Regulation of EGFR Signal Transduction Figure 5. DHA impairs EGF-induced activation of Ras. A) YAMC cells were treated with 50 mM BSA-complexed fatty acids for 72 h. Twenty-four h after initiating fatty acid treatment, cells were transfected with Grb2-YFP. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) with the same concentration of fatty acids and imaged using TIRF microscopy. Cells were stimulated with 100 ng/mL EGF and imaged every 5 sec. Images are representative of 4 independent experiments (n = 22225 cells/treatment). Changes in total surface intensity were quantified using Nikon Elements AR 3.2. Fluorescence images and the respective surface intensity plots are shown. Surface intensity plots were generated in Nikon Elements AR 3.2 (the scale is from blue (lowest intensity) to red/pink (highest intensity). Data are presented as mean6SEM normalized to control. Bars, 10 mM. B) YAMC cells were treated with 50 mM BSA-complexed fatty acids for 72 h. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were stimulated with 25 ng/mL EGF for 2 min and harvested. GTP-bound Ras was isolated using a GST pull-down assay. Isolated GTP-bound Ras was then analyzed by Western blotting for pan Ras. Isolated GTP-bound Ras was additionally analyzed by Western blotting for H, K, and N-Ras. Blots are representative of 3 independent experiments. Quantification of band volume was performed. Data are expressed as mean6SEM (n = 3), normalized to control. Statistical significance between treatments (P,0.05) as indicated by different letters was determined using ANOVA and Tukey’s test of contrast. C, control; LA, linoleic acid; DHA, docosahexaenoic acid. doi:10.1371/journal.pone.0039682.g005 increased by DHA treatment (Figure 6C). Additionally, EGFR expression was reduced in DHA treated cells 30 min after stimulation. Together, these data suggest that DHA further regulates EGFR signaling capacity by increasing EGFR internal- ization and degradation. Suppression of Cell Proliferation by DHA is EGFR- dependent In order to document a functional endpoint of the DHA- induced decrease in EGFR signaling, we chose to measure cell proliferation, which is regulated in part by signaling through EGFR. DHA treatment of wild-type YAMC cells resulted in an approximately 40% decrease in cell proliferation compared to control and LA treated cells (Figure 7A). We additionally assessed whether expression of a constitutively active form of H-Ras (GFP- H-RasG12V) could rescue the DHA-induced suppression of cell proliferation. We found that DHA-treated cells expressing constitutively activated H-Ras recovered partially from the DHA-induced suppression of cell proliferation but still exhibited approximately a 15% decrease in cell proliferation compared to control cells expressing GFP-H-RasG12V. Additionally, the effect of DHA on proliferation of an isogenic cell line that does not express EGFR (EGFR2/2) was assessed. Interestingly, DHA treatment had no effect on cell proliferation in EGFR2/2 YAMC cells compared to control. Together, these data demonstrate that DHA suppresses cell proliferation in an EGFR-dependent manner. Based on these findings, we have developed a putative model illustrating the effects of DHA on EGFR (Figure 7B). Fish Oil Inhibits EGFR Signaling in Mouse Colonic Mucosa In order to further corroborate the effects of DHA on EGFR activation status, we evaluated the status of total and phosphor- ylated EGFR in colonic mucosa from carcinogen-injected (azoxymethane), chronically-inflamed (dextran sulfate sodium treated) C57BL/6 mice fed diets enriched in fish oil (containing DHA) or corn oil (control, contains primarily LA and no n-3 polyunsaturated fatty acids). Protein lysates from the uninvolved colonic mucosa were collected and probed for total and phosphorylated EGFR (Tyr1068) by Western blotting. Addition- ally, we performed immunoprecipitation for EGFR followed by Western blotting for phosphorylated tyrosine residues. Consistent with the cell culture data, phosphorylation of EGFR increased approximately two-fold in mice fed a fish oil diet compared to mice fed a corn oil diet, while no difference in the total expression of EGFR was observed (Figure 8A). We further probed tissue lysates for phosphorylation of ERK1/2, STAT3, and Akt. Similar to Figure 1 data, phosphorylation of ERK1/2 and STAT3 decreased by approximately 40% and 50%, respectively, in fish oil fed mice compared to corn oil fed mice with no difference in phosphorylation of Akt between diets (Figure 8A). Additionally, tumors from the animals were stained and typed. Interestingly, fish oil fed animals developed fewer tumors compared to animals fed a corn oil diet (Figure 8B). Overall, these data indicate a protective effect of dietary fish oil against colon tumorigenesis by suppressing EGFR signal transduction. Discussion EGFR is a transmembrane receptor tyrosine kinase involved in transmitting external cues to the intestinal epithelium, thereby modulating cell proliferation, migration, and survival. Recent evidence suggests that the membrane lipid microenvironment can significantly modulate EGFR localization and function [62]. Here, we report that membrane incorporation of DHA alters the lateral organization of EGFR (Figure 1). The lateral organization of the receptor has been shown to directly regulate ligand binding, dimerization, and receptor phosphorylation [5,6,9]. Consistent with these observations, we demonstrated that DHA treatment resulted in increased ligand-stimulated EGFR dimerization and phosphorylation (Figures 2A and 4B). Therefore, the DHA- induced shift of EGFR localization within the plasma membrane alters the ability of the receptor to dimerize and transpho- sphorylate. These findings favor a model according to which receptor modulation by fatty acids is mediated by the membrane. According to the canonical process of signal transduction, the observed increase in EGFR phosphorylation upon treatment with DHA is expected to be correlated with enhanced downstream signaling. Two previous studies have observed that DHA increases EGFR phosphorylation, but they reported conflicting results regarding downstream signaling [54,55]. Additionally, the flawed design of one of the studies renders the results difficult to interpret. Therefore, in our study, we evaluated the effect of DHA on activation of multiple distinct signaling cascades that radiate from EGFR within the colon. Although DHA was found to increase EGFR phosphorylation, we noted that this fatty acid uniquely inhibited EGF-stimulated activation of downstream signaling from EGFR through ERK1/2, STAT3, and mTOR/S6K (Figure 2B). A substantial body of work has documented the central role of lateral membrane organization in mediating EGFR signal transduction [73]. Integral to EGFR signaling is its localization to lipid raft domains. These specialized membrane domains have the ability to assemble the molecular machineries necessary for intracellular propagation of EGFR effector signals. Stimulation with EGF induces coalescence of lipid raft domains and promotes the formation of these signaling platforms, which suggests a central role for these domains in EGFR signal propagation [74]. Collectively, these data suggest that by altering the lateral organization of EGFR, DHA can modulate multiple cellular signaling cascades. In addition to regulating the localization of EGFR within the plasma membrane, DHA also altered the subcellular distribution of EGFR. Under unstimulated conditions, DHA treated cells contained significantly less EGFR at the plasma membrane PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39682