[Show abstract][Hide abstract] ABSTRACT: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events.
We analyzed paired PDA-normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA.
COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells.
We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
Surgery 06/2011; 150(2):306-15. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Substantial evidence indicates that exposure to cigarette smoke is associated with an elevated risk of 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. In this study, 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.
MMP-9 and VEGF mRNA and protein were analyzed in PDA cells treated with or without nicotine (3-300 nM). 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.
Nicotine significantly enhanced the expression of MMP-9 and VEGF 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 expression levels and induced their promoter activities. In invasive PDA lesions, MMP-9 mRNA levels were significantly (p < 0.005) higher in smokers vs. nonsmokers. VEGF protein co-localized with MMP-9 and OPN in the malignant ducts and correlated well with their higher levels in invasive PDA lesions.
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.
Journal of Gastrointestinal Surgery 10/2010; 14(10):1566-77. · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein (MCP)-1 and evaluated the role of OPN in mediating these effects.
MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3-300 nmol/L) or OPN (0.15-15 nmol/L) were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-1 transcription. Intracellular and tissue colocalization of OPN and MCP-1 were examined by immunofluorescence and immunohistochemistry.
Nicotine treatment significantly increased MCP-1 expression in PDA cells. Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P < .05) increased MCP-1 mRNA and protein and induced its promoter activity. MCP-1 was found in 60% of invasive PDA lesions, of whom 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA.
Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation by inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects.
Surgery 08/2010; 148(2):298-309. · 3.37 Impact Factor