Abstract A68: Hypoxia-induced CHK1 repression may enhance the mutator phenotype of pancreatic cancer cells

Abstract

Abstract Background: Dysregulation of DNA repair mechanisms are known to play a major role in tumor progression. Moreover, the tumor microenviroment itself has been shown to enhance this phenomenon. For example, studies using reporter genes and endogenous loci have demonstrated increased mutation rates in cells grown in tumors compared to the identical cells grown in culture, or a higher frequency of point mutations in cancer cells cultured under hypoxic conditions, a common feature during tumor growth. An accepted mechanism for these observations is hypoxia-induced down-regulation of MLH1, MSH2 or MSH6 leading to an increased mutation rate, or to hypoxia-induced repression of DNA double strand break repair. Methods: Expression microarrays were performed using RNA isolated from nine pancreatic cancer cell lines cultured under normoxic (Nx, 20% O2) or hypoxic conditions (Hx, 1% O2) for 24 hours to identify differentially expressed genes. Retroviruses encoding oligos for HIF-1 or HIF-2 knockdown were used to generate stable pancreatic cancer cell lines in which HIF-1 or HIF-2, respectively, expression was blocked. Alternatively, Hx was induced by culture of cells inside an air-tight chamber with inflow and outflow valves that was infused with a mixture of 1% O2, 5% CO2 and 94% N2. Protein levels of HIF-1, HIF-2, MLH1, MSH2, MSH6 and CHK1 were determined by western blotting. Cell proliferation was measured by BrdU incorporation and cell cycle analysis by flow cytometry of propidium iodide stained cells. G2/M check point was studied analyzing Histone H3 phosphorylation by flow cytometry and DNA damage was determined by immunofluorescence of cells stained with anti phospho γ-H2AX antibody (γ-H2AX foci formation) Results: We first compared the gene expression profiles of 9 different pancreatic cancer cell lines each grown under normoxic versus hypoxic conditions. As previously described, hypoxia led to down-regulation of the mRNA levels of the mismatch repair proteins MLH1, MSH2 and MSH6 as well as down-regulation of several genes involved in the DNA damage response to double strand breaks such as Brca1, Rad51, Ku70 and Ku80. Of interest, a novel downregulated gene identified was CHK1, a master coordinator of DNA repair following genotoxic stress. Growth of pancreatic cancer cells under hypoxic conditions was confirmed to decrease CHK1 mRNA and protein levels and stable knockdown of HIF-1a but not HIF-2 abolished these effects. In contrast to what have been previously reported in other cancer cell types, hypoxia had no effects on cell proliferation in pancreatic cancer cells as assessed by cell cycle analysis or by BrdU incorporation.To determine if hypoxic down-regulation of CHK1 protein levels has functional effects, we determined the ability of pancreatic cancer cells to arrest in the G2/M checkpoint after ionizing radiation. We found that cells cultured under hypoxia were significantly less arrested on G2/M check point after radiation compared to the same irradiated cells grown under normoxic conditions. This hypoxic effect on G2/M checkpoint was abolished on HIF-1 knockdown cells. Finally, pancreatic cancer cells grown under hypoxic conditions accumulated more DNA damage 24 hours after radiation compared to the same irradiated cells grown under normoxic conditions. Conclusion: Hypoxic conditions decrease CHK1 mRNA and protein levels in pancreatic cancer cells in a HIF1-dependent manner. Impairment on CHK1 function may promote the mutator phenotype of pancreatic cancer cells during tumor progression by allowing unchecked cell divisions despite ongoing DNA damage. This finding also has significance for the use of CHK1 inhibitors for this tumor type. Citation Format: Salvador Naranjo-Suarez, Elsa Callen, Yi Zhong, Alvin Makohon-Moore, Andre Nussenzweig, Christine Iacobuzio-Donahue. Hypoxia-induced CHK1 repression may enhance the mutator phenotype of pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A68.