The biology of the CAK complex and a functional analysis of its role in cancer

Abstract

Deregulation of normal CDK activity can lead to cell transformation through enhanced growth signalling. CDKs are therefore attractive drug targets but the propensity for CDKs to functionally compensate as a failsafe mechanism makes selecting the most efficient CDK to inhibit a difficult task. Many drugs aimed at inhibiting CDK action are non-selective, eliciting pleiotropic effects and therefore a more selective approach may be advantageous. Given its dual importance in both the cell cycle and transcription, CDK7 has recently become the focus for drug development. Previous work from the group has identified a CDK7-specific small molecule inhibitor, named BS181, which inhibits cancer cell growth in vitro and in vivo and leads to cell cycle arrest and apoptosis. A second compound, named BS194, is a selective inhibitor of CDK1, CDK2, CDK5 and CDK9. siRNA-mediated knockdown of each component of the CAK complex was undertaken in HCT116 colorectal cancer cells and verified by western blotting and qRT-PCR. Cell cycle FACS analysis showed an increase in apoptosis when CDK7, cyclin H or MAT1 were knocked-down. Western blotting revealed a curious relationship between each member of the CAK complex; knockdown of any one component of the CAK complex resulted in a reduction in levels of the other components. As these effects were not observed at the mRNA level, these findings are indicative of ubiquitin/proteasome-mediated degradation. Finally, a gene expression microarray was performed on RNA preparations made from HCT116 cells treated with BS181 or BS194. It is clear that the two inhibitors elicit their effects in subtly different ways leading to distinct genes and pathways being perturbed. Although p53 signalling is a common theme, it appears that at the concentrations used, the two agents modulate this axis with different kinetics. I conclude that, contrary to the potential advantages of selectively inhibiting CAK, pan-inhibition with an agent capable of targeting several CDKs may prove more efficacious in cancer therapy.