The expression patterns and the prognostic roles of PTPN family members in digestive tract cancers

Background Non-receptor protein tyrosine phosphatases (PTPNs) are a set of enzymes involved in the tyrosyl phosphorylation. The present study intended to clarify the associations between the expression patterns of PTPN family members and the prognosis of digestive tract cancers. Method Expression profiling of PTPN family genes in digestive tract cancers were analyzed through ONCOMINE and UALCAN. Gene ontology enrichment analysis was conducted using the DAVID database. The gene–gene interaction network was performed by GeneMANIA and the protein–protein interaction (PPI) network was built using STRING portal couple with Cytoscape. Data from The Cancer Genome Atlas (TCGA) were downloaded for validation and to explore the relationship of the PTPN expression with clinicopathological parameters and survival of digestive tract cancers. Results Most PTPN family members were associated with digestive tract cancers according to Oncomine, Ualcan and TCGA data. For esophageal carcinoma (ESCA), expression of PTPN1, PTPN4 and PTPN12 were upregulated; expression of PTPN20 was associated with poor prognosis. For stomach adenocarcinoma (STAD), expression of PTPN2, PTPN12 and PTPN22 was upregulated in cancer tissue; pathological grade was significantly correlated with PTPN3, PTPN5, PTPN7, PTPN11, PTPN13, PTPN14, PTPN18 and PTPN23; PTPN20 expression was related with both TNM stage and N stage; PTPN22 was associated with T stage and pathological grade; decreased expression of PTPN5 and PTPN13 was associated with worse overall survival of STAD, while elevated PTPN6 expression indicated better prognosis. For colon adenocarcinoma (COAD), expression of PTPN1, PTPN11, PTPN12 and PTPN13 was upregulated, while expression of PTPN18, PTPN21 and PTPN22 was decreased; expression of PTPN5, PTPN12, and PTPN14 was correlated with TNM stage and N stage; analysis. The Pearson X 2 test was performed to evaluate the relationship between different PTPN expression status and clinicopathological features. The effects of different PTPN expressions on overall survival were estimated through univariate and multivariate Cox proportional hazards models


Background
Tyrosyl phosphorylation is a dynamic and reversible process which plays a pivotal part in many cellular signaling pathways (1). The dephosphorylation of tyrosine residues are catalyzed by a series of enzymes named protein tyrosine phosphatases (PTPs) (2). Encoded by 107 genes, PTPs are sorted into four major superfamily classes (3), and every single PTP was denominated an official gene name by The Human Genome Organization in Nomenclature Committee. According to the latter system, 17 non-receptor PTPs which belong to the biggest family class I, are designated PTPN, followed by a number (3). There is mounting evidence suggesting that the cross-talk of the PTPN gene family members is involved in extensive physiological processes, such as cell proliferation, survival, immune response, migration, and metabolism (3)(4)(5)(6). Previously published study stated that PTPN family members play an essential part in numerous diseases. For example, the expression of PTPN6 with the loss of pSTAT3 expression could be chosen as a biomarker for the prognosis of peripheral-T cell lymphoma (7). One research published recently noted that a deficiency of PTPN2 could enhance antitumor immunity and the therapeutic efficacy of CAR T cells to solid cancers (8). PTPN22 plays an important part in regulating autophagy and NLR family pyrin domain containing 3 inflammasome activation (9). It is hopeful that PTPN genes have potential to be served as prognostic and diagnostic indicators (10), and even therapeutic targets.
As main malignancies of gastrointestinal tract, esophagus cancer (EC), gastric cancer (GC) and colorectal cancer (CRC) are responsible for a large portion of cancer-related deaths worldwide, of which the data are 5.3%, 8.2% and 9.0% respectively (11). All of the three digestive tract cancers are ranked in the top 10 for incidence rates of tumors and have poor prognosis (11). Although a couple of diagnostic biomarkers have been observed, robust biomarkers to predict clinical outcomes are still in urgent need (12). Former investigations reported that the expression pattern of individual PTPNs and its correlations with patients in various digestive tract neoplasms, but research to date has not yet observed the whole picture of the entire PTPN family, from the aspects of prognostic role and expression characteristics. Personalized therapies based upon the genetics of individual cancer will be prior to other treatments in the near future. This research therefore is intended to illuminate the prognostic value of different PTPN genes to support potential biomarkers and new individualized targets for patients. In the current study, we analyzed the expression status of different PTPN members and their clinical and prognostic values to comprehensively evaluate the role of PTPNs in various digestive tract cancers.

Methods
Oncomine Database Analysis for the expression patterns of PTPN family in digestive tract cancers Oncomine database is a cancer microarray database (https://www.oncomine.org/) which shows the expression information of genes in cancer and normal samples (13). Oncomine provides both microarray information from 715 datasets and a set of online data-mining functions. The expression levels of individual PTPN family members in different types of cancer were obtained from the Oncomine database. Student's t test was applied to calculate the P value for expression differences of PTPN family genes between normal controls and cancer samples. The threshold parameters of P value and fold change were demarcated as 0.05 and 2 respectively.

Ualcan Database Analysis For The Validation
Ualcan is a publicly available web-portal (http://ualcan.path.uab.edu) that offers online analysis of data from The Cancer Genome Atlas (TCGA) (14). In this study, we used it to analyze the relative expression of PTPN family genes in esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), rectum adenocarcinoma (READ) and normal samples. The expression level of PTPN members was normalized as transcript per million reads, and a P value of no more than 0.01 conducted through Student's t test was considered to be significant.
TCGA Data Analysis for the association between PTPN family and prognosis of digestive tract cancers.
The Cancer Genome Atlas (TCGA) is a cancer genomics database (https://cancergenome.nih.gov/) which contains genomic information of over 2000 primary neoplasms and matched normal samples (15,16). In our study, case information of 115 ESCA, 347 STAD and 273 COAD were extracted for further analysis. The Pearson X 2 test was performed to evaluate the relationship between different PTPN expression status and clinicopathological features. The effects of different PTPN expressions on overall survival were estimated through univariate and multivariate Cox proportional hazards models with or without adjustment for confounding factors. Variables including TNM stage, sex and age were further adjusted during the evaluation. The medical data obtained was managed by R language (Version 3.6.1). P value < 0.05 was regarded as statistically significant.  (19). The Cytoscape software was applied to visualize network diagrams for PPI analysis (20).

PTPN Genes Expression Patterns in Digestive Tract Cancers.
Before expression profiling analysis, we refined the chromosome location of all PTPN gene family members through published literature review. The detailed information was summarized in Table 1. All of these seventeen PTPN family members have been analyzed in our study. According to the analysis results of ONCOMINE, the expression of PTPN genes were different in all types of cancer and its matched normal tissues (Fig. 1). For EC samples, PTPN1, PTPN4, PTPN12, PTPN18 and PTPN21 were over-expressed, while PTPN3, PTPN11, PTPN13 and PTPN21 were downregulated. In GC tissues, the expression of PTPN5 and PTPN13 was decreased while at the same time PTPN2, PTPN12 and PTPN22 were highly expressed in patients. As for CRC patients, the expression of PTPN1, PTPN3, PTPN7, PTPN11, PTPN12, PTPN13 and PTPN14 was upregulated in patients with CRC, while PTPN2, PTPN18, PTPN20, PTPN21 and PTPN22 were expressed in a lower level in tissues. Comprehensively, the expression of PTPN12 was higher in EC, GC and CRC samples.

Correlations between PTPN Members and Clinicopathological Parameters.
Association of PTPN family genes expression status with different clinicopathological features of ESCA, STAD and COAD were analyzed in the study. For ESCA, no statistically association was found between PTPN members and clinicopathological parameters (Additional file 1: Table S1). For STAD, grade was significantly correlated with PTPN3, PTPN11, PTPN13, PTPN14, and PTPN18 (p = 0.015, 0.021, 0.005, 0.002 and 0.034, resp.) (Additional file 1: Table S2). In addition, PTPN5, PTPN7 and PTPN23 showed statistically association with pathological grade of STAD with a p value of no more than 0.001.
Prognostic Roles of PTPN Family Genes.
The analysis results of the relationship between PTPN family genes and the prognosis of digestive tract cancers showed that for ESCA, elevated expression of PTPN20 was associated with a worse overall survival (OS) of ESCA in multivariate model (p = 0.038, adjusted HR = 1.982, 95% CI = 1.039-3.780; Table 2).   GO analysis was basically grouped into three terms including molecular function groups, cellular component groups and biological process groups. The top 5 enriched categories obtained from the analysis results of each group were showed in Fig. 6a. GO analysis revealed that PTPN proteins were mainly related to cytoplasmic side of plasma membrane. PTPN genes exert their functions primarily on peptidyl-tyrosine dephosphorylation and protein tyrosine phosphatase activity. Further, the interaction analysis of PTPN genes at the gene level was performed by GeneMANIA to clarify the correlations among colocalization, shared protein domains, co-expression, prediction and pathways (Fig. 6b). As the protein-protein interaction network of STRING analysis result revealed, interrelationships among PTPN gene family members were intricate (Fig. 6c).  (28). Consistent with our results, Slattery ML et al (29) reported that PTPN11 expression is upregulated in CRC. PTPN11 was also suggested to impact the tumorigenesis and metastasis of CRC(30) and termed as a potential prognostic marker (31). The data in one report showed that in patients with gastric B-cell non-Hodgkin's lymphoma, PTPN21 is over expressed (32). In ESCA, the expression levels of PTPN1, PTPN4, and PTPN12 were increased. PTPN12 expression was upregulated in both ESCA, STAD, COAD and READ. Recently PTPN12 is characterized as a tumor suppressor which antagonizes EGFR/HER2 signaling (33,34). In hepatocellular carcinoma cells, PTPN12 regulates epithelial-mesenchymal transition which contributes to chemoresistance and metastasis (35). All of these indicate that many PTPNs function in the progress of cancer, and particularly, PTPN12 may play a crucial role for its increased levels in all of the three types of digestive tract cancers.

Discussion
There may also exist associations between differentially expressed PTPNs and prognosis of digestive tract cancers. To further figure out the associations, we then analyzed the interrelationships between PTPN expressions and the clinical outcomes of digestive tract cancers. As revealed by the current study, PTPN5, PTPN13 and PTPN22 were also associated with clinicopathological parameters of STAD.
And decreased expression of PTPN5 and PTPN13 indicated worse OS of STAD patients, while high PTPN6 expression was associated with a favorable STAD OS. It is worth noting that SHP-1 protein encoded by PTPN6 mediates the tumor-suppressive function of TMEFF2 in STAD (36), which indicates expression of PTPN6 might influence the carcinogenesis of STAD patients. PTPN13 has been reported to regulate the resistance of human lung fibroblasts to Fas-induced apoptosis in previous study (37).
Mutated PTPN13 was suggested to be a tumor suppressor gene in colorectal cancer(27). PTPN22 gene encodes an enzyme called lymphoid-specific tyrosine phosphatase, which functions as a master regulator in the biological process of relevant immune responses (38). Upregulation of PTPN22 could result in impairment of regulatory T-cell differentiation in patients with non-ST-segment elevation acute coronary syndromes (39). As for COAD, expression levels of PTPN5, PTPN12, and PTPN14 were correlated with clinicopathological parameters. Besides, elevation of PTPN5 and PTPN7 was significantly associated with increased death hazards of COAD patients respectively. PTPN5 could regulate tyrosine dephosphorylation needed for the activation of BAK, a noteworthy cell-death mediator in apoptosis (40). One study presents PTPN12 as a novel candidate that contributes to the heterogeneous susceptibility to colorectal cancer (41). It was once reported that PTPN14 regulated phosphorylation of p130Cas Y128 plays a crucial role in colorectal carcinogenesis (42). And mutated PTPN14 is suggested to be a tumor suppressor gene for colorectal cancer, regulating cellular pathways that are appropriate for therapeutic intervention(27). As for ESCA, our analysis indicated that only PTPN20 expression was related with a worse OS of patients. However, former research indicated that PTPN12 may serve as a potential prognostic indicator for esophagus cancer patients (43). Altogether, our results revealed that PTPN5 expression status was significantly related to clinical features and prognosis of both STAD and COAD, which warrants further investigation for its function in progress of digestive tract cancers.
In this study, GO analysis revealed that PTPN proteins were mainly related to cytoplasmic side of plasma membrane. PTPN genes exert their functions primarily on peptidyl-tyrosine dephosphorylation and protein tyrosine phosphatase activity, which was illustrated by dozens of publications.  (44,48). Multiple efforts demonstrated that through binding and inactivating the mitogen activated protein kinase Erk2 and p38, PTPN5 and PTPN7 could negatively regulate cell proliferation as well as differentiation (49)(50)(51)(52). Promoter hypermethylation of PTPN6 and PTPN13 was reported to inhibit the progression of diffuse large B cell lymphomas (53). In our study, PTPN5 and PTPN7 were found to be correlated with prognosis of colon adenocarcinoma patients, while PTPN6 and PTPN13 were statistically associated with the prognosis of STAD. All of these implied that there may exist alliance mechanisms in biological process of PTPN members and even in the progress of digestive tract cancers.

Conclusions
In summary, findings of our study illustrated the expression status and prognostic values of PTPN members in digestive tract cancers. The results indicated that several PTPN members were differentially expressed and related to clinical outcomes of patients with digestive tract cancers.
Especially, level of PTPN12 expression was upregulated in both ESCA,STAD, COAD and READ. PTPN5 was associated with the clinical features and prognosis of both STAD and COAD. Future well-designed investigations are in required to elucidate the significance of our findings and thus develop the clinical utility of PTPNs.

Funding
This work was supported by the National Natural Science Foundation of China (31800124).

Authors' contributions
JC collected the data and wrote the paper; JC, JJ and XZ performed the statistical analysis; JJ and YY conceived the study and revised the manuscript.All authors read and approved the final manuscript. Figure 1 The expression level of PTPN family genes in different types of cancers. Red and blue stand for the numbers of datasets with statistically significant (P<0.05) increased and decreased levels of PTPN family genes, respectively.

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