ENO1 is a key enzyme in the glycolytic pathway and plays multiple roles in various physiological and pathophysiological processes, including the occurrence and development of tumors[10–26]. Our previous studies have shown that ENO1 is related to both HBV replication and the development of hepatocellular carcinoma. Furthermore, knockdown ENO1 could significantly inhibit the proliferation, migration, and invasion abilities of HepG2 cells. The inhibition of migration and invasion abilities in HepG2 cells may be related to the activation of the Notch signaling pathway[23, 24]. In this study, we found that silencing ENO1 can inhibit cellular proliferation in HepG2 and prolong the G2/M and S phases of the cell cycle. These results suggest that ENO1 may play a crucial role in the proliferation and cell cycle of hepatoma carcinoma cells. Up-regulated ENO1can promote various types of human cancer, such as bladder cancer cells, oral squamous cell carcinoma, and breast cancer, by regulating β-catenin [12–19, 30–33]. However, the mechanism of ENO1 regulating proliferation and cell cycle in the HCC remains unknown.Recent studies have shown that ENO1 interacts with a diverse range of proteins and plays a key role in regulating the occurrence and progression of various diseases[29, 34, 35]. However, there is no report on ENO1-interacting proteins regulating the proliferation and cell cycle of HCC.
To further clarify the role and possible mechanism of ENO1 in hepatocellular carcinoma (HCC), we used a targeted proteomic strategy combined with RNA interference (RNAi), co-immunoprecipitation (co-IP), and iTRAQ to identify the ENO1-interacting proteins from HepG2 cell line. Results showed that there were 40 proteins that interact with ENO1 in HepG2 cells, of which 15 have been reported. Functional categories of the identified proteins are mainly related to metabolic enzymes, transduction-related proteins, molecular chaperones, natural oxidant-related proteins, signaling cell skeleton-related proteins, protease-related proteins, and others.
Based on recent studies, it appears that several proteins, including PCNA, HSP90, PCBP1, PSME1, and PEBP1, may be significant factors contributing to the development, differentiation, invasion, and metastasis of malignant tumors [36–40]. In this study, the interaction of ENO1 with PCNA, HSP90, PCBP1, PSME1, and receptor (PPAR)-binding protein (PEBP1) was confirmed by Immunofluorescence microscopy and co-immunoprecipitation.
Proliferation and cell cycle rating cell nuclear antigen (PCNA) is crucial for cell replication and serves as a marker for cell proliferation, playing a vital role in both the G1 and S phases of the cell cycle[36, 41]. PCNA serves as a molecular platform for protein-protein interactions and is involved in many cellular processes, such as DNA replication, DNA repair, cell cycle control, chromatin remodeling, and sister chromatid cohesion [36, 41]. In mammals, the function of PCNA in the cell cycle can be regulated by binding with p21KIP1 protein (a protein that inhibits G1 Cyclin-CDK complexes) [42]. Additionally, PCNA can interact with cell cycle regulators, forming a quaternary complex that includes p21, a cyclin-dependent kinase (CDK), and Cyclin D (CycD) [43]. Furthermore, PCNA can serve as a docking protein to facilitate protein-protein interaction during DNA replication. The binding of CycA/CDK2 to PCNA may stimulate the phosphorylation of DNA ligase I, Replication Factor C (RFC), and Flap endonuclease-1 (FEN1) [44]. In this study, the silencing of ENO1 led to the downregulation of the expression of PCNA, Cyclin A2, Cyclin B1, Cyclin D1, and Cyclin E2, while the expression of p21 was upregulated. These findings indicate that ENO1's interaction with PCNA regulates the proliferation and cell cycle of hepatoma carcinoma cells through the ENO1/PCNA/p21 signaling pathway.
It has been reported that the ERK/MAPK/AKT signaling pathways play a crucial role in regulating cell cycle and proliferation [45]. There are three parallel MAPK signaling cascades: the JNK, ERK, and p38 MAPK pathways. It is widely acknowledged that the inhibition of both JNK and ERK pathways can suppress cell proliferation [46], while the cell cycle is regulated by the ERK and p38 MAPK pathways. ERK1/2 plays an essential role in tumor cell apoptosis, with Bcl-2/Bax serving as a downstream component in the ERK1/2 signal transduction pathway [47]. PI3K/Akt is a classical signal pathway, and its activation status is known to stimulate cell growth [48]. PEBP1 can prevent the phosphorylation and activation of MEK mediated by the RAF-1 and MAPK signaling pathways [49]. In this study, ERK1/2, p38MAPK, and AKT phosphorylation were upregulated after silencing ENO1. Results showed that ENO1 interaction with PEBP1 was also involved in the tumorigenesis of hepatoma carcinoma by regulating the phosphorylation of the MAPK signaling pathway.
Hsp90 is a crucial component of an essential cytoplasmic chaperone complex that plays a critical role in various cellular processes, including cell proliferation, differentiation, and apoptosis[50]. The full functional potential of Hsp90 is realized through its collaboration with other co-chaperones. In addition, Hsp90 interacts with a diverse set of client proteins, and the need for co-chaperones varies depending on the specific client. It has been reported that certain molecules play crucial roles in tumor development signaling pathways, including epidermal growth factor receptor (EGFR) [51], human epidermal growth factor receptor-2 (HER2), mesenchymal-epithelial transition (MET), and protein kinase B (AKT), all of which are Hsp90 client proteins [52, 53]. Hsp90 interacts with these client proteins and participates in various pathophysiological processes of cells [53]. In this study, the interaction of ENO1 with HSP90 was confirmed. Therefore, we hypothesized that ENO1 is one of the client proteins, but the mechanisms for participating in HCC progression need further study.
Poly (rC)-binding protein 1 (PCBP1, also known as hnRNP E1) is a splicing factor that has been identified as a promising tumor suppressor. It is commonly downregulated in various types of cancer and has been shown to inhibit both tumor formation and metastasis [54]. In cancer cells, the PCBP1 protein is frequently degraded or rendered non-functional due to phosphorylation [55], despite the fact that messenger RNA levels of PCBP1 are generally not significantly reduced across most types of cancer. According to reports, the excessive expression of PCBP1 has been shown to trigger apoptosis by reducing the levels of Bcl-2 [56]. PCBP1 has the ability to down-regulate the expression of various crucial elements within the TGF-β pathway[57, 58]. This indicates that PCBP1 can prevent tumor metastasis by obstructing the TGF-β pathway. TGF-βcan inhibit cell growth and early tumor formation, but in later stages, it can promote EMT, invasion, and metastasis[57]. PCBP1 plays a crucial role in regulating the alternative splicing, translation, and RNA stability of numerous genes associated with cancer[58]. Overall, PCBP1 appears to have unique tumor suppressive properties, which suggests that boosting its expression levels could be a promising strategy for treating cancer. Furthermore, PCBP1 is very similar to its family member PCBP2[56, 59]. It's interesting to note that PCBP2 seems to function as an oncogenic splicing factor, while the splicing factor heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is also frequently over-expressed in cancerous tissues [60]. This study found that PCBP1, PCBP2, and hnRNP A1 are ENO1 interacting proteins. The interaction of ENO1 with PCBP1 was further confirmed by Immunofluorescence microscopy and co-immunoprecipitation, which implied that ENO1 could regulate tumor formation and metastasis through directly interacting with various splicing factors, such as PCBP1, PCBP2, and hnRNP A1. However, ENO1 interaction with PCBP1 remains to be elucidated.