PDA is an aggressive disease that has a strong etiological association with cigarette smoking and high blood levels of nicotine (2
). MCP-1 is the most frequently found CC chemokine in several tumors, including those of the pancreas (10
). Although several studies investigated the role of MCP-1 in cancer progression, very few have investigated its relationship to smoking and the upstream factors involved in its regulation in PDA cells. In this study, we show for the first time that nicotine treatment both dose- and time-dependently increased MCP-1 expression in PDA cells. We also demonstrate a previously undescribed role for OPN as a mediator for these effects.
Our data show that nicotine induced MCP-1 accumulation rapidly in PDA cells and with significant magnitude. Dose-response studies demonstrated a significant induction of MCP-1 mRNA and protein levels at the physiological range of blood levels of nicotine in smokers (3–300 nM). The maximal effective concentration (at 30 nM) is similar to other nicotine actions that have been reported (22
Our data show that the MCP-1 promoter was not activated by nicotine when it was added at (3–300 nM) to PDA cells at different time points. Since we showed previously that nicotine directly induces OPN transcription in PDA cells (15
), and since OPN was shown to increase MCP-1 expression in other cells (19
), we tested the hypothesis that OPN mediates the upregulation of MCP-1 by nicotine.
We conducted four sets of experiments to evaluate the role of OPN. First, we inhibited OPN synthesis by siRNA () or blocked its function by a polyclonal antibody against human OPN (). In both studies, nicotine was unable to increase MCP-1 expression in PDA cells, suggesting that OPN may play a role in mediating the effects of nicotine. Next, we treated the PDA cells with recombinant human OPN protein () or overexpressed an OPN isoform (OPNc) (), which has been shown to promote metastasis in cancer cells (23
). In both experiments, we show that increased constitutive OPN or addition of exogenous OPN allowed the increase of MCP-1 expression by nicotine (). Furthermore, OPN also increased the basal levels of MCP-1 expression in PDA cells () and increased its transcription by activating its promoter activity (). Previous studies have shown that nicotine induces the expression of total OPN (15
) and selectively induce the expression of OPNc in PDA cells (16
). This is the first report to demonstrate a relationship between nicotine, OPN/OPNc and MCP-1. Confocal microscopy analysis also revealed intracytoplasmic colocalization of OPN and MCP-1 in PDA cells (), providing more evidence for the paracrine/autocrine relationship between the two molecules. Additional studies are now required to delineate the details of this relationship and the signaling pathways involved in mediating the increase of MCP-1 by OPN. Furthermore, the effect of the nicotine-mediated increase of OPNc on PDA cell inflammatory behavior and function is the subject of our currently ongoing studies in the laboratory.
Numerous studies have correlated high levels of MCP-1 expression with tumor progression in many cancers, including pancreatic cancer (20
). MCP-1 promotes cell survival (25
) and induces the expression of vascular endothelial growth factor (26
). These functions are similar to those reported to be mediated by OPN (27
). Our in vitro data suggest that OPN might be acting upstream of MCP-1 to mediate these effects.
Studies in colorectal cancer have correlated higher levels of MCP-1 with tumor stage and high metastatic potential (26
). Our analyses reported here have found that MCP-1 was found in 60% of invasive PDA lesions, of which 66% were smokers. This is the first report to examine the relationship between tumor MCP-1 and the status of smoking in PDA patients. Our analysis also reveals that higher levels of MCP-1 were seen in invasive PDA as compared to premalignant lesions. Immunohistochemical analysis of PDA showed that MCP-1 colocalizes with OPN in the malignant ducts and its mRNA levels correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA (). It remains to be determined, however, whether MCP-1 levels correlate with pathologic stage, survival or recurrence. Furthermore, additional studies are required to determine whether similar findings could be obtained from Endoscopic Ultrasound and Fine Needle Aspiration (EUS FNA) samples.
Interestingly, our studies show that high levels of MCP-1 and OPN exist in invasive lesions from non-smokers. Factors such as second hand smoke could contribute to this. In addition, a previous history of pancreatic inflammation (pancreatitis), which has been linked to pancreatic carcinogenesis (29
) could create a tumor microenvironment with higher levels of OPN and MCP-1. Additional studies addressing these possibilities are currently ongoing in our laboratory.
In summary, our study suggests that cigarette smoking and nicotine may contribute to PDA inflammation through inducing MCP-1 and provides a novel insight into a unique role for OPN in mediating these effects. Although the role of OPN-mediated induction of MCP-1 in pancreatic carcinogenesis remains to be defined, the existence of OPN as a downstream effector of nicotine, capable of mediating proinflammatory effects in PDA cells is novel and could provide a unique potential target to control pancreatic cancer aggressiveness, especially in the cigarette smoking population.