Abstract
Background: Renal cell carcinoma (RCC) is a common urological cancer, and its risk correlates with environmental factors such as obesity, smoking and hypertension. Microarray technology enables analysis of the expression pattern of the whole phosphatome, members of which are involved in many cellular pathways and may act as either tumour suppressors or oncogenes in cancers. Materials and Methods: We analysed data for the expression level of 87 out of 107 known protein phosphatase genes included in the Hugo Gene Nomenclature Committee Website for 72 RCC tissues and paired healthy tissues obtained from the GEO Database. Results: Our analysis revealed overexpression of DUSP1, DUSP4, PTP4A3, PTPRC and PTPRE genes at all examined stages of RCC. Moreover, we found overexpression of PTPN12 at stage 2, overexpression of CDKN3 at stages 3 and 4, and overexpression of DUSP10 and PTPN22 at stages 2, 3 and 4. Lower expression of DUSP9, PTPR9 and PTPRO was also observed at all stages. Conclusion: Significant changes in expression patterns of protein tyrosine phosphatase genes confirm the involvement of this group in crucial carcinogenesis pathways underlying RCC. Thus, we postulate that protein tyrosine phosphatases play an important role in RCC promotion and progression, and may be considered as potential therapeutic targets.
Microarray technology enables the analysis of genetic events of selected genes in various groups of cancers and therefore can show the impact of particular enzymatic processes or whole biochemical pathways on cancer promotion and progression. Analysis of the expression pattern of the whole phosphatome may reveal whether this group of genes is pivotal in carcinogenesis and allow selection of some genes from the group which should be intensively studied in order to improve and develop cancer prognosis and treatment strategies.
Protein tyrosine phosphatases (PTPs) have been widely described, and their role in cancer cells is well documented (1, 2). It has been shown that dysregulation of dephosphorylation leads to uncontrolled cell growth and proliferation, apoptosis arrest and cancer development (3). Because of their involvement in many cellular pathways, PTPs may act either as tumour suppressors or oncogenes, and their dual role has been explored before (1, 3).
Renal cell carcinoma (RCC) is one of the common urological cancers, and its risk correlates with environmental factors such as obesity, smoking and hypertension (4). RCC constitutes about 2-3% of all cancers in adult patients and is classified according to histological characteristics with more than 15 subtypes (5-7). The most common subtype of sporadic RCC is clear cell renal cell carcinoma (ccRCC), which accounts for about 75% of all renal cancers. Metastases of RCC appear in about 30% of patients (5). About 3-5% of RCCs are hereditary. The study of one of the hereditary forms of RCC, von Hippel-Lindau (VHL) syndrome, revealed the importance of the 3p chromosome region (5, 8). Among all critical genes, genes such as VHL, BAP1, PBMR1, TECB1, MTOR, PTEN, SETD2, and TP53 are reported to be frequently altered in both sporadic and hereditary forms of RCC (4, 5, 9).
The importance of downstream kinase pathways in RCC initiation and progression, i.e. the MAPK and the PI3K/AKT/mTOR pathways, along with inactivation of PTEN, indicates the essential role of kinase activity for this type of cancer, and therefore it can be concluded that the opposite phosphatase activity is also crucial for RCC development and progression (4).
The majority of previous studies addressing the role of PTPs in the development of RCC focused on selected genetic events, e.g. pathogenic variants (point mutations), single nucleotide polymorphisms (SNPs), loss of heterozygosity (LOH), expression pattern or methylation status (3, 6, 10-13). Therefore, our study aimed to evaluate the whole phosphatome expression, and thus we analysed the microarray expression data available for 87 out of 107 known phosphatase genes in 72 normal and 72 cancer tissues obtained from the GEO Database of the National Center for Biotechnology Information.
Materials and Methods
We analysed data for the expression levels of 87 out of 107 known protein phosphatase genes included in the Hugo Gene Nomenclature Committee (HGNC) Website (access date: August 7 2018). Microarray expression data were obtained from the GEO Database of the National Center for Biotechnology Information (NCBI) (GSE53757). mRNA was isolated from 72 tissues of RCC at various stages: stage 1 (n = 24), stage 2 (n=19), stage 3 (n=14) and stage 4 (n=15), as well as 72 normal paired renal tissues obtained during surgery (14).
RCC tumour samples were hybridized to the Affymetrix HGU133 Plus 2.0 expression arrays (Platform GPL570, Santa Clara, CA, USA). Datasets were normalized by the robust multi-array average (RMA) method (15). Data were presented as log2-transformed values by RMA. Log2 fold change (log2FC) was calculated as the difference between two means: the log2 RMA signal obtained from cancer tissue and the log2 RMA signal obtained from normal tissue. A log2FC value below-1 corresponds to down-regulated expression and log2FC >1 corresponds to overexpression. The t-test was used to calculate the significance of the difference between the expression levels in tumour and healthy tissues. p-Values<0.05 were considered significant.
Results and Discussion
The regulatory role of the phosphatome in normal cells and its dysregulation in cancer cells has been previously shown (1, 3). Therefore, following the expression pattern of genes encoding for tyrosine phosphatases in renal cell carcinoma might bring crucial information about its epidemiology.
The analysis of 87 protein tyrosine phosphatase genes using Affymetrix HGU133 Plus 2.0 expression arrays revealed statistically significant results for 12 genes. Expression of 5 genes including 2 dual-specificity phosphatases: DUSP1 and DUSP4, and 3 protein tyrosine phosphatases: PTP4A3, PTPRC and PTPRE was higher in all examined RCC stages, while expression of DUSP10 and PTPN22 was higher at all stages except stage 1. At stages 3 and 4 we found overexpression of one dual-specificity phosphatase - CDKN3 and only at stage 2 overexpression of PTPN12 (Table I).
Three examined phosphatase genes had lower expression levels in cancer than in normal cells. Decrease in expression of DUSP9, PTPR9 and PTPRO was observed at all stages of RCCs (Table I).
Our findings suggest that these genes play an important role in the initiation of RCC development.
The role of dual-specific phosphatases (DUSPs) in inhibition of mitogen-activated protein kinase (MAPK) signalling means that DUSPs belong to the key group of genes involved in carcinogenesis (16-18). Among DUSPs that target MAPKs, the nuclear-inducible DUSP1, DUSP2, DUSP4 and DUSP5 dephosphorylate ERK, JNK (except DUSP5) and/or p38; the cytoplasmic DUSP6, 7 and 9 mainly target ERK while DUSP8, 10 and 16 catalyse JNK/p38 dephosphorylation (18). Decreased expression of DUSP9 was previously reported in clear cell RCC (ccRCC) using real-time qPCR and immunohistochemical analysis (p<0.001) (19). Moreover, expression of DUSP9 was positively correlated with tumour size, pathologic state, Fuhrman grade, T stage, N classification, and metastasis and negatively correlated with recurrence risk (19). The association of DUSP9 expression with poor prognosis was suggested to be useful as a prognostic marker for ccRCC (19). DUSP9 negatively regulates MAPK, p38 and SAPK – members of the mitogen-activated protein (MAP) kinase superfamily which promote cellular proliferation and migration (20). It has also been shown that epigenetic silencing of DUSP9 in human gastric cancer activates JNK signalling and therefore induces proliferation of cancer cells (19). Moreover, increased DUSP9 expression leads to inhibition of ERK in triple-negative breast cancer chemotherapy (21). The analysis of the REpository for Molecular BRAin Neoplasia DaTa (REMBRANDT) also showed changes in expression of DUSP4, DUSP6 (induction), DUSP2, DUSP7-9 (repression) and DUSP1, DUSP5, DUSP10, DUSP15 (mixed) in glioblastoma cells (22). Also, DUSP10 and DUSP16 were described as positive regulators of activation by modulating cross-talk between the p38 and ERK pathways in HeLa cells (18). Therefore our results showed induction of expression of DUSP1, DUSP4 and DUSP10 along with repression of DUSP9 expression and confirmed an important role of these genes in RCC development.
Results of phosphatome expression analysis in renal cell carcinoma.
CDKN3 was previously reported to promote ovarian carcinoma cell proliferation (23). Correlations of CDKN3 overexpression with stage, tumour size, risk of tumour recurrence, and poor patient survival in ovarian cancer has been described. Moreover the overexpression of this gene was also observed in lung adenocarcinoma patients with poor survival, nasopharyngeal carcinoma patients with advanced tumour stage, and in hepatocellular carcinoma, gastric cancer and pancreatic ductal adenocarcinoma patients with tumour cell proliferation (23-28). The important role of this gene in cancer development and progression has been confirmed by the fact that CDKN3 may influence p53 activity via forming a complex with MdM2-P53, thus leading to inhibiting the expression of p21 (26). Our results are consistent with these reports and revealed overexpression of CDKN3 in more advanced RCC stages.
Previous analysis of microarray expression profiling using the GEO database of transcription factors and their associations with target genes has revealed that the most targeted genes were mainly involved in the ErbB and MAPK cancer pathways (29). Also, PTP4A3 negatively regulates p38 MAPK, which is involved in modulating the transcription of inflammatory cytokine genes (30). Expression analysis of phosphatase and phosphatase-interacting genes in 102 breast cancers from Affymetrix microarray revealed that 146 phosphatases were significantly differentially expressed in triple-negative breast cancer (TNBC) as compared to estrogen receptor (ER)-positive tumours. In a group of 19 up-regulated genes with ≥1.5-fold protein tyrosine phosphatase 4A3 (PTP4A3) and CDKN3 were present (31). Further analysis of PTP4A3 revealed that high expression of PTP4A3 may be an independent prognostic indicator of worse overall survival in TNBC patients (31). Another genome-wide analysis of differentially expressed genes and splicing isoforms conducted in ccRCC cells showed over 2000 differentially expressed genes with overexpression of PTP4A3 among them (32). Furthermore, overexpression of PTP4A3 was positively correlated with the pathological tumor (pT) status, nodal metastasis and lymphovascular and perineural invasion in upper tract urothelial carcinoma (33). All these findings are consistent with our results for RCC.
Protein tyrosine phosphatase non-receptor type 22 (PTPN22) was previously reported to be overexpressed in chronic lymphocytic leukaemia, and it was also shown that suppression of this gene induces apoptosis in a T-cell leukaemia cell line via the AKT and ERG pathways (34, 35). Our analysis also revealed overexpression of PTPN22 in RCC, which may suggest the important role of this gene in cancer.
Protein tyrosine phosphatase non-receptor type 12 (PTPN12) was reported as a tumour suppressor presenting with lower expression in breast cancer (36). It was also suggested that this gene may be methylation-silenced and plays a crucial role in this type of cancer (36). In another study on PTPN12 in nasopharyngeal carcinoma cell lines it was found that PTPN12 may negatively regulate EGFR and therefore suppress cell proliferation and migration (37). Similarly, PTPN12 down-regulation as compared to healthy renal tissues was revealed for RCCs, although PTPN12 overexpression was observed in larger tumours, metastasis and high grades (38). This result is consistent with our findings, as we observed overexpression of this gene in RCCs stage 2, but not at stage 1. However, we did not observe higher PTPN12 expression in stages 3 and 4. The importance of PTPN12 was also confirmed by a study on the association of missense polymorphism of this gene modifying the inhibitory effect on the Ras/MEK/ERK pathway and the risk of colorectal cancer (39). Also, the correlation between overexpression of PTPN12 and ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1) phosphorylation inhibition in hereditary leiomyomatosis and renal cell carcinoma was reported (40).
Many protein tyrosine phosphatase receptor type genes such as PTPRC, PTPRE, PTPR9 and PTPRO have been reported to be involved in cancer development and progression. The dual role of PTPs either as tumour suppressors or as oncogenes has been widely identified and discussed (1, 3). We have reported the overexpression of PTPRC and PTPRE as well as down-regulation of PTPR9 and PTPRO in RCCs. However, although currently the knowledge of the role of PTPRC, PTPRE and PTPR9 in cancers is still incomplete, many members of this group are suspected to act as proto-oncogenes or tumour suppressor genes (1).
Protein tyrosine phosphatase receptor type O (PTPRO) acts as a tumour suppressor and is epigenetically silenced, thus presenting with lower mRNA levels in both human lung squamous cell carcinomas (LSCC) and breast cancer. PTPRO is also down-regulated in CRCs in patients with poor prognosis (41-43). Reduced expression of this gene was also observed in B-cell chronic lymphocytic leukaemia (44). Moreover, the re-activation of PTPRO in tumour tissues can either contribute to tumour reduction or improve its susceptibility to chemotherapy (42, 45). It is postulated that PTPRO regulates insulin and lipid metabolism via the PI3K/Akt/MDM4/MDM2/P53 pathway by influencing autophagy (46). It has also been found that PTPRO negatively regulates SRC through dephosphorylation of Y416. Therefore, down-regulation of this gene induces SRC activation and subsequent phosphorylation of EGFR, which leads to hyperactivation of EGFR (41). Thus, our results, along with former knowledge, confirm the high importance of this gene in cancer development and progression and suggest the crucial role of PTPRO in RCC.
Conclusion
Microarray technology allowed us to examine the expression levels of many genes simultaneously. The analysis of available data from different studies listed in the GEO database revealed significant changes in protein tyrosine phosphatase gene expression in renal cell carcinoma. This result, along with the recently confirmed involvement of this group of genes in crucial carcinogenesis pathways, allows them to be considered as potential targets in tailored therapy.
Acknowledgements
The Authors are grateful to Blazej Misiak for his support in preparing the manuscript.
Footnotes
Authors' Contributions
IL conceived the presented idea, was involved in data analysis, wrote the manuscript. LL collected and analysed the data, performed the calculations, was involved in writing of the manuscript (wrote Materials and Methods and Results). MS was involved in planning and supervised the writing of the manuscript.
Conflicts of Interest
The Authors declare that there are no conflicts of interest.
- Received June 25, 2020.
- Revision received July 10, 2020.
- Accepted July 15, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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