Cdc7 is a potent anti-cancer target in pancreatic cancer due to abrogation of the DNA origin activation checkpoint

// Matthew T. Huggett 1, 2 , Slavica Tudzarova 2 , Ian Proctor 2 , Marco Loddo 2, 3 , Margaret G. Keane 1 , Kai Stoeber 2 , Gareth H. Williams 2, 3 , Stephen P. Pereira 1 1 UCL Institute for Liver and Digestive Health and UCL Cancer Institute, University College London, London, UK 2 The Research Department of Pathology, UCL Cancer Institute, University College London, London, UK 3 Oncologica Ltd, The Science Village, Chesterford Research Park, Cambridge, UK Correspondence to: Gareth Williams, e-mail: gareth.williams@oncologica.com Keywords: pancreatic cancer, cell cycle, DNA replication, Cdc7 Received: July 22, 2015      Accepted: January 23, 2016      Published: February 23, 2016 ABSTRACT Purpose: Cdc7 is a serine/threonine kinase which is responsible for the ‘firing’ of replication origins leading to initiation of DNA replication. Inhibition or depletion of Cdc7 in normal cells triggers a DNA origin activation checkpoint causing a reversible G1 arrest. Here we investigate Cdc7 as a novel therapeutic target in pancreatic cancer. Experimental design: Cdc7 target validation was performed by immunoexpression profiling in a cohort of 73 patients with pancreatic adenocarcinoma including 24 controls. Secondly Cdc7 kinase was targeted in Capan-1 and PANC-1 pancreatic cancer cell line models using either an siRNA against Cdc7 or alternatively a small molecule inhibitor (SMI) of Cdc7 (PHA-767491). Results: Cdc7 was significantly overexpressed in pancreatic adenocarcinoma compared to benign pancreatic tissue (median LI 34.3% vs. 1.3%; P<0.0001). Cdc7 knockdown using siRNA in Capan-1 and PANC-1 cells resulted in marked apoptotic cell death when compared with control cells. A prominent sub-G1 peak was seen on flow cytometry (sub-G1 51% vs. 3% and 45% vs. 0.7% in Capan-1 and PANC-1 cells, respectively). Annexin V labelling confirmed apoptosis in 64% vs. 11% and 75% vs. 8%, respectively. Western blotting showed cleavage of PARP-1 and caspase-3 and presence of γH2A.X. TUNEL assay showed strong staining in treated cells. These results were mirrored following Cdc7 kinase inhibition with PHA-767491. Conclusions: Our findings show that Cdc7 is a potent anti-cancer target in pancreatic adenocarcinoma and that Cdc7 immunoexpression levels might be used as a companion diagnostic to predict response to therapeutic siRNAs or SMIs directed against this kinase.