Hyperthermia Inhibits Recombination Repair of Gemcitabine-Stalled Replication Forks

Primary hepatocellular carcinoma (HCC) is an aggressive disease. Globally, about one million new cases of HCC are diagnosed each year with an identical cause-specific mortality rate (1). Most patients are not eligible for curative intent local-regional therapies (1). Conventional cytotoxic therapy has been shown to be of minimal benefit. This has largely been attributed to over-expression of multidrug resistance-associated efflux protein (2–4). The clinical response of HCC to sorafenib, a multi-kinase inhibitor, is limited and transient (5). We purposely focused our investigation on gemcitabine, which is not used for the treatment of HCC because of substantial chemoresistance. We believe it is a priority to understand the mechanisms of gemcitabine chemoresistance and to develop methods to overcome this resistance. Gemcitabine (2′, 2′ - difluoro 2′-deoxycytidine, dFdC) is a nucleoside analogue and a prodrug that is incorporated into the DNA of replicating cancer cells after activation. Despite resistance of HCC to gemcitabine, the mechanisms of resistance are largely elusive. Study of gemcitabine resistance in other cancers has focused on pathways involving its transport and metabolism, or those of altered apoptosis and survival. It is unclear if mechanisms engaged in repairing gemcitabine-stalled replication forks are important in resistance. Aberrant mismatched nucleotides are removed from the DNA by 3′-5′ exonuclease activity of DNA polymerase e (6). It was demonstrated that dFdCMP residues are difficult to excise from the DNA, in part because of masked-chain termination in comparison with dCMP residues (7). Recent data suggests that restart requires regression of the stalled fork into a chicken-foot structure (8). This replication fork intermediate is sensed by poly (ADP-ribose) polymerase 1 (PARP1). Poly-ADP ribose residues associate with the chromatin recruit Mre11, a 3′-5′ exonuclease for DNA end processing (9). The DNA end processing is essential for loading of Rad51 recombinase on the DNA that forms a RAD51 nucleoprotein filament assisted by BRCA-2 (10). This complex subsequently catalyzes sister chromatid homology search and strand invasion to complete homologous recombination repair (HRR) (8). The HRR pathway attempts to restore error-free replication; however, once malignant transformation has occurred, cancer cells may rely on DNA repair pathways such as HRR to propagate the mutated genome. Importantly, it has been clearly demonstrated that cells treated with replication inhibitors exhibit pronounced activation of HRR and that this pathway is essential for survival during recovery from stalled replication forks (10). At least one report suggests localization of HRR pathway proteins to sites of gemcitabine-stalled replication forks (11). Recent studies have demonstrated that hyperthermia has pronounced inhibitory effects on HRR pathways, mainly mediated through inhibition of PARP-1, MRN-complex, or BRCA-2 in the context of DNA double strand break repair (12–18). It is not known, however, if HRR of stalled replication forks is an important mechanism that contributes to chemoresistance of gemcitabine. We hypothesize that hyperthermia can inhibit homologous recombination after gemcitabine-stalled replication forks through its effects on key components of the HRR pathway, hence contributing to chemosensitivity in hepatocellular cancer, a malignancy not usually treated with gemcitabine because of drug resistance.