Downregulated expression of plakophilin‐2 gene in patients with colon adenocarcinoma predicts an unfavorable prognosis and immune infiltrate

Plakophilin 2 gene (PKP2) has been revealed to be differentially expressed in various cancer types and is correlated with prognosis. However, the role of PKP2 in colon adenocarcinoma remains indistinct.

Based on colorectal cancer (CRC) statistics, CRC is ranked the third most common cause of cancer-correlated mortality in men and the second in women. 1 Although the incidence and mortality of CRC have decreased over the past decades owing to achievements in medical management, the 5-year survival rate is still dismal ($55%), 2,3 which brings a huge economic burden to both the patients and the society.
Colon adenocarcinoma (COAD) is the most common subtype of CRC. 4 Treatment for CRC constitutes surgery operation and/or chemotherapy, but surgery is curative only for a small number of patients with CRC, especially those with cancer in the early stages. 5Albeit many therapeutic efforts have been made for COAD, including targeted therapy, neoadjuvant chemotherapy and immunotherapy, the survival rate has not completely benefited, which might be at least in part attributable to individual heterogeneity.Therefore, it is necessary to seek identifying biomarkers which better predict the prognosis of COAD cases, which would be of high clinical relevance.
Plakophilin-2 (PKP2), encoded by PKP2 gene, belongs to the plaquebound plakophilin family of proteins. 6PKP2 gene has been reported to be expressed in epithelial cells. 7PKP2 has been validated to play a vital role in cardiac junction formation and heart morphogenesis. 8P2 gene mutation is associated with inherited arrhythmogenic right ventricular cardiomyopathy. 9regulated expression of PKP2 has been confirmed to be correlated with reinforced invasion and metastasis of bladder cancer cells. 10PKP2 has been reported to promote tumor progression of cancer cells by inducing and promoting phosphorylation and the activation of epidermal growth factor receptor (EGFR). 11,12In addition, the PKP2 expression drives oncogenicity of glioma cells as knockdown of PKP2 can inhibit the proliferation and migration of murine glioma. 13wever, expression of PKP2 has not been correlated with clinical data and outcome in cancers of the colon and/or rectum.Considering that abnormal levels of PKP2 can influence cell proliferation and/or migration, the expression level of PKP2 might be correlated with clinical parameters as well as the outcome of CRC patients.To test this hypothesis, the prognostic effect of PKP2 was assessed on the basis of data downloaded from The Cancer Genome Atlas (TCGA)-COAD cohort.In this analysis, PKP2 expression was found to be lower in COAD than in normal colonic epithelium.This lower expression was significantly correlated with unfavorable clinical outcomes and risk factors.Further, PKP2 expression in COAD was correlated with levels of immune infiltrating cells.

| Data collection from TCGA
Colon adenocarcinoma patient datasets, with PKP2 expression information and corresponding clinical data, were downloaded from the publicly available official website, TCGA. 14The 21 enrolled cancer types contained at least five samples in the tumor group.Finally, the RNA-sequencing expression data of PKP2 with TPM format and log2 conversion were performed for analysis.

| RNA-sequencing data of PKP2 in COAD
The RNA-sequencing expression data of PKP2 in colon adenocarcinoma were also downloaded from TCGA, including 480 colon adenocarcinoma and 81 adjacent normal tissue data.The selected samples contained expression data of PKP2 gene and paired clinical data, including age, gender, T stage, N stage, M stage and pathological stage.The data for PKP2 expression were characterized as mean ± standard deviation (SD).

| Protein-protein interaction network construction and functional enrichment analysis
In this analysis, a protein-protein interactionI network of PKP2 was conducted with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database to explore PKP2-related proteins.
Subsequently, the prediction pathway of PKP2 and its related proteins were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO).

| Tumor-immune system interaction database
The Tumor-immune System Interaction Database (TISIDB) is a public online website for systemically analyzing tumor-immune interaction. 15 our analysis, the TISIDB was used to determine PKP2 expression and tumor-infiltrating cells in various human cancers.On the basis of the gene expression profile, we inferred the relative abundance of tumor-infiltrating cells with gene set variation analysis.The correlations between tumor-infiltrating cells and PKP2 expression were determined by Spearman's test.

| Tumor Immune Estimation Resource database
The Tumor Immune Estimation Resource (TIMER) is an online database for systematically analyzing tumor immune infiltrating in a variety of cancer types.In our analysis, TIMER was conducted to determine the correlation between PKP2 expression in COAD and six immune infiltrate cells compromising B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils and dendritic cells.
The correlations between PKP2 expression and corresponding clinical information were statistically analyzed by logistic regression.
Wilcoxon rank sum tests and t-tests were performed to confirm the variance between colon adenocarcinoma and adjacent normal colon tissues.The COX regression analysis was performed to evaluate survival-related clinical information collected from TCGA.The correlation of gene expression was evaluated using Spearman's R and statistical significance.In our analytic results, p-values <0.05 were considered as statistical significance.

| PKP2 expression pattern in pan-cancer analysis
Expression of the PKP2 transcript was compared between the tumor and a matching normal in 21 cancer entities.PKP2 had significantly lower expression in six and higher expression in another six cancer entities (Figure 1).This indicated abnormal mRNA expression of PKP2 across the majority of cancers as well as a context-dependent biological role for PKP2 in cancer cell development.

| Association between PKP2 expression and clinicopathologic variables
The underlying mechanism of PKP2 expression in cancer requires further study, hence we analyzed and correlated it with certain clinical aspects in cases of COAD.COAD cases with eligible clinical information were analyzed using R-3.5.3.As shown in Table 1, univariate analysis using logistic regression with PKP2 expression as a categorical dependent variable revealed that increased expression of PKP2 correlated significantly with the TNM stage (N1 vs. N0, p = 0.042; N2 vs. N0, p < 0.001; M0 vs. p < 0.001), pathological stage (III vs. I, p = 0.007; IV vs. I; p < 0.001).As shown in Figure 3A-F, downregulated expression levels of PKP2 were observed in patients with lymph node metastases (p < 0.001), patients with advanced M stage (p = 0.01) and patients with more advanced pathological stage (p < 0.001).However, no statistically significant correlation was found between the expression levels of PKP2 and other clinical pathological characteristics, such as age, gender and T stage.Taken together, these results suggested that PKP2 is correlated with advanced pathological stage, with lymph node metastases further suggesting PKP2 may act as a biomarker of poor prognosis for colon adenocarcinoma.

| Differential RNA-Seq levels of PKP2 as a prospective biomarker to distinguish COAD tissues from normal tissues
To investigate the value for PKP2 to distinguish COAD samples from normal samples, we performed a receiver operating curve (ROC) curve analysis. 17As showed in Figure 4, the ROC analysis showed that PKP2 had an area under the curve (AUC) value of 0.787 (95% confidence interval, 0.721-0.854).At a cutoff of 6.034, PKP2 had a sensitivity and specificity of 90.2 and 66.5%, respectively.The positive predictive value was 18.7% and the negative predictive value was 98.8%.These findings indicate that PKP2 could be a promising biomarker to differentiate COAD tissues from normal tissues.

| Downregulated mRNA expression of PKP2 is associated with longer median survival time
To explore the relationship between PKP2 mRNA expression and OS in COAD patients with supporting data, 18 Kaplan-Meier curves were performed.As shown in Figure 5, the median survival time of COAD patients with low levels of PKP2 was significantly longer than those with high levels of PKP2 (101.4 vs. 68.2months, p = 0.018).The data suggest that high PKP2 mRNA expression is associated with poor prognosis in COAD.

T A B L E 1
The results of COX regression analysis.F I G U R E 3 Relationships between PKP2 mRNA levels and clinical pathological characteristics.PKP2 mRNA expression was significantly correlated with lymph node metastases (B), advanced M stage (C) and more advanced pathological stage (D).However, no statistically significant correlation was found between the expression levels of PKP2 and T stage (A), age (E), and gender (F) (ns, no significance, **p < 0.01, ***p < 0.001).

| Protein-protein interaction networks and enrichment analysis
In our study, the interactive network of PKP2 and its corrected genes was constructed with STRING.PKP2-correlated genes included CTNNB1, DSC2, DSC3, DSG1, DSG2, DSP, GJA1, JUP, SCN5A and CTNNA3.The cumulative scores were >0.9.The information is exhibited in Figure 6 and Table 2. Combined with enrichment analysis with GO/KEGG, it revealed that CTNNB1 and CTNNA3 are enriched in the Hippo signaling pathway (Figure 7).

| Correlation analysis between PKP2 expression and immune cell infiltration in COAD
We analyzed the correlation between PKP2 expression and the six and dendritic cells (r = 0.333, p = 6.90 Â 10 À12 ).We also evaluated the correlation between LIMK1 expression and 28 types of tumor infiltrating lymphocytes (TILs) in the TISIDB database.Figure 8B shows the relations between the expression of PKP2 and 28 types of

| DISCUSSION
In our analysis, the expression of PKP2 is found to be downregulated in COAD tissues compared with adjacent normal tissues.The downregulated expression of PKP2 is significantly correlated with more advanced pathological stage and lymph node metastases.The ROC curve analysis revealed that PKP2 could be a diagnostic biomarker to differentiate COAD tissues from normal tissues.In univariate analysis and Kaplan-Meier curve analysis, downregulated expression of PKP2 is confirmed to be correlated with long survival and PKP2 can be Protein-protein interaction analysis of PKP2 from Search Tool for the Retrieval of Interacting Genes/Proteins (STRING).

T A B L E 2
The information for PKP2-related genes.Plakophilin-2 (PKP2) is a member of desmosomal protein family.
PKP2 has been confirmed to have a significant effect on accelerating cell proliferation, migration and tumor cell growth. 8,19Recent research on the oncogenic effect of PKP2 in human cancers of different types has been reported, such as lung cancer, 20 ovarian cancer 21 and breast cancer. 223][24][25] Nevertheless, the prognostic and diagnostic values of PKP2 expression have not been thoroughly validated in COAD.The results in our study are accordant with the demonstration that PKP2 is abnormally expressed in different types of tumors.
We also confirmed that PKP2 is significantly downregulated in COAD.Downregulation of PKP2 is positively correlated with more advanced pathological stage and lymph node metastases.These discoveries suggest that PKP2 can be used as a biomarker of unfavorable prognosis to identify COAD with unfavorable survival outcomes.
Until now, the function of PKP2 in cancers has not been fully understood and reported.Niell et al. 26  was found in 87.5% of cancer tissues by immunohistochemistry and also correlated with local recurrences and unfavorable survival outcomes. 29In lung adenocarcinoma, overexpression of PKP2 is demonstrated to be positively correlated with enhancing focal adhesion and epithelial-mesenchymal transition as well as unfavorable survival outcomes. 23,30In gastric cancer, compared with normal gastric tissues, PKP2 was significantly downregulated and decreased PKP2 had a negative impact on disease-free survival. 31However, the prognostic effect of PKP2 has not been demonstrated in COAD.Owing to the downregulation of PKP2 being significantly correlated with more advanced pathological stage and lymph node metastases, PKP2 is inferred to play a vital role in the development of COAD.In addition, as more advanced pathological stage and lymph node metastases are correlated with unfavorable survival outcomes, the downregulation of PKP2 is predicted to be a biomarker of unfavorable prognosis of COAD.Moreover, based on Kaplan-Meier curves, COAD patients with down-regulated expression of PKP2 are correlated with a more unfavorable survival rate than those with high expression levels of PKP2.In accordance with our data analysis, PKP2 can be regarded as a promising biomarker of unfavorable prognosis for predicting prognosis in COAD.
In our analysis, some tumor-infiltrating immune cells, such as B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophil and dendritic cells were found to be correlated with PKP2 expression in COAD on the basis of analytic results with TIMER.These discoveries indicate that a potential correlation exists between PKP2 expression and immune infiltration in COAD.Therefore, it is necessary to design further research to demonstrate the correlation between them.

| CONCLUSIONS
In our analysis, the expression of PKP2 is demonstrated to be upregulated in COAD for the first time and downregulation of PKP2 expression is positively correlated with more advanced pathological stage and lymph node metastases.This analysis reveals that PKP2 can be used as a biomarker of unfavorable prognosis to identify COAD patients with unfavorable clinical outcomes.Meanwhile, downregulated expression of PKP2 may have an influence on immune infiltration.

AUTHOR CONTRIBUTIONS
Zhixiong Wu and Yuantao Su are responsible for the design and critical revision of the manuscript.Meng Dai analyzed the data and wrote the manuscript.

F
I G U R E 2 The mRNA expression of PKP2 in colon adenocarcinoma (COAD).(A) The mRNA expression levels of PKP2 in 480 COAD samples and 81 normal samples.(B) The mRNA expression levels of PKP2 in 41 COAD samples and 41 matched-adjacent normal samples (***p < 0.001).

F I G U R E 4
Receiver operating curve (ROC) curve: PKP2 had an area under the curve (AUC) value of 0.787 to discriminate colon adenocarcinoma (COAD) tissues from healthy controls.With a cutoff of 6.034, the sensitivity and specificity were 90.2 and 66.5%, respectively.FPR, False positive rate; TPR, true positive rate.F I G U R E 5 Kaplan-Meier survival curves indicated that colon adenocarcinoma (COAD) patients with high PKP2 mRNA expression had a longer median survival time than those with low levels of PKP2 (101.4 vs. 68.2months, p = 0.018).HR, hazard ratio.
as a promising biomarker of unfavorable prognosis for COAD.Moreover, PKP2 may have a specific influence on immune infiltration in COAD.

F I G U R E 7
The enrichment of PKP2.(A) The bubble plot revealed the signaling pathway of PKP2.(B) CTNNB1 and CTNNA3 are enriched in the Hippo signaling pathway.F I G U R E 8 Correlations of PKP2 expression with immune infiltration level.(A) PKP2 expression is negatively related to tumor purity and has correlations with B cells, CD8+ T cells, CD4+ T cells, dendritic cells, neutrophils and macrophages in colon adenocarcinoma (COAD).(B) Relationship between the expression of PKP2 and 28 types of tumor infiltrating lymphocytes (TILs) across human cancers.(C) PKP2 was correlated with an abundance of Act_DC cells, iDC cells, macrophages, mast cells, Tcm_CD8 cells, Act_CD4 cells, Tgd cells, Th2 cells, mem_B cells and CD56bright cells.