The progression of the cell cycle is regulated by many factors. There are two main regulation points: one at the G1/S restriction point, which controls cells from entering the S phase, and the other at the G2/M turning point. Non-proliferative cells are in the G0 phase, and when stimulated by growth factors, etc
., they can enter the S phase through the G1/S restriction point, exiting the G1 phase. In mammalian cell cycle regulation, the regulation points between G1-S phase are key points for the control of cell proliferation. Abnormalities in these regulation points are closely related to the formation and development of tumors. Our previous work had found that Lewis y overexpression significantly reduces ovarian cancer cell number in G0-G1 phase, and significantly increases the percentage of cells in S and G2 phases compared to that in untransfected cells [1
], indicating that the cell cycle is in a proliferative state. In this study, we found that the mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E significantly increased in Lewis y-overexpressed cells and that the mRNA and protein expression levels of CDK2, CDK4 and CDK6 did not change. Cyclin A, E and D1 are positive regulatory proteins that promote G1/S transition in the cell cycle, and CDKs are the core of the cell cycle regulatory network. Different cyclin proteins bind to their corresponding CDKs and form corresponding cyclin-CDK complexes to activate the activities of CDK kinases, and they are thus involved in cell cycle regulation. By binding to CDK2 to form a complex, cyclin A initiates S-phase DNA replication to complete the S phase, and assists in G2/M phase transition [17
]. In our in vitro
culture system, the cyclin A mRNA and protein expression levels in Lewis y-overexpressed cells significantly increased compared to those in the untransfected cells, which was consistent with the increased proportion of cells in the G2/M phase. Cyclin D1 binds to corresponding CDK4 or CDK6 to form a complex, activates Rb proteins, initiates the transcription of S phase-related genes, and then allows the cells to enter into an autonomous division and proliferation program, resulting in CDK kinase activation and the cell division entering S phase from G1 phase [18
]. Currently, cyclin D1 protein overexpression has been found in the majority of tumors (breast, gastric, esophageal and ovarian cancer, and B-cell lymphoma). Cyclin E mainly forms a complex with CDK2 to drive the G1-S phase, initiate DNA synthesis in S phase, and promote cell cycle progression [19
]. Gene amplification and protein expression disorder of cyclin E can be found in breast, colon, gastric and ovarian cancer, among others [20
]. This study found that the increased mRNA and protein expression levels of cyclin D1 and cyclin E caused by Lewis y overexpression were consistent with the decreased proportion of G1 phase in ovarian cancer cells resulting from Lewis y overexpression. After the cells were treated with anti-Lewis y monoclonal antibody, the mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E were significantly reduced, which further confirmed our conclusions.
Negative regulatory factors also play important roles in cell cycle control. At present, many studies have shown that p16, p21 and p27 most likely directly act on the regulation of the G1/S phase restriction point [21
]. P16 can bind to CDK4/6, and the cyclin D1-CDK complex, thereby inhibiting the phosphorylation of Rb proteins and the release of transcription factor E2F, which in turn induces G1-S phase arrest. The P21 protein is an inhibitor that binds to almost every cyclin-CDK complex, and makes the complex lose its kinase activity, which leads to cell cycle arrest in G2 phase. P27 arrests the cell cycle in G1 phase and inhibits cell proliferation by binding to the activated cyclin-CDK2 complex and cyclin-CDK4 complexes and then inhibiting their activities. In this study, it was found that in Lewis y-overexpressed cells, both mRNA and protein expression levels of P16 and P21 were reduced; although reduction in P27 mRNA was not obvious, its protein level was significantly decreased. After the cells were treated with anti-Lewis y monoclonal antibody, the above changes were reversed, which further confirmed that the promotion of G1-S transition by Lewis y was related to the inhibition of p16, p21 and p27.
The synthesis, assembly, modification and activation, disassembly, degradation and conversion of cyclins, CDKs, and CKIs are the core mechanisms that lead to the progression of cell cycle phases and the phase transitions and are regulated by signal transduction from extracellular growth factors. These signaling molecules, including mitogens and anti-mitogens, transduce the corresponding signals downward after binding to their receptors, and ultimately affect the cell cycle through a complex network system. The PI3K/Akt and ERK/MAPK signal transduction pathways are important pathways for cell survival and apoptosis inhibition. Studies have shown that IGF-1 upregulates cyclin D1 and cyclin E expression via the MAPK signaling pathway [24
]; p21 is a direct substrate for Akt proteins, and activated Akt phosphorylates the Thr145 site in p21 proteins, which promotes the extranuclear transport of p21, leads to the accumulation of the proteins in cytoplasm, and thus facilitates p21 degradation [25
]. TGF-β is an anti-mitogen that can arrest cells in late G1 phase, which is caused by p27 binding to the CDK2-cyclin E complex and inhibiting the activity of the latter [26
]. The ERK/MAPK and PI3K/Akt signaling pathway inhibitors were used in the transfected cells for corresponding inhibition, and it was found that the differences in cyclin and CKI expression levels were significantly reduced. Combined with our previous findings that Lewis y overexpression leads to a significant increase in phosphorylation levels of Akt and ERK1/2 [5
], increase in Lewis y antigen, as a part of the structure of IGF-1R, EGFR and other cell surface receptors [6
], not only activates the receptor tyrosine kinase activities but also further activates its downstream PI3K/Akt and Raf/MEK/MAPK signaling pathways. We speculate that increased expression of Lewis y antigen accelerates gene transcription in nuclei by activating the above pathways, stimulates DNA synthesis, alters expression levels and activities of cycle-related proteins, and ultimately promotes cells to skip the G1 phase restriction point and enter into S phase for cell proliferation.
The results of this study provide a detailed theoretical basis of how Lewis y overexpression causes accelerated proliferation of ovarian cancer cells. Lewis y regulates the expression of cell cycle-related factors through the ERK/MAPK and PI3K/Akt signaling pathways, thereby affecting cell proliferation.