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EPDR1 correlates with immune cell infiltration in hepatocellular carcinoma and can be used as a prognostic biomarker

Abstract Hepatocellular carcinoma (HCC) has high mortality rate and is a serious disease burden globally. EPDR1 (ependymin related 1) is a member of piscine brain glycoproteins and is involved in cell adhesion. The gene expression, prognostic, and clinicopathological related data for EPDR1 were obtained from multiple transcriptome databases. Protein level of EPDR1 in HCC was verified using human protein atlas and CPTAC databases. EPDR1 co‐expressed genes were identified using LinkedOmics. Functional analysis of the co‐expressed genes was performed using gene set enrichment analysis, Gene Ontology, and KEGG. Statistical analysis was conducted in R. The relationship between EPDR1 expression and immune cell infiltration was analyzed using TIMER and CIBERSORT. The expression of EPDR1 was found to be significantly higher in HCC than in normal tissues. Further, EPDR1 level was correlated with advanced stage of HCC. EPDR1 was associated with multiple signaling, as well as cancer and apoptotic pathways. Further, EPDR1 expression was significantly correlated with purity and infiltration levels of various immune cells as well as immune signatures. This is the first study to report the role of EPDR1 in HCC. EPDR1 can be used as a novel prognostic biomarker as well as an effective target for diagnosis and treatment in HCC.

of HCC. Further, the prognosis of HCC patients is very poor, and the 5-year survival rate is as low as 30%. 9,10 Therefore, identifying specific and sensitive biomarkers related to early diagnosis, treatment, and prognosis of HCC is of great significance.
The ependymin-related 1 (EPDR1) gene encodes for a protein that is similar to ependymins, which belong to a family of piscine brain glycoproteins. 11 Ependymins, encoded by Epd genes, are transmembrane proteins that play a crucial role in adhesion of neural cells. 12 For the first time, mammalian ependymin-related transcript was discovered in 2001 in two colorectal cancer (CRC) cell lines, 13 and was designated as UCC1 (upregulated in colon cancer 1). Following this discovery, other studies identified and characterized the expression of MERP1 (mammalian ependymin-related protein 1) in hematopoietic cells, non-hematopoietic tissues, and in several malignant cell lines and tissues, 14,15 which was later confirmed to be UCC1, and currently known as EPDR1 gene. EPDR1 is highly expressed in the brain and also detected in other tissues, such as muscle, heart and in extracellular fluids. [14][15][16][17][18] The function of EPDR1 is not well known, although several studies have reported its differential expression and single-nucleotide polymorphisms associated with its locus in different pathological and developmental processes, which are known to affect the cell adhesion. [19][20][21][22][23][24] Furthermore, genetic variants in EPDR1 have been reported to be linked to several diseases, including Dupuytren's disease 21,25,26 and primary angle closure glaucoma 23,27 ; however these observations do not provide obvious insight into the molecular functions of the protein. In 2016, Valiente et al reported that EPDR1 and its spliced isoforms are differentially expressed in human CRC cell lines, and the up-regulation of EPDR1 in human colorectal cancer was reported to promote cell growth, proliferation, and invasiveness. 28 Genome-wide DNA methylation profiling studies revealed that methylation-mediated epigenetic silencing of EPDR1 may play an important role in preventing CRC progression. 29 In this context, exploring the role of EPDR1 in the onset and/or development of tumors is particularly appealing.
Our study reported that EPDR1 correlates with immune cell infiltration and can be used as a prognostic biomarker in hepatocellular carcinoma. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of EPDR1-coexpressed genes were performed to explore the associated processes and pathways. In addition, relationship between EPDR1 expression and tumor-infiltrating immune cells, as well as immune-related genes was explored through multi-dimensional analysis. The current study will provide novel insights into the underlying mechanisms associated with HCC development, thus help in discovering novel potential targets and strategies for HCC diagnosis and treatment.

| Survival analysis using R and Kaplan-Meier plotter
The prognostic significance of EPDR1 in HCC was explored using the Kaplan-Meier plotter (http://kmplot.com/analy sis/) based on TCGA data and then validated using ICGC database using survival package in R software (version 3.5.2). The hazard ratio 30 with 95% confidence intervals (CI) and log-rank P-value was also calculated.

| Expression level of EPDR1 in human normal tissues
The gene expression and phenotype data corresponding to human normal tissues in GTEx were obtained from UCSC Xena project (http://xena.ucsc.edu/). The downloaded dataset included TOIL RSEM FPKM (n = 7862) and phenotype (n = 9783) data corresponding to different human normal tissues. The Ensembl gene identifiers in the RNA-seq data were converted to official gene symbols using an in-house perl script. Further, the expression data of EPDR1 in different organs of males and females were extracted based on GTEx RNA-seq and phenotype data using perl scripts and visualized using gganatogram and ggpubr R packages.

| Correlation between EPDR1 expression and clinical characteristics of HCC patients
The correlation between EPDR1 expression and different clinicopathological characteristics of HCC patients, such as age, race, gender, survival, histopathological grade, AJCC stage, malignancy, cancer history, and stage was analyzed using R software and then validated using UALCAN (http://ualcan.path.uab.edu). T-test was used to estimate the significant differences in the gene expression levels between the groups.

| Co-expression analysis using LinkedOmics database
The genes co-expressed with EPDR1 in HCC were identified using the LinkedOmics database (http://www.linke domics.org/login.php), a thirdparty online tool containing TCGA data. Co-expressed genes were statistically analyzed and their expression patterns were shown using volcano plots and heatmaps. Heatmaps were generated using the heatmap R package. Pearson correlation coefficient was used to evaluate the significant correlation of the co-expressed genes with EPDR1 expression. FDR < 0.01 was considered for representing significant expression, whereas, P < .05 was considered for significantly related genes.

| Relationship between EPDR1 expression and immune cell infiltration
The correlation between EPDR1 level and infiltration of immune cells in HCC was explored using Tumor Immune Estimation Resource

| EPDR1 protein levels in HCC
The expression of EPDR1 protein in HCC was explored based on the immunohistochemistry (IHC) data from Human Protein Atlas (HPA) database (https://www.prote inatl as.org/), as well as the mass spectrometric data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) database (https://cptac-data-portal.georg etown.edu/cptac Publi c/).

| Statistical analysis
Statistical analyses for TCGA data were performed using different packages in R (version 3.6.1). The correlations between clinical characteristics and EPDR1 expression were analyzed using logistic regression. Univariate and multivariate Cox regression analyses were performed to reveal the relationship between EPDR1 and the clinical parameters and the immune cell infiltration with overall survival of HCC using survival package in R software. Time-dependent receiver operating characteristic (ROC) curves, with AUC values were quantified with the survival ROC package. The values with P < .05 were considered statistically significant.

| EPDR1 expression is significantly elevated in HCC tissues
First, we compared the expression of EPDR1 in HCC and normal liver tissues using multiple datasets obtained from TCGA, ICGC, GEPIA and GEO databases. Analysis of several HCC cohorts in TCGA and ICGC databases revealed that EPDR1 mRNA expression was significantly higher in HCC tissues than in the adjacent normal tissues ( Figure 1A and B. Further, the expression data in the GEPIA database indicated up-regulation of EPDR1 in the HCC tissues compared to the normal tissues from TCGA ( Figure 1C) or GTEx database ( Figure 1D). We also compared the expression of EPDR1 in tumor and paracancerous tissues from the same patient ( Figure S1).
The expression of ERDR1 was significantly higher in tumor versus paired normal tissues (P < .0001). Additionally, three independent microarray studies obtained from the GEO database validated the  Figure 1E).
Finally, we verified the protein levels of EPDR1 in HCC tissues using HPA and CPTAC databases. The IHC data in HPA database revealed that the expression of EPDR1 was significantly higher in HCC tissue than the normal tissues ( Figure 1F). Further, EPDR1 level was found to be significantly increased in the HCC group compared to that in normal group (P = 9.938-e04) based on CPTAC data, as shown in Figure 1G.

| Prognostic significance of EPDR1 expression in HCC
Next, we sought to investigate the prognostic significance of EPDR1  Figure 2C, univariate analysis using Cox regression revealed that EPDR1 expression and T stage are significantly associated with overall survival (P < .05). Multivariate analysis and ROC curve revealed that up-regulated expression of EPDR1 is an independent prognostic factor of poor prognosis based on TCGA data (P < .005). The prognostic significance of EPDR1 verified using the data obtained from ICGC database is shown in Figure 2D

| Correlation between EPDR1 expression and clinical characteristics of HCC patients
The correlation between EPDR1 expression and various clinicopathological parameters in HCC patients based on TCGA and ICGC data are shown in Figure 3A,B, respectively. We observed significantly higher levels of EPDR1 in male patients (P = .015), and shown to be significantly increased gradually from stage I to stage IV (P = .001), and was found to be significantly higher in patients with follow-up death (P = .003). We further compared the expression of EPDR1 considering age, gender, race, weight, grade, stage, histology subtypes, metastasis status and stage of HCC patients ( Figure   S2). Of note, EPDR1 expression was higher in stage I-IV patients compared to healthy individuals. We also assessed and compared the overall survival of the EPDR1-high-expression and EPDR1-lowexpression groups, per stage, grade, and T of HCC patients ( Figure   S3). In stage III, grade III and T3 groups, the overall survival was significantly worse in EPDR1-high-expression compared to EPDR1low-expression patients (P < .05). Thus, these results indicate that EPDR1 is overexpressed and positively associated with advanced tumor stage in HCC.

| Expression of EPDR1 in human normal tissues
EPDR1 expression in normal human tissues was determined using the data obtained from the GTEx database. Red represents high expression, black represents median expression, and green represents low expression ( Figure 4A). EPDR1 was found to be highly expressed in adrenal gland, blood vessel, lung, and nerve tissues, among others.
However, the blood, and stomach, pancreas and skin tissues showed EPDR1 expression levels similar to those of liver tissues, as represented in Figure 4A and B. Further, no significant gender-based difference was observed in the expression of EPDR1 in most tissues or organs, except in case of blood and skeletal muscle (P < .05 and P < .01, respectively), Figure 4B.

| EPDR1 co-expressed genes in HCC
To gain insight into the biological importance of EPDR1 in HCC, the function module of LinkedOmics was used to examine the EPDR1 co-expression in liver HCC (LIHC) cohort. Figure 5A Figure 5D. Further, 24 of the top 50 positively correlated genes were shown to be high-risk genes; whereas 8 of the top 50 negatively correlated genes were low-risk genes ( Figure 5D). Gene Ontology (GO) enrichment analysis showed that EPDR1 co-expressed genes were significantly associated with the activation of integrin-mediated signaling pathway, antigen processing, and presentation and leukocyte apoptotic process, while the processes, such as fatty acid metabolism, protein activation cascade were inhibited ( Figure 5E). KEGG pathway analysis showed enrichment of pathways such as, hippo signaling, pathways related to various infections, small cell lung cancer, nucleotide excision repair, and DNA replication ( Figure 5F whereas, pathways associated with glycine, serine and threonine metabolism were represented by EPDR1 low expression phenotype. One study on colorectal carcinoma demonstrated that DNA methylation might play a critical role in EPDR1 expression-regulation. 29 However, no regulators of EPDR1 were identified so far. Since IDH1/H2 and TP53 mutations are found in a large percentage of HCC patients, 31 we further analyzed the correlation between IDH1, IDH2, and p53 mutations and the expression of EPDR1 in HCC using TCGA database ( Figure S4). No significant correlation was observed between EPDR1 expression and IDH1, IDH2, or p53 mutations.

| EPDR1 expression and tumor-infiltrating immune cells (TIICs)
The survival of patients in several cancers is determined by the number and activity of tumor-infiltrating lymphocytes. 32 Therefore, we explored the relationship between EPDR1 expression and immune cell infiltration in HCC using the TIMER database. Figure 6A shows that EPDR1 expression significantly correlates with purity, and infil-

| D ISCUSS I ON
HCC is the most common type of liver cancer. Most HCC patients are diagnosed at an advanced stage, which lacks effective treatment. 33,34 Therefore, early screening and diagnosis of HCC patients is beneficial for the treatment and prognosis of patients. However, currently there are no effective biomarkers for the early diagnosis of HCC, and the molecular mechanisms underlying HCC metastasis remains unclear. 35,36 EPDR1, a member of piscine brain glycoproteins, plays a role in cell adhesion. To gain detailed insights into the potential functions of EPDR1 and its regulatory network in HCC, we performed bioinformatics analysis using publicly available data, which we hope will benefit future research related to HCC.
In the current study, we explored the expression of EPDR1 in HCC patient data obtained from various database to assess its prognostic value. High expression of EPDR1 was significantly correlated with worse RFS, PFS, OS, and DSS of HCC patients. Further, the relationship between EPDR1 expression and various clinicopathological characteristics of HCC patients highlights the role of elevated EPDR1 as an independent prognostic factor for poor OS. The HCC patients with high EPDR1 expression are more likely to present advanced grade, stage, and poor prognosis than those with low EPDR1 expression. Therefore, our findings suggest that EPDR1 overexpression occurs in most HCC cases and deserves further clinical validation as a potential diagnostic and prognostic marker.
To probe the signaling pathways associated with EPDR1 expression, we constructed the EPDR1 co-expression network and performed functional analysis of the co-expressed genes. GO and KEGG pathway analysis revealed that the co-expressed genes were mainly related to processes and pathways associated with different signaling, DNA replication and metabolism. To further analyze the biological role of EPDR1 in HCC, GSEA was performed, which revealed that EPDR1 overexpression was implicated in multiple signaling pathways, such as WNT, MAPK, NOD like receptor, and cancer, and apoptosis. These signaling pathways and processes have been reported to be associated with HCC carcinogenesis. 37 However, in vivo studies are needed to further validate the association of these processes and pathways in regulating EPDR1 functions in HCC.
The liver is the largest immune organ of the body and harbors various immune cells. HCC is a typical inflammatory related tumor, and immune tolerance and escape plays a key role in the carcinogenesis process. 38  In conclusion, our study identifies EPDR1 as a novel biomarker with prognostic significance in HCC patients. Our results indicate an association between EPDR1 expression and immune cell infiltration and unveil the potential molecular mechanism underlying the carcinogenesis in HCC. Thus, we conclude that EPDR1 can be used as an effective tool or target for the diagnosis or treatment of HCC, respectively, in the future.

ACK N OWLED G EM ENTS
We would like to thank Editage (www.edita ge.cn) for English lan-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in multiple databases and repositories. These data were derived from the