Coordinated expression of cyclin-dependent kinase-4 and its regulators in human oral tumors.

Carcinogenesis is a multi-step process through which normal cells are sequentially transformed via the activation of proto-oncogenes and inactivation of tumor suppressor genes into their malignant derivatives, and most of these tumor-prone events occur in cellular signaling pathways involving p53 (the TP53 gene product) and pRb (the RB1 gene product) (1–4). While p53 acts as a pivotal regulator in apoptosis and senescence (2, 5), pRb functions primarily in cell proliferation, especially in progression through the G1-to-S transition of the cell cycle (1, 4). In G0 and early G1 phases, pRb is hypo-phosphorylated and forms complexes with members of the E2F family of transcription factors. These complexes sequester E2Fs and prevent their access to the promoters of proliferation-associated genes, such as cyclin B1 (CCNB1) and jun B proto-oncogene (JUNB) (1). Once committed to cell proliferation in late G1, pRb is progressively hyper-phosphorylated by cyclin-dependent kinases-4 and -6 (CDK4/6; CDK4 is the primary kinase), resulting in the entry into S phase (6). Consequently, CDK4-mediated phosphorylation of pRb is under strict homeostatic controls in order to maintain biomolecular equilibrium and promote cell cycle progression. Intricate molecular machinery and mechanisms are present in cells to modulate pRb phosphorylation. On one hand, p16INK4A (p16), a member of the INK4 inhibitors (specific inhibitors of CDK4, including p15INK4B, p16, p18INK4C, and p19INK4D), binds to CDK4 and inhibits its kinase activity, thus maintaining pRb in its hypophosphorylated E2F-associated state (6). On the other hand, CDK4 itself exhibits minimal pRb-phosphorylating activity and becomes fully functional only after being charged with cyclins, especially cyclin D1, D2, and D3 (1). Moreover, it has been reported that a number of proteins are able to up-regulate CDK4 in cells through different mechanisms (1). For example, gankyrin competes with p16 for binding to CDK4, thus blocking the CDK4-inhibitory activity of p16 (7); p34SEI1 (SERTAD1) is able to bind to the CDK4-cyclin D1 complex and enhance the latter’s kinase activity (8, 9); BMI1 (B lymphoma Mo-MLV insertion region 1 homolog, a polycomb ring finger protein) down-regulates the expression of p16 (CDKN2A) and p15 (CDKN2B) genes, which consequently promotes CDK4-mediated phosphorylation of pRb (10). In addition, while cyclin D1 physically interacts with both CDK4 and CDK2 in cells, it has been reported that in some tumor cells, elevated expression of cyclin E, a cyclin primarily binding to CDK2, is able to influence the distribution of cyclin D1 between CDK4 and CDK2 in favor of CDK4-mediated phosphorylation of pRb (11, 12). In normal cells, all the aforementioned positive and negative regulators work coordinately to ensure the control at the G1-S transition (1). Nonetheless, in most human malignancies, the balance among these regulators is impaired through diverse mechanisms, ultimately leading to the de-regulation of the pRb pathway and aberrant cell proliferation (2). While genetic inactivation of the CDKN2A/p16 gene has been found in nearly 50% of all human cancers (4), over-expression of cyclin D1 (13, 14), gankyrin (15, 16), p34SEI1 (17–19), and BMI1 (20, 21) has been found prevalently in breast, lung, liver cancers and oral squamous cell carcinoma. In regard to the fact that all these molecular events have the potential to impact CDK4-mediated phosphorylation of pRb in cancer cells, the interesting question is whether/how these molecular events are coordinated with each other in carcinogenesis. In a recent study (22) on a cohort of 30 squamous cell carcinomas of the head and neck (SCCHN) and patient-matched high at-risk mucosa (HARM, phenotypically “normal” tissues beyond the surgical resection margin and distant to the SCCHN foci), we found a positive association between down-regulation of p16 mRNA and up-regulation of BMI1 in SCCHN specimens, supporting the model that alterations in CDK4-related tumor suppressors (p16) and oncoproteins (BMI1) are coordinated in SCCHN. In the present study, we continued to investigate the potential correlations among alterations in the mRNA levels of CDK4, CCND1/cyclin D1 (hereafter, cyclin D1) CCNE1/cyclin E (cyclin E) PSMD10/gankyrin (gankyrin), SERTAD1/p34SEI1 (SEI1), and BMI1 oncogenes in SCCHN, HARM, and healthy controls. Our results revealed coordinated over-expression of these oncogenes in SCCHN and HARM. Further studies using principal component analyses (PCA) showed that the mRNA expression profiles of CDK4, cyclin D, cyclin E, gankyrin, SEI1, and BMI1 in SCCHN and HARM are distinct from those in healthy controls, but not between themselves, indicating that in spite of their differences in histology, HARM and SCCHN are similar in some molecular biological characteristics.