Knockdown or inhibition of CDK4 sensitizes pancreatic cancer cells to TRAIL-induced apoptosis

Knockdown or inhibition of CDK4 sensitizes pancreatic cancer cells to TRAIL-induced apoptosis

Aims: Pancreatic cancer is lethal because of its poor response to most treatment modalities. The key factor preventing responses to therapies is the resistance of pancreatic cancer to apoptotic stimuli. The resistance to apoptosis is due to activation or upregulation of anti-apoptotic proteins. Furthermore, the distribution of cells in the different phases of the cell cycle is known to confer sensitivity or resistance towards apoptosis. In this regard, almost all genetic changes occuring in pancreatic cancer lead to an altered CDK4/6-pRb pathway resulting in a shorthened G1 phase and uncontrolled proliferation. Methods: MiaPaCa2 pancreatic adenocarcinoma cell lines were used as a model system. CDK4 was inhibited by functinal genomics using RNA interference technology or a specific chemical CDK4 inhibitor. Expression of CDK4 was determined by Western blot analysis. Cell proliferation was determined by BrdU incorporation assay. Cell cycle distribution was quantified by propidium iodide staining and flow cytometry. TRAIL-induced apoptosis was determined by hoechst staining, flow cytometry or PARP cleavage. Pro- and anti-apoptotic proteins were analysed by Western blot and RT-PCR. Results: CDK4 knockdown or inhibition led to Rb hypophosphorylation, G1 arrest and reduced cell proliferation. Furthermore, suppression of CDK4 expression by RNA interference induced caspase-dependent apoptosis in MiaPaCa2 cells. The percentage of apoptotic cells 48h after transfection analysed by hoechst and propidium iodide staining was 10–25%. In addition, sensitization towards TRAIL, but not chemotherapeutics like 5-FU and gemcitabine, induced apoptosis was detected after inhibition of CDK4. Here, a correlation between sensitization towards TRAIL and reduced protein abundance of the anti-apoptotic protein survivin after the inhibition of CDK4 was found. Conclusion: This study defines CDK4 as a potential target for therapeutic intervention. Inhibition of CDK4 results in impaired proliferation as well as in induction of apoptosis. Furthemore, this study offers a rational for a sensitizer (CDK4 knockdown/inhibition)/inducer (e.g. TRAIL) strategy for the treatment of pancreatic cancer.

Keywords: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), apoptosis, cdk (cyclin dependent kinase), cell cycle, pancreatic cancer