Abstract A33: γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma
Authors: Honnenahally Kumaraswamy , Shi Chanjuan , Chen Xi , Castellanos Jason , Merchant Nipun , Blackwell Timothy , Nagathihalli Nagaraj 
Published: October 2015

Abstract
Introduction: Cigarette smoking is an important risk factor for the development pancreatic ductal adenocarcinoma (PDAC). Nicotine and nitrosamines cause pancreatic cell injury and contribute to a cascade of oncogenic events. Among multiple tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic and induces DNA double strand-breaks (DSBs). However, identification of potential prognostic biomarkers and experimental models of smoking-induced PDAC remain unknown. We have studied phosphorylated histone H2AX (γH2AX), a marker for double-stranded DNA breaks, as a new biomarker for detecting cigarette smoke-induced pathogenesis of PDAC.
Experimental Procedure: We have characterized the expression of γH2AX protein in human pancreatic tissues, human pancreatic duct epithelial (HPDE)6, PDAC cell lines, and PanIn, primary PDAC (PDA) and liver metastasis (LMP) cell lines generated from a genetically engineered mice model of PDAC. Kaplan-Meier survival analysis was performed to determine the significance of γH2AX expression amongst smokers and non-smokers with PDAC. HPDE6 and PanIN cells were cultured for 4 days in the presence or absence of cigarette smoke (CS) (0-25μg/ml) or NNK (0-10μM). Cell cycle analysis and apoptosis were assessed through flow cytometry with the markers propidium iodide and annexin V respectively. HPDE6 and PanIN cells treated with CS or NNK for up to 50 days were analyzed for rapid activation of DNA damage response (DDR) pathway proteins γH2AX, RAD51, BRCA1, BRCA2, phospho-CHK2 and phospho-CHK1. Cells were treated with either CS or NNK along with a CHK2 inhibitor and analyzed for activation of γH2AX. To determine DNA integrity, accumulation of γH2AX was measured by ELISA in cells treated with CS or NNK. Immunofluorescence confocal microscopy was utilized to examine the role of γH2AX on nuclear foci formation with DNA repair complexes by quantifying the amount of co-localization. To replicate chronic smoke exposure, we treated HPDE6 and PanIn cells with 1 and 10 µg/ml CS or 10nM and 1µM NNK for 10 passages, and examined these cell lines for DSBs by comet assay, apoptosis markers by protein array and in vitro tumorigenicity by colony formation assay.
Results: γH2AX expression was significantly higher (p&lt;0.0001) in smokers when compared to non-smokers. Overall survival of patients with high γH2AX expression in their primary tumor was associated with significantly decreased median survival (11 months) when compared to low γH2AX expression (36 months, p=0.0007). HPDE6 cells exposed to CS or NNK, showed more than 80% survival. The surviving cells had altered morphology, acquired mesenchymal characteristics and displayed increased tumorigenicity suggesting that these cells acquire resistance to apoptosis after CS or NNK exposure. In addition, exposure of cells to CS or NNK resulted in generation of γH2AX and induction of phospho-CHK2 expression. RAD51 and phospho-CHK2 co-localize with γH2AX after DNA damage with CS or NNK exposure. Inhibition of CHK2 reduced expression of γH2AX. The product of the tumor suppressor gene BRCA2 co-localized with γH2AX indicating that γH2AX is necessary for the recruitment of DNA repair factors to the site of DNA damage. BRCA2 mutant cell line CAPAN1 exhibited a substantially reduced ability to form co-localization with RAD51 and γH2AX, consistent with a role of γH2AX in DNA damage repair.
Conclusion: This work supports the role of γH2AX as a new molecular biomarker to assess the contribution of cigarette smoking-induced PDAC. γH2AX is necessary for DNA damage repair in PDAC and its activation is dependent on CHK2. Therapies that can block γH2AX expression by inhibiting upstream kinases may be a new potential therapeutic approach to treat and/or prevent PDAC.
Citation Format: Kumaraswamy Honnenahally, Chanjuan Shi, Xi Chen, Jason Castellanos, Nipun Merchant, Timothy Blackwell, Nagaraj Nagathihalli. γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma. [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 A33.