Alterations of pRb1-cyclin D1-cdk4/6-p16^INK4A pathway in endometrial carcinogenesis

Abstract

The retinoblastoma protein pathway (pRb1-cyclin D1-cdk4/6-p16^INK4A) participates in the regulation of the cellular processes at the transition of G1/S phases of the cell-cycle. Derailments of this pathway, caused either by lack of pRb1 or p16^INK4A expression or overexpression of cyclin D1 and/or cdk4/6, are implicated in the deregulation of the cell-cycle machinery, resulting in uncontrolled cell proliferation, tumor heterogeneity, invasion and metastasis. Several studies conducted so far have assessed the deregulation of the pRb1-pathway components in various human tumors and cell-lines, provided these pathway alterations play an obligatory role in tumorigenesis. This review briefly summarizes the current information on the pRb1-cyclin D1-cdk4/6-p16^INK4A alterations in sporadic uterine cancer, placing emphasis on the influence on the dualistic model of endometrial carcinogenesis.

Introduction

Tumor development and progression have been shown to be dependent on cellular accumulation of various genetic and epigenetic events, including alterations in the cell-cycle machinery at the G1/S checkpoint [1], [2]. Four important pathways, the p21^WAF1-p27^KIP1-cyclin E-cdk2, catherinβ-catenin-Tcf-myc-telomerase, ARF-MDM2-p53 and pRb1-cyclin D1-cdk4/6-p16^INK4A, are mediated through the appropriate function of the cell-cycle mechanisms [1], [3]. The pRb1-pathway consists of Rb1 protein, cyclin D1, cdk4/6, and p16^INK4A protein. Each of these components plays either a positive or a negative role in cell-cycle control mechanisms [4], [5]. RB1 gene, mapping to human chromosome 13q14.2, was the first TSG to be discovered and cloned, and it was associated with the development of tumor of the retina (retinoblastoma) at childhood [6], [7], [8]. Genetic studies confirmed Knudson's ‘two-hit’ hypothesis that two successive genetic alterations gave rise to retinoblastoma and the two events corresponded to inactivation of both copies of the TSG [9], [10], [11]. RB1 encodes a 110-kDa nuclear phosphoprotein (pRb1), actively participating in the transcriptional control mechanisms mediated through G1 restriction point of the cell-cycle [12]. pRb1 is permanently present within the cell nuclei, although the phosphorylation status of this protein fluctuates in a cell-cycle-dependent manner [13]. Hypophosphorylated pRb1 forms a stable complex with transcriptional activatory members of the E2F-family and this complex halts the progression of the cell cycle through G1 phase [4]. The phosphorylation of pRb1 is generally achieved by cyclin D1 complex with cyclin-dependent-kinases (4 and 6) at G1/S checkpoint. This results in the release of the pRb1-bound E2F-members and transcription of the S-phase genes [2] (Fig. 1). Conversely, expression of cyclin D1 is also dependent on the phosphorylation status of pRb1 [14]. The potential inhibitors of cdks (p15^INK4B, p16^INK4A, p18^INK4C and p19^ARF), inhibit cdk4 and, in particular, cdk6, whereas p21^WAF1/p27^KIP1/p57^KIP2-inhibitors halt the broad spectrum of various cyclins (including cyclin E) and cyclin-dependent kinases (cdk2 and cdk4/6) [15]. Interestingly, it has also been demonstrated that inactivation of pRb1 may lead to increased expression of p16^INK4A [16]. However, if the cyclin D-cdk4/6 complex is inhibited, pRb1 is in a state of low phosphorylation and is tightly bound to E2F, inhibiting its activity. According to Serrano [17], four mammalian cdk-inhibitors bind to cdk4/6 and inhibit the kinase activity of the cyclin D1-cdk4/6 complex, although the relevant feature that distinguishes each protein is connected with their different transcriptional regulation.

It has been suggested previously that the pRb1-cyclin D1-cdk4/6-p16^INK4A pathway behaves as a single mutagenic target during the process of human carcinogenesis [4]. Moreover, Michalides [18] reported that pRb1-signal transduction pathway derailments, commonly identified in various human malignant tumors and cell-lines, are achieved at each step of the hierarchy. In general, pRb1-pathway components are inactivated by several mechanisms. The RB1 gene can undergo point mutations, gene deletions, promoter hypermethylation and functional inactivation. The functional inactivation occurs through binding of viral proteins at the site of interactions with cyclins/cdks. Cyclin D1 gene amplification, inter- and intra-chromosomal recombination, loss of destabilising sequences in mRNA by deletion or alternative splicing have been documented. In addition, overexpression of cyclin D1 as a result of aberrant growth factor stimulation has been reported. cdk4/6 genes amplification and point mutations of the interaction sites with p16^INK4A are the two known mechanisms that have been identified so far. Inactivation of p16^INK4A by gene deletions, point mutations and promoter methylation at the 5′-CpG islands have been reported in the literature. In general, in most of the studies, only one or a few of the G1-components have been investigated in detail [18]. With regards to endometrial cancer, it is not as yet clear which of them are most important in the process of tumorigenesis. Finally, distinct sets of various genetic alterations within oncogenes, TSG, mismatch repair genes and growth factors have been briefly reviewed in human endometrial carcinomas [19], [20], [21], [22], [23]. Based on the Medline® database search, there is only one review by Milde-Langosch and Riethdorf [3] that has attempted to summarize the role of cell-cycle regulatory proteins in malignant human tumors of the ovary, endometrium, cervix uteri and vulva.

In this review, we briefly summarize the current knowledge on the role of pRb1-cyclin D1-cdk4/6-p16^INK4A pathway alterations in human endometrial cancer development and progression. Attention will be paid on the influence of G1 distortions of cell-cycle regulatory protein on a dualistic model of endometrial tumorigenesis.