Invasion and metastasis are considered as complex processes involving detachment of cancer cell from primary tumour site and formation of new tumour foci in distant organs. [3][4][5] Epithelial-mesenchymal transition (EMT), a developmental regulatory programme, identified as transformed epithelial cells loss epithelial constraints and then acquire the abilities to invade, resist apoptosis and metastasis, which is believed to play a vital role in broadly regulating invasion and metastasis. 2,[6][7][8][9] Cadherins are a family of calcium-dependent glycoproteins, which responsible for Ca 2+ -dependent cell-cell adhesions. Of the members of this family, N-cadherin (neuronal cadherin or cadherin-2), a transmembrane glycoprotein, which is normally expressed in neuroectodermal and mesenchymal-derived tissues, plays crucial role in lots of processes, such as cell-cell adhesion, embryogenesis, gastrulation, neurulation, migration and invasion. 4,[10][11][12][13] Today, N-cadherin is regarded as a vital marker of EMT. 14 Neoexpression or upregulated expression of N-cadherin has been reported to accelerate migration and invasion of cancer cells, which showed a contrary function to those of E-cadherin. [15][16][17][18] This process of reciprocally downregulating Ecadherin and upregulating N-cadherin is known as cadherin switching that characterizes EMT. 19 Cadherin switching is a necessary procedure during the course of development, while it will lead to an aggressive tumour cell phenotype and enable tumour cells obtain ability of migration from primary site and metastasis to distant tissues. 20 Study reveals that Ncadherin-positive cancer cells are capable of building cellcell adhesion with stromal cells that express N-cadherin, thus facilitating invasion process. 21 Some studies indicate that Ncadherin boosts the combination of fibroblast growth factor (FGF) with the receptor to initiate FGFR signal transduction, thereby inducing signalling cascades that promote migration and invasion of cancer cells. 22 Furthermore, studies have shown that N-cadherin promotes cells survival and protects cancer cells from apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)-AKT (also known as protein kinase B) pathway. 17,23,24 Therefore, N-cadherin presents distinct functions from its adhesion activity.
Nevertheless, the prognostic role of N-cadherin in tumours still remains controversially. Some studies suggested that expression of N-cadherin was generally upregulated in prostate, breast cancer and urothelial tumours, and was associated with poorer clinical outcomes. 10,11,25 In addition, the upregulation of N-cadherin was significantly related with postoperative recurrence in hepatocellular carcinoma (HCC) patients. 26 However, it was also reported that the expression of N-cadherin was decreased in glioblastoma, ovarian carcinoma, osteosarcoma and HCC, which may provide a hypothesis that N-cadherin serves as a tumour suppressor and its downregulation links to poor surgical outcomes. 15,[27][28][29] Therefore, this meta-analysis was conducted to assess the relationship of N-cadherin expression with clinicopathological features and prognosis of different carcinomas, especially in epithelial-derived solid tumours.

| Primary search strategy
The Pubmed/MELINE and EMBASE databases were used for related publications search by variably combining the terms "cancer [Title/Abstract]," "tumour [Title/Abstract]," "carcinoma [Title/Abstract]," "neoplasm [Title/Abstract]" and "N-cadherin [Title/Abstract]," without any limitation of publication date. The most recent search update was conducted on 23 February 2017. To avoid missing additional studies, the references of all eligible studies were further explored in these databases. PRISMA and the broader EQUATOR guidelines were referred to in the reporting of our study to comply with the standard of meta-analysis. 30

| Inclusion and exclusion criteria
The included studies were required to meet the following criteria: (i) the patients with solid tumours included in these studies were diagnosed histopathologically; (ii) the correlation of N-cadherin expression and solid tumours was evaluated; (iii) clinicopathological features, 3/5-year survival, OS or Kaplan-Meier survival curves were investigated; and (iv) full text was written in English. Studies were excluded if (i) the study was published in conference abstract, letters, editorials, reviews, expert opinion or case reports; (ii) studies of animals or cell lines; (iii) the relevant clinicopathological features or outcomes of patients were not able to be acquired for necessary analysis; (iv) overlapped or duplicated studies.

| Data extraction
Data were carefully retrieved independently by 2 reviewers from all the eligible studies, using a standardized form with the following characteristics: author, year, country, study design, number of patients, mean age, method used for evaluating expression of N-cadherin, antibody source, definition of N-cadherin positive, expression rate of N-cadherin, median follow-up time, clinicopathological features and survival data. Discrepancies between 2 reviewers were solved by discussing until agreement was reached with the third reviewer. Quality assessment of included studies was conducted on the basis of the Newcastle-Ottawa quality assessment scale (NOS scale). Six points or higher were regarded as high quality.

| Statistical analysis
Included studies were divided into 7 groups for analysing the prognostic value of N-cadherin expression in solid tumour patients regarding histological grade, lymph node status, angiolymphatic invasion, distant metastasis, clinical stage, 3year survival, 5-year survival and OS. Clinicopathological features were synthesized using the risk ratio (RR) as the calculating measure, while the survival outcome data using the hazard ratio (HR) as calculating measure. RR/HR with 95% confidence interval (CI) value was performed to quantitatively evaluate how increased N-cadherin expression impacts the prognosis of solid tumour patients. Reported RR/HR and their 95% CI were directly extracted from included studies. If RR/HR and 95% CIs were not available in the original articles, the methods demonstrated by Parmer et al 30 and Tierney et al 31 were applied to calculate RR/HR and 95% CIs. Moreover, when the exploitable data were presented in the figure form, Engauge Digitizer version 4.1 (free software downloading from http://sourceforge.net) was applied to extract data from Kaplan-Meier curves thereby obtaining HR and 95% CIs. Heterogeneities of included studies were assessed using I 2 metric and Q statistic. If heterogeneity was significantly obvious (I 2 > 50% or P < .1), a random effect model was used. Otherwise, a fixed effect model would be chosen (I 2 < 50% or P > .1).The pooled RRs/HRs and 95% CIs were presented in forest plots. If the 95% CIs of RR/HR did not cover the value 1 with P < .05, the difference between groups would be regarded as statistically significant. In addition, funnel plots (Begg's test) and Egger's linear regression method (Egger's test) were performed to evaluate the publication bias through assessing the asymmetry of funnel plots (if P < .05, considering to be statistically significant). 32 All statistical analyses in this meta-analysis were performed using STATA version 12.0 (STATA corporation, College Station, TS, USA).

| Study selection and characteristics
According to the initial search algorithm, total 4476 studies were screened out, and 389 candidate studies were read in full. Eventually, a total of 36 publications were included in this meta-analysis, and the other 353 studies were out of scope, as shown in Figure 1. A total of 5705 patients were included in this study, ranging from 38 to 1035 patients per study, the main characteristics and NOS scale of eligible studies were presented in Table 1. Most of studies were prospective cohort studies (n = 25), and 11 studies were retrospective cohort studies. The median follow-up time varied from 10.1 months to 125 months.
Among the eligible studies, 36 studies compared the correlation of clinicopathological features and N-cadherin expression, including lymph node metastasis (n = 21), histological grade (n = 21), angiolymphatic invasion (n = 8), distant metastasis (n = 7) and clinical stage (n = 14). In addition, 10 studies assessed the association of 3-year survival and N-cadherin positivity, 8 studies for 5-year survival and 22 studies for OS. In terms of NOS scale, 3 articles scored 6 points, 16 articles scored 7 points, 13 articles scored 8 points and 4 articles scored 9 points, hence, all of these inclusive studies were regarded as qualified enough for meta-analysis.

| Clinical Stage
A total of 14 studies with 1479 patients discussed the relationship between N-cadherin upregulation and clinical stage (III/IV vs I/II). 36,37,39,40,[43][44][45][46]48,50,54,55,58,59 15,18,20,69 In addition, the upregulation of N-cadherin was also noticed in different cancer types and was also hinted to link to higher histological grade and metastatic tendency. [69][70][71] Hence, N-cadherin has been implicated as potential target for anticancer therapy. 72 Monoclonal antibody targeting of N-cadherin has been reported to be effective in inhibiting proliferation, metastasis and castration resistance of prostate cancer. 10 Moreover, a kind of Ncadherin antagonist has been developed by researchers, a synthetic cyclic pentapeptide (known as ADH-1). In an animal model of pancreatic cancer, ADH-1 showed the inhibitory effect against tumour growth and metastasis. 73 Importantly, a phase I/II clinical trial has shown combination of ADH-1 and melphalan could suppress tumour growth in patients with locally advanced melanoma. 74 Furthermore, ADH-1 was evaluated to be well tolerable both in animal models and humans. 75,76 However, according to current clinical evidences, the clinical significance of Ncadherin still was unclear. The majority of studies demonstrated N-cadherin as an unfavourable prognostic marker in a variety of tumours, while some studies showed the tumour suppressor role of N-cadherin. Therefore, an integrated study is urgently needed to answer this question. As far as we know, this study is the first comprehensive and most full-scale meta-analysis to investigate the prognostic value of N-cadherin upregulation in epithelialderived solid tumours. At the beginning, 38 studies were involved in this meta-analysis, while 2 studies were about nonepithelial solid tumours, including melanoma and sarcoma. 77,78 Thus, we excluded these 2 studies for making our conclusions more applicable to epithelia-derived solid tumours. In summary, upregulation of N-cadherin was shown to be significantly associated with lymph node metastasis, higher histological grade, angiolymphatic invasion and advanced clinical stage. According to different cancer types, subgroup analyses further explored relationships between some special cancer types and clinicopathological features. Results suggested upregulation of Ncadherin was associated with lymph node metastasis in  prostate cancer, higher histological grade in breast cancer, advanced clinical stage in HNC, positive angiolymphatic invasion and distant metastasis in cancers of digestive system. Importantly, pooled HRs showed upregulation of Ncadherin was an obvious unfavourably prognostic factor in analyses of 3-year survival, 5-year survival and OS, especially for NSCLC and cancers of digestive system according to results from subgroup analyses of OS. Therefore, patients with higher N-cadherin expression may need more aggressive treatment, especially for NSCLC and cancers of digestive system.

| DISCUSSION
In spite of the inspiring outcomes, some limitations could not be ignored. First, although all of the included studies used IHC method to measure expression level of N-cadherin, different antibody sources or various cut-off values could lead to potential bias. However, limited by insufficient number of studies, the subgroup analyses of antibody sources or cut-off values were unable to be carried out. Second, obvious heterogeneity was existed in analyses of lymph node metastasis, histological grade, clinical stage, 5-year survival and OS, which may be derived from different cancer types, cancer stages, follow-up time, etc. Random effect model was employed to reduce the effect of heterogeneity on outcomes. In addition, corresponding subgroup analyses were also conducted to make outcomes more reliable and decrease potential bias. Finally, it would be better if we could conduct a comprehensive analysis about prognostic value of cadherin switching in tumours. Loss of E-cadherin always accompanied with upregulation of N-cadherin in tumours. Besides, loss of E-cadherin has been proved to be associated with unfavourable outcomes in various tumours. [79][80][81][82] The analysis in combination of E-cadherin and N-cadherin might be more valuable in prognostic evaluation of some special tumours, while only a few studies assessed the impact of cadherin switching. 83 In conclusion, our study suggests that positivity of Ncadherin is associated with more aggressive behaviour of epithelial-derived solid malignancies, linked to lymph node metastasis, higher histological grade, angiolymphatic invasion and advanced clinical stage. Moreover, N-cadherin could be regarded as a useful predictor for unfavourable prognosis of 3-year survival, 5-year survival and OS in epithelial-derived solid malignancies. The expression status of N-cadherin can provide a reference for treatment strategy and prognostic prediction of epithelial-derived solid malignancies.