> Research & Grants
> Grants Program
> Research Grants
> Research Grants Awarded
Research Grants Awarded
The Potential Of Cdk1 Inhibition To Sensitize Breast Cancer Cells To Dna Damaging Treatments Through Loss Of Brca1 Function.
Previous studies in our laboratory demonstrated that depletion of either Cdk2 or Cdk1 alone did not prevent cell cycle progression in a range of cancer cell lines. Both Cdk2 and Cdk1 were able to compensate for one another. In contrast, loss of both Cdk2 and Cdk1 resulted in G2/M arrest and cell death. As Cdk proteins are key mediators of S phase progression and DNA replication, we hypothesize that there may be specific S phase functions of either Cdk2 or Cdk1 which can not be compensated for by one another and may sensitize cells to DNA damaging treatments.
Preliminary data from our laboratory demonstrated that shRNA mediated depletion of Cdk1 resulted in sensitization to a range of DNA damaging treatments in several cancer cell lines. In contrast, Cdk2 depletion did not affect DNA damage sensitivity. In addition, we have found that in the absence of Cdk1, BRCA1 is not recruited to chromatin and does not become hyperphosphorylated by ATR/ATM kinases. We therefore propose that Cdk1 inhibition could be a novel way to sensitize BRCA1 positive breast cancer cells to DNA damaging treatments by mimicking the BRCA1 mutant/loss of function phenotype. Specifically, we hypothesize that - 1. Reduced Cdk1 activity will sensitize wild-type BRCA1 breast cancer cells to DNA damaging agents, and - 2. Cdk1 depletion or inhibition will abrogate BRCA1 mediated DNA damage signaling pathways in BRCA1 wild type breast cancer cell lines
We will first measure the ability of breast cancer cell lines to survive a range of DNA damaging treatments in the presence or absence of Cdk1 by colony forming assays. Cells will be treated with shRNA to deplete Cdk1 and RO-3306 or Purvanalol to inhibit Cdk1 activity. We will combine these treatments with DNA damage - including ?-irradiation, doxorubicin, cisplatin, mitomycin C. To measure the effects of loss of Cdk1 activity on BRCA1 mediated DNA damage responses we will investigate the ability of BRCA1 to form foci by immunofluorescence after DNA damage. We will also look at a range of other DNA damage responses downstream of BRCA1 activation including checkpoint protein phosphorylation and Cdk2 activity, also the effects of Cdk1 inhibition on S phase and G2 checkpoint arrest. We will investigate the ability of Cdk1 to phosphorylate recombinant BRCA1 in vitro and mutate potential phosphorylated residues. Finally we propose to investigate if Cdk1 loss also sensitizes cells to PARP inhibitors through loss of BRCA1 function.
The successful combination of these agents with DNA damaging treatments will suggest in vivo experiments to be performed in order to justify the development of Cdk1-specific agents and ultimately novel phase I trials in the breast cancer population. If small molecule Cdk1 inhibitors are found to block BRCA1function, this will demonstrate a novel way of sensitizing breast cancers to DNA damage, also providing novel mechanisms of regulation of BRCA1 function. This work may also extend the utility of PARP inhibitors to breast cancer patients whose cancers are not BRCA1 deficient.
The multiplication of cells depends on the activity of a class of enzymes called cyclin-dependent kinases or Cdk?s. We have recently found that reducing the levels of either Cdk1 or Cdk2 proteins alone does not affect the ability of a cancer cell to multiply but if both are reduced together, the cell can no longer produce daughter cells but do not die. We found that when Cdk1 was depleted by itself, cells were extremely sensitive to conventional chemotherapies which kill cells by damaging the cells DNA and there were high levels of cell death.
Interestingly, our preliminary studies suggest that Cdk1 affects the function of the BRCA1 protein (Breast Cancer susceptibility gene 1). BRCA1 gene mutations leading to loss of function are found in 5% of breast cancer cases. Although defects in this gene are associated with the development of breast cancer, the tumors are, at least initially, sensitive to DNA damaging chemotherapy. This is because BRCA1 is part of the cell?s response to DNA damage and promotes DNA repair and hence cell survival. We therefore propose to confirm that loss of Cdk1 activity leads to loss of BRCA1 function and DNA damage treatment sensitivity. We will investigate how Cdk1 regulates the BRCA1 protein and affects its function. Then we will see if by mimicking BRCA1 loss by reducing Cdk1 activity we can make breast cancers which are not mutant for the BRCA1 gene more sensitive to chemotherapies. This will give us a greater understanding of ways in which BRCA1 protein can be targeted to improve cancer treatments. The successful combination of Cdk1 down regulation and chemotherapy will encourage the development of Cdk1 inhibitor drugs to be used for clinical trials in the breast cancer population.