Background: Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor.
Results: A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy.
Conclusion: These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic biomarker and a putative functional site for kinase-unrelated activities of Cdk4.
Bibliographical noteWe are grateful to Mikhail Lomosonov for Western blotting of Cdk1/Cdk4 in RT112 cells, Bill Primrose (Theryte Ltd) for designing THR79, Kyla Grimshaw (Horizon Discovery Services, 260 Cambridge Science Park, Milton Road, Cambridge, CB4 0WE) for some of the clonogenic assays and photomicrography, Kyla Grimshaw and John Goodall for carrying out sulphorhodamine, Alamar Blue, Cell Titre-Glo and autophagy assays and Amanda Howarth for data analysis and preparation of figures.
This work was supported by the Cancer and Polio Research Fund, Hoylake, Wirral, UK and Theryte Ltd., William Russell House, The Square, Lightwater, Surrey, GU18 5SS, UK.
- programmed cell death
- selective anticancer
- broad spectrum
- SKIN TUMOR-DEVELOPMENT
- HUMAN CANCER-CELLS
- KERATINOCYTE PROLIFERATION