Affiliations of authors: Medicine Branch, Center for Cancer Research, National Cancer Institute, and National Naval Medical Center, Bethesda, MD.
Correspondence to: Frederic J. Kaye, M.D., National Naval Medical Center, Bldg. 8, Rm. 5101, Bethesda, MD 20889 (e-mail: fkaye{at}helix.nih.gov).
The retinoblastoma protein (pRb)/cyclin/cyclin-dependent kinase (Cdk)/p16 tumor-suppressor pathway participates in the regulation of cellular proliferation and undergoes mutational or epigenetic inactivation in essentially 100% of selected human malignancies, including lung cancer (1,2). Since this pathway is frequently altered by inactivation of either the RB gene or the upstream Cdk4/6-inhibitor gene, Cdkn2a/p16ink4a, it is commonly referred to as the RB/p16 tumor-suppressor pathway. Within this tumor-suppressor pathway, the loss of p16 function results in constitutive Cdk4/6 activation that confers aberrant pRb hyperphosphorylation. pRb hyperphosphorylation, in turn, locks the protein in an "inactive" conformation that disrupts the function of downstream cellular-binding partners, such as members of the E2F family of transcription factors. Surprisingly, the deregulation of E2F by aberrant Cdk activation appears to be similar to the deregulation of E2F observed in tumor cells that lack pRb protein but retain p16 activity. Since only one component within the pRb/p16 circuit needs to be inactivated for tumor development, this pathway has been an attractive target for novel cancer-treatment strategies.
In fact, the observation that many advanced and highly aneuploid cancer cell lines could undergo growth arrest following the ectopic expression of either pRb or p16 offered hope that gene-replacement therapy would serve as the paradigm for a rational, molecular cancer treatment (3). The predictable difficulties in optimizing gene delivery and gene expression by use of current technologies, however, have led investigators to aggressively pursue the design and synthesis of small, diffusible molecules that may instead exert a quantitative alteration in enzyme activity to restrain cell growth and, under appropriate conditions, to induce cell death (4,5). In the case of the pRb/p16 pathway, the enzymatic target has been clear: Reduce the activity of the Cdk4/6 and/or Cdk2 kinase molecules that mediate the sequential phosphorylation of the pRB product (6,7), and it may be possible to prevent the expression of a cassette of E2F-regulated genes that drive cells to initiate cell division.
The most common approach, so far, has been the identification of a group of molecules that can nonspecifically inhibit endogenous Cdk catalytic activity by competitive binding within the adenosine triphosphate (ATP) cleft of various Cdk-like proteins (8,9). In this issue of the Journal, Soni et al. (10) report that they have screened a library of potential Cdk4 inhibitors by use of a partial C-terminal fragment of pRb that contains at least two Cdk4 phosphorylation sites as substrate. A major point of this study is that the authors have developed a high-throughput screening assay by use of a 96-well format that does not require sodium dodecyl sulfatepolyacrylamide gel electrophoresis, thin-layer chromatography, or filter-binding methods (10,11). This screening effort resulted in the identification of a new pharmacologic agent, a triaminopyrimidine derivative called CINK4 (chemical inhibitor of Cdk4), which they found mediated-selective Cdk4 inhibitory activity in vitro by competitive ATP binding with a reduced ability to block Cdk6, Cdk2, or other unrelated kinase activities. Soni et al. also demonstrated the ability of CINK4 to 1) induce both G1 growth arrest and apoptosis in pRb-expressing tumor cells, depending on the dose and schedule tested, 2) inhibit pRb phosphorylation in vivo at two Cdk4 phosphorylation sites, 3) inhibit dephosphorylation at tyrosine-15 on the Cdk4 molecule, and 4) exert a modest inhibition of tumor xenograft growth in mice with the use of the HCT116 colon cancer cell line. In conclusion, the authors suggest that CINK4, or some closely related molecule, offers the future promise of important therapeutic value as a rational and selective in vivo cancer treatment.
This effort is another example of the modern era in cancer research that is focused on identifying critical molecular targets with defined biochemical properties to design new and testable models for cancer treatments. To critically assess these findings, however, a few questions should be raised. First, it would be interesting for the readers to know the origin and nature of the collection of potential Cdk4 inhibitory molecules that was screened by the authors, whether many or a few related compounds were identified in this process, and the specificity of the high-throughput screening assay for identifying validated Cdk inhibitors. Second, because the mechanism of action of these compounds is important for planning future studies, it would be more helpful to present the dissociation constant (Ki) value for CINK4 in standard ATP competition experiments and to reserve molecular modeling and docking methods as hypothesis-generating tools for further mutagenesis experiments.
Finally, although pRb is the logical substrate for the study of Cdk4-specific inhibitors (since it is, to date, the exclusive substrate for Cdk4/6 members), the analysis of its phosphorylation status in vivo is quite complex. As the authors noted, pRb contains 16 consensus sites for Cdk-mediated phosphorylation, which are thought to undergo sequential rounds of phosphorylation during the G1 and S phases of the cell cycle (12). However, the literature regarding the specificity of many of these sites for either Cdk2, cyclin E, Cdk4, Cdk6, and cyclins D1, D2, and D3 has been conflicting. Part of the confusion arises from the use of ectopic Cdk and/or cyclin products incubated with endogenous or recombinant pRb fragments. Recent data (6,7,12) suggest that the in vivo regulation of pRb phosphorylation is dependent on the orderly expression of Cdk/cyclin and a native pRb structure that includes a large pocket conformation, as well as Cdk2, Cdk4, and, perhaps, cyclin, docking domains that are located at a distance from both the pocket and the actual phosphorylation sites. The selection of serine residues 780 and 795 as sites to monitor the effects of CINK4 on pRb by Soni et al. minimizes these concerns, since there is substantial evidence that both of these residues are bona fide Cdk4 phosphorylation sites that do not seem to be dependent on earlier phosphorylation events (13,14). The experiments measuring Cdk4 inhibition on the endogenous pRb substrate, however, show an unexpected decrease in steady-state levels of pRb protein after CINK4 exposure that may contribute to the loss of phosphorylation at S780 and S795. What is the molecular basis for this finding? One explanation is that pRb is known to undergo a caspase-specific cleavage event at codon 886 during early apoptosis, resulting in the loss of a 5-kd (42-residue) carboxy-terminal fragment (15,16). This event could complicate the analysis of Cdk4 inhibition in vivo as pRb loses a critical Cdk4 docking site (17) and undergoes further degradation. Since CINK4 can induce both growth arrest and apoptosis, it would, therefore, be important to correlate the association of loss of phosphorylation at the Cdk4 sites with the onset of caspase-mediated cleavage and protein destabilization versus direct Cdk4 inhibition.
In summary, the continued search for selective small-molecule inhibitors will help, in the laboratory, to define more precisely key molecular processes in tumor growth control and, at the bedside, to offer the possibility of impacting on tumor growth and patient survival with a reduced risk to other critical homeostatic processes. Cdk4, with a substrate range that is surprisingly limited to RB and, perhaps, the related RBL1/p107 and RBL2/p130 family members, may be an ideal molecular target for selective enzyme inhibition. Other approaches to identify novel regulators of Cdk4 activity have been published recently by the use of 1) high-throughput screening of a library with 100 000 different chemicals (18), 2) the COMPARE algorithm to analyze cytotoxicity and genotype data from the National Cancer Institute (Bethesda, MD) drug screen panel to identify candidate compounds (19), and 3) a synthetic peptide-based strategy to mimic p16 binding to Cdk4 (20). In addition, Soni et al. (21) have identified previously another potential Cdk4 inhibitory compound by use of a similar strategy as outlined in the Journal.
Ultimately, the challenge will be to rigorously confirm these, and other, preclinical findings and to prioritize the most promising agents for human clinical studies. However, in our search for novel agents to rationally interrupt the functions of key molecular targets, we must also remain sober to the reality that our insight into the underlying biology of tumorigenesis is still incomplete and there will remain important questions about the molecular specificity of many of these treatments (22,23). In this new era of molecular targets in cancer clinical trials, the need for close cooperation between clinical and bench research personnel has never been greater.
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