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Characterization of the prognostic values of the NDRG family in gastric cancer

Background: The N-myc downstream-regulated gene (NDRG) family, NDRG1-4, has been involved in a wide spectrum of biological functions in multiple cancers. However, their prognostic values remain sparse in gastric cancer (GC). Therefore, it is crucial to systematically investigate the prognostic values of the NDRG family in GC. Methods: The prognostic values of the NDRG family were evaluated by Kaplan–Meier Plotter and SurvExpress. The mRNA of the NDRG family was investigated in The Cancer Genome Atlas (TCGA). Transcription factors (TFs) and miRNAs associated with the NDRG family were predicted by NetworkAnalysis. The prognostic values of DNA methylation levels were analyzed by MethSurv. The correlation between immune cells and the NDRG family was evaluated by the Tumor Immune Estimation Resource (TIMER) database. Results: High levels of mRNA expression of NDRG2 and NDRG3 were associated with a favorable prognosis in all GCs. In HER2− GC, NDRG1 was significantly associated with a poor prognosis of GC [hazard ratio (HR) = 1.65, 95% confidence interval (CI) = 1.16–2.33, p = 0.0046]. In HER2+ GC, NDRG4 showed a poor prognosis (HR = 1.4, 95% CI: 1.06–1.85, p = 0.017). NDRG4 was an independent prognostic factor in recurrence-free survival by TCGA cohort. The low-risk NDRG-signature group displayed a significantly favorable survival outcome than the high-risk group (HR = 1.76, 95% CI: 1.2–2.59, p = 0.00385). The phosphorylated protein NDRG1 (NDRG1_pT346) displayed a favorable overall survival and was significantly associated with HER2 and phosphorylated HER2. Epidermis development was the top biological process (BP) for coexpressed genes associated with NDRG1 and NDRG4, while mitotic nuclear division and mitotic cell processes were the top BPs for NDRG2 and NDRG3, respectively. Overall, 6 CpGs of NDRG1, 4 CpGs of NDRG2, 3 CpGs of NDRG3 and 24 CpGs of NDRG4 were associated with significant prognosis. CD4+ T-cells showed the highest correlation with NDRG4 (correlation = 0.341, p = 2.14e−11). Furthermore, BCL6 in follicular helper T-cells (Tfh) cells showed the highest association with NDRG4 (correlation = 0.438, p = 00e+00). Conclusions: This study analyzed the multilevel prognostic values and biological roles of the NDRG family in GC.


Introduction
Gastric cancer (GC) is one of the leading deathcausing, malignant diseases in eastern Asia. [1][2][3] Although improved dietary habits, solid diagnostic screening systems, multiprincipled therapeutic regimes and updated surgical techniques have reduced both the incidence and mortality rates of GC, 4-6 the prognosis of GC remains unsatisfactory. 3 Thus, identification of reliable biomarkers for the prognostic prediction of GC could facilitate individualized clinical management.
The N-myc downstream-regulated gene (NDRG) family consists of four members, NDRG1, NDRG2, NDRG3 and NDRG4, located on chromosomes 8q24.3, 14q11.2, 20q11.21-23 and 16q21-q22.1 respectively. [7][8] Although the four members share 57-65% of amino acid sequences with an alpha/beta hydrolase-fold and an NDR region, they lack catalytic motifs and therefore do not have a hydrolase function. 8 NDRG1-4 have been found to be widely expressed in human organs and multiple biological functions have been recently discovered. 9 The molecular functions of the NDRG family cover a wide spectrum of biological processes, including cell development and differentiation, stress responses and proliferation, tumor progression and metastasis. 7,10-19 NDRG1 has been implicated in embryonic placentation, organ development and cellular skeleton modification, 7,10,11 and is induced by hypoxia and DNA damage. 12 Global gene expression analysis of breast epithelial cells indicates that NDRG1 is closely associated with cellular vesicle transport and regulation of membrane proteins, such as low-density lipoprotein and E-cadherin endosomal trafficking. [13][14][15] The prognostic values of NDRG1 in solid tumors have been intensively investigated. In esophageal cancer, low NDRG1 mRNA expression indicates a worse prognosis. 20 It is also negatively correlated with tumor progression and metastasis in colorectal, breast and prostate cancers, 12,21 while associated with an unfavorable prognosis for hepatocellular carcinoma. 22 NDRG2, regulated by maturation-associated stimuli, is strongly expressed in dendritic cells 16 and is able to maintain activated leukocyte cell adhesion during the entire differentiation progress of dendritic cells. 17 NDRG2 expression is found significantly reduced in pancreatic, breast and hepatocellular carcinomas compared with normal counterparts. [23][24][25] Specifically, reduced expression of NDRG2 is correlated with aggressive tumor behavior, higher recurrence and distant metastasis ratio in hepatocellular carcinoma. 24 Of note, NDRG2 expression has been found to be negatively associated with a worse prognosis in GC and prostate cancer. 26,27 NDRG3 promotes angiogenesis and cell growth and is also involved in the lactate-dependent hypoxia signaling pathway. 18 High levels of NDRG3 are associated with shorter overall survival (OS) and relapse-free survival (RFS) in advanced prostate cancer. 27 NDRG4 is exclusively expressed in the central nervous system and heart in the embryonic stage, highlighting its essential role of regulating growth and proliferation. 19 NDRG4 is reduced in both mRNA and protein expression in colorectal cancer tissues and functionally suppressed in tumor invasion and cell proliferation, 28 and is associated with a favorable survival. 29 Collectively, the prognostic values of the NDRG family have been noticed in various types of cancers. However, the whole picture of the prognostic value of the entire NDRG family remains poorly investigated in GC. Hereby, based on updated public resources and integrative bioinformatics analysis, the prognostic value of the NDRG family was comprehensively assessed.

Survival analysis in Kaplan-Meier plotter
The prognostic values of mRNA expression of each NDRG family member to OS were analyzed based on Kaplan-Meier (KM) plotter, a website database based on resources from the Gene Expression Omnibus, including GSE14210, GSE15459, GSE22377, GSE29272, GSE51105 and GSE62254. In fact, GSE62254 was excluded from the total sample survival analysis given its markedly different clinical and genomic data, as suggested by KM plotter. Survival data in each subgroup, including pathological stage, Lauren classification, histological differentiation and human epidermal growth factor receptor 2 (HER2) status, were collected respectively. All four members of the NDRG family were analyzed with various parameters in KM plotter (http:// kmplot.com/analysis/index.php?p=service&cancer =gastric). 30 The best cutoff values were determined by algorithms embedded in KM plotter. 30 The final prognostic KM plots were presented with a hazard ratio (HR), 95% confidence interval (CI) and log-rank p value. A p value <0.05 was considered statistically significant. provides a comprehensive resource for the assessment, visualization and analysis of cancer proteomic data based on TCGA tumor tissue sample sets. Reverse-phase protein array (RPPA) data are used as a high-throughput antibodydependent experimental procedure with increased quality and robust quantification. 35 The phosphorylated NDRG1 (NDRG1_pT364) and epidermal growth factor receptor (EGFR), HER2, as well as corresponding phosphorylated data (EGFR_pY1068, EGFR_pY1173, HER2_ pY1248) were extracted for correlation. 35 Prognostic value of NDRG1_pT346 via TRGAted platform The TRGAted platform (https://nborcherding. shinyapps.io/TRGAted/), is a web tool for survival analysis based on RPPA data retrieved from TCGA. 36 Given only NDRG1_pT346 was available in the RPPA of STAD, we only accessed the prognostic value of NDRG1_pT346, including OS and disease-free survival (DFS), between high and low expression groups. The optimal cutoff was determined based on the surv-cutpoint function in the survminer package via TRGAted. 36 HR was determined by the Cox proportional hazard regression model. 36 Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the coexpressed genes of NDRG1-4 The genomic alterations of NDRG1-4 were analyzed by cBioPortal, an integrative analytic platform of TCGA. 37,38 The coexpressed genes of NDRG1-4 with a Pearson correlation (⩾0.3 or ⩽−0.3) were subject to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis in the Database for Annotation, Visualization and Integrated Discovery (DAVID). [39][40][41] The cutoff value was a false discover rate (FDR) <0.05 for significant GO and KEGG data.

Prediction of transcription factors and miRNAs for NDRG1-4
Potential transcription factors (TFs) and miR-NAs of NDRG1-4 were predicted by Network-Analysis (http://www.networkanalyst.ca). 42,43 The prediction of TFs for NDRG1-4 was based on the ENCODE database with ChIP-seq data. Only the data with a peak intensity signal value <500 4 journals.sagepub.com/home/tag and a potential score value <1 was screened for further analysis. The miRNA-gene interaction data were retrieved from TarBase and miRTar-Base via the NetworkAnalysis platform.

Tumor-immune infiltrating cells associated with the NDRG family via the Tumor Immune Estimation Resource database
Correlations between all tumor-immune infiltrating cells (TIICs) and the NDRG family were analyzed via the Tumor Immune Estimation Resource (TIMER) platform (https://cistrome. shinyapps.io/timer/), a web tool for gene-specific correlational analysis with TIICs. TIICs included B-cells, CD4 + T-cells, CD8 + T-cells, dendritic cells, macrophages and neutrophils. 45 Tumor purity was used for the correction of Spearmanbased correlation analysis. 45 Moreover, TIICs with the highest correlation to the NDRG family were selected for further subtype-based biomarker analysis. 46

Prognostic values of NDRG members in the whole group of patients with GC
The prognostic values of NDRG mRNA expression in the whole group of patients with GC from KM plotter were collected [ Figure 1(a-e)]. NDRG2 and NDRG3 were significantly associated with a better OS prognosis [ Figure 1

NDRG4 was validated as an independent prognostic factor
The prognostic values of the NDRG family had been studied in KM plotter. Furthermore, they were further validated in the TCGA database (STAD). The Cox regression was analyzed for both univariate and multivariate process, including sex, age, TNM stage and mRNA expression of the NDRG family (Table 1 and 2). The results indicated that only NDRG4 was determined as an independent prognostic factor for GC in recurrence-free survival results (HR = 1.247, 95% CI: 1.057-1.470, p = 0.009; Table 2).  The mRNA and protein correlation between HER2/EGFR and NDRG1 Given the fact from Figure 2 that NDRG1, and NDRG4 may feature inverse prognostic values in HER2 +/− groups, we further explored the mRNA expression correlation between NDRG1, NDGR4 and HER2. Moreover, given the close functional relationship between HER2 and EGFR, EGFR was also included for correlational analysis. Interestingly, no significant result was identified [ Figure 6(a-d)]. Next, the protein expression correlation was investigated in the RPPA data of    . Moreover, given the relative weak mRNA correlation among each NDRG member, this study further explored potential TFs and miRNA that predicted to be connected with NDRG members. However, no miRNA or TF was predicted to synchronously correlate with all NDRG members or at least three of them [ Figure  7(j, k)].

Prognostic values of NDRG1-4 DNA methylation in MethSurv
The DNA methylation levels of NDRG1-4 with the prognostic values of each single CpG in TCGA were analyzed by MethSurv [ Figure 8(a-d), Table 1]. In fact, cg15393676 of NDRG1, cg16409562 of NDRG2, cg26287101 of NDRG3  (Table 3).

Correlation between TIICs and NDRG members
Given the increasing association between immunological feature and prognosis in cancer, we further explored the correlation between TIICs and NDRG members. In fact, only CD4 + T-cells showed the highest correlation with NDRG4 [correlation = 0.341, p = 2.14e −11 ; Figure 9(a)]. Given a variety of immune cells were defined by CD4 + T-cells, we further examined the gene markers in each subtype [ Figure 9(b-g)]. Of note NDRG4 was highly associated with BCL6 in Tfh cells [correlation = 0.438, p = 00e + 00; Figure 9(c)].

Discussion
The increasing availability of published mRNA data, clinical outcomes and standardized analysis NDRG1 had been implicated in the regulation of embryonic placentation and organ development, 7 the cellular vesicle transport system, 13 endocytosis and recycling of membrane proteins. 14,15 The reduced expression of NDRG1 had been associated with a worse prognosis in esophageal, 20 colorectal 21 and breast cancers 12 while leading to favorable clinical outcomes in hepatocellular carcinoma. 22 This paradoxical fact may be tumor typespecific, further highlighting the complicated biological function and processes that NDRG1 is involved with. In fact, NDRG1 was associated with a decrease in the proliferation and induction of apoptosis of cancer cells by the regulation of Bcl-2 and Ca 2+ -associated protein 43, 48,49 and the dysregulation of epithelial-mesenchymal transition (EMT). [50][51][52] Nonetheless, NDRG1 might exert inconsistent effects in GC prognosis. 49,50,53,54 In this study, although NDRG1 was not significantly associated with overall prognosis in all cases, 6 CpGs of NDRG1 were associated with significant prognosis. Of note, inverse prognostic values of NDRG1 in HER2 +/− groups indicated a potential correlation between NDRG1 and HER2. However, no significant mRNA expression correlation was identified between NDRG1 and HER2/ EGFR. Furthermore, the protein expression of NDRG1 phosphorylation level, NDRG1_pT346, was found to be significantly associated with EGFR, EGFR_pY1068, EGFR_pY1173, HER2 and HER2_pY1248. Previous study indicated that, in colon cancer and pancreatic cancer, NDRG1 significantly reduced the expression of HER2 (general expression, heterodimerization and phosphorylation) and the activation of downstream MAPKK in response to the epidermal growth factor ligand. 55 For the first time, our study highlighted a potential role of NDRG1 associated with HER2 status in GC. Given the HER2 targeting drug, trastuzumab, has been widely used in GC, 56 digging into the NDRG1-related mechanisms may shed light upon further biological and pharmacological values.
Collectively, although NDRG1 was not validated as an independent general prognostic factor in  The NDRG2 expression had been found significantly reduced in pancreatic, breast cancers and hepatocellular carcinoma compared with normal tissues, accompanied by more aggressive features and a high ratio of relapse. [23][24][25] In this study, NDRG2 was associated with favorable prognosis in all and significantly reduced in tumors compared with normal tissues, consistent with previous studies. 26 However, no significance was found in the stage-specific pattern. Of note, increased chemo-resistance and decreased Fas-mediated cell death had been validated due to the inhibition of NDRG2. 26 Interestingly, the promoter methylation of NDRG2 was frequently hypermethylated, leading to decreasing expression of NDRG2 at both mRNA and protein level and further associated with worse prognosis of GC. 57 Similar prognostic role of NDRG2 had been validated in prostate cancer. The downregulation of NDRG2 was associated with advanced pathological stages and identified as an independent prognostic factor for short recurrence-free survival and OS. 27 Of note, overexpression of NDRG2 could decrease the radiosensitization of Hela cells by the regulation of Bax signaling. 58 NDRG3 was found to be significantly upregulated in prostate cancer, and was associated with advanced pathological stage and a worse prognosis of prostate cancer, contrary to NDRG2. 27 Currently the role of NDRG3 had not been fully investigated in GC. Our study had revealed that NDRG3 was significantly associated with a favorable prognosis for the OS of all patients, as well as the HER2 + and diffuse type subgroups. Furthermore, NDRG3 was significantly increased in tumor compared with normal, with no significant distribution in various stages. In fact, although NDRG3 showed a favorable outcome based on the outcome from the KM plotter, a significant upregulation of NDRG3 was found in tumors compared with normal tissues in TCGA. Moreover, upregulation of NDRG3 was also associated with the high-risk group in the NDRG signature. There are a few issues that need to be clarified. First, clinical heterogeneity may account for the controversial outcome between TCGA and KM plotter (GSE14210, GSE15459, GSE22377, GSE29272, GSE51105 and GSE62254). Second, multivariate Cox analysis of the NDRG family using TCGA also eluded the potential independent prognostic value of NDRG3, both in OS and RFS. However, given the current studies remained sparse, the biological and prognostic values of NDRG3 warrant further intensive investigation. It may be insightful to systematically explore the prognostic value of NDRG3 using meta-analysis.
Previously, NDRG4 was found reduced in both mRNA and protein expression in colorectal cancer tissues compared with normal counterparts, and significantly suppressed tumor invasion and proliferation. 28    an independent prognostic factor in TCGA dataset, further indicating a possible association between NDRG4 and recurrence in GC. For the rest of the NDRG family, it remained far from conclusive due to possible race diversity and other confounding factors such as radiochemotherapy.
Interestingly, although NDRG1 and NDRG4 did not show significantly differential expression between tumor and normal tissues in STAD using the GEPIA platform, the high/low-risk groups exhibited distinct expression patterns of NDRG1 and NDRG4 using the same dataset [ Figure 4(a, b)]. In fact, the NDRG member signature may provide insightful clues on the prognostic values of combinational analysis, rather than a single gene.
The current network regulation of the NDRG family associated with GC was summarized ( Figure 10). 26 Although NDRG2 was not an independent prognostic indicator in this manuscript, it was determined as an independent risk factor by Choi and colleagues. 26 Moreover, silencing of NDRG2 increased the proliferation and resistance of cisplatin in GC cell lines. 26 Up to now, studies focusing on the association between NDRG3 and 4 and GC remain limited. Interestingly, in this manuscript, NDRG4 was highly associated with BCL6 in Tfh cells. Up to now, this is the first study reporting the correlation between BCL6 and NDRG4 in GC, indicating a potential role of NDRG4 in follicular helper CD4 + T-cells. Moreover, potential inverse prognostic values of NDRG1 between HER2 +/− GC and the significant association between protein expression of NDRG1 (NDRG1_pT346) and HER2/HER2_pY1248 indicated possible connection as well. However, in silico findings warrant further experimental validation.
Up to now, methylation-related study of the NDRG family remains limited. High levels of NDRG1 promoter methylation in the CpG islands were found in both GC cell lines and tissues. 64 Interestingly, no mutation of NDRG1 was detected in this study. 64 Consistently, our finding also indicated rare cases of NDRG1 mutation. For NDRG2, hypermethylation status was detected in the NDRG2 promoter both in GC cell lines and tissues. 62 In fact, the reduced expression of NDRG2 in GC compared with normal tissues was highly correlated with the promoter hypermethylation. 62 For NDRG4, both promoter and gene body methylation levels were increased in GC tissues. 63 Interestingly, opposite clinical results of NDRG4 were found between the Chinese samples from Chen and colleagues and TCGA data, highlighting the race difference beneath the prognostic values of NDRG4. 63 The limitation of this study was the lack of experimental validation and externally clinical cohort validation. The limited number of some subgroups of KM plotter for prognostic analysis and potential sample heterogeneity could bias the results. Further validation on a larger sample size is also required.

Conclusions
This in silico study investigated the biological and prognostic values of the NDRG family in GC based on KM plotter and TCGA, providing insights for further investigation of NDRG family as potential targets in GC.