ccRCC is the most common pathological subtype of renal cell carcinoma, with high malignancy, easy metastasis and poor prognosis, accounting for the majority of kidney cancer deaths. Despite localized renal cancer can be cured by nephrectomy, up to 30% patients with localized tumors ultimately develop recurrence and metastasis [25, 26]. With regard to the patients who have lost the opportunity for surgery, systemic treatment encompassing targeted therapy and immunotherapy is primary approach [27, 28]. Until now, mutations of certain genes such as VHL, PBRM1, SETD2, BAP1, have been found to be related to the oncogenic driving events of ccRCC, but the contributions of their mutations in the tumorigenesis and progression of ccRCC and their values as prognostic biomarkers are still not entirely clear [29, 30]. With the extensive applications of genomics technology, researchers have gained access to vast amounts of genetic data. Accordingly, bioinformatics emerged and helped researchers to dig out vital information in the gene regulatory networks of diversified cancers from abundant biological data.
In this research, we combined four mRNA microarray series which containing 106 pairs of ccRCC tissues and adjacent normal tissues from GEO database to obtain the significant DEGs in ccRCC. The results showed that 159 and 223 genes are up-regulated or down-regulated by more than four times in ccRCC, respectively. Subsequently, GO analysis of the DEGs indicated that they are principally involved in response to hypoxia, regulation of immune response, angiogenesis, inflammatory response, cell adhesion, excretion, gluconeogenesis. The KEGG analysis also showed that the DEGs mainly participate in focal adhesion, HIF-1 signaling pathway, Toll-like receptor signaling pathway, metabolic pathways, glycolysis/gluconeogenesis. The results of functional annotation of DEGs, on the other hand, suggested that many classical metabolic pathways are reprogrammed in order to empower frantic proliferation of cancer cells in ccRCC, as previous studies have revealed [31, 32]. After construction of protein-protein interaction network of DEGs, we used CytoHubba to identify the hub genes with degree > 10 and further screened the hub genes to obtain candidate genes related with the prognoses of ccRCC by synthesizing the results of UALCAN, GEPIA and HPA. The results showed that TIMP1, PCK1, HMGCS2, G6PC, FBP1, ACAA1, HADH, HAO2, TGFBI, RRM2 and SUCLG1 are of prognostic significance in ccRCC patients, which were consistent with results of previous researches [33–37]. Whereafter, we verified the mRNA and protein expression of these genes in different ways, thus obtained three target genes, namely PCK1, HMGCS2 and RRM2. Survival analysis with three websites indicated that low expression of PCK1 or HMGCS2 and high expression of RRM2 have significantly correlated to poorer prognoses in ccRCC patients regardless of overall survival or disease free survival. This result hinted that PCK1, HMGCS2 and RRM2 could be potential prognostic biomarkers for ccRCC patients. Furthermore, we investigated the relationships between target genes and immune infiltration and immunological checkpoints in ccRCC emphatically. Analysis of TIMER showed that RRM2 expression is related to infiltration levels of B cells, CD8 + T Cells, macrophage, neutrophil and dendritic cells, as well as the expression of PDL-1 and CTLA4. The results were almost the same when analysing with TISIDB website, suggesting that RRM2 plays an important role in the occurrence and progression of ccRCC and is a potential target for immunotherapy of ccRCC patients.
PCK1(Phosphoenolpyruvate Carboxykinase 1) is the first rate-limiting enzyme of gluconeogenesis in cytoplasm, catalyzing the transformation fromoxaloacetate to phosphoenolpyruvate. The isozyme is PCK2, located in mitochondria, catalyzes the same reaction. Studies have found that PCK1 is increased in gastric cancer, colon carcinoma, and tumor-repopulating cells from liver cancer, melanoma and lymphoma [38–40]. However, the expression of PCK1 was also found down-regulated in hepatocellular carcinoma and renal cancer, and low expression of this gene is related to worse survival in hepatocellular carcinoma patients [35, 41–43]. HMGCS2 (3-Hydroxy-3-Methylglutaryl-CoA Synthase 2) encodes a rate-limiting enzyme that catalyzes the first reaction of ketogenesis in mitochondrial. Researchers have discovered that HMGCS2 is reduced in colorectal cancer, prostate cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma and correlated with the prognoses of patients in these cancers [44–47]. Herein, we discovered both PCK1 and HMGCS2 are reduced in ccRCC and correlated to the prognoses of ccRCC patients. All these discoveries indicated that cancer cells have different metabolic pathways compared to normal cells and the metabolic rewiring appears to be advantageous for cancer proliferation and progression [48]. Notably, ccRCC is one of the most studied malignancies characterized by metabolic reprogramming [31]. Such alterations of metabolism in ccRCC have provided us new thoughts for the exploitation of new targets for oncotherapy [49, 50].
RRM2 (Ribonucleotide Reductase Regulatory Subunit M2) is one of the two subunits of Ribonucleotide reductase (RR), which is the rate-limiting enzyme catalyzing the formation of deoxyribonucleotides from ribonucleotides, playing a pivotal role in DNA replication and repair. Different from the constant expression of RRM1 throughout the whole cell life, RRM2 expression changes dynamically with stimulus [51]. Considerable researches have demonstrated that RRM2 is dysregulated in multiple cancer types, including liver cancer, breast cancer, lung adenocarcinoma and glioblastoma [52–56]. As a tumor promotor, RRM2 enhances the proliferation, invasion of cancer cells and the resistance to chemotherapeutic drugs, could become the predictor for chemosensitivity and prognosis [57]. In this study, we discovered the expression of RRM2 in ccRCC is significantly increased, and patients with high expression had relative poor prognosis, which was consistent with previous results in other cancer types, suggesting that it could be a promising biomarker for the prognostic assessment for ccRCC patients. Additionally, we also found a significant correlation between RRM2 and immune cell infiltration and immunological checkpoints, suggesting that RRM2 may plays an important role in immune response of ccRCC. Numerous studies have shown that ccRCC is an immune tumor with a synergistic effect of angiogenesis and immuno-suppression [58]. Combination of antiangiogenics and targeted immunotherapy to overcome resistance has been proposed as an option in first line treatment of advanced ccRCC currently [59–60]. Our findings revealed that RRM2 is probably to be a new oncogene involved in the immune regulation mechanism and promising biomarker for ccRCC immunotherapy.