BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)
Cells divide through a systematic and precisely regulated process with the ultimate goal of producing viable daughter cells that each possesses a set of faithfully duplicated chromosomes (Fig. 7.1). The majority of cells exist in G0 phase of the cell cycle, which is also known as quiescence or senescence. In the quiescent state, cells no longer replicate but have the potential to re-enter the cell cycle, whereas senescence refers to a cellular response to various types of stress (e.g. DNA damage, oncogene activation, oxidative stress, etc.) in which a cell is primarily arrested in G1 phase and has irreversibly lost the capability to replicate [48, 4]. The ability of cells to enter senescence in response to oncogene activation is believed to be a potential barrier to tumorigenesis . Upon receiving mitogenic signals, a cell leaves G0 phase and enters G1 phase in which there is growth in preparation for S phase. In S phase, DNA is replicated with high fidelity, and is followed by G2 phase where cells continue to grow and make final preparations for M phase where mitosis and later cytokinesis occur. Depending on the cellular and signaling milieu, cells may either return to G1 phase to continue dividing or enter G0 phase [49, 83, 112].
Regulation of the cell cycle: cyclin dependent kinases
The cell cycle is tightly regulated by a series of serine/threonine kinases known as cyclin-dependent kinases (CDKs) that form heterodimers with regulatory cyclins [83, 112]. According to the “classical” model, each phase of the cell cycle is controlled by the cyclic expression and activation of specific cyclins and CDKs
Fig. 7.1. Phases of the cell cycle. G0 represents quiescent or senescent cells. Upon receiving mitogenic signals, cells enter G1 phase and proceed through S, G2 and M phases. Cells may then either re-enter G1 phase to continue dividing or enter G0 phase. Based on the “classical” model of the cell cycle, each phase is controlled by the expression of specific cyclins and cyclin-dependent kinases. In addition, cyclin-dependent kinase inhibitors such as p16INK4a and p21Cip1/Waf1 play important roles in helping to regulate the cell cycle. CDK cyclin-dependent kinase
Fig. 7.2. The retinoblastoma protein (?pRb) pathway. The transcription factor E2F is normally bound to and repressed by pRb. E2F plays a critical role in controlling the transcription of numerous genes involved in cell cycle regulation, apoptosis and maintaining genome stability. In addition, E2F is also involved in regulating chromatin structure and in promoting senescence. CDK4 and CDK6 that have been activated by D-type cyclins phosphorylate pRb which causes the release of E2F and transcription of E2F target genes. A positive feedback loop exists where E2F-mediated transcription leads to increased levels of A-type cyclins and eventual activation of CDK2. Activated CDK2 then further phosphorylates pRb leading to release of additional E2F and passage through the “restriction point” of the cell cycle. In contrast, a negative feedback loop also exists where E2F activation leads to increased pRb levels, via transcription of the RB1 gene, and sequestration of E2F. CDK inhibitors p16INK4a and p21Cip1/Waf1 play pivotal roles in regulating the pRb pathway by inhibiting CDK4/CDK6 and cyclin-CDK2/CDK1 complexes, respectively. In melanoma, prominent alterations in the pRb pathway are seen and include loss of p16INK4a and amplification of cyclin D1, CDK2, CDK4 and CDK6. CDK cyclin-dependent kinase, P phosphorylation, pRb retinoblastoma protein (Fig. 7.1). In response to mitogenic signals, D-type cyclins are expressed in early G1 phase and activate CDK4 and CDK6. Activated CDK4 and CDK6 then phosphorylate retinoblastoma protein (pRb) causing the release of transcription factor E2F, which is normally bound to and repressed by pRb (Fig. 7.2). This allows E2F to proceed with transcription of target genes including E-type and A-type cyclins. Expression of E-type cyclins during G1 phase activates CDK2, which then further phosphorylates pRb leading to amplification of E2F-mediated transcription. These steps ultimately result in G1 to S phase transition and passage through the “restriction point” at which point the cell has committed to cellular division. During S phase, CDK2 associates with A-type cyclins to allow for progression from S to G2 phase. Eventually, CDK1 binds to A-type cyclins to initiate mitosis (G2 to M phase). A-type cyclins are degraded during mitosis and CDK1 then binds to B-type cyclins to complete mitosis.
Regulation of the cell cycle: cyclin-dependent kinase inhibitors
In addition to its regulation by cyclins, CDK activity is also regulated by two families of specific CDK inhibitors [22, 83, 112, 144]. The first family consists of the INK4 proteins (p16INK4a, p15INK4b, p18INK4c, p19INK4d) that inhibit CDK4 and CDK6 during G1 phase and therefore primarily affect the pRb pathway . In addition to its role in promoting cell cycle arrest, p16INK4a has also been associated with cellular aging and senescence particularly in melanocytes, however the exact role of p16INK4a in promoting cellular senescence is still debated [91, 53, 22, 46, 121]. The second family consists of the Cip/Kip family of proteins (p21Cip1/Waf1, p27Kip1, p57Kip2) which inhibit CDK2 and CDK1 when complexed with E-type, A-type and/ or B-type cyclins . Inhibition of CDK2 leads to decreased pRb phosphorylation and sequestration of E2F. In addition, p21Cip1/Waf1 further antagonizes pRb function by promoting proteosomal degradation of pRb . Of note, levels of p21Cip1/ Waf1 are under the transcriptional control of activated p53 that utilizes p21Cip1/Waf1 to arrest the cell cycle and to activate senescence pathways [140, 121, 94].
Regulation of the cell cycle: retinoblastoma protein pathway
The retinoblastoma gene family consists of three members and encodes for the proteins pRb, p107 and p130 [56, 20, 29, 51]. Of these three proteins, pRb (encoded by the RB1 gene) has been extensively studied due to its key role in regulating the cell cycle and in functioning as a tumor suppressor gene. It is a 928 amino acid protein that consists of tandem cyclin fold regions separated by spacers and a C-terminal domain. These domains form a “pocket” which is the basis of pRb function. Targets that interact with the pRb pocket include E2F transcription factors and regulators of pRb, such as CDK-cyclin complexes. The affinity of the binding pocket is regulated by post-translational modifications, most commonly phosphorylation of serine and threonine residues in N-terminal and C-terminal domains and in spacer regions, which alter the conformation of the pocket and the binding affinity for specific targets.
CDK inhibitors such as p16INK4a and p21Cip1/Waf1 also play critical roles in regulating pRb function by directly inhibiting CDK4/CDK6 or inhibiting cyclin-CDK2/ CDK1 complexes, respectively (Fig. 7.2) [56, 22, 20, 1, 29, 51, 144]. Feedback loops exist that also regulate pRb function . Phosphorylation of pRb releases E2F and allows for transcription of E-type and A-type cyclins that leads to further phosphorylation of pRb via CDK2. This positive feedback loop allows the cell to progress through the “restriction point” of the cell cycle. However, E2F that has been freed of pRb repression also initiates a negative feedback loop by promoting RB1 gene transcription. This results in an increase in pRb levels, sequestration of E2F and concomitant downregulation of E2F. Epigenetic signaling may also play a role in regulating pRb activity specifically through promoter hypermethylation and silencing of the RB1 gene .
Despite the key role played by pRb in regulating the G1 phase of the cell cycle, it is interesting to note that control of cell cycle arrest requires cooperation between pRb and p53 as shown by the fact that RB null mouse embryonic fibroblasts still transition from G1 to S phase but arrest in G2 phase under conditions of serum starvation due to upregulation of p21Cip1/Waf1 via p53 . Other studies have shown that combined heterozygous loss of pRb and p53 result in the development of a wider range of tumors compared with mice with heterozygous pRb loss alone . Small cell lung cancer (SCLC) is induced in the lung epithelium of mice deficient in pRb and p53, while mice with CDKN2A loss and functional inactivation of pRb and p53 via loss of p16INK4a and p19ARF (mouse homologue of p14ARF in humans) respectively, develop sarcomas and lymphomas [115, 90]. Another study showed that over 75% of melanoma cell lines had defects in both the p53 and pRb pathways . These results highlight the importance of the interaction between the pRb and p53 pathways and the potential role of pRb in tumor initiation and the critical role played by p53 in acting as a failsafe cell cycle checkpoint [79, 82].
In addition to regulation of the cell cycle, pRb plays a role in several other related cellular functions. pRb is known to bind factors that regulate chromatin structure such as DNA methyltransferases, histone methyltransferases, histone demethylases and histone deacetylases [29, 51]. Through chromatin modification and interaction with E2F, pRb plays a fundamental role in regulating the transcription of an array of genes. In addition, pRb may play a role in promoting cellular senescence, through chromatin remodeling and formation of senescence associated heterochromatin foci, and in regulating apoptosis through E2F-1 which can transcribe genes necessary for apoptosis such as APAF-1 [29, 51, 121]. Another essential role of pRb that has been recently brought to light is its ability to help maintain genomic stability and to prevent aneuploidy . Loss of pRb is associated with accumulation of DNA damage and with defects in the mitotic spindle, kinetochores and centrosomes. It is believed that dysregulation of E2F and its target genes such as MAD2 may help explain some of these mitotic defects.
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