A-1. Definition of the RB-Pathway
The RB-pathway that is discussed in this article consists of five families of proteins () – CDKN (e.g., Ink4a), D-type cyclins, cyclin-dependent protein kinases (cdk4, cdk6), RB-family of pocket proteins (RB, p107, p130), and the E2F-family of transcription factors (heterodimers of E2F1–7, DP1, 2). This pathway plays a central role in the regulation of cell proliferation as its constituents are activated and/or inhibited by growth-promoting as well as growth-suppressing signals. Furthermore, several components of this pathway, i.e., p16Ink4a, cyclin D1 and RB, are frequently altered in cancer cells including, the deletion/silencing of the p16Ink4a locus, the amplification of the cyclin D1 focus, and the bialleleic mutation of the RB1 gene. Thus, components of this RB-pathway are rational targets in cancer therapy.
The RB-Pathway in Cancer Therapy
The functional interactions among the five families of proteins in this pathway are well established. The Ink4-family of proteins, p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d are small heat-stable proteins containing the AKN (ankyrin repeat) domain. Each of the Ink4 proteins can bind to and inhibit the activity of cdk4 and cdk6. The cdk4/6 are D-cyclin-dependent protein kinases. Each of the D-cyclin proteins can associate with cdk4 or cdk6 to form the active kinase complex. The Ink4 proteins compete with the D-cyclins for cdk4/6 to prevent the formation of the active kinase complex. During regulated cell proliferation, the complex of D-cyclin/cdk4/6 is activated as cells respond to mitogenic signals and commit to cell cycle entry. The major cellular targets of the D-cyclin/cdk4/6 complexes are the RB-family of pocket proteins, which contain multiple peptide-binding pockets and assemble nuclear protein-complexes to regulate chromatin structures and transcription factor activities. The RB-family proteins are recruited to specific promoters through their interactions with sequence-specific DNA binding proteins. In the pathway discussed here (), the critical interactions are between the RB-pocket proteins and the E2F-family of transcription factors. When recruited to E2F-regulated promoters, RB-pocket proteins inhibit transcription by directly suppressing the transactivation function of E2F and by recruiting factors that mediate transcriptional repression. Phosphorylation of the RB-pocket proteins by D-cyclin/cdk4 and 6 invariably disrupts the RB•E2F interaction, leading to the activation of E2F-regulated gene expression. E2F binds to and regulates the promoters of multiple genes involved in cell cycle progression (e.g. cyclin E and cyclin A), nucleotide biosynthesis (e.g. thymidylate synthase and ribononucleotide reductase), DNA replication (e.g. MCM7 and cdc6), and mitotic progression (e.g. cyclin B1 and cdk1). As will be discussed below, E2F also stimulates the expression of pro-apoptotic genes (e.g., caspases and Apaf-1) (), and thus alterations in the RB-pathway can affect tumor cell response to cytotoxic agents.
A-2. Alterations in the RB-Pathway in Cancer Cells
Cancer researchers have been interested in the RB-pathway because it is consistently altered in cancer cells to promote deregulated cell proliferation. In this pathway, the Ink4-family and the RB-family proteins function as tumor suppressors, whereas the D-cyclins, cdk4/6 and E2F promote tumor cell proliferation. Recently, a comprehensive analyses of the genome and transcriptome of 206 primary glioblastoma tumors together with the selected sequencing of 601 genes in 91 of the 206 tumor samples have shown that the RB-pathway is altered in 78% of the primary glioblastoma tumor samples. These alterations in the RB-pathway include homozygous deletion and mutation of CDKN2A
(p16Ink4a) and RB1
(RB) in 52% and 11% of the samples, respectively, and homozygous deletion of CDKN2B
(p15Ink4b) and CDKN2C
(p18Ink4c) in 47% and 2% of the tumor samples, respectively. On the other hand, the CDK4
(cyclin D2) genes are amplified in 18%, 1% and 2% of the glioblastoma tumors examined (1
). Taken together, the frequent yet distinct alterations of components of the RB-pathway in cancer raise the possibility for rationally designed therapeutic strategies that exploit defects in this pathway.
A-3. Role of RB-Pathway in Cellular Responses to Genotoxins
Cytotoxic chemotherapeutic agents and ionizing radiation remain the mainstay therapeutic approaches in the treatment of cancer. These agents almost always cause DNA damage, and the molecular mechanisms underlying the cellular response to genotoxic stresses have been the subject of intense research (2
). In this context, it is well appreciated that the RB-pathway is regulated at multiple points to instill the appropriate cell cycle inhibition that is induced by DNA damage. For example, cyclin D1 is rapidly degraded following DNA damage (4
) (). Correspondingly, blockade of cyclin D1 degradation in damaged cells leads to aberrant cell cycle progression that is associated with a breakdown in genome integrity (4
). The degradation of cyclin D1, together with p53-mediated induction of p21Cip1 and the activity of protein phosphatases cause the dephosphorylation and activation of RB to block cell cycle progression (). The bialleleic loss of RB1
results in a proclivity of deregulated DNA replication in the presence of DNA damage, and thus resulting in additional secondary DNA lesions and enhanced cellular death (6
). In addition to cyclin D1 and RB, p16ink4a is implicated in enforcing senescence-like growth arrest in response to the DNA damage (8
). Together, several interesting features of the RB-pathway have emerged from these analyses: First,
the amplification of cyclin D1 does NOT equate with RB loss in the context of DNA damage response, because the overproduced cyclin D1 can still be efficiently attenuated through proteolytic degradation (5
RB loss may affect the DNA damage response in ways that are not found with either the loss of p16ink4a loss or the gain of cyclin D1. In other words, RB function can be controlled by factors beyond those comprising the canonical RB-pathway (9
). Thus, in the context of genotoxic response, the individual components of the RB-pathway are important, yet their defects are likely to have common as well as distinct biological consequences.
A-4. Role of RB-Pathway in Cellular Responses to Anti-Mitogens
There is a growing class of therapeutic agents that target intrinsic oncogenic or growth stimulatory pathways that are required for tumor maintenance or growth. As such, the plethora of studies that evaluated the importance of the RB-pathway in anti-mitogenic signaling (10
) may have applicability to therapeutic agents that perturb these pathways. In general, attenuation of mitogenic signaling results in reduced cyclin D1 levels, limited CDK4/6 activity, and the resultant dephosphorylation/activation of RB (). In this context, cyclin D1 is typically down regulated via a combination of transcriptional and post-transcriptional mechanisms (12
). Some anti-proliferative stresses also cause the proteolytic turnover and/or the nuclear exclusion of cyclin D1 (12
). Importantly, formation of the cyclin D1-CDK4/6 complex is also dependent on mitogenic signaling. Because of these multiple mechanisms of regulation, the functional impact of deregulated cyclin D1 expression can be highly context dependent.
Unlike cyclin D1 and Cdk4/6, p16ink4a is not generally responsive to mitogenic factors and it is not strongly implicated in the response to antimitogenic perturbations. The loss of RB alone is not sufficient to render cells mitogen-independent, in part due to the activity of the RB-related pocket proteins p107 and p130 that can inhibit cell cycle progression via compensatory mechanisms (13
). However, the deletion of RB can limit the effectiveness of specific anti-proliferative signals. For example, TGF-beta mediated cell cycle arrest and the anti-proliferative effect of ERK-inhibitors are largely dependent on RB in simple genetic models (15
). Thus, there are clear distinctions through which the RB-pathway functions in relation to anti-mitogenic signaling that would be expected to modulate therapeutic response and have implications for clinical response.