Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, about 43,920 people will be diagnosed with pancreatic cancer and about 37,390 people will die of this disease. Unfortunately, because of high fatality rates, pancreatic cancer incidence rates are almost equal to mortality rates. About 90% of pancreatic tumors bharbor activating mutations in the KRAS2 oncogene. Furthermore, 95% of pancreatic tumors display inactivation of the CDKN2A gene (encoding the protein inhibitor of cyclin-dependent kinase 4), resulting in the loss of p16 protein, an important regulator of the G1 to S phase cell-cycle transition.
Cdc7 is a highly conserved serine-threonine kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint pathway, and has an important role in promoting a proper response to DNA damage in eukaryotic organisms. Downregulation or inhibition of Cdc7 kinase activity resulted in slowing of S-phase progression and cell cycle arrest followed by accumulation of nuclear damage. This kinase has been shown to be over-expressed in many different tumors including the majority of solid tumors and hematologic malignancies. In our laboratory a novel natural product small molecule Cdc7 kinase inhibitor (MSK-777) has been identified, developed and shown to be efficacious in standard cell based cytotoxicity assays (solid and liquid tumors) and multiple different animal models of cancer.
We have begun to examine to efficacy of Cdc7 kinase inhibition as a possible therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in three different human pancreatic carcinoma cell lines, Capan-1, BxPC3, and PANC-1. These cells were treated at different time points with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS) of DNA, and protein studies using western blotting. Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours.
We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells and will be presenting these results. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.
Citation Format: Lucia Regales, Ruth Santos, Diana Carrillo, Mark G. Frattini. Cdc7 Inhibition as a novel approach for pancreas cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2153. doi:10.1158/1538-7445.AM2013-2153