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A novel scoring system for gastric cancer risk assessment based on the expression of three CLIP4 DNA methylation-associated genes

Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-associated mortality worldwide. In the current study, comprehensive bioinformatic analyses were performed to develop a novel scoring system for GC risk assessment based on CAP-Gly domain containing linker protein family member 4 (CLIP4) DNA methylation status. Two GC datasets with methylation sequencing information and mRNA expression profiling were downloaded from the The Cancer Genome Atlas and Gene Expression Omnibus databases. Differentially expressed genes (DEGs) between the CLIP4 hypermethylation and CLIP4 hypomethylation groups were screened using the limma package in R 3.3.1, and survival analysis of these DEGs was performed using the survival package. A risk scoring system was established via regression factor-weighted gene expression based on linear combination to screen the most important genes associated with CLIP4 methylation and prognosis. Genes associated with high/low-risk value were selected using the limma package. Functional enrichment analysis of the top 500 DEGs that positively and negatively associated with risk values was performed using DAVID 6.8 online and the gene set enrichment analysis (GSEA) software. In total, 35 genes were identified to be that significantly associated with prognosis and CLIP4 DNA methylation, and three prognostic signature genes, claudin-11 (CLDN11), apolipoprotein D (APOD), and chordin like 1 (CHRDL1), were used to establish a risk assessment system. The prognostic scoring system exhibited efficiency in classifying patients with different prognoses, where the low-risk groups had significantly longer overall survival times than those in the high-risk groups. CLDN11, APOD and CHRDL1 exhibited reduced expression in the hypermethylation and low-risk groups compare with the hypomethylation and high-risk groups, respectively. Multivariate Cox analysis indicated that risk value could be used as an independent prognostic factor. In functional analysis, six functional gene ontology terms and five GSEA pathways were associated with CLDN11, APOD and CHRDL1. The results established the credibility of the scoring system in this study. Additionally, these three genes, which were significantly associated with CLIP4 DNA methylation and GC risk assessment, were identified as potential prognostic biomarkers.


Introduction
Gastric cancer (GC) is the fifth most common cancer in both sexes and the third most common cause of cancer-associated mortality worldwide (1). Due to the advances in diagnostic and therapeutic approaches, long-term survival for patients with early stage of GC has improved; however, the outlook for individuals with advanced GC is still disappointing because of poor prognosis and limited treatment options (2,3). Poor prognosis is frequently explained by the lack of early diagnostic biomarkers and effective therapeutic treatments (3). As the prognosis of GC is closely associated with the stage of the disease at diagnosis, novel diagnostic modalities for early stages and novel therapeutics are urgently required (4). Several diagnostic biomarkers, such as aberrantly methylated DNA, have aided the diagnoses and disease monitoring efforts in GC.
Changes in DNA methylation have crucial roles during the early stages of GC; therefore, aberrant DNA methylation is highlighted as the main change differentiating GC subtypes from the very first stage (2,5,6). Over 550 studies have demonstrated that aberrant epigenetic changes of >100 genes have crucial roles during the early stages of GC (7,8). For example, a recent report associated promoter methylation of CAP-Gly domain containing linker protein family member 4 (CLIP4) with the increase in severity of gastritis with no metaplasia to gastritis with metaplasia and GC, which may be a potentially useful molecular tool for GC risk stratification in endoscopic biopsies (9).
CLIP4, also known as UBASH3A or TULA, is a member of the T-cell ubiquitin ligand family. These proteins can suppress T-cell signaling, facilitate growth factor withdrawal-induced apoptosis in T-cells and promote the accumulation of various activated target receptors, such as T-cell receptors and epidermal growth factor receptors (EGFRs), which can induce cell invasiveness and metastasis (10)(11)(12). Additionally, CLIP4 is involved in regulating the expression of several tumor-associated genes, such as spleen associated tyrosine kinase, a member of the protein tyrosine kinase family associated with cell motility and increased cell migration (13)(14)(15), and Cbl proto-oncogene, which downregulates EGFR and activates the epithelial-mesenchymal transition-associated EGFR signaling pathway (10,16). CLIP4 expression is also suggested to stimulate tumor metastasis in certain tumor types (17).
Considering the involvement of CLIP4 in tumor metastasis and the association between its promoter methylation and GC, it is worth exploring the CLIP4 DNA methylation-associated genes that may facilitate further understanding of the function of CLIP4 in the pathogenesis of GC and provide potential diagnostic biomarkers for clinical treatment.
In the present study, large quantities of methylation sequencing information and the mRNA expression profiling data from GC samples published in The Cancer Genome Atlas (TCGA) database were used to screen out the significant differentially expressed genes (DEGs) associated with CLIP4 DNA methylation. A prognostic scoring system was established based on the screened genes and simultaneously validated by the datasets from the independent Gene Expression Omnibus (GEO) database. The reliability of the prognostic scoring system was further validated by correlation analysis between clinical characteristics and prognosis. Functional enrichment analysis of DEGs related to prognosis was performed using gene ontology (GO) and gene set enrichment analysis (GSEA).  (18). The 157 GC patients consisted of 100 males and 57 females with 63.242±12.607 years (mean ± SD). There were 23 stage I, 25 stage II, 59 stage III and 20 stage IV cases, and the remaining stages were unavailable. The average survival time for 81 patients was 699.88±728.17 days (mean ± SD). Information on the samples in the validation dataset is summarized in Table I.

Materials and methods
Selection of candidate expression factors. Samples were divided into CLIP4 hypermethylation and CLIP4 hypomethylation groups according to the median CLIP4 methylated value of 0.326. The gene expression differences between the two groups were compared using the limma package in the R software 3.3.1 (19), and genes with |log fold change (FC)| >1 and Benjamini-Hochberg (BH)-adjusted P<0.01 were considered to be significant DEGs. Subsequently, survival analysis associated with these DEGs was performed by Kaplan-Meier analysis and univariate Cox regression analysis using the 'survidiff' function in the survival package of R 3.3.1 (20,21) and P<0.05 was set as the significance threshold. As mortality of patients with an overall survival (OS) of <30 days may due to other factors, these patients and those without survival data were excluded from the survival analysis. The KM diagram was generated using 'ggsurvplot'. The top three genes that were significantly associated with OS (P<0.005) were selected as signature genes.
Establishment of the risk assessment model. Each risk value was calculated as a linear combination of the mRNA expression value (expr) following weighting by regression coefficients (β) (22)(23)(24). The risk score for each patient was calculated according to the following formula: Risk score = β gene1 x expr gene1 + β gene2 x expr gene2 + β gene3 x expr gene3 β represents the gene risk coefficient, expr represents the gene expression level and gene1, gene2 and gene3 represents the three genes. The high-and low-risk groups were classified based on the median of the risk values.
Functional annotation of the prognostic genes. BH-adjusted P<0.01 was used as the threshold to screen out genes significantly associated with high-and low-risk groups using the limma package. According to the association between the genes and their risk values, the genes positively or negatively associated with the risk value were defined as the high-risk group and high expression genes, or low-risk group and low expression genes, respectively. The top 100 (alternatively 50) genes with high and low expression were chosen to generate a heatmap plot using the ggplot2 drawing package. Subsequently, functional enrichment analysis and mapping of the top 500 genes with high and low expression levels were performed using the DAVID 6.8 online software (https://david.ncifcrf. gov) (25,26).
Verification of gene functions was performed via the screening standard, nominal P<0.05, using the GSEA software (software.broadinstitute.org/gsea/index.jsp) (27,28). GSEA analysis is a statistical method for calculating the enrichment of a gene list in a pathway. Briefly, all the genes in a particular gene list are scored and ranked by a statistical method based on their expression levels. The primary result of GSEA is the enrichment score (ES), which reflects the degree to which a pathway is overrepresented at the top or bottom of the ranked list of genes. The ES was calculated by walking down the ranked list of genes, increasing a runningsum statistic when a gene is in the pathway while decreasing it when it is not. The ES is the maximum deviation from zero encountered in walking the list. The score at the peak of the plot is the ES for the gene set. Gene sets with a distinct peak at the beginning or end of the ranked list are generally the most interesting. For this process, the significant P-values calculated by permutation 1,000 times determined whether the genes were enriched or not.
Statistical analysis. In addition to the statistical methods noted above, the statistical method used in this study was t-test. Univariate Cox regression was used to analyzed the clinical features and risk score for association with patient OS. Multivariate Cox regression analysis were conducted to evaluate whether the clinical features and risk score was independent of other clinical variables, with hazard ratios were calculated. P<0.05 was considered to indicate a statistically significant difference using R 3.3.1.

Results
Identification and validation of a three-gene prognostic signature in two datasets. The workflow of the current study is presented in Fig. 1. In the training dataset, the samples were divided into the hypermethylated and hypomethylated groups, each with 168 individuals, based on 0.326 as the median of the CLIP4 methylated values. The gene expression differences between the two groups were compared using the limma package, and 279 DEGs were filtered via the threshold |logFC| >1 and BH-adjusted P<0.01. This revealed the expression level of 35 genes were significantly associated with prognosis, obtained using the univariate Cox regression analysis (data not shown). High expression levels of 32 genes out of 35 were associated with shorter OS; while higher expression levels of the other 3 genes were associated with longer OS. The top three genes with lowest P-value (P<0.005), claudin-11 (CLDN11), apolipoprotein D (APOD), and chordin like 1 (CHRDL1), were selected as the prognostic gene signatures in the CLIP4 DNA hypermethylation patients. Using these three genes, a risk assessment system for cancer patients was established via regression-weighted gene expression based on linear combination. The risk score for each patient was calculated according to the following formula: Risk score = 0.30 x CLDN11 expression + 0.16 x APOD expression + 0.14 x CHRDL1 expression.
Patients in the hypermethylation group were divided into high-and low-risk groups according to median risk score of 0.4289. Fig. 2 indicated that the OS of the patients in the low-risk group was significantly improved compared with those in the high-risk group (P= 0.00744; KM analysis and log-rank test). The OS median values of the low-and high-risk groups were 538.5 days and 422 days, respectively.
The median of methylation in the training set was used as the standard for dividing samples in validation dataset into hypomethylation and hypermethylation groups. In the validation dataset, 48 patients were classified into the hypermethylation group. The risk scores of samples in the hypermethylation group of validation set were calculated according to the risk assessment system, and the samples were also divided into high-risk group and low-risk group according to the median risk score. Using the median risk score of 5.04 as the dividing point, the samples were divided into high-and low-risk groups with 24 individuals in each group. Fig. 3 demonstrated that the OS of the patients in the low-risk group was significantly improved compared with those in the highrisk group (P=0.0083, KM analysis and log-rank test). The OS median values of the low-and high-risk groups were 462 days and 345 days, respectively. This result suggested that the risk assessment was also effective in the validation dataset.
Clinical and molecular features of the low-and high-risk patients with CLIP4 promoter methylation. Fig. 4 indicated that the expression levels of the three DEGs, CLDN11, APOD and CHRDL1, in the hypermethylation group were significantly lower than those in the hypomethylation group. The P-values, determined via Student's t-test, were 1.09x10 -13 (CLDN11), 4.12x10 -8 (APOD) and 0.00128 (CHRDL1). Further, their expression levels were significantly different between the high-and low-risk groups in the CLIP4 DNA hypermethylation patients, as presented in Fig. 5.
The independence of the three important factors was also evaluated. In the training dataset, univariate Cox regression analysis of patient age, sex, race, chemotherapy, targeted molecular therapy, radiotherapy and risk value were analyzed for association with patient OS. Targeted molecular therapy, radiotherapy and risk value were associated with GC patient overall survival time (P<0.05; Table II). Multivariate Cox analysis was also performed on targeted molecular therapy,    radiotherapy and risk value. The results showed radiotherapy and risk value to be independent prognostic factors. Fig. 6 demonstrated the risk values, OS and expression levels of the three genes in the training (left) and validation (right) datasets.
The impact of radiotherapy on risk assessment was also examined. Fig. 8 results indicated significant prognostic differences between the high-and low-risk groups for nonradiotherapy patient, with no difference in survival for those that had received radiotherapy.
Functional enrichment analysis of the DEGs related to prognosis. In the training dataset, DEGs were screened between the high-and low-risk groups using BH-adjusted P<0.01 as the threshold via the limma package. The top 500 DEGs that were positively and negatively associated with risk value were functionally enriched and DEG expression patterns were analyzed using hierarchical clustering. In Fig. 9, the upper and lower heatmaps represented 500 genes that were positively and negatively associated with risk values, respectively. Fig. 9 also presents the top 12 biological process terms involving DEGs that had a significant positive or negative association with the risk values. Functional enrichment analysis showed that CLDN11, APOD and CHRDL1 are involved in six functional terms ('cell adhesion', 'cell-cell adhesion', 'nervous system development', 'signal transduction', 'cell proliferation' and 'negative regulation of cell proliferation').
The pathways significantly enriched in the high-and low-risk groups were stored in the GSEA folder, of which six and four pathways were respectively enriched in the two groups shown in Fig. 10. The increasing curve trends demonstrated that the top-ranked genes were preferentially enriched in the aforementioned pathways. However, the declining curves showed a gradual decrease in the number of genes that were enriched in pathways. CLDN11, APOD and CHRDL1 are involved in a total of five GSEA pathways     The top curve in the graph represents the ES, which reflects the degree to which a pathway is overrepresented at the top or bottom of the ranked list of genes. The ES was calculated by walking down the ranked list of genes, increasing a running-sum statistic when a gene is in the pathway while decreasing it when it is not. The ES is the maximum deviation from zero encountered in walking the list. The score at the peak of the plot is the ES for the gene set. Gene sets with a distinct peak at the beginning or end of the ranked list are generally the most interesting. The x-axis represent all the genes in a particular gene list which were firstly scored and ranked by a statistical method based on their expression levels. ES, enrichment score.

Discussion
Changes in DNA methylation have crucial roles during the early stages of GC; therefore, aberrant DNA methylation is a major change differentiating GC subtypes from the very first stage (2,5,6). Among the 100 genes with aberrant epigenetic changes that have crucial roles during the early stages of GC (7,8), CLIP4 is of particular interest as it is involved in tumor metastasis and its promoter methylation is associated with an increase in GC severity (9). Thus, it is worth exploring CLIP4 DNA methylation-associated genes that may help to further understand the function of CLIP4 in the pathogenesis of GC and provide potential diagnostic biomarkers for clinical treatment.
In the present study, the methylation sequencing information and mRNA expression profiling data of 393 GC samples were downloaded from the TCGA database and used as the training dataset to screen for significant DEGs associated with CLIP4 DNA methylation. The TCGA database is a large-scale cancer genomics project that has generated an overwhelming amount of cancer genomics data from multiple technical platforms that is of great value to cancer research (30). Consequently, several elegant studies have demonstrated the value of analyzing networks based on this database (31). Of the DEGs with differential expression between samples with CLIP4 hyper-and hypo-methylation, 35 genes were identified to be significantly associated with GC prognosis (OS) using the univariate Cox regression analysis, out of which three genes (CLDN11, APOD and CHRDL1) were significantly associated with OS. These genes were selected as prognostic gene signatures to establish a risk assessment system, which indicated that the prognosis of patients in the low-risk group was significantly improved compared with those in the high-risk group. Reliability tests were performed using a validation dataset that included the methylation sequencing information and mRNA expression profiling data of the other 157 GC samples (18) downloaded from the GEO database (32).
All three genes were associated with CLIP4 DNA methylation and prognosis of patients with GC. CLDN11 is a member of the claudin family of proteins, which are transmembrane proteins that have crucial roles in tight junction (TJ) formation and function (33). TJs are intercellular junctions that are crucial for paracellular solute transport and cell polarity maintenance. Tumor cells commonly exhibit structural and functional deficiencies in their TJs (34), and aberrant expression of claudin proteins is also observed in various cancer types (35,36). For example, CLDN11 is silenced in GC via hypermethylation of its promoter region, which contributes to GC by increasing cellular motility and invasiveness (33). DEGs associated with risk were confirmed to be enriched in 'cell adhesion' and 'cell-cell adhesion' GO terms, and enriched in the 'Hallmark_Apical_Junction' pathway in GSEA analysis. ApoD is a small, soluble lipid carrier expressed in most human tissues, particularly in the glia of the nervous system (37,38). It is elevated in various pathological situations, particularly in patients with nervous system diseases, including Alzheimer's disease, Parkinson's disease and schizophrenia (39,40). It has also been indicated in the transport of membrane lipids and may be involved in the clearance and/or repair of damaged membranes, potentially by the quenching of harmful materials released by neurons and glial cells in response to damage or recruitment of lipids to expanding membranes (37). GO analysis indicated that the DEGs were enriched in 'nervous system development'. CHRDL1 is a secreted protein that acts as an antagonist of bone morphogenetic protein (BMP), which activates BMP receptor (BMPR) II (41,42). The activation of intracellular signaling via BMPR induces a series of responses, including proliferation, migration and invasion in various tumor types (43). Furthermore, direct experimental evidence suggests that CHRDL1 has an important role in embryonic cell differentiation and in the adult brain (44,45), and that CHRDL1 expression is significantly downregulated in GC tissues and associated with poor survival (39). In the current study, DEGs were confirmed to be enriched in 'signal transduction', 'cell proliferation', and 'negative regulation of cell proliferation' via GO analysis, and enriched in 'Hallmark_Mtorc1_Signaling', 'Hallmark_Kras_Signaling_Up', 'Hallmark_Hedgehog_ Signaling', and 'Hallmark_Kras_Signaling_Dn' via GSEA analysis. As CLDN11 and CHRDL1 are established factors involved in GC (31,39), it is reasonable to hypothesize that the risk assessment system constructed in the present study is reliable and may prove useful in clarifying the pathogenic process of GC.
However, there are several limitations in the present study. The predictive capability of the present model was established by bioinformatics analysis and it has not been validated in direct experiments. Thus, this model may only provide a direction for further investigation of GC patients with CLIP4 promoter methylation. Additionally, the survival time and pathological staging of different individuals in the two datasets were different, and the therapy data was not available in the validation dataset. Some data bias between training and validation cohort may exist and may impact the accuracy of the analysis. Furthermore, certain information was not available from the original dataset, including the type of chemotherapy or radiotherapy, and whether surgery was performed. However, from Table II, the P-value of chemotherapy in univariate Cox analysis and that of targeted molecular therapy in multivariate Cox analysis are both >0.05. Therefore, these two factors were not considered as independent risk factors for OS. Whereas, the risk score established by our study can be considered as independent risk factor for prognosis as the produced P<0.05 in univariate and multivariate Cox analyses. Therefore, the scoring system established in the current study may be useful in predicting the prognosis of GC although further studies are required prior to clinical use.
In summary, the current study study provided a credible risk assessment model for the predicting GC prognosis based on comprehensive bioinformatic analysis of three CLIP4 DNA methylation-associated genes (CLDN11, APOD and CHRDL1) in two independent datasets. The risk value may be useful as an independent prognostic factor. CLDN11, APOD and CHRDL1 expression was significantly associated with CLIP4 DNA methylation and GC diagnosis and thus, may be potential prognostic biomarkers.