The combination of EPA and Naproxen prevents iCAF polarization and attenuates inflammation associated with Pirc tumor development

We recently reported that eicosapentaenoic free fatty acid (EPA-FFA) or a chemically stable EPA analog (TP-252), when combined in the diet with naproxen, reduces colon tumor development by up to 98% in the highly penetrant Pirc rat model. Cancer suppression is dependent, in part, upon modifications of eicosanoid biosynthesis. The following study expands upon these initial findings using genome-wide RNA-sequencing (RNA-seq) analysis to identify specific molecular pathways in Pirc tumors affected by drug treatment. Colon tumors isolated from untreated control Pirc rats maintained on AIN93G diet exhibited gene expression profiles associated with the presence of inflammatory cancer-associated fibroblasts (iCAFs). Following a twenty-week treatment with a combination diet of EPA-FFA (2% w/w) and naproxen (200 ppm), there was a marked reduction in inflammatory and proliferative gene signatures. Further analysis of these drug combination-treated tumors showed expression profiles resembling a less inflammatory fibroblast phenotype, suggesting the presence of myofibroblasts (myCAFs). To further establish potential mechanisms, we isolated CAFs from colon tumors of untreated Pirc rats. CAFs were then stimulated for 12 hours with individual cytokines (TNF-a, IL-1beta or IL-6) to promote iCAF activation. The secretion of a panel of inflammatory cytokines (IL-6, Cxcl2, Csf3 and Nos2) determined in IL-1beta activated CAFs were similar to those found by RNA-seq analysis of colon tumors from untreated Pirc diet controls. In addition, the combination treatment of EPA-FFA and naproxen attenuated polarization of CAFs towards an iCAF phenotype upon IL-1beta stimulation. The present work contributes further to our understanding of intestinal tumor protection afforded by a combination of dietary EPA and naproxen. These findings further uncover potential mechanisms that can be exploited in future clinical chemoprevention studies.