Abstract
PurposeCdc7 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 designCdc7 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).ResultsCdc7 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.ConclusionsOur 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.
Highlights
Adenocarcinoma of the pancreas is one of the top 10 leading causes of cancer deaths [1, 2]
Cdc7 was significantly overexpressed in pancreatic adenocarcinoma compared to benign pancreatic tissue
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 small molecule inhibitor (SMI) directed against this kinase
Summary
Adenocarcinoma of the pancreas is one of the top 10 leading causes of cancer deaths [1, 2]. The six Mcm proteins function as a replicative helix, unwinding the template DNA, with Cdc and Cdt acting as clamp loaders for this ring-shaped heterohexameric complex [13,14,15]. The formation of an active helicase leads to recruitment of additional factors, including Cdc and the four subunit GINS complex [16,17,18]. Once activated the MCM helicase unwinds double-stranded DNA at replication origins to generate a single-stranded DNA template required to recruit the DNA synthesis machinery including RPA, PCNA and DNA polymerase α-primase [12]. During S phase, Cdc kinase and cyclin-dependent kinases induce a conformational change in the prereplicative complex, resulting in recruitment of additional initiator proteins that collectively promote DNA unwinding, recruitment of DNA polymerases and the establishment of bi-directional replication forks
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