Silencing of CDK2, but not CDK1, separates mitogenic from anti-apoptotic signaling, sensitizing p53 defective cells for synthetic lethality

Abstract

Small molecule inhibitors targeting CDK1/CDK2 have been clinically proven effective against a variety of tumors, albeit at the cost of profound off target toxicities. To separate potential therapeutic from toxic effects, we selectively knocked down CDK1 or CDK2 in p53 mutated HACAT cells by siRNA silencing. Using dynamic, cell cycle wide proteome arrays, we observed minor changes in overall abundance of proteins critically involved in cell cycle transition despite profound G₂/M or G₁/S arrest, respectively. Employing phospho site specific analyses, we identified uncoupled mitogenic, yet pro-apoptotic signaling from counter balancing anti-apoptotic activity in CDK2 disrupted cells. Moreover, a crucial role of CDK2 activity in early serum response was observed, extending well-established roles of CDKs outside their cell cycle regulating functions. In contrast, disruption of CDK1 only marginally affected phosphorylation events of crucial signaling nodes prior to G₂/S transition. The data presented here suggest that the temporal separation of pro- and anti-apoptotic pathways by selective inhibition of CDK2 disrupts coherent signaling modules and may synergize with anti-proliferative drugs, averting toxic side effects from CDK1 inhibition.

Keywords: CDK1; CDK2; HACAT; cell cycle; p53; phosphorylation; serine-threonine kinase; signaling pathway; tyrosine kinase.

Figure 1.

Kinetics of CDK1 and CDK2 inhibition by siRNA. HACAT cells were transiently transfected with CDK1 or CDK2 siRNA (75 nM) and non-target control siRNA, synchronized for 48 hours by serum starvation, followed by G1-release after addition of serum-containing medium. Protein levels of CDK1 and CDK2 were analyzed by western blot. Tubulin was used as loading control. The images are representative of at least 3 series of different experiments with various concentrations of siRNA (25-75nM) and time points (24 to 84 hours).

Figure 2.

Cell cycle analysis in CDK1 and CDK2-deficient cells. HACAT cells were transiently transfected with CDK1 or CDK2 siRNA (75 nM) and non-target control siRNA, synchronized for 48 hours by serum starvation, followed by G₁-release after addition of serum-containing medium. Distribution of cells into distinct cell cycle phases was analyzed by propidium iodide staining using flow cytometry techniques. Histogram plots are representative of 3 comparable experiments.

Figure 3.

Cell cycle wide dynamics of proteins and phosphorylated proteins in CDK1 and CDK2 depleted HACAT cells. Heat map analysis with k-means clustering of 248 phospho- and total proteins, expressed as log2-values, row-wise scaling. Each row corresponds to the same protein order. Expression levels of low intensities are colored in blue, high intensities are colored in yellow. The results are from one single representative experiment.