The root cause of cancer is the accumulation of genetic mutations(12),It is speculated that more than two-thirds of the mutations in tumor tissue result from DNA replication errors during cell proliferation(13). Uncontrolled proliferation, apoptotic disorder, immortalized replication, long-lasting angiogenesis, local invasion, distant metastasis, escape from immune and growth inhibitors and so on are the biological capabilities that cancer acquires during the development process. The basis of these capabilities is genetic instability and chronic inflammation(14). The E2F family is downstream of CDK-Rb-E2Fs in a cell cycle regulation network(15), and is a crutial transcriptional regulatory factor in the cell cycle. It has been reported that they not only play an important role in cell proliferation and maintain gene stability(13), but also have critical effects on apoptosis, metabolism, differentiation, DNA damage and repair, angiogenesis and so on(16, 17). The role of the E2F family is very complex, they seem to act as tumor suppressors or promoters depending on their environment, target genes and coenzyme factors(18).
As an important member of the E2F family, E2F2 is considered to be a transcriptional activator of the target gene of E2F. It regulates the transcriptional activity of the target gene by binding to the promoter of the target gene, and plays a key role in regulating G1 / S phase transition and the beginning of DNA replication(19). Previous studies have shown that E2F2 is an oncogene in many tumor types, for instance, it has been discovered that E2F2 is prominently up-regulated in NSCLC, and can be serve as a therapeutic target to prevent the proliferation and invasion of NSCLC(3). Quan Zhou et al. reported that overexpressed E2F2 is closely related to poor post progression survival in ovarian cancer patients, and can be used for targeted treatment and prognosis prediction(4). Additionally, Hang Song et al. provided evidence that Let-7b can inhibit the malignant proliferation of glioma cells by down-regulating the expression of E2F2(5). Similar results can be found in osteosarcoma(6), gastric cancer(7) and melanoma(8).
However, so far, there has been little research on the role of E2F2 in HCC.An experimental study reported that mir-218 and mir-520a could inhibit the proliferation of HCC cells by down-regulating the expression of E2F2(20),It implied that highly expressed E2F2 is associated with proliferation of HCC. Another study provided evidence that overexpression of mir-490-5p inhibited the metastasis of HCC cells by down-regulating the expression of E2F2 and ECT2 (21)༌this study indirectly suggests that E2F2 may be involved in the metastasis of HCC cells. Nevertheless, none of them systematically investigated the role of E2F2 in HCC. Seong Hwi Hong et al. concluded that E2F2 was highly expressed in HCC based on the data analysis of GEO database, and suggested that high E2F2 expression was associated with poor OS by kaplan-meier plot (22). Unfortunately, this study has its limitations. It did not analyze the correlation between E2F2 and other clinicopathological characteristics of HCC patients. In terms of survival analysis, E2F2 was only proposed to be associated with poor OS, other survival outcome like DFI, PFI and DSS were not considered .In addition, the most importantly, they did not use multivariate regression analysis.There have been no other reports on the value of E2F2 in predicting the prognosis of HCC.The underlying mechanism by which E2F2 is closely associated with HCC has not been elucidated completely. Our study investigated the expression of E2F2 in HCC based on TCGA database, we found that E2F2 is overexpressed(Fig. 1). Subsequently, we further analyzed the relationship between E2F2 expression and the clinicopathological characteristics of HCC patients, and the effect of high E2F2 expression on the prognosis of HCC patients. Our study revealed that high E2F2 expression was closely related to worse tumor grade, advanced tumor stage, more lymph node metastasis, and higher serum AFP value (Fig. 2, Table 2). Moreover, our study uncovered that elevated E2F2 was negatively correlated with OS, DFI, PFI and DSS (Fig. 3). Most importantly, multivariate regression analysis provided evidence that highly expressed E2F2 was strikingly associated with poor OS and DSS even after other factors were excluded, suggesting that E2F2 can independently predict the prognosis of HCC patients.
In the present study, cell cycle, RNA degradation, pyrimidine metabolism, base excision repair, aminoacyl tRNA biosynthesis, DNA replication, p53 signaling pathway, nucleotide excision repair, ubiquitin mediated proteolysis and citrate cycle TCA cycle were the major pathway regulated by E2F2 based on GSEA.
The possible role of abnormal E2F2 in the regulation of cell cycle and DNA replication in HCC have been described above. Besides, pyrimidine metabolism, p53 signaling pathway and ubiquitin mediated protease are pathways that are also closely correlated with regulation of cell cycle. p53 is by far one of the most important tumor suppressors. p53 and its target genes constitute a complex p53 signaling pathway that regulates various biological functions, such as DNA repair, cell cycle regulation, cell apoptosis, aging, and energy metabolism, in order to maintain gene integrity and prevent tumor formation. Almost all types of tumors and more than 50% of human tumor cells have p53 mutations.The p53 mutation and subsequent regulation of its target genes cause p53 signaling pathway not only lose effect of tumor inhibition, but also acquire carcinogenic functions, such as promoting cell proliferation, metastasis, anti-apoptosis, angiogenesis and metabolic changes(23). Literature has been reported that genes in the p53 signaling pathway and cell cycle signaling pathway are often mutated in HCC(24). In other words, the p53 signaling pathway and cell cycle signaling pathway are often dysregulated in HCC. Dysregulation of these signaling pathways are frequently involved in the development and progression of HCC. In addition, E2F2 has been previously reported to have regulatory effects on p53. Abnormal DNA replication in E2F1/2 knockout cells will activate the p53 pathway and then generate p53-dependent apoptosis to prevent the occurrence of tumor, but when p53 is also inactivated, it promotes tumor development.The powerful E2F-p53 regulatory axis has the function of maintaining tissue homeostasis and preventing tumorigenesis (25). Another reports suggested that the targeted inactivation of E2F1-3 leads to cell cycle stagnation at G1 / S and G2 / M, and when p53 and p21 are also inactivated, cells resume cell cycle progression and continue to grow. The inactivation of E2F1-3 activates the p53-p21 axis, they together control the process of cell cycle, and prevents the occurrence of tumors (26). Besides, as a component of many key molecules, pyrimidines are involved in important biological processes such as the synthesis of DNA,RNA, saccharides and lipid (27). Abnormal pyrimidine pathways can promote the characteristics of cancer stem cells in poorly differentiated HCC, which can be used as a potential therapeutic target for anti-HCC tumor progression(28). Additionally, ubiquitin-mediated proteolysis has the function of regulating and controlling the normal evolution of cell cycle, and the maladjustment of this pathway can lead to abnormal cell proliferation, gene instability and the occurrence of cancer(15). Studies have revealed that genes in this signaling pathway, such as HUWE1, are often mutated in HCC and are associated with the proliferation of HCC(29). Previous studies have explored the regulation of E2F2 expression on cell cycle, DNA replication and p53 signaling pathway, nevertheless, at present, no literature has revealed the relationship between E2F2 expression and pyrimidine metabolism,ubiquitin mediated protease. Our study is the first to report the regulatory effects of E2F2 on pyrimidine metabolism, ubiquitin mediated protease and p53 signaling pathway in HCC, and this regulatory mechanism needs to be further verified by experiments.
TCA cycle, also known as the citric acid cycle or Krebs cycle, is an important pathway for substance metabolism and energy supply in the human body. About two-thirds of the organic substances in the human body, including three major nutrients (sugar, fat and protein) are decomposed by TCA cycle. It is also a common pathway for the complete oxidation and decomposition of the three major nutrients to provide energy. Early studies suggested that cancer cells bypass the TCA cycle and use aerobic glycolysis, but emerging evidence suggests that some cancer cells, particularly those with maladjusted expression of oncogenes and tumor suppressors, rely heavily on the TCA cycle to produce energy and synthesize large molecules(30). In a variety of cancers, including HCC, the expression or activity levels of the TCA cycle and related enzymes are generally dysregulated, which is a pivotal driver of cancer development and progression(31, 32). In addition, wild-type P53 alos has an important effect on metabolism,the mutation of P53 will lead to the enhancement of glycolysis and the reduction of oxidative phosphorylation in tumor cells. As a result, tumor cells digest a large amount of glucose but cannot produce energy efficiently. Our study is the first to report the relationship between E2F2 and TCA cycle in HCC and it needs more work to be verified in the future.