Characterisation of the cellular responses to CDC7 inhibition in breast-derived human cells

Abstract

CDC7 plays a role in DNA replication initiation and cell cycle regulation. Inhibition of CDC7 kinase by siRNA triggers a p53-independent apoptotic cell death in cancer cells but not in human fibroblast suggesting this Ser/Thr kinase as one of potential therapeutic targets for cancer treatment. To investigate the role of CDC7 kinase activity in DNA replication and cell cycle progression, we employed small molecule CDC7 inhibitors XL413 and PHA-767491 in testing the biological effects on human cell lines. We also performed high-throughput screenings with the Public Kinase Inhibitor Set (PKIS) for the purpose of finding a potential kinase inhibitor that is potentiated by XL413 or the knock-out of CDC7 regulatory subunit DBF4. Finally, we generated stably expressing TREx-293 cell lines to characterise the phenotype of overexpressing DBF4 and its fragments in human cells and to detect novel DBF4 interactors. We found that XL413 does not affect the cell growth of MCF10A but slightly restrains cell proliferation of MDA-MB-231 during a long-term treatment. XL413 delays S-phase but no effect on replication fork progression was observed. In contrast, PHA-767491 blocks DNA synthesis and prevents cells from going through mitosis to enter the next cell cycle suggesting a potential inhibitory effect of combination of kinase inhibitors. PKIS screening performed in the presence of XL413 and in the absence of DBF4 revealed several potential kinases which are involved in controlling cell proliferation. PLK1 was identified as one of those candidates. Inhibition of PLK1 causes a blockade in G2/M-phase and apoptosis. However, cells lacking DBF4 become less sensitive to PLK1 inhibition suggesting an unknown role of CDC7-DBF4 in humans. The overexpression of DBF4 or its domains does not affect cell cycle progression nor cell proliferation of TREx-293 cells. Two new DBF4 interactors were found. In summary, XL413 and PHA-767491 have distinct biological effects on human cells. PKIS screenings revealed potential kinase inhibitors that are potentiated by CDC7 inhibition. The lack of DBF4 enhances the drug resistance of human cells to PLK1 inhibitor BI2676. Novel DBF4 interactors were identified.