Dysfunction of cell cycle signaling is one of main features in NP carcinogenesis [10, 19]. CDK4, a member of the cyclin-dependent kinase family, is a key factor of cell cycle signal affecting cell cycle progression and its overexpression has been described in many tumors, including NPC. However, CDK4-mediated molecular mechanisms linked to the initiation and development of NPC are not completely understood.
In previous studies, CDK4 had been shown to promote cell proliferation by driving cell cycle progression [20–26]. To understand the biological functions of CDK4 in NPC, we first constructed NPC cells with stable suppression of CDK4 protein. We observed that knocking down CDK4 inhibited cell growth and G1 to S phase cell cycle progression. Our results are similar with these previous reports of CDK4 function in other tumors. CDK4 mediating miRNA expression to modulate the pathogenesis of NPC was not been reported. In this study, we examined the expression of tumor-suppressive miRNA let-7c by qPCR in NPC cells after knockdown of CDK4. Interestingly, let-7c expression was significantly upregulated after suppressing CDK4 expression which strongly suggested that CDK4 regulated let-7c expression in NPC. In previous reports, CDK4 was observed to be a nuclear expressed protein that forms a complex with CCND1, CDK6, and p21, and modulates the expression of pRB activating transcription factor E2F1 . In further analyses, predicted binding sites of transcription factor E2F1 were found in the putative let-7c promoter, suggesting that E2F1 might modulate let-7c expression. Therefore, we suspected that knocking down CDK4 stimulated let-7c expression might be attributed to the suppression of E2F1. Consistent with this expectation, we discovered that knocking down CDK4 elevated tumor suppressor p21 and inactivated oncocogenic factors CCND1, CDK6, and E2F1 in NPC cells. Subsequently, we observed that inhibiting E2F1 by specific siRNA increased the expression of let-7c in NPC cells.
Let-7c has been identified as a tumor suppressor in some tumors [15, 16]. However, its roles in NPC have not been yet reported. In this study, we found that let-7c was significantly decreased in NPC tissues compared to nasopharyngeal tissues. Further, we observed that let-7c inhibited cell growth, migration, and invasion of NPC cells. These results suggested that let-7c functions as a potential tumor suppressor in NPC. In a previous study, let-7c was reported to directly target C-Myc-mediated CDK4 suppression blocking cell growth in some tumors. In this investigation, we observed that p15 and p16 [28–31], two tumor suppressors that are upstream regulators of CDK4  were positively modulated by let-7c, whereas CDK4 and E2F1 were negatively regulated by let-7c in NPC cells. These results suggested that let-7c suppressed cell growth through p15/p16/CDK4/E2F1 signaling in NPC. More interestingly, a positive feedback loop of CDK4-E2F1-let-7c was observed, which was similar to our previous report for CTGF-C-Jun/C-Myc-miR-18b in NPC which promoted NPC pathogenesis .
Increased expression of CDK4 has been reported in NPC . However, the correlation of CDK4 expression with clinical features and prognosis of NPC has not been documented. In this study, we observed that CDK4 was mainly coexpressed in the nucleus and cytoplasm in lung cancer and normal lung tissues. Furthermore, we found that total protein levels of CDK4 were overexpressed in NPC tissues compared to normal NP tissues. These results were analogous to our previous reports in lung cancer , suggesting that CDK4 participates in the pathogenesis of NPC.
CDK4 expression patterns had been reported to be associated with clinical pathology parameters in some tumors including lung cancer, osteosarcomas, colorectal cancer, and chondrosarcomas [12, 33–35]. In this study, we observed that overexpressed CDK4 was positively associated with clinical stage, but not correlated with patient's age, sex, smoking, or T classification, N classification, and M classification in NPC. Further, we observed that the level of CDK4 protein expression was significantly correlated with the overall survival of NPC patients. Patients with higher levels of CDK4 expression had poorer survival rates than those with lower levels of CDK4 expression. NPC is highly sensitive to radiotherapy. In this study, we also observed that NPC patients with the treatment of radiotherapy had markedly better overall survival rates than those without the treatment of radiotherapy, which indicated the significance of radiotherapy for NPC patients. Finally, we found that although CDK expression was not significantly associated with overall survival of NPC patients according to univariate analyses, it seemed that its overexpression showed a tendency as an independent prognostic factor for NPC patients regardless of its patients' disease status based on multivariate analyses.