Overexpression of a novel oncogene Cadherin 6 correlates with tumor progression and poor prognosis in gastric cancer

Background: Gastric cancer (GC) is one of the most common and fatal cancers worldwide and effective biomarkers aids in GC management and prognosis. Hence, we explored the role and function of cadherin 6 (CDH6) in diagnosis and prognosis of gastric cancer. Methods: The expression level of CDH6 in GC tissue and normal gastric tissue were analyzed using multiple public databases. Gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas dataset (TCGA). The diagnostic efficiency of CDH6 expression in GC patients was determined through receiver operating characteristic (ROC) curve analysis. The associations between clinical variables and expression of CDH6 were evaluated statistically and the prognostic factors for overall survival were analyzed by univariate and multivariate Cox regression. Forty-four GC tissues, corresponding adjacent normal tissues (n=20), and detailed clinical information were collected from Tianjin Medical University General Hospital, CDH6 expression level was detected for further validation. Results: CDH6 was upregulated in GC samples compared with normal gastric tissue, and GSEA identified the citrate cycle tricarboxylic (TCA) cycle, extracellular matrix (ECM) receptor interaction, glyoxylate and dicarboxylate metabolism oxidative phosphorylation, and pentose phosphate pathway as differentially enriched in GCs. According to the area under the ROC curve (AUC) (AUC=0.829 in TCGA and 0.966 in GSE54129), CDH6 had high diagnostic efficiency. Patients with high expression of CDH6 was associated with higher T classification and worse prognoses than those with low CDH6 expression in GC. Univariate and multivariate Cox regression analysis showed that CDH6 was an independent risk factor for overall survival (univariate: HR = 1.305, P = 0.002, multivariate: HR = 1.481, P < 0.001). Conclusion: CDH6 was upregulated in GC and high CDH6 expression indicated higher T classification and worse prognoses. CDH6 could be a potentially independent molecular biomarker for diagnosis and prognosis of GC. and a member of the CDH family. Recent studies have shown that CDH6 can be aberrantly over expressed in cancer. In thyroid cancer, CDH6 expression is


Background
Gastric cancer (GC) is one of the most common and lethal cancers worldwide. It is the third most-common cancer type and second common cause of cancer-related deaths in China [1]. In addition, the most common type of stomach cancer is adenocarcinoma. With improvements in surgical techniques, traditional radiotherapy, chemotherapy, and the implementation of neoadjuvant therapy, satisfactory therapeutic effects have been achieved for early GC [2].
Because of the nonspecific symptoms of GC, most patients with the disease are diagnosed only when the tumor has reached an advanced stage, at which point the disease has likely metastasized, leading to poor prognosis, low 5-year overall survival rates, and missed opportunity of radical operation [3,4]. Therefore, further research is urgently needed to identify biomarkers with high sensitivity and specificity for early and accurate diagnosis of GC in order to increase long-term survival of patients.
Cadherins (CDHs) are a multigene family of proteins that mediate homophilic calciumdependent cell adhesion and are considered to play critical roles in morphogenesis by mediating specific intercellular adhesion and organization of the cytoskeleton [5]. In addition, CDHs can also serve as sensors of the surrounding microenvironment and as signaling centers for cellular pathways [6]. Recently, several studies have found that CDHs can participate in the promotion of tumorigenesis, tumor growth and malignant progression, diagnosis, and survival prediction of cancer, and even be therapeutic targets [7][8][9]. For example, transcriptional silencing or mutation of E-cadherin is correlated with familial diffuse GC, which may serve as biomarkers for early diagnosis [10]. Besides, CDH2 was considered a potential prognostic and predictive biomarker for the grading and treatment of gliomas [11].
Cadherin-6 (CDH6) is a class II CDH, mainly involved in the morphogenesis of the central nervous system and kidney [12,13]. Previous studies have declared that CDH6 can be abnormally upregulated and promotes epithelial mesenchymal transition (EMT) and cancer metastasis by restraining autophagy in papillary thyroid carcinomas [14,15]. In addition, increased expression of CDH6 has been reported in several malignancies, including nasopharyngeal cancers, ovarian cancers, oral squamous cell cancers, and renal cancers, and is associated with lymph node metastasis and poor prognosis [16][17][18]. However, the clinical significance, diagnosis, and prognostic value of CDH6 in GC remains unclear, and further investigations are required to understand whether it can be used as a novel biomarker for GC diagnosis, prognosis, and as a therapeutic target in GC. In this report, we provide a comprehensive and systematic analysis of CDH6 expression level in GC tissues as compared to normal gastric tissues. To further study the function of CDH6, we used Gene Set Enrichment Analysis (GSEA) to evaluate the biological pathways involved in GC pathogenesis. Survival analyses (Cox regression analyses) were also performed to assess the prognostic value of CDH6 expression and other clinicopathological features.

Data collection
The gene expression profiles and associated clinicopathological data of patients with gastric adenocarcinoma were downloaded from the TCGA Genomic Data Commons data portal

Gene set enrichment analysis
The GSEA is a computational method to detect whether a priori defined gene sets have statistically significant and consistent differences between two biological states [19]. Datasets and phenotype label files from TCGA were generated and uploaded into the GSEA software.
The phenotype labels were CDH6-high expression and CDH6-low expression. Gene set permutations were conducted 1000 times for each analysis. Gene sets with |ES| > 0.6, FWER P values < 0.05 were considered as enriched.

Survival analysis and COX analysis
We divided the TCGA samples into two groups by the median value of CDH6 gene expression to construct the survival curve. Univariate and multivariate Cox analysis were used to investigate the role of CDH6 expression and other clinical characteristics (age, stage, grade, distant metastasis status, and lymph node status) in overall survival. In addition, survival analysis was directly verified using the Kaplan-Meier Plotter (http://kmplot.com/) online.

Statistical analysis
R3.5.2, Bioconductor (https://www.bioconductor.org/) and GraphPad Prism 8 were used for statistical analysis. Survival curves were plotted using the Kaplan-Meier method and were compared using the log-rank test. Cox regression analyses were completed by using the 'survival' R package. The relationship of clinical pathologic features and CDH6 expression were completed using Wilcox test and Kruskal-Wallis test. P<0.05 was considered to indicate statistical significance.

GSEA identifies functions and signaling pathways
To analyze biologic characteristics shared by the different CDH6 expression levels and predict the functions and pathways in which CDH6 may be involved, we performed the GSEA assay.
GO analysis indicated that ATP metabolic process, cellular respiration, inner mitochondrial membrane protein complex, intrinsic component of the mitochondrial inner membrane, mitochondrial matrix, mitochondrial protein complex, mitochondrial respiratory chain complex assembly, and mitochondrial transmembrane transport, oxidoreductase complex, ribosome biogenesis were mainly enrichment items (Fig. 2a). In addition, KEGG analysis found that the TCA cycle, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, and pentose phosphate pathway were significant enrichment items in the CDH6 high-expression phenotype. On the other hand, ECM receptor interaction was significant in the CDH6 lowexpression phenotype (Fig. 2b).

High CDH6 expression is associated with tumor progression
We analyzed the clinical pathologic data of 343 patients with GC from TGCA, including the patients' age, sex, clinical stage, histological grade, and tumor-lymph node-metastasis (TNM) classification. As shown in Figure 4a, expression of CDH6 was only significantly associated with T stage (p=0.046). And CDH6 was unrelated to age, sex, clinical stage, histological grade, lymph node metastasis, and distant metastasis. The same analysis outcomes were observed in the 44 patients with GC from Tianjin Medical University General Hospital (T1-2 VS T3-4, p=0.031) (Fig. 4b).

Discussion
The primary function of CDH family is considered to determine the cell-cell and cell-matrix adhesion, define the structure and organization of the cellular interactions with the surrounding microenvironment [6]. In cancer, any disfunction in the cell-cell and cell-matrix adhesion are related to tumor progression, lymph node infiltration, and distant metastases [20]. Tumor growth, malignant progression, and distant metastasis were associated with cellular adhesion molecules such as CDHs, integrins, and immunoglobulins [21,22]. With the deepening of the research on cadherins, several studies have indicated that cadherins not only have structural function but also can regulate complex biological signals and participate in the promotion of tumorigenesis, tumor growth, and malignant progression. For example, CDH1 gene is associated with familial diffuse gastric cancer and the process of EMT [10,23]. In glioma, low CDH2 expression had an improved prognosis and benefited from temozolomide therapy [11]. In thyroid cancer cell line, downregulated CDH3 inhibited proliferation, migration, and invasion [24].Cadherin-6 (CDH6) is a transmembrane glycoprotein and a member of the CDH family. Recent studies have shown that CDH6 can be aberrantly over expressed in cancer. In thyroid cancer, CDH6 expression is strongly associated with EMT, metastatic behavior, and worse outcome [14]. In other malignancies, CDH6 was reportedly associated with tumor growth and poor prognosis [16,18]. However, studies regarding the function of CDH6 in GC are inadequate.
Our research showed that CDH6 was highly expressed in GC compared with normal gastric tissues based on multiple public databases, which was verified in cell lines and 20 paired gastric tissues by qRT-PCR. To analyze biologic functions of CDH6 in GC, GSEA was completed. As shown in GSEA (Fig. 2a, 2b). ECM receptor interaction was enriched significantly in CDH6 low expression groups, which was consistent with several previous bioinformatic studies of patients with GC [25][26][27]. This result might be related to the decrease of intercellular adhesion and instability of cellular interactions. In addition, CDH6 was closely related to energy metabolism such as citrate cycle TCA cycle, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, and pentose phosphate pathway. In addition, GO analyses suggested that CDH6 might participate in the formation of mitochondrial membrane structure including the intrinsic component of mitochondrial inner membrane, mitochondrial matrix, mitochondrial protein complex, and mitochondrial respiratory chain complex assembly.
To evaluate the diagnostic value of CDH6, the AUC from TCGA was 0.829, and the AUC from GSE54129 was 0.966, which indicated that the diagnostic efficacy of CDH6 was credible.
Some conventional biomarkers such as CEA, CA199, and CA72-4 that have shown limited diagnostic efficacy for early detection of GC [28]. However, CDH6 played a good diagnostic value for the early stage (stage I, AUC=0.747). In particular, CDHs have been found not only between the tumor cells but also in body fluids (mainly in blood), which indicated that CDH6 has high clinical diagnostic significance [29]. According to the associations of CDH6 and clinical pathologic features and survival outcome, higher expression level of CDH6 was found more frequently in advanced tumors with advanced T stage and poor prognosis. Our univariate and multivariate Cox analyses indicated that the CDH6 expression level was a potential independent marker for poor prognosis in GC. Besides, survival analyses of the 44 patients with GC from Tianjin Medical University General Hospital and Kaplan-Meier plots all supported the same conclusion.
In this work, we mainly focused on the gene expression of CDH6 and clinical pathologic features and survival outcome. Although the Human Protein Atlas (HPA) showed that gene expression of CDH6 was consistent with protein expression results, further protein evidences and functional experiments also need to be completed.
Noticeably, a previous study reported that expression of CDH6 in the oral squamous cell carcinoma was relative to lymph node metastasis and poor prognosis [18]. Gugnoni M, Sancisi V et al reported that CDH6 was associated with the structure and function of mitochondria and promotes EMT and cancer metastasis in papillary thyroid carcinomas [14]. On the contrary, Benjamin Goeppert reported that CDH6 was a putative tumor suppressor and downregulation of CDH6 and association with poor patient outcome in cholangiocarcinoma [30]. These results alert a different role of CDH6 in specific tumors that needs to be further elucidated.