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Introduction: Substantial evidence indicates that exposure to cigarette smoke is associated with an elevated risk for pancreatic ductal adenocarcinoma (PDA). However, the mechanisms underlying the effects of nicotine on the development or progression of PDA remain to be investigated. Previously, we showed that nicotine promotes the expression of osteopontin c (OPNc), an isoform of OPN protein that confers on cancer cells a migratory phenotype. Here, we explored the potential prometastatic role of nicotine in PDA through studying its effect on the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF), and evaluated the role of OPN in mediating these effects. We also analyzed the expression of MMP-9 and VEGF in PDA specimens from smokers and nonsmokers.Methods: MMP-9 and VEGF mRNA and protein in MiaPaca, AsPC-1 and HS766T PDA cell lines treated with or without nicotine (3-300 nM) were analyzed by real time PCR and ELISA, respectively. Transient transfection and luciferase-labeled promoter studies evaluated the effects of OPNc and OPN protein on the transcription and translation of MMP-9 and VEGF. Real time PCR and immunohistochemistry were used to analyze the mRNA expression levels and localization of OPN, MMP-9 and VEGF proteins in matched invasive human PDA and surrounding nonmalignant tissues (n=52; 27 smokers and 25 nonsmokers) and intraductal papillary mucinous neoplasms, IPMN (n=6; 2 smokers, 4 nonsmokers). Results: Nicotine significantly enhanced the expression of MMP-9, VEGF, and OPN mRNA and protein in PDA cells. Blocking OPN with siRNA or OPN antibody prevented the nicotine-mediated increase of both MMP-9 and VEGF. Transient transfection of OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P<0.05) increased MMP-9 and VEGF mRNA and protein expression levels and induced their promoter activities. In invasive PDA lesions, MMP-9 mRNA levels were significantly (p<0.005) higher than their matching controls, and in smokers vs. nonsmokers. VEGF protein colocalized with MMP-9 and OPN in the malignant ducts and correlated well with their higher levels in invasive PDA lesions. Conclusions: Our data show for the first time that cigarette smoking and nicotine may contribute to PDA metastasis through inducing MMP-9 and VEGF, and suggest that OPN plays a central role in mediating these effects. The presence of OPN as a downstream effector of nicotine that is capable of mediating its prometastatic effects in PDA cells is novel and could provide a unique therapeutic target to control pancreatic cancer aggressiveness, especially in the cigarette smoking population.