Tobacco Carcinogen Induces Persistent DNA Damage and Homologous Recombination Dysregulation in Pancreatic Ductal Carcinogenesis

Abstract

Cigarette smoking is a well-established risk factor for the development of pancreatic ductal adenocarcinoma (PDAC), yet the mechanisms by which tobacco carcinogens, such as Nicotine-Derived Nitrosamine Ketone (NNK), drive pancreatic carcinogenesis remain incompletely understood. We investigated the impact of NNK on pancreatic epithelium by treating human pancreatic ductal epithelial cells (HPDE6-E6E7; H6c7) with NNK for 5 or 50 days followed by genome-wide RNA sequencing, qRT-PCR, immunofluorescence, comet assay, and {gamma}H2AX ELISA. We assessed {gamma}H2AX by immunohistochemistry in human PDAC tissue microarrays and in TCGA-PAAD datasets to identify clinical correlates. Acute NNK exposure (5 days) induced DNA double-strand breaks and activated ATM-{gamma}H2AX signaling, accompanied by increased RAD51 and BRCA2 recruitment, consistent with activation of homologous recombination (HR)-mediated DNA repair. In contrast, chronic NNK exposure (50 days) resulted in persistent DNA damage signaling and transcriptional upregulation of HR-associated genes, including RAD51, BRCA1, BRCA2, FANCD2, and PLK1. Despite sustained accumulation of RAD51- and BRCA2-positive repair foci, chronic NNK exposure was associated with altered RAD51 localization, persistent DNA damage, and chromosomal instability, suggesting dysregulation of HR repair fidelity during prolonged genotoxic stress. {gamma}H2AX expression was significantly elevated in NNK-treated cells, KRAS-driven premalignant pancreatic lesions, and pancreatic tissues from smokers. Increased {gamma}H2AX expression correlated with tumor progression and poor overall survival, while elevated RAD51 expression was associated with adverse outcomes in patients with PDAC. Collectively, these findings reveal a biphasic DNA damage response to NNK characterized by early activation of ATM-{gamma}H2AX-dependent DNA repair followed by persistent DNA damage signaling, dysregulated HR repair, and genomic instability during chronic exposure. Tobacco-induced disruption of DNA repair homeostasis may represent a key mechanism driving pancreatic carcinogenesis and supports {gamma}H2AX as a clinically relevant prognostic biomarker in tobacco-associated PDAC.