Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

Abstract

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.

Figure 1

A potential protein-protein non-kinase functional binding site in Cdk4 not present in Cdk2 or Cdk6. A - Relative proteomic expression by Western blotting, of Cdk4, Cdk6 and Cdk2 compared to Cdk1 on 16 human in vitro cancer cell lines of widely differing histological subtype by quantitative Western blotting as previously described [20]. Primary antibodies were Cdk1 - mouse monoclonal sc-54 (1/250), Cdk2 - (M2) rabbit polyclonal sc-163 (1/250), Cdk4 - rabbit polyclonal sc-260 (1/250) Cdk6 - rabbit polyclonal sc-260 at 1/250, (see methods). B - Upper panels: Endogenous Cdk1 follows a similar time course and increase in proteomic expression as exogenous Cdk4 induced by 1 μM muristerone in 2780 pvrg-pINDK4 clone 1D cells (see methods). Lower panels Western blotting for total retinoblastoma protein (pRb 105) and hyperphosphorylated retinoblastoma protein (p110) following exposure of 2780 pvrgpINDK4 clone 1D cells to 1 μM muristerone. C - Location of the PRGPRP central hexameric amino acid sequence (residues 249 - 260) of FPPRGPRPVQS within an externalised loop in the C' terminal region, distant from the classically described kinase domain of Cdk4.

Figure 2

Selective in-vitro cell killing of human cancer cells by exposure to linear N'-capped PRGPRP-amide and the derived cyclic amphiphilic analogue, THR53. (cyc-[FPPRGPRPVKLALKLALK]) A Photomicrographs illustrating typical in-vitro morphological appearances of RT112 human bladder cancer cells (Left hand panels) and normal human fibroblasts (Right hand panels) following exposure to 5.0 mM PRGPRP (Ac-Pro-Arg-Gly-Pro-Arg-Pro-NH₂) or control nonsense peptide. PRGPRP causes complete necrosis of RT112 bladder cancer cells but increases the confluent cell density of normal diploid fibroblasts (Magnification ×40). B. Photomicrographs illustrating typical in-vitro morphological appearances of NCI-H460 human non-small cell lung cancer cells (Left hand panels) and normal diploid human keratinocytes (Right hand panels) following exposure to 200 μM THR53 or control nonsense peptide. (Magnification × 40) C. Macroscopic appearances of clonogenic assay tissue culture wells of RT112 human bladder cancer cells exposed to 5 mM PRGPRP (left hand panels) and H460 human non-small cell lung cancer cells exposed to 200 μM THR53 (right hand panels).

Figure 3

Clonogenic cell survival assays of RT112 human bladder cancer cells and H460 human non-small cell lung cancer cells exposed to PRGPRP vs PRRPGP as linear hexapeptides or in cyclic amphiphilic cassettes. A. Comparison of response of RT112 human bladder cancer cells to PRGPRP and THR53. B. Clonogenic cell survival of H460 human non-small cell lung cancer cells to THR53 (cyc-[FPPRGPRPVKLALKALAK]); THR54 (cyc-[PRGPRPVALKLALKLAL]) and THR79 (cyc-[PRGPRPvalklalkalal]). (Capitals signify L-amino acids, lower case signify D-amino-acids). C. Comparative clonogenic cell survival of RT112 human bladder cancer cells exposed to the linear end-capped hexapeptides PRGPRP or PRRPGP. D. Comparative clonogenic cell survival of H460 human non-small cell lung cancer cells exposed to the cyclic amphiphilic peptides: THR 53 (cyc-[FPPRGPRPVKLALKALAK]) or THR 53C (cyc-[FPPRRPGPVKLALKALAK]).

Figure 4

Time course of cell death and morphological appearances of a range of cancers of different histologies exposed to 200 μM THR53. A. Time course of loss of viability in H460 human non-small cell lung cancer, H1299 human non-small cell lung cancer and MCF-7 human breast cancer cells exposed to 200 μM THR53. MRC5-hTERT cells were unaffected by 200 μM THR53. Viability assessed by Trypan Blue exclusion of MRC5-hTERT, H460, H1299 and MCF-7 cells in the presence or absence of the compound, trypsinised off and stained at the appropriate time point. Each data point is the mean of results from triplicate wells (Solid lines - Live cells, Broken lines - Dead Cells; Blue - Control, Red - THR53 200 μM). B. Morphgological appearances of ubiquitous response to 200 μM THR53 of: SK-BR-3 human breast cancer, H460 human non-small cell lung cancer, SW620 human colorectal cancer and G361 malignant melanoma cells. C. Side by side quantification of H460 and MRC-5 cell viability assays at a single, 7 day, time point comparing THR53 (PRGPRP in cyclic amphiphilic cassette) and THR53C (PRRPGP in cyclic amphiphilic cassette) after initial exposure to compounds at a concentration of 200 μM.

Figure 5

Time Lapse Photomicrography of MRC5-hTERT, RT112 human bladder cancer and H5460 human non-small cell lung cancer following exposure toTHR53. Cells were incubated in Ham's F12 + 10% FCS (control medium) or medium + 200 μM THR53. Morphological changes of vacuolation + cellar swelling are seen in RT112 at 33 hours after exposure to THR53 and in H460 at 90 hours after exposure to THR53. There was no evidence of cell blebbing or fragmentation. (see also videos)

Figure 6

THR53 cancer cell killing requires cells to have entered the cell division cycle, does not cause perturbation of cycling cells but does alter relative Cdk1/Cdk4 co-expression. A. Macroscopic appearances of cycling THR53-treated MCF-7 human breast cancer cells in medium + 10% FCS (left hand panels) compared to the same cells held outside the cell cycle in medium + 0.1%FCS (right hand panel) B. DNA histograms of H460 human non-small cell lung cancer cells exposed to THR53 or an autophagy-inducing dose of 50 μM chloroquine. C. Sequential Western blots of Cdk1 and Cdk4 expression in RT112 human bladder cancer cells at 0 - 10 days after exposure to 5 mM linear, end-capped, PRGPRP. D. Western blot of Cdk1 and Cdk4 expression in H460 human non-small cell lung cancer cells 24 hours after exposure to 100 μM and 200 μM THR53.

Figure 7

Induction of changes in mitochondrial function, total cellular protein, ATP activity and autophagy in H460 human non-small cell lung cancer cells following exposure to THR53 or chloroquine. A. Treatment with THR53 showing selective inhibition of ATP at 96 hours after initial exposure. B. Treatment of H460 with Chloroquine results in non-specific changes in all cell death parameters at 96 hours after initial exposure. C. THR53 treatment of H460 non-small cell lung cancer cells does not induce autophagy.