demonstrating that EGFR-mediated activation of all three isoforms of Ras was suppressed by DHA. In addition, DHA had the strongest inhibitory effect on activation of H-Ras. This is intriguing due to the fact that both H-Ras and EGFR are localized to lipid rafts [59], and that EGF-stimulated interactions between Ras and its downstream partner Raf-1 have been shown to occur in lipid rafts [3]. A potential explanation for the observed DHA- mediated suppression of activation of Ras, specifically H-Ras, is based on the altered localization of EGFR within the plasma membrane. Convincing evidence illustrates the importance of lateral segregation of signaling cascades on the plasma membrane. Indeed, for signal transduction to occur, phosphorylated EGFR must form a transient complex with Grb2, Sos, and Ras [76]. Due to the altered plasma membrane localization of EGFR upon treatment with DHA, this could result in a spatial abrogation of signal transduction by reducing the formation of these transient complexes. Since DHA antagonizes the activation of all Ras isoforms regardless of their plasma membrane localization, it is likely that an additional mechanism mediates the suppression of EGFR signal transduction by DHA. Therefore, we hypothesize that the observed increased endocytosis of the receptor contributes to the suppressive phenotype. The majority of activation of Ras by EGFR has been shown to occur at the plasma membrane [77]. Therefore, the observed DHA-induced increase in EGFR internalization likely contributes to the reduced activation of all Ras isoforms. We are currently determining whether the altered plasma membrane localization of EGFR and/or enhanced EGFR endocytosis are responsible for the suppression of downstream signaling. Additionally, further work is required to determine how the altered plasma membrane localization of EGFR contributes to enhanced receptor internalization and degradation. Previous work by Sigismund et al. indicates that high levels of EGFR phosphorylation can lead to activation of additional forms of EGFR endocytosis which sort EGFR for degradation instead of recycling [66]. Therefore, it is possible that DHA could function by altering EGFR plasma membrane localization, resulting in higher levels of EGFR phosphorylation and activation of a non- canonical form of receptor endocytosis that sorts the receptor for degradation as opposed to recycling. Current studies in our lab are focused on testing this hypothesis. These results have substantial biological relevance because Ras plays a central role in the development of human colon cancer and is commonly hyperactivated by somatic mutation or signaling through growth factor receptors [78]. Hence, by suppressing EGF- stimulated activation of Ras, DHA can provide protection against colonic transformation. In addition to suppressing activation of the Ras-ERK1/2 pathway, DHA suppressed EGF-stimulated activa- tion of STAT3. STAT3 can be activated downstream of EGFR by several pathways, and Ras signaling has been shown to be intimately linked to STAT3 activation [79,80,81]. Therefore, the reduction in STAT3 activation could be a direct result of reduced activation of Ras. Additionally, lipid rafts have been shown to play a central role in the activation of STAT3 [82], which may explain the DHA-induced suppression of STAT3 activation. Clearly, further studies are required to determine the exact mechanism of action. To analyze the consequence of the suppression of EGFR signaling by DHA, we measured cell proliferation in wild-type and EGFR-null colonocytes. Interestingly, DHA suppressed cell proliferation only in cells with a functional EGFR (Figure 7A). These results indicate that the DHA modulates cell proliferation in an EGFR-dependent manner. It is important to note that DHA does not induce apoptosis in this cell line [83], so the changes Regulation of EGFR Signal Transduction observed are due to suppressed proliferation and not increased cell death. Additionally, expression of a constitutively active form of H- Ras partially rescued the DHA-induced suppression of cell proliferation. The limited ability of constitutively active H-Ras was likely due to the inhibitory effect that DHA has on other downstream pathways from EGFR that are independent of Ras signaling. Based on these data, we have developed a putative model according to which DHA alters the cellular localization and signaling capacity of EGFR (Figure 7B). By altering the lateral organization of EGFR, DHA enhances the ability of EGFR to dimerize and transphosphorylate. DHA also increases EGFR internalization and degradation, which suppresses the ability of EGFR to activate downstream signaling cascades. In order to determine the in vivo effect of DHA on EGFR signaling, we assessed whether dietary consumption of fish oil, enriched in DHA, modulates EGFR phosphorylation and signaling in mice. Consistent with our cell culture data, feeding a DHA-enriched diet resulted in an increase in EGFR phosphor- ylation and a suppression of ERK1/2 and STAT3 activation in mouse colonic epithelium (Figure 8). The suppression of EGFR signaling in the colon was associated with a reduction in tumor- incidence in mice fed a DHA-enriched diet. These data highlight the unique mechanism by which DHA suppresses colon tumor- igenesis. The differential effects of DHA and EPA, the two n-3 PUFAs enriched in fish oil, are often overlooked and underappreciated. Therefore, we assessed whether EPA had the same effect as DHA on EGFR phosphorylation. Interestingly, in contrast to DHA, neither EPA nor AA, another long-chain PUFA, exerted an effect on EGFR phosphorylation (Figure 3D). This is consistent with a previous study showing that DHA, but not EPA, suppressed EGF- stimulated activation of AP-1 [84]. Collectively, these results highlight the uniqueness of DHA, which has been shown to significantly alter numerous membrane properties [48,49]. The effects of DHA on EGFR signaling are reversible when supplementation with DHA is discontinued and the fatty acid is washed out of the plasma membrane (Figure 4D). This is consistent with our hypothesis that DHA enrichment in the plasma membrane directly modulates EGFR signaling. DHA is a structurally unique fatty acid. It is slightly polar due to its six double bonds, and it rapidly reorients through multiple confor- mational states [48]. This flexible structure renders DHA incompatible with ordered saturated acyl chains and cholesterol, two major constituents of lipid rafts. Multiple biophysical studies utilizing model membranes and molecular dynamic simulations have shown that DHA acyl chains do not pack efficiently with cholesterol and saturated acyl chains [85,86]. Additionally, studies using cell lines have demonstrated that DHA can disrupt lipid rafts and alter protein distribution [52,53,87]. Although, the exact effect that DHA has on lipid rafts remains to be fully elucidated, an abundance of data clearly demonstrate that DHA can impede lipid raft mediated processes and alter lipid raft composition [47,54]. Our data demonstrate that DHA inhibits the lateral organization of EGFR within the plasma membrane. Furthermore, the data suggest that DHA modifies lipid rafts because it altered the overall localization of the lipid raft marker, RFP-tH, within the plasma membrane. Overall, this work provides further evidence of the global effect of DHA on lipid rafts. A seminal finding of this work is the DHA-induced severance of EGFR phosphorylation from downstream signaling. Phosphory- lation of EGFR has previously been considered a marker for activation of downstream signaling. In contrast, our data describe a mechanism whereby DHA induces an increase in EGFR phosphorylation by altering its localization but suppresses down- PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e39682