Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction Harmony F. Turk1, Rola Barhoumi2, Robert S. Chapkin1,3* 1 Program in Integrative Nutrition and Complex Diseases, Texas A & M University, College Station, Texas, United States of America, 2 Image Analysis Laboratory, Texas A & M University, College Station, Texas, United States of America, 3 Center for Environmental and Rural Health, Texas A & M University, College Station, Texas, United States of America Abstract The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR- Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA. Citation: Turk HF, Barhoumi R, Chapkin RS (2012) Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction. PLoS ONE 7(6): e39682. doi:10.1371/journal.pone.0039682 Editor: David Holowka, Cornell University, United States of America Received March 29, 2012; Accepted May 29, 2012; Published June 27, 2012 Copyright: 2012 Turk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants CA59034 and CA168312. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: r-chapkin@tamu.edu Introduction The epidermal growth factor receptor (EGFR;ErbB1) is a transmembrane receptor tyrosine kinase, which contains an extracellular binding domain, a single transmembrane spanning domain, and a cytoplasmic tyrosine kinase domain [1,2]. Ligands for EGFR, including EGF, bind to the extracellular domain of EGFR, stimulating conformational changes that support receptor dimerization. Receptor dimerization results in the activation of the intracellular tyrosine kinase domain, which phosphorylates the dimerization partner on specific tyrosine residues. The phosphorylated tyrosine residues then function as docking sites for adaptor proteins, which serve to activate intracellular signaling cascades. Ultimately, these cascades result in alterations of gene expression, which determines the biological response to receptor activation. Key to the ability of EGFR to activate downstream pathways is its localization in lipid raft domains of the plasma membrane [3,4,5,6,7]. Lipid rafts are highly-ordered, detergent-resistant membrane domains enriched in cholesterol, sphingolipids, and saturated fatty acyl chains that function as signaling platforms [8]. Localization of EGFR to lipid rafts is crucial for efficient EGFR signaling, partially due to colocalization with downstream mediators within lipid rafts [9,10]. In addition, disruption of lipid rafts results in the relocalization of EGFR to bulk membrane regions, which alters EGFR activation and signaling [5,6,9,11,12,13]. Therefore, it is likely that these specialized membrane domains provide a mechanism for spatial and temporal control of EGFR signaling. Aberrant expression or activation of EGFR has been strongly linked to the etiology of several human epithelial cancers, including colon cancer [14]. Colon cancer is a major public health concern, being the third leading cause of cancer related deaths in the United States [15]. Signaling through EGFR activates various cellular processes involved in carcinogenesis, such as cell proliferation, inhibition of apoptosis, angiogenesis, cell motility, and metastasis [16,17]. The numerous signaling cascades that radiate from EGFR, including the Akt, extracellular signal regulated kinase (ERK) 1/2, and signal transducer and activator of transcription (STAT) 3 pathways, mediate a variety of mitogenic, metastatic, and other tumor-promoting cellular activities. Signal- ing through EGFR is up-regulated in colon cancer [18], and inhibition of signaling through EGFR has been shown to prevent colon tumor formation [19]. Additionally, overexpression of EGFR has been reported in up to 85% of human colon cancers [20,21,22,23,24], and expression of EGFR in colon cancer is correlated with a more aggressive disease and poor patient prognosis [25,26,27,28]. Collectively, these data implicate EGFR as a master signal capable of driving colon tumorigenesis. For these reasons, EGFR is an attractive target for therapeutic intervention; thus, intense efforts have been made to inhibit the PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39682