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Membrane therapy using DHA suppresses epidermal growth factor receptor signaling by disrupting nanocluster formation

  • Natividad R. Fuentes
    Affiliations
    Program in Integrative Nutrition and Complex Diseases, Texas A&M University, College Station, TX, USA

    Department of Nutrition, Texas A&M University, College Station, TX, USA

    Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, TX, USA
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  • Mohamed Mlih
    Affiliations
    Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, Bryan, TX, USA
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  • Xiaoli Wang
    Affiliations
    Program in Integrative Nutrition and Complex Diseases, Texas A&M University, College Station, TX, USA

    Department of Nutrition, Texas A&M University, College Station, TX, USA
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  • Gabriella Webster
    Affiliations
    Program in Integrative Nutrition and Complex Diseases, Texas A&M University, College Station, TX, USA

    Department of Nutrition, Texas A&M University, College Station, TX, USA
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  • Sergio Cortes-Acosta
    Affiliations
    Program in Integrative Nutrition and Complex Diseases, Texas A&M University, College Station, TX, USA

    Department of Nutrition, Texas A&M University, College Station, TX, USA
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  • Michael L. Salinas
    Affiliations
    Program in Integrative Nutrition and Complex Diseases, Texas A&M University, College Station, TX, USA

    Department of Nutrition, Texas A&M University, College Station, TX, USA
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  • Ian R. Corbin
    Affiliations
    Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA
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  • Jason Karpac
    Affiliations
    Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, Bryan, TX, USA
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  • Robert S. Chapkin
    Correspondence
    For correspondence: Robert S. Chapkin
    Affiliations
    Program in Integrative Nutrition and Complex Diseases, Texas A&M University, College Station, TX, USA

    Department of Nutrition, Texas A&M University, College Station, TX, USA

    Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, TX, USA

    Center for Translational Environmental Health Research, Texas A&M University, College Station, TX, USA
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Open AccessPublished:January 27, 2021DOI:https://doi.org/10.1016/j.jlr.2021.100026

      Abstract

      Epidermal growth factor receptor (EGFR) signaling drives the formation of many types of cancer, including colon cancer. Docosahexaenoic acid (DHA, 22∶6Δ4,7,10,13,16,19), a chemoprotective long-chain n-3 polyunsaturated fatty acid suppresses EGFR signaling. However, the mechanism underlying this phenotype remains unclear. Therefore, we used super-resolution microscopy techniques to investigate the mechanistic link between EGFR function and DHA-induced alterations to plasma membrane nanodomains. Using isogenic in vitro (YAMC and IMCE mouse colonic cell lines) and in vivo (Drosophila, wild type and Fat-1 mice) models, cellular DHA enrichment via therapeutic nanoparticle delivery, endogenous synthesis, or dietary supplementation reduced EGFR-mediated cell proliferation and downstream Ras/ERK signaling. Phospholipid incorporation of DHA reduced membrane rigidity and the size of EGFR nanoclusters. Similarly, pharmacological reduction of plasma membrane phosphatidic acid (PA), phosphatidylinositol-4,5-bisphosphate (PIP2) or cholesterol was associated with a decrease in EGFR nanocluster size. Furthermore, in DHA-treated cells only the addition of cholesterol, unlike PA or PIP2, restored EGFR nanoscale clustering. These findings reveal that DHA reduces EGFR signaling in part by reshaping EGFR proteolipid nanodomains, supporting the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane organization, to reduce EGFR signaling and cancer risk.

      Supplementary key words

      Abbreviations:

      DHA (docosahexaenoic acid), DPBS (Dulbecco's phosphate buffered saline), EGF (epidermal growth factor), EGFR (epidermal growth factor receptor), FIPI (5-fluoro-2-indolyl des-chlorohalopemide), FLIM (fluorescence lifetime imaging), FRET (fluorescence resonance energy transfer), IMCE (immortalized murine colonic epithelial), ISC (intestinal stem cell), LA (linoleic acid), LSD (least significant difference), MβCD (methyl-beta-cyclodextrin), OA (oleic acid), PA (phosphatidic acid), PAO (phenylarsine oxide), PIP2 (phosphatidylinositol-4,5-bisphosphate), STED (stimulated emission depletion), STORM (stochastic optical reconstruction microscopy), YAMC (young adult mouse colonic)
      The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that is mutated or overexpressed in many cancerous tissues, including the colon (
      • Thomas R.
      • Weihua Z.
      Rethink of EGFR in cancer with its kinase independent function on board.
      ). EGFR signaling mediates many cellular processes involved in epithelial tissue homeostasis (
      • Jiang H.
      • Grenley M.O.
      • Bravo M.J.
      • Blumhagen R.Z.
      • Edgar B.A.
      EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila.
      ). Inhibition of EGFR signaling has been shown to reduce uncontrolled cell growth (
      • Seshacharyulu P.
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      • Haridas D.
      • Jain M.
      • Ganti A.K.
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      Targeting the EGFR signaling pathway in cancer therapy.
      ), and EGFR targeted pharmaceuticals are used in the treatment of colorectal cancer (
      • Markman B.
      • Javier Ramos F.
      • Capdevila J.
      • Tabernero J.
      EGFR and KRAS in colorectal cancer.
      ). However, undesirable side effects (
      • Fakih M.
      • Vincent M.
      Adverse events associated with anti-EGFR therapies for the treatment of metastatic colorectal cancer.
      ) and acquired resistance (
      • Dienstmann R.
      • Salazar R.
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      Overcoming resistance to anti-EGFR therapy in colorectal cancer.
      ,
      • Zhao B.
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      • Yuan X.
      Mechanisms of resistance to anti-EGFR therapy in colorectal cancer.
      ) to these therapeutics highlight the need for the development of alternative strategies.
      Mounting experimental, epidemiological, and clinical evidence suggest that consumption of n-3 polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA, 22∶6Δ4,7,10,13,16,19), is protective against colon tumorigenesis (
      • Hall M.N.
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      A 22-year prospective study of fish, n-3 fatty acid intake, and colorectal cancer risk in men.
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      Intake of polyunsaturated fatty acids and distal large bowel cancer risk in Whites and African Americans.
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      • Chan A.T.
      Marine ω-3 polyunsaturated fatty acid intake and survival after colorectal cancer diagnosis.
      ). Furthermore, preclinical evidence support the role of DHA as an adjuvant therapeutic for colon cancer (
      • Cockbain A.J.
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      • Munarini A.
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      ,
      • Cockbain A.J.
      • Toogood G.J.
      • Hull M.A.
      Omega-3 polyunsaturated fatty acids for the treatment and prevention of colorectal cancer.
      ,
      • Volpato M.
      • Hull M.A.
      Omega-3 polyunsaturated fatty acids as adjuvant therapy of colorectal cancer.
      ,
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ,
      • Rogers K.R.
      • Kikawa K.D.
      • Mouradian M.
      • Hernandez K.
      • McKinnon K.M.
      • Ahwah S.M.
      • Pardini R.S.
      Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association.
      ,
      • Fuentes N.R.
      • Mlih M.
      • Barhoumi R.
      • Fan Y.Y.
      • Hardin P.
      • Steele T.J.
      • Behmer S.
      • Prior I.A.
      • Karpac J.
      • Chapkin R.S.
      Long-chain n-3 fatty acids attenuate oncogenic kras-driven proliferation by altering plasma membrane nanoscale proteolipid composition.
      ,
      • Ding X.
      • Ge L.
      • Yan A.
      • Ding Y.
      • Tao J.
      • Liu Q.
      • Qiao C.
      Docosahexaenoic acid serving as sensitizing agents and gefitinib resistance revertants in EGFR targeting treatment.
      ). Of particular interest is the paradoxical ability of DHA to increase EGFR phosphorylation yet attenuate epidermal growth factor (EGF)-mediated Ras activation and subsequent ERK phosphorylation (
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ,
      • Rogers K.R.
      • Kikawa K.D.
      • Mouradian M.
      • Hernandez K.
      • McKinnon K.M.
      • Ahwah S.M.
      • Pardini R.S.
      Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association.
      ). However, the underlying cause for this disruption is unclear, highlighting the need to elucidate the molecular mechanism by which DHA suppresses EGFR signaling.
      EGFR signaling is initiated by the binding of ligands, such as EGF, which induces conformational changes that support receptor dimerization, autophosphorylation, the subsequent recruitment of signaling adaptors Grb2 and Sos1, downstream effectors such Ras (
      • Margolis B.
      • Skolnik E.Y.
      Activation of Ras by receptor tyrosine kinases.
      ), and ultimately the activation of ERK (
      • Shatos M.A.
      • Gu J.
      • Hodges R.R.
      • Lashkari K.
      • Dartt D.A.
      ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture.
      ). However, it was recently established that EGFR dimerization, phosphorylation, and recruitment of Grb2 and Sos1 is not sufficient for Ras activation (
      • Liang S.I.
      • van Lengerich B.
      • Eichel K.
      • Cha M.
      • Patterson D.M.
      • Yoon T.Y.
      • von Zastrow M.
      • Jura N.
      • Gartner Z.J.
      Phosphorylated EGFR dimers are not sufficient to activate Ras.
      ). A prevailing hypothesis argues that EGFR signaling is influenced by the formation of nanoscale clusters (referred to as nanoclusters) in the plasma membrane, which are maintained by protein-lipid interactions between EGFR, phosphatidic acid (PA), phosphatidylinositol-4,5-bisphosphate (PIP2), and cholesterol (
      • Ariotti N.
      • Liang H.
      • Xu Y.
      • Zhang Y.
      • Yonekubo Y.
      • Inder K.
      • Du G.
      • Parton R.G.
      • Hancock J.F.
      • Plowman S.J.
      Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation.
      ,
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ,
      • Gao J.
      • Wang Y.
      • Cai M.
      • Pan Y.
      • Xu H.
      • Jiang J.
      • Ji H.
      • Wang H.
      Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging.
      ,
      • Liang H.
      • Estes M.K.
      • Zhang H.
      • Du G.
      • Zhou Y.
      Bile acids target proteolipid nano-assemblies of EGFR and phosphatidic acid in the plasma membrane for stimulation of MAPK signaling.
      ). The clinical relevance of EGFR nanocluster formation is supported by observations indicating that the number and size of EGFR nanoclusters is increased in epithelial cancers (
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ), providing rationale for the targeted disruption of EGFR nanocluster formation as a novel therapy. Thus, the development of membrane therapy could provide a complementary tool against EGFR-driven cancer.
      Herein, we report the reduction of EGFR nanocluster formation as an underlying cause for DHA-mediated EGFR signal attenuation. By incorporation into membrane phospholipids, DHA reduces the rigidity of the plasma membrane, interfering with EGFR-cholesterol interactions, leading to the collective reduction in EGFR cluster formation, downstream Ras activation, and ERK signaling. Together, these results support the feasibility of utilizing DHA in the modulation of EGFR nanoscale spatial organization and signaling.

      Materials and methods

      Cell culture

      Conditionally immortalized young adult mouse colonic (YAMC) epithelial (RRID: CVCL_6E40) and immortalized murine colonic epithelial (IMCE) cells were originally obtained from R.H. Whitehead, Ludwig Cancer Institute (Melbourne, Australia). YAMC (passages 12–20) and IMCE (passages 19–26) cells were cultured under permissive conditions, 33°C, and 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium, no glutamine (Gibco, 21870076) supplemented with 5% fetal bovine serum (FBS; Hyclone, SH300084.03), 2 mM GlutaMAX (Gibco, 35050061), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenious acid (Corning, 354351), and 5 IU/mL of murine interferon-g (Roche, 11276905001). SW48 cells (RRID:CVCL_1724) were obtained (10/08/14) from Horizon Discovery (Cambridge, United Kingdom), where they were authenticated by gDNA and cDNA genotyping. SW48 cells (passages 8–13) were maintained at 37°C and 5% CO2 in McCoy's 5A medium supplemented with 10% FBS. YAMC cells were authenticated (07/24/15) by STR profiling (CellCheck Plus) by IDEXX BioResearch (Westbrook, ME). All cell lines used tested negative for mycoplasma bacteria (05/09/18) as assessed by a Universal Mycoplasma Detection Kit (ATCC, 30-1012K). Select cultures were treated for 24 or 72 h with 50 μM fatty acid [oleic acid (OA, 18:1n9), linoleic acid (LA, 18:2n6), arachidonic acid (AA, 20:4n6), or DHA (22:6n3); Nu-Chek Prep, Inc., Elysian, MN] complexed with fatty acid-free bovine serum albumin (BSA) or low density lipoproteins (LDLs). Select cultures were treated for 24 h with LDL-OA or LDL-DHA. Incorporation of unesterified DHA (Nu-Chek Prep, Inc., Elysian, MN) into LDL was performed by the reconstitution method, as described in our previous publication (
      • Reynolds L.
      • Mulik R.S.
      • Wen X.
      • Dilip A.
      • Corbin I.R.
      Low-density lipoprotein-mediated delivery of docosahexaenoic acid selectively kills murine liver cancer cells.
      ). BSA-DHA, LDL-OA, and LDL-DHA nanoparticle lipid composition is described in supplemental Table S1.

      EGF-dependent colonoid growth assay

      Ninety IMCE cells were seeded in 3 mL Matrigel per well in a 96-well plate using RPMI supplemented with 10% FBS and IFN-γ at 33°C. On the following day medium containing EGF (25 ng/mL) was added to allow colonies to establish. After 48 h, medium containing EGF and the indicated treatments (50 μM) was added. Organoids were grown for 10 days while changing media every 3–4 days. Cells were imaged with a 2× objective on a Keyence microscope (BZ X-710 fluorescent microscope, RRID:SCR_017202). Keyence analyzer software (BZ Analyzer software, RRID:SCR_017205) was used to generate a full focused image from a Z-stacks of 30 planes at 20 μm steps. For analysis, full focused images were opened in National Institutes of Health ImageJ software (ImageJ, RRID:SCR_003070; Fiji, RRID:SCR_002285), and a custom macro was used to quantify colonoid surface coverage.

      Spatiotemporal Ras biosensor fluorescence resonance energy transfer imaging

      YAMC cells were untreated or treated with indicated fatty acids (50 μM) for 24 h, then transfected with plasmid encoding the KRas-Raichu biosensor. Cells were subsequently incubated an additional 48 h and starved in Phenol-free RPMI (0.5%, FBS), 1% Glutamax, 1% Pen/Strep, with IFN-γ, no insulin-transferrin-selenium, for 4 h before stimulation with EGF (25 ng/mL), and images were taken at 40× magnification with 4 × 4 binning every 2 min. The fluorescence resonance energy transfer (FRET)/cyan fluorescent protein ratio of each cell was normalized by dividing by the averaged FRET/cyan fluorescent protein value before stimulation, as previously described (
      • Aoki K.
      • Matsuda M.
      Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors.
      ).

      Drosophila genetics, stocks, and culture

      The following strains were obtained from the Bloomington Drosophila Stock Center: w1118, EGFR (9535), UAS-he-EGFR-GFP (58415) and tub-Gal80ts (DGGR Cat# 130454, RRID:DGGR_130454). esg-Gal4 was kindly provided by Dr Shigeo Hayashi, Riken Center for Developmental Biology. All flies were reared on standard yeast- and cornmeal-based diet at 25°C and 65% humidity on a 12 h light/dark cycle, unless otherwise indicated. The standard laboratory diet (cornmeal based) was made with the following protocol: 14 g agar/165.4 g malt extract/41.4 g dry yeast/78.2 g cornmeal/4.7 mL propionic acid/3 g methyl 4-hydroxybenzoate/1.5 liters water. All analyses were exclusively done in female flies because of sex-specific differences in midgut regeneration.

      Mouse genetics, husbandry, and diet

      All animal experiments were approved and conducted in strict accordance with the Texas A&M University Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines. Fat-1 transgenic mice were generated and backcrossed onto a C57BL/6 background as previously described (
      • Kang J.X.
      • Wang J.
      • Wu L.
      • Kang Z.B.
      Transgenic mice: Fat-1 mice convert n-6 to n-3 fatty acids.
      ). The colony of fat-1 mice used for this study was generated by breeding heterozygous mice. The genotype and phenotype of offspring of each animal were characterized using isolated DNA and total lipids from mice tail clips (
      • Jia Q.
      • Lupton J.R.
      • Smith R.
      • Weeks B.R.
      • Callaway E.
      • Davidson L.A.
      • Kim W.
      • Fan Y.-Y.Y.
      • Yang P.
      • Newman R.A.
      • Kang J.X.
      • McMurray D.N.
      • Chapkin R.S.
      Reduced colitis-associated colon cancer in Fat-1 (n-3 fatty acid desaturase) transgenic mice.
      ).
      Mice were housed in cages in a temperature- and humidity-controlled animal facility with a 12 h light/dark cycle and fed a 10% safflower oil diet (Research Diets) ad libitum. The diet contained (g/100 g diet) 40 sucrose, 20 casein, 15 corn starch, 0.3 dl-methionine, 3.5 AIN 76A salt mix, 1.0 AIN 76A mineral mix, 0.2 choline chloride, 5 fiber (cellulose), and 10 safflower oil.

      Super-resolution microscopy labeling

      For labeling cells for nanocluster analysis using stochastic optical reconstruction microscopy (STORM), YAMC cells were seeded in cell imaging 8 chamber coverglass slides (Cellvis, C8-1.5H-N) and allowed to attach for 24 h. Cells were subsequently treated with control RPMI media (5% FBS), LDL-OA (50 μM), or LDL-DHA (50 μM) for 24 h (
      • Reynolds L.
      • Mulik R.S.
      • Wen X.
      • Dilip A.
      • Corbin I.R.
      Low-density lipoprotein-mediated delivery of docosahexaenoic acid selectively kills murine liver cancer cells.
      ). After 24 h, cells were fixed with prewarmed (37°C) 4% cytoskeleton stabilizing buffer-paraformaldehyde for 15 min at room temperature (
      • Leyton-Puig D.
      • Kedziora K.M.
      • Isogai T.
      • van den Broek B.
      • Jalink K.
      • Innocenti M.
      PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins.
      ,
      • Pereira P.M.
      • Albrecht D.
      • Culley S.
      • Jacobs C.
      • Marsh M.
      • Mercer J.
      • Henriques R.
      Fix your membrane receptor imaging: Actin cytoskeleton and cd4 membrane organization disruption by chemical fixation.
      ). After rinsing with Dulbecco's phosphate buffered saline (DPBS), cells were blocked with 5% BSA-DPBS for 30 min and subsequently incubated with 0.5 μg/mL EGF-Alexa647 (ThermoFisher, E35351) in 1% BSA-DPBS for 30 min at room temperature. Cells were rinsed with 1% BSA-DPBS twice and then with DPBS twice prior to imaging.
      For labeling primary murine colonic cells for nanocluster analysis using STORM, colonic crypts were isolated as previously described (
      • Fan Y.-Y.
      • Davidson L.A.
      • Callaway E.S.
      • Wright G.A.
      • Safe S.
      • Chapkin R.S.
      A bioassay to measure energy metabolism in mouse colonic crypts, organoids and sorted stem cells.
      ). Isolated crypts were incubated in TrypLE™ Select Enzyme (10×) (ThermoFisher, A1217701) for 30 min at 37°C, pipetted up and down every 5–10 min, and then passed through a 40 μm cell strainer. Cells were suspended in live cell imaging solution (ThermoFisher, A14291DJ) and seeded in cell imaging eight chamber coverglass slides (Cellvis, C8-1.5H-N) coated with poly-d-lysine (ThermoFisher, A3890401) and allowed to attach for 30 min on ice before fixing with 4% cytoskeleton stabilizing buffer-paraformaldehyde (ice cold) for 15 min on ice (
      • Leyton-Puig D.
      • Kedziora K.M.
      • Isogai T.
      • van den Broek B.
      • Jalink K.
      • Innocenti M.
      PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins.
      ). After rinsing with DPBS, cells were blocked with 5% BSA-DPBS for 30 min. Cells were subsequently incubated with 0.5 μg/mL EGF-Alexa647 (ThermoFisher, E35351) in 1% BSA-DPBS for 30 min at room temperature. Cells were rinsed with 1% BSA-DPBS twice and then with DPBS twice prior to imaging.
      For labeling Drosophila gut esgG4 cells for nanocluster analysis using STORM, intact fly guts were fixed at room temperature for 20 min in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, and 4% formaldehyde. After rinsing with DPBS, guts were blocked with 5% BSA-DPBS for 30 min. Subsequently, guts were incubated with 2.5 μg/mL cetuximab-Alexa594 (R&D Systems, FAB9577T) overnight at 4°C. Guts were rinsed with DPBS 1% BSA twice and then with DPBS twice more prior to mounting in Mowiol medium.

      Direct stochastic optical reconstruction microscopy and stimulated emission depletion super-resolution microscopy imaging and nanocluster analysis

      For details regarding STORM and stimulated emission depletion (STED) super-resolution imaging please see supplemental methods.

      Membrane order measurement via image-based flow cytometry

      Cells were stained with Di-4-ANEPPDHQ (Invitrogen, D36802) for membrane order determination as previously described (
      • Fan Y.-Y.
      • Fuentes N.R.
      • Hou T.Y.
      • Barhoumi R.
      • Li X.C.
      • Deutz N.E.P.
      • Engelen M.P.K.J.
      • McMurray D.N.
      • Chapkin R.S.
      Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.
      ,
      • Owen D.M.
      • Rentero C.
      • Magenau A.
      • Abu-Siniyeh A.
      • Gaus K.
      Quantitative imaging of membrane lipid order in cells and organisms.
      ,
      • Fuentes N.R.
      • Salinas M.L.
      • Kim E.
      • Chapkin R.S.
      Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization.
      ,
      • Salinas M.L.
      • Fuentes N.R.
      • Choate R.
      • Wright R.C.
      • McMurray D.N.
      • Chapkin R.S.
      AdipoRon attenuates wnt signaling by reducing cholesterol-dependent plasma membrane rigidity.
      ). In brief, cells were stained with 1 μM Di4 and imaged via image-based flow cytometry, Amnis FlowSight (
      • Fuentes N.R.
      • Salinas M.L.
      • Kim E.
      • Chapkin R.S.
      Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization.
      ,
      • Salinas M.L.
      • Fuentes N.R.
      • Choate R.
      • Wright R.C.
      • McMurray D.N.
      • Chapkin R.S.
      AdipoRon attenuates wnt signaling by reducing cholesterol-dependent plasma membrane rigidity.
      ). Laser light at 488 nm was used to excite Di4 and emission wavelengths and subsequently collected in two preset channels representing ordered (O: 480–560 nm) and disordered (D: 640–745 nm). Generalized Polarization (GP) was calculated using the equation below: GP = (Intensity(O) − G × Intensity(D))/(Intensity(O) + G × Intensity(D)). Because there is no way to acquire a calibration image, the G factor was omitted and GP was calculated as stated above using Amnis IDEAS software.

      Statistical analysis

      Statistical significance between treatments as indicated by uncommon letters (P < 0.01) was analyzed using one-way ANOVA and uncorrected Fisher's least significant difference (LSD) tests. All analyses were conducted using Prism statistical software (GraphPad Software, Inc.).

      Results

      DHA attenuates EGFR-mediated phenotypes

      To assess the ability of DHA to modulate EGFR-driven proliferation, we utilized a 3D model of EGF-dependent growth, i.e., the (IMCE) cell line, which is a nontransformed normal colonic epithelial cell line, carrying one mutated APC allele, which is conditionally immortalized by expression of a temperature-sensitive simian virus 40 (SV40) large T antigen (
      • D'Abaco G.M.
      • Whitehead R.H.
      • Burgess A.W.
      Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.
      ). IMCE cells do not form colonies in soft agar without the addition of an oncogenic Ras mutation (
      • D'Abaco G.M.
      • Whitehead R.H.
      • Burgess A.W.
      Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.
      ). Since oncogenic Ras is downstream of EGFR, we hypothesized that the addition of exogenous EGF would allow IMCE cells to form 3D colonies, i.e., colonoids, in Matrigel™. Supplementing media with EGF (25 ng/mL) induced the formation of IMCE colonoids (supplemental Fig. S1A, B). Colonoid size was EGF dependent, since EGF withdrawal produced smaller colonoids as determined by surface area assessment (supplemental Fig. S1C, D). To determine the effect of DHA supplementation on EGFR-dependent growth, we utilized a DHA-based therapeutic consisting of DHA free fatty acid inserted into human LDL particles (
      • Reynolds L.
      • Mulik R.S.
      • Wen X.
      • Dilip A.
      • Corbin I.R.
      Low-density lipoprotein-mediated delivery of docosahexaenoic acid selectively kills murine liver cancer cells.
      ). Each LDL nanoparticle is devoid of cholesterol, contains approximately 1,100 unesterified DHA fatty acid molecules (LDL-DHA), and is approximately 22 nm in diameter (
      • Mulik R.S.
      • Zheng H.
      • Pichumani K.
      • Ratnakar J.
      • Jiang Q.X.
      • Corbin I.R.
      Elucidating the structural organization of a novel low-density lipoprotein nanoparticle reconstituted with docosahexaenoic acid.
      ). These nanoparticles have been shown to preferentially and efficiently target liver cancer versus noncancer cells in vitro (
      • Reynolds L.
      • Mulik R.S.
      • Wen X.
      • Dilip A.
      • Corbin I.R.
      Low-density lipoprotein-mediated delivery of docosahexaenoic acid selectively kills murine liver cancer cells.
      ,
      • Moss L.R.
      • Mulik R.S.
      • Van Treuren T.
      • Kim S.Y.
      • Corbin I.R.
      Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells.
      ) and reduce proliferation of xenografts of hepatocellular carcinoma (HepG2) in vivo. LDL-DHA treatment reduced colonoid size as compared with untreated and monounsaturated fatty acid (OA) controls (Fig. 1A, B). This is consistent with our previous studies demonstrating the EGFR dependency of DHA with regard to cell proliferation by comparing its effects in wild type versus EGFR null YAMC cells (
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ). Next, we assessed the impact of DHA on EGF-mediated spatiotemporal activation of K- and H-Ras. Nontransformed conditionally immortalized YAMC cells, which contain a temperature-sensitive mutation of the SV40 large T antigen gene, were treated with fatty acid complexed with BSA and subsequently transfected with plasmids encoding K- or H-Ras-Raichu FRET-based biosensors (
      • Fukano T.
      • Sawano A.
      • Ohba Y.
      • Matsuda M.
      • Miyawaki A.
      Differential Ras activation between caveolae/raft and non-raft microdomains.
      ,
      • Mochizuki N.
      • Yamashita S.
      • Kurokawa K.
      • Ohba Y.
      • Nagai T.
      • Miyawaki A.
      • Matsuda M.
      Spatio-temporal images of growth-factor-induced activation of Ras and Rap1.
      ). DHA treatment attenuated temporal activation of K- and H-Ras biosensors compared with untreated and PUFA (e.g., LA)-treated controls (Fig. 1C–E). Collectively, these findings demonstrate that in vitro delivery of DHA functionally impairs EGFR-dependent signaling in colonic cells, consistent with previous observations (
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ,
      • Rogers K.R.
      • Kikawa K.D.
      • Mouradian M.
      • Hernandez K.
      • McKinnon K.M.
      • Ahwah S.M.
      • Pardini R.S.
      Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association.
      ).
      Figure thumbnail gr1
      Fig. 1Exogenous supplementation with DHA reduces EGFR-dependent proliferation and downstream signaling in vitro. A: Representative images of colonoids grown with indicated treatments. Scale bar, 300 and 100 μm. B: Quantification of colonoid surface area after 10 days of treatment. Data represent mean ± SE. Number of organoids examined per treatment, Untreated No EGF = 112, Untreated + EGF = 109, LDL-OA + EGF = 109, LDL-DHA + EGF = 112, from eight wells per group from two independent experiments. Statistical significance between treatments as indicated by uncommon letters (P < 0.05) was examined using one-way ANOVA and uncorrected Fisher's LSD tests. C: Spatiotemporal activation of Ras was determined by monitoring activation of FRET biosensors targeted to (D) K- or (E) H-Ras domains. C: Representative intensity-modulated images of KRas-Raichu–expressing cells at various time points following EGF stimulation. Scale bar, 20 μm. D: Data represent mean ± SE, FRET ratio for each cell. Number of cells examined per treatment, Untreated = 8, BSA-LA = 11, and LDL-DHA = 8. All points after 4 min are statistically significant (P < 0.05) between BSA-DHA and untreated (control) as indicated by bar and (∗). E: Data represent mean ± SE, FRET ratio for each cell. Number of cells examined per treatment, Untreated = 26, BSA-LA = 10, and LDL-DHA = 22. All points after 4 min are statistically significant (P < 0.05) between BSA-DHA and untreated (control) as indicated by bar and (∗).
      In complementary experiments, we determined whether dietary DHA modulates EGFR-mediated phenotypes in vivo. For this purpose, the Drosophila intestinal (midgut) epithelium model was utilized. The presence of somatic intestinal stem cells (ISCs) within the fly midgut allows for the use of a wide range of genetic tools to assay signaling events that govern proliferative homeostasis in vivo (
      • Biteau B.
      • Hochmuth C.E.
      • Jasper H.
      Maintaining tissue homeostasis: Dynamic control of somatic stem cell activity.
      ). This barrier epithelium, with functional and morphological similarities to the mammalian small intestine and mouse airway epithelia (
      • Biteau B.
      • Hochmuth C.E.
      • Jasper H.
      Maintaining tissue homeostasis: Dynamic control of somatic stem cell activity.
      ), contains ISCs that can asymmetrically divide, forming an enteroblast that directly differentiates into functional enterocytes. Thus, the Drosophila ISC lineage provides an excellent model to study signaling mechanisms regulating stem cell maintenance and dysfunction, including EGFR-mediated proliferative signaling (
      • Jiang H.
      • Grenley M.O.
      • Bravo M.J.
      • Blumhagen R.Z.
      • Edgar B.A.
      EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila.
      ,
      • Jiang H.
      • Edgar B.A.
      EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors.
      ). To extend our findings in IMCE cells, we assessed the effects of a DHA-enriched diet on EGFR-dependent proliferation and signaling in Drosophila using transgenic flies that overexpress dEGFR specifically within somatic ISC and enteroblasts (using the EsgGal4, GFP driver). Flies were fed a low-PUFA diet (
      • Ziegler A.B.
      • Ménagé C.
      • Grégoire S.
      • Garcia T.
      • Ferveur J.-F.
      • Bretillon L.
      • Grosjean Y.
      Lack of dietary polyunsaturated fatty acids causes synapse dysfunction in the drosophila visual system.
      ) with or without the addition of 0.5% w/w DHA or OA, a control monounsaturated fatty acid. As expected, overexpression of dEGFR targeted to ISCs in the adult fly increased stem cell proliferation and ERK signaling (Fig. 2A–D). Flies fed DHA verses low PUFA (basal) or OA control diets exhibited a reduction in stem cell proliferation (Fig. 2A, B) and ERK activation (Fig. 2C, D). Collectively, these data demonstrate that DHA suppresses EGFR-dependent proliferative phenotypes in vivo.
      Figure thumbnail gr2
      Fig. 2Dietary DHA reduces EGFR overexpression–driven proliferation and ERK activation. Adult Drosophila were placed on control (PUFA Free) or OA- or DHA-enriched diets for 5 days at 18°C (permissive temperature) before switching to 29°C for 2 days to induce EGFR overexpression in gut esgG4 cells. A: Representative merged and pH3 images. Scale bar, 20 μm. B: Quantitative analysis of proliferation as assessed by pH3 at 48 h post EGFR induction. Data represent mean ± SE from 16–21 guts from three independent experiments. C: Representative merged maximum image projection and masked esgG4 stem cell pERK. Scale bar, 20 μm. Quantitative analysis of (D) mean pERK in esgG4 cells per field of view (FOV) from flies fed the experimental diets. Data represent mean ± SE from 39–51 FOV from 30 guts from three independent experiments. Statistical significance between treatments as indicated by uncommon letters (B, P < 0.05; D, P < 0.01) was determined using one-way ANOVA and uncorrected Fisher's LSD tests.

      Plasma membrane biophysical properties are altered by DHA

      We next investigated the mechanism by which DHA impairs EGFR function. With 22 carbons and six double bonds, DHA is the longest and most unsaturated fatty acid commonly found in human membranes. When incorporated into membrane phospholipids, including caveolae and lipid raft domains (
      • Ma D.W.L.
      • Seo J.
      • Davidson L.A.
      • Callaway E.S.
      • Fan Y.-Y.Y.
      • Lupton J.R.
      • Chapkin R.S.
      n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon.
      ,
      • Fan Y.Y.
      • Ly L.H.
      • Barhoumi R.
      • McMurray D.N.
      • Chapkin R.S.
      Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production.
      ), the unique biophysical characteristics of DHA can influence membrane structure (
      • Fan Y.-Y.
      • Fuentes N.R.
      • Hou T.Y.
      • Barhoumi R.
      • Li X.C.
      • Deutz N.E.P.
      • Engelen M.P.K.J.
      • McMurray D.N.
      • Chapkin R.S.
      Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.
      ,
      • Shaikh S.R.
      • Locascio D.S.
      • Soni S.P.
      • Wassall S.R.
      • Stillwell W.
      Oleic- and docosahexaenoic acid-containing phosphatidylethanolamines differentially phase separate from sphingomyelin.
      ,
      • Williams J.A.
      • Batten S.E.
      • Harris M.
      • Rockett B.D.
      • Shaikh S.R.
      • Stillwell W.
      • Wassall S.R.
      Docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains.
      ). Since the biophysical properties of the plasma membrane can modulate plasma membrane receptor function (
      • Salinas M.L.
      • Fuentes N.R.
      • Choate R.
      • Wright R.C.
      • McMurray D.N.
      • Chapkin R.S.
      AdipoRon attenuates wnt signaling by reducing cholesterol-dependent plasma membrane rigidity.
      ) and downstream cellular signaling (
      • Sezgin E.
      • Levental I.
      • Mayor S.
      • Eggeling C.
      The mystery of membrane organization: Composition, regulation and roles of lipid rafts.
      ), we initially explored the effects of LDL-DHA on plasma membrane rigidity. For this purpose, IMCE cells were labeled with a polarity-sensitive dye, Di-4-ANEPPDHQ (Di4) (
      • Owen D.M.
      • Rentero C.
      • Magenau A.
      • Abu-Siniyeh A.
      • Gaus K.
      Quantitative imaging of membrane lipid order in cells and organisms.
      ,
      • Fuentes N.R.
      • Salinas M.L.
      • Kim E.
      • Chapkin R.S.
      Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization.
      ), and individually imaged (Fig. 3A). LDL-DHA treatment reduced plasma membrane rigidity (Fig. 3B). To determine if DHA modulates plasma membrane biophysical properties in vivo, colonocytes were isolated from Fat-1 transgenic mice, a genetic model in which phospholipids are enriched via de novo synthesis of DHA (
      • Kang J.X.
      • Wang J.
      • Wu L.
      • Kang Z.B.
      Transgenic mice: Fat-1 mice convert n-6 to n-3 fatty acids.
      ,
      • Jia Q.
      • Lupton J.R.
      • Smith R.
      • Weeks B.R.
      • Callaway E.
      • Davidson L.A.
      • Kim W.
      • Fan Y.-Y.Y.
      • Yang P.
      • Newman R.A.
      • Kang J.X.
      • McMurray D.N.
      • Chapkin R.S.
      Reduced colitis-associated colon cancer in Fat-1 (n-3 fatty acid desaturase) transgenic mice.
      ). Of note, plasma membrane rigidity was also reduced in primary colonic cells isolated from Fat-1 mice versus wild-type controls (Fig. 3C, D). Overall, these findings underscore the ability of DHA to modulate plasma membrane biophysical properties by reducing plasma membrane rigidity.
      Figure thumbnail gr3
      Fig. 3DHA reduces plasma membrane rigidity. IMCE cells were incubated with the indicated treatments (50 μM) for 24 h before labeling with Di-4-ANEPPDHQ, followed by assessment of membrane order using imaged based flow cytometry. A: Representative images and (B) quantitative analysis of membrane order in IMCE cells. Data represent mean ± SE from individual cells from untreated (13,240), LDL-OA (12,202), and LDL-DHA (14,674), from three independent experiments. Statistical significance between treatments as indicated by uncommon letters (P < 0.0001) was examined using one-way ANOVA and uncorrected Fisher's LSD tests. Single cells from wild-type or Fat-1 mice were labeled with Di-4-ANEPPDHQ and imaged via imaging flow cytometry. C: Representative images and (D) quantitative analysis of membrane order in isolated primary murine colonic cells. Data represent mean ± SE from individual cells from WT (12,655) and Fat-1 (16,675) from 3 and 4 mice, respectively. Statistical significance between groups (∗P < 0.0001) was examined using an unpaired t-tests.

      DHA attenuates EGFR nanocluster formation

      The observed effect of DHA on plasma membrane biophysical properties is relevant since EGF stimulation can induce the formation of cholesterol-enriched rigid plasma membrane domains, i.e., lipid raft components (
      • Hofman E.G.
      • Bader A.N.
      • Gerritsen H.C.
      • van Bergen En Henegouwen P.M.
      EGF induces rapid reorganization of plasma membrane microdomains.
      ,
      • Hofman E.G.
      • Ruonala M.O.
      • Bader A.N.
      • van den Heuvel D.
      • Voortman J.
      • Roovers R.C.
      • Verkleij A.J.
      • Gerritsen H.C.
      • van Bergen en Henegouwen P.M.P.
      EGF induces coalescence of different lipid rafts.
      ,
      • Golfetto O.
      • Hinde E.
      • Gratton E.
      Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes.
      ). Since plasma membrane rigidity is influenced by many factors including lipid saturation (
      • Fan Y.-Y.
      • Fuentes N.R.
      • Hou T.Y.
      • Barhoumi R.
      • Li X.C.
      • Deutz N.E.P.
      • Engelen M.P.K.J.
      • McMurray D.N.
      • Chapkin R.S.
      Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.
      ,
      • Wang B.
      • Rong X.
      • Palladino E.N.D.
      • Wang J.
      • Fogelman A.M.
      • Martín M.G.
      • Alrefai W.A.
      • Ford D.A.
      • Tontonoz P.
      Phospholipid remodeling and cholesterol availability regulate intestinal stemness and tumorigenesis.
      ,
      • Levental K.R.
      • Surma M.A.
      • Skinkle A.D.
      • Lorent J.H.
      • Zhou Y.
      • Klose C.
      • Chang J.T.
      • Hancock J.F.
      • Levental I.
      ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis.
      ), cholesterol content (
      • Sezgin E.
      • Sadowski T.
      • Simons K.
      Measuring lipid packing of model and cellular membranes with environment sensitive probes.
      ,
      • Fuentes N.R.
      • Kim E.
      • Fan Y.-Y.
      • Chapkin R.S.
      Omega-3 fatty acids, membrane remodeling and cancer prevention.
      ), and cytoskeletal interactions (
      • Dinic J.
      • Ashrafzadeh P.
      • Parmryd I.
      Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains.
      ), its modulation may not be correlated to protein clustering in the plasma membrane (
      • Alvarez-Guaita A.
      • Vilà de Muga S.
      • Owen D.M.
      • Williamson D.
      • Magenau A.
      • García-Melero A.
      • Reverter M.
      • Hoque M.
      • Cairns R.
      • Cornely R.
      • Tebar F.
      • Grewal T.
      • Gaus K.
      • Ayala-Sanmartín J.
      • Enrich C.
      • et al.
      Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains.
      ). Therefore, we used super-resolution microscopy to determine if DHA incorporation into membrane phospholipids reduces EGFR cluster size. We first validated the functionality and specificity of fluorophore complexed EGF. EGF-488 showed strong internalization at 37°C but not 4°C (supplemental Fig. S2A, B) and was specific for EGFR, e.g., it failed to label EGFR-negative cells (supplemental Fig. S2C). In subsequent experiments, EGFR clustering in YAMC cells was assessed by STORM (supplemental Fig. S3). EGFR nanocluster size was reduced by LDL-DHA (Fig. 4A, B; supplemental Fig. S4A–C) and BSA-DHA (supplemental Fig. S5) treatment as compared with LDL-OA and untreated controls. Of note, EGFR clustering was reduced by LDL-DHA and BSA-DHA treatment in SW48 colorectal adenocarcinoma cells (supplemental Fig. S6), which express high levels of EGFR (
      • Yuan Z.
      • Shin J.
      • Wilson A.
      • Goel S.
      • Ling Y.-H.
      • Ahmed N.
      • Dopeso H.
      • Jhawer M.
      • Nasser S.
      • Montagna C.
      • Fordyce K.
      • Augenlicht L.H.
      • Aaltonen L.A.
      • Arango D.
      • Weber T.K.
      • et al.
      An A13 repeat within the 3’-untranslated region of epidermal growth factor receptor (EGFR) is frequently mutated in microsatellite instability colon cancers and is associated with increased EGFR expression.
      ) containing a G719S mutation, which is constitutively active and resistant to kinase inhibitors (
      • Cho J.
      • Bass A.J.
      • Lawrence M.S.
      • Cibulskis K.
      • Cho A.
      • Lee S.-N.
      • Yamauchi M.
      • Wagle N.
      • Pochanard P.
      • Kim N.
      • Park A.K.
      • Won J.
      • Hur H.-S.
      • Greulich H.
      • Ogino S.
      • et al.
      Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab.
      ,
      • Osoegawa A.
      • Hashimoto T.
      • Takumi Y.
      • Abe M.
      • Yamada T.
      • Kobayashi R.
      • Miyawaki M.
      • Takeuchi H.
      • Okamoto T.
      • Sugio K.
      Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation.
      ). Because delivery of DHA by LDL nanoparticles is pharmacological in nature, we verified that LDL-DHA treatment enhanced the DHA composition of cellular phospholipids to a similar degree as compared with the physiological method of DHA delivery using BSA-DHA (supplemental Table S2). Furthermore, isolated colonocytes from Fat-1 (genetically enriched with DHA) versus wild-type litter-mate control mice contained smaller EGFR clusters (Fig. 4C, D; supplemental Fig. S7A, B). Therefore, we investigated the in vivo effects of dietary DHA on EGFR clustering in Drosophila using STED microscopy. For this purpose, a chimeric EGFR protein containing human extracellular and Drosophila intracellular domains (
      • Inaki M.
      • Vishnu S.
      • Cliffe A.
      • Rorth P.
      Effective guidance of collective migration based on differences in cell states.
      ) was expressed in ISCs. This model allows for the labeling of EGFR in intact gut ISCs in a manner compatible with STED imaging (supplemental Fig. S8A–E). Utilizing the same dietary enrichment strategy as previously described, flies fed a DHA-enriched diet as compared with OA and low PUFA controls exhibited reduced EGFR cluster size (Fig. 4E–I). Collectively, these results demonstrate the ability of DHA to attenuate EGFR cluster formation across multiple in vitro and in vivo models.
      Figure thumbnail gr4
      Fig. 4DHA reduces EGFR nanoclustering. YAMC cells were incubated with the indicated treatments (50 μM) for 24 h prior to fixation and subsequent labeling with EGF-Alexa647 for STORM imaging. A: Quantitative analysis of EGFR cluster diameter and (B) relative cluster size in YAMC cells. Data are presented as (A) mean ± SE of average EGFR cluster diameter per FOV and (B) individual cluster distribution. Number of FOVs examined per treatment, untreated = 46, LDL-OA = 46, LDL-DHA = 46, and individual clusters, untreated = 4,824, LDL-OA = 8,305, LDL-DHA = 2,921, from four wells per group from two independent experiments. C: Quantitative analysis of EGFR cluster diameter and (D) relative cluster size in isolated primary murine colonic cells. Data are presented as (C) mean ± SE of average EGFR cluster diameter per FOV, and (D) individual cluster size distribution. Number of FOVs examined per group, wild type = 55 and Fat-1 = 69, and individual clusters, wild type = 4,742 and Fat-1 = 3,872, from 3 or 4 mice, respectively. Adult Drosophila were placed on control (PUFA Free) or OA- or DHA-enriched diets for 5 days at 18°C (permissive temperature) before switching to 29°C for 2 days to induce chimeric human EGFR expression in gut esgG4 cells. E–G: Representative processed STED images from indicated diet. Scale bar, 1 μm and 500 nm. H: Quantitative analysis of EGFR cluster diameter and (I) relative frequency in Drosophila gut esgG4 cells. Data are presented as (H) mean ± SE of average EGFR cluster diameter per FOV, and (I) individual cluster size distribution. Number of FOVs examined per group, Low PUFA = 68, OA = 80, and DHA = 82, and individual clusters, Low PUFA = 51,442, OA = 56,471, and DHA = 35,103, from three independent experiments. Unless otherwise indicated, statistical significance between groups as indicated by uncommon letters (P < 0.001) was analyzed using one-way ANOVA and uncorrected Fisher's LSD tests.

      PA, PIP2, and cholesterol maintain EGFR nanocluster formation

      The acidic lipids PA and PIP2 as well as cholesterol are key structural components of EGFR signaling proteolipid nanodomains (
      • Ariotti N.
      • Liang H.
      • Xu Y.
      • Zhang Y.
      • Yonekubo Y.
      • Inder K.
      • Du G.
      • Parton R.G.
      • Hancock J.F.
      • Plowman S.J.
      Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation.
      ,
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ,
      • Gao J.
      • Wang Y.
      • Cai M.
      • Pan Y.
      • Xu H.
      • Jiang J.
      • Ji H.
      • Wang H.
      Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging.
      ,
      • Michailidis I.E.
      • Rusinova R.
      • Georgakopoulos A.
      • Chen Y.
      • Iyengar R.
      • Robakis N.K.
      • Logothetis D.E.
      • Baki L.
      • et al.
      Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation.
      ,
      • Zhang F.
      • Wang Z.
      • Lu M.
      • Yonekubo Y.
      • Liang X.
      • Zhang Y.
      • Wu P.
      • Zhou Y.
      • Grinstein S.
      • Hancock J.F.
      • Du G.
      Temporal production of the signaling lipid phosphatidic acid by phospholipase D2 determines the output of extracellular signal-regulated kinase signaling in cancer cells.
      ,
      • Zhou Y.
      • Liang H.
      • Rodkey T.
      • Ariotti N.
      • Parton R.G.
      • Hancock J.F.
      Signal integration by lipid-mediated spatial cross talk between Ras nanoclusters.
      ,
      • Nishioka T.
      • Frohman M.A.
      • Matsuda M.
      • Kiyokawa E.
      Heterogeneity of phosphatidic acid levels and distribution at the plasma membrane in living cells as visualized by a Föster resonance energy transfer (FRET) biosensor.
      ,
      • Nishioka T.
      • Aoki K.
      • Hikake K.
      • Yoshizaki H.
      • Kiyokawa E.
      • Matsuda M.
      Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells.
      ). To investigate the role of PA, PIP2, and cholesterol in EGFR nanocluster formation in our models, we treated YAMC cells with 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), phenylarsine oxide (PAO), or methyl-beta-cyclodextrin (MβCD) to reduce levels of PA, PIP2, and cholesterol, respectively (
      • Liang H.
      • Estes M.K.
      • Zhang H.
      • Du G.
      • Zhou Y.
      Bile acids target proteolipid nano-assemblies of EGFR and phosphatidic acid in the plasma membrane for stimulation of MAPK signaling.
      ,
      • Salinas M.L.
      • Fuentes N.R.
      • Choate R.
      • Wright R.C.
      • McMurray D.N.
      • Chapkin R.S.
      AdipoRon attenuates wnt signaling by reducing cholesterol-dependent plasma membrane rigidity.
      ,
      • Nishioka T.
      • Frohman M.A.
      • Matsuda M.
      • Kiyokawa E.
      Heterogeneity of phosphatidic acid levels and distribution at the plasma membrane in living cells as visualized by a Föster resonance energy transfer (FRET) biosensor.
      ,
      • Nishioka T.
      • Aoki K.
      • Hikake K.
      • Yoshizaki H.
      • Kiyokawa E.
      • Matsuda M.
      Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells.
      ). As expected, the suppression of PA, PIP2, and cholesterol reduced the size of EGFR nanoclusters (Fig. 5A, B). In addition, we determined the functional consequences of inhibitor-mediated EGFR cluster size reduction. Downstream signaling, e.g., spatiotemporal Ras activation (supplemental Fig. S9A, B), was also inhibited by FIPI and PAO. Overall, these findings support the premise that EGFR nanocluster formation and signal propagation are in part dependent on lipid structural components.
      Figure thumbnail gr5
      Fig. 5Influence of PIP2, PA, and cholesterol on EGFR nanocluster size in mouse colonic cells. YAMC cells were incubated with DMSO (0.1%), FIPI (1 μM), PAO (1 μM), or MβCD (10 mM) for 30 min at 33°C, before fixation and labeling with EGF-Alexa647 for STORM imaging. A: Quantitative analysis of EGFR cluster diameter and (B) relative frequency in YAMC cells. Data are presented as (A) mean ± SE of average EGFR cluster diameter per FOV and (B) individual cluster size distribution. Number of FOVs examined per treatment, DMSO = 32, FIPI = 32, PAO= 34, and MβCD = 31, and individual clusters, DMSO = 3,559, FIPI = 2,443, PAO = 1,512, and MβCD = 2,700, from 3 wells per group from 3 independent experiments. Statistical significance between treatments as indicated by uncommon letters (P < 0.05) was analyzed using one-way ANOVA and uncorrected Fisher's LSD tests.

      DHA disrupts EGFR proteolipid composition

      Because PA, PIP2, and cholesterol play an important role in maintaining EGFR clustering and signaling, we hypothesized that exogenous addition of these components would rescue EGFR clustering in the presence of DHA. Of note, only the addition of cholesterol, but not PA or PIP2, restored EGFR cluster formation (Fig. 6A, B; supplemental Fig. S10). We have previously established that DHA can displace EGFR from cholesterol-rich lipid raft domains (
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ). In the present study, we use fluorescence lifetime imaging combined with fluorescence resonance energy transfer (FLIM-FRET) microscopy to monitor nanoscale (<10 nm) interactions between fluorescently tagged EGFR and a cholesterol binding probe (
      • Zhou Y.
      • Hancock J.F.
      Chapter 2 – Ras proteolipid nanoassemblies on the plasma membrane sort lipids with high selectivity.
      ,
      • Maekawa M.
      • Fairn G.D.
      Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.
      ). When coexpressed with a corresponding FRET pair, such as red fluorescent protein, a reduction of GFP lifetime is indicative of more extensive FRET as a result of a smaller distance between GFP and red fluorescent protein, which is correlated with more extensive nanoclustering. Lifetime values were then converted to apparent FRET efficiency %, where an increase is indicative of enhanced nanoclustering (
      • Najumudeen A.K.
      • Jaiswal A.
      • Lectez B.
      • Oetken-Lindholm C.
      • Guzmán C.
      • Siljamäki E.
      • Posada I.M.D.
      • Lacey E.
      • Aittokallio T.
      • Abankwa D.
      Cancer stem cell drugs target K-ras signaling in a stemness context.
      ). Of note, YAMC cells treated with DHA showed a reduction in FRET efficiency between EGFR and cholesterol (supplemental Fig. S11). These observations indicate that DHA-mediated reduction of EGFR clustering involves cholesterol-EGFR interactions.
      Figure thumbnail gr6
      Fig. 6Exogenous cholesterol restores EGFR cluster formation in DHA-treated cells. YAMC cells were untreated or incubated with LDL-DHA (50 μM) for 24 h before the addition of media alone or media supplemented with PA (100 μM), PIP2 (100 μM), PS (100 μM), or cholesterol (1 mM) for 30 min at 33°C, before fixation and labeling with EGF-Alexa647 for STORM imaging. A: Quantitative analysis of EGFR cluster diameter and (B) relative frequency in YAMC cells. Data are presented as (A) mean ± SE of average EGFR cluster diameter per FOV and (B) individual cluster size distribution. Number of FOVs examined per treatment, untreated = 52, LDL-DHA = 52, LDL-DHA + PA = 40, LDL-DHA + PIP2 = 40, LDL-DHA + PS = 40, LDL-DHA + cholesterol = 52, and individual clusters, untreated = 6,196, LDL-DHA = 5,435, LDL-DHA + PA = 3,451, LDL-DHA + PIP2 = 2,319, LDL-DHA + PS = 2,870, LDL-DHA + cholesterol = 5,790, from five wells per group from three independent experiments. Statistical significance between treatments as indicated by uncommon letters (P < 0.005) was analyzed using one-way ANOVA and uncorrected Fisher's LSD tests.

      Discussion

      There is a critical need for the development of novel anti-EGFR targeted therapies as patients utilizing current therapies often exhibit undesirable side effects (
      • Fakih M.
      • Vincent M.
      Adverse events associated with anti-EGFR therapies for the treatment of metastatic colorectal cancer.
      ) or develop acquired resistance (
      • Dienstmann R.
      • Salazar R.
      • Tabernero J.
      Overcoming resistance to anti-EGFR therapy in colorectal cancer.
      ,
      • Zhao B.
      • Wang L.
      • Qiu H.
      • Zhang M.
      • Sun L.
      • Peng P.
      • Yu Q.
      • Yuan X.
      Mechanisms of resistance to anti-EGFR therapy in colorectal cancer.
      ). A large body of epidemiological, preclinical, and clinical evidence supports the role of DHA as an adjuvant therapy for colon cancer (
      • Cockbain A.J.
      • Volpato M.
      • Race A.D.
      • Munarini A.
      • Fazio C.
      • Belluzzi A.
      • Loadman P.M.
      • Toogood G.J.
      • Hull M.A.
      Anticolorectal cancer activity of the omega-3 polyunsaturated fatty acid eicosapentaenoic acid.
      ,
      • Cockbain A.J.
      • Toogood G.J.
      • Hull M.A.
      Omega-3 polyunsaturated fatty acids for the treatment and prevention of colorectal cancer.
      ,
      • Volpato M.
      • Hull M.A.
      Omega-3 polyunsaturated fatty acids as adjuvant therapy of colorectal cancer.
      ,
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ,
      • Rogers K.R.
      • Kikawa K.D.
      • Mouradian M.
      • Hernandez K.
      • McKinnon K.M.
      • Ahwah S.M.
      • Pardini R.S.
      Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association.
      ,
      • Fuentes N.R.
      • Mlih M.
      • Barhoumi R.
      • Fan Y.Y.
      • Hardin P.
      • Steele T.J.
      • Behmer S.
      • Prior I.A.
      • Karpac J.
      • Chapkin R.S.
      Long-chain n-3 fatty acids attenuate oncogenic kras-driven proliferation by altering plasma membrane nanoscale proteolipid composition.
      ,
      • Ding X.
      • Ge L.
      • Yan A.
      • Ding Y.
      • Tao J.
      • Liu Q.
      • Qiao C.
      Docosahexaenoic acid serving as sensitizing agents and gefitinib resistance revertants in EGFR targeting treatment.
      ). However, if DHA is to be pursued as an alternative/complementary strategy for anti-EGFR therapy, the mechanism by which it suppresses EGFR signaling must be elucidated.
      In this study, we utilized complementary super-resolution microscopy techniques to demonstrate that cellular membrane phospholipid enrichment of DHA by therapeutic nanoparticle delivery, endogenous synthesis, or diet suppresses EGFR-mediated phenotypes and reduces EGFR nanocluster formation across a variety of in vitro and in vivo models. This is noteworthy, because EGFR nanocluster formation influences the efficiency of signal propagation (
      • Ariotti N.
      • Liang H.
      • Xu Y.
      • Zhang Y.
      • Yonekubo Y.
      • Inder K.
      • Du G.
      • Parton R.G.
      • Hancock J.F.
      • Plowman S.J.
      Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation.
      ,
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ,
      • Liang H.
      • Estes M.K.
      • Zhang H.
      • Du G.
      • Zhou Y.
      Bile acids target proteolipid nano-assemblies of EGFR and phosphatidic acid in the plasma membrane for stimulation of MAPK signaling.
      ). Dimerization and subsequent phosphorylation of EGFR does not sufficiently induce ERK phosphorylation (
      • Liang S.I.
      • van Lengerich B.
      • Eichel K.
      • Cha M.
      • Patterson D.M.
      • Yoon T.Y.
      • von Zastrow M.
      • Jura N.
      • Gartner Z.J.
      Phosphorylated EGFR dimers are not sufficient to activate Ras.
      ,
      • Yoshida T.
      • Okamoto I.
      • Okabe T.
      • Iwasa T.
      • Satoh T.
      • Nishio K.
      • Fukuoka M.
      • Nakagawa K.
      Matuzumab and cetuximab activate the epidermal growth factor receptor but fail to trigger downstream signaling by Akt or Erk.
      ). These observations are consistent with reports that DHA-induced hyperphosphorylation of EGFR paradoxically suppressed Ras activation and ERK phosphorylation by reducing Ras/Sos1 interaction (
      • Turk H.F.
      • Barhoumi R.
      • Chapkin R.S.
      Alteration of EGFR spatiotemporal dynamics suppresses signal transduction.
      ,
      • Rogers K.R.
      • Kikawa K.D.
      • Mouradian M.
      • Hernandez K.
      • McKinnon K.M.
      • Ahwah S.M.
      • Pardini R.S.
      Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association.
      ). The mechanism by which EGFR nanocluster formation supports signal propagation is still not fully understood; however, it likely involves interactions with Ras, Sos1, and critical structural lipids (
      • Liang H.
      • Estes M.K.
      • Zhang H.
      • Du G.
      • Zhou Y.
      Bile acids target proteolipid nano-assemblies of EGFR and phosphatidic acid in the plasma membrane for stimulation of MAPK signaling.
      ,
      • Jun J.E.
      • Rubio I.
      • Roose J.P.
      Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells.
      ). Sos1 is recruited to the plasma membrane by activated EGFR, which subsequently engages Ras. This process is subject to complex regulation by the temporal and spatial production of phosphoinositides through interactions with SOS1 Dbl homology/pleckstrin homology regulatory domains (
      • Sondermann H.
      • Soisson S.M.
      • Boykevisch S.
      • Yang S.-S.
      • Bar-Sagi D.
      • Kuriyan J.
      Structural analysis of autoinhibition in the Ras activator son of sevenless.
      ). The acidic lipids PA and PIP2 regulate Sos1 recruitment to the plasma membrane where it interacts with and activates Ras to propagate downstream signaling (
      • Zhang F.
      • Wang Z.
      • Lu M.
      • Yonekubo Y.
      • Liang X.
      • Zhang Y.
      • Wu P.
      • Zhou Y.
      • Grinstein S.
      • Hancock J.F.
      • Du G.
      Temporal production of the signaling lipid phosphatidic acid by phospholipase D2 determines the output of extracellular signal-regulated kinase signaling in cancer cells.
      ,
      • Yadav K.K.
      • Bar-Sagi D.
      Allosteric gating of Son of sevenless activity by the histone domain.
      ,
      • Gureasko J.
      • Kuchment O.
      • Makino D.L.
      • Sondermann H.
      • Bar-Sagi D.
      • Kuriyan J.
      Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless.
      ,
      • Zhao C.
      • Du G.
      • Skowronek K.
      • Frohman M.A.
      • Bar-Sagi D.
      Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos.
      ). Furthermore, PA production by phospholipase D2 and the nanoscale organization of Ras are influenced by cholesterol (
      • Lisboa F.A.
      • Peng Z.
      • Combs C.A.
      • Beaven M.A.
      Phospholipase D promotes lipid microdomain-associated signaling events in mast cells.
      ,
      • Diaz O.
      • Mébarek-Azzam S.
      • Benzaria A.
      • Dubois M.
      • Lagarde M.
      • Némoz G.
      • Prigent A.-F.
      • et al.
      Disruption of lipid rafts stimulates phospholipase D activity in human lymphocytes: Implication in the regulation of immune function.
      ,
      • Prior I.A.
      • Muncke C.
      • Parton R.G.
      • Hancock J.F.
      Direct visualization of Ras proteins in spatially distinct cell surface microdomains.
      ,
      • Plowman S.J.
      • Muncke C.
      • Parton R.G.
      • Hancock J.F.
      H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton.
      ). Overall, this supports a model where the formation of EGFR nanoclusters is driven by lipid-protein interactions whose proteolipid composition acts as a niche to recruit the appropriate components necessary to activate Ras and propagate downstream signaling. Our data indicate that the removal of any one of these lipid components is sufficient to reduce EGFR cluster formation, which is consistent with previous reports (
      • Ariotti N.
      • Liang H.
      • Xu Y.
      • Zhang Y.
      • Yonekubo Y.
      • Inder K.
      • Du G.
      • Parton R.G.
      • Hancock J.F.
      • Plowman S.J.
      Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation.
      ,
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ).
      Here, we extend previous studies focusing on membrane biophysical properties by addressing the impact of DHA on the nanoscale structure of EGFR. With regard to the molecular mechanism by which DHA reduces EGFR cluster size, we focused on lipids that serve as key structural components of EGFR signaling proteolipid nanodomains. We previously reported that DHA influences both PA and PIP2-related protein interactions (
      • Fuentes N.R.
      • Mlih M.
      • Barhoumi R.
      • Fan Y.Y.
      • Hardin P.
      • Steele T.J.
      • Behmer S.
      • Prior I.A.
      • Karpac J.
      • Chapkin R.S.
      Long-chain n-3 fatty acids attenuate oncogenic kras-driven proliferation by altering plasma membrane nanoscale proteolipid composition.
      ,
      • Hou T.Y.
      • Barhoumi R.
      • Fan Y.-Y.
      • Rivera G.M.
      • Hannoush R.N.
      • McMurray D.N.
      • Chapkin R.S.
      n-3 polyunsaturated fatty acids suppress CD4+ T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization.
      ). However, neither the exogenous addition of PA or PIP2 restored DHA-induced EGFR cluster reduction. Instead, only cholesterol supplementation restored EGF-dependent EGFR clustering. This is supported by the FLIM-FRET-based observation that DHA reduces the interaction of EGFR and cholesterol occurring at the <10 nm scale (supplemental Fig. S11). Furthermore, the mutual aversion between DHA-containing phospholipids and cholesterol is well documented (
      • Williams J.A.
      • Batten S.E.
      • Harris M.
      • Rockett B.D.
      • Shaikh S.R.
      • Stillwell W.
      • Wassall S.R.
      Docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains.
      ,
      • Wassall S.R.
      • Leng X.
      • Canner S.W.
      • Pennington E.R.
      • Kinnun J.J.
      • Cavazos A.T.
      • Dadoo S.
      • Johnson D.
      • Heberle F.A.
      • Katsaras J.
      • Shaikh S.R.
      Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation.
      ,
      • Wassall S.R.
      • Stillwell W.
      Polyunsaturated fatty acid–cholesterol interactions: Domain formation in membranes.
      ). In regard to how exogenous cholesterol restores EGFR cluster formation, we posit that the high levels of exogenous cholesterol simply overcome the biophysical effects of DHA. Another possibility is that cholesterol restores the interaction of PA and PIP2 with EGFR. Further work is needed to address this putative model.
      EGFR nanoclusters are reported to contain ∼5–30 receptors and range in size from ∼20 to 630 nm depending on the methodology used (
      • Liang S.I.
      • van Lengerich B.
      • Eichel K.
      • Cha M.
      • Patterson D.M.
      • Yoon T.Y.
      • von Zastrow M.
      • Jura N.
      • Gartner Z.J.
      Phosphorylated EGFR dimers are not sufficient to activate Ras.
      ,
      • Ariotti N.
      • Liang H.
      • Xu Y.
      • Zhang Y.
      • Yonekubo Y.
      • Inder K.
      • Du G.
      • Parton R.G.
      • Hancock J.F.
      • Plowman S.J.
      Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation.
      ,
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ,
      • Gao J.
      • Wang Y.
      • Cai M.
      • Pan Y.
      • Xu H.
      • Jiang J.
      • Ji H.
      • Wang H.
      Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging.
      ,
      • Van Lengerich B.
      • Agnew C.
      • Puchner E.M.
      • Huang B.
      • Jura N.
      EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms.
      ,
      • Needham S.R.
      • Roberts S.K.
      • Arkhipov A.
      • Mysore V.P.
      • Tynan C.J.
      • Zanetti-Domingues L.C.
      • Kim E.T.
      • Losasso V.
      • Korovesis D.
      • Hirsch M.
      • Rolfe D.J.
      • Clarke D.T.
      • Winn M.D.
      • Lajevardipour A.
      • Clayton A.H.A.
      • et al.
      EGFR oligomerization organizes kinase-active dimers into competent signalling platforms.
      ,
      • Clayton A.H.A.
      • Tavarnesi M.L.
      • Johns T.G.
      Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding.
      ). Similar to the reported size for EGFR clusters, we observed that individual EGFR clusters occur on a large spectrum ranging from ∼40 to 700 nm. Of note, the effect of DHA on average EGFR cluster size was modest (∼10%) across all models; however, the effect of DHA on large clusters (>150 nm) was more pronounced (Fig. 4). We speculate that these larger clusters may be the most efficient activators of Ras. This is noteworthy, because small (<10 nm) changes to EGFR organization driven by EGFR-lipid interactions may influence effector recruitment similar to what occurs with Ras signaling (
      • Ariotti N.
      • Liang H.
      • Xu Y.
      • Zhang Y.
      • Yonekubo Y.
      • Inder K.
      • Du G.
      • Parton R.G.
      • Hancock J.F.
      • Plowman S.J.
      Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation.
      ,
      • Wang Y.
      • Gao J.
      • Guo X.
      • Tong T.
      • Shi X.
      • Li L.
      • Qi M.
      • Wang Y.
      • Cai M.
      • Jiang J.
      • Xu C.
      • Ji H.
      • Wang H.
      Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.
      ,
      • Zhou Y.
      • Hancock J.F.
      Chapter 2 – Ras proteolipid nanoassemblies on the plasma membrane sort lipids with high selectivity.
      ,
      • Prior I.A.
      • Muncke C.
      • Parton R.G.
      • Hancock J.F.
      Direct visualization of Ras proteins in spatially distinct cell surface microdomains.
      ,
      • Plowman S.J.
      • Muncke C.
      • Parton R.G.
      • Hancock J.F.
      H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton.
      ,
      • Cho K.-J.J.K.J.
      • Hancock J.F.J.F.
      Ras nanoclusters: A new drug target?.
      ).
      The ability of DHA to reduce membrane rigidity is conserved across many cell types (
      • Fan Y.-Y.
      • Fuentes N.R.
      • Hou T.Y.
      • Barhoumi R.
      • Li X.C.
      • Deutz N.E.P.
      • Engelen M.P.K.J.
      • McMurray D.N.
      • Chapkin R.S.
      Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.
      ,
      • Levental K.R.
      • Lorent J.H.
      • Lin X.
      • Skinkle A.D.
      • Surma M.A.
      • Stockenbojer E.A.
      • Gorfe A.A.
      • Levental I.
      Polyunsaturated lipids regulate membrane domain stability by tuning membrane order.
      ,
      • Kim W.
      • Fan Y.-Y.Y.
      • Barhoumi R.
      • Smith R.
      • McMurray D.N.
      • Chapkin R.S.
      n-3 polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation.
      ). This is likely driven by the incompatibility of DHA and major lipid raft components such as cholesterol and sphingomyelin (
      • Ma D.W.L.
      • Seo J.
      • Davidson L.A.
      • Callaway E.S.
      • Fan Y.-Y.Y.
      • Lupton J.R.
      • Chapkin R.S.
      n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon.
      ,
      • Fan Y.Y.
      • Ly L.H.
      • Barhoumi R.
      • McMurray D.N.
      • Chapkin R.S.
      Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production.
      ,
      • Williams J.A.
      • Batten S.E.
      • Harris M.
      • Rockett B.D.
      • Shaikh S.R.
      • Stillwell W.
      • Wassall S.R.
      Docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains.
      ,
      • Wassall S.R.
      • Leng X.
      • Canner S.W.
      • Pennington E.R.
      • Kinnun J.J.
      • Cavazos A.T.
      • Dadoo S.
      • Johnson D.
      • Heberle F.A.
      • Katsaras J.
      • Shaikh S.R.
      Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation.
      ), as both these components play a large role in influencing plasma membrane biophysical properties as well as EGFR activity (
      • Pike L.J.
      • Han X.
      • Gross R.W.
      Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids.
      ). Altering the biophysical properties of the plasma membrane may have broad effects on many membrane proteins. Although this study focused only on EGFR, DHA is known to affect the nanoscale architecture of other membrane proteins including Ras, Akt, Lck, and Lat (
      • Fuentes N.R.
      • Mlih M.
      • Barhoumi R.
      • Fan Y.Y.
      • Hardin P.
      • Steele T.J.
      • Behmer S.
      • Prior I.A.
      • Karpac J.
      • Chapkin R.S.
      Long-chain n-3 fatty acids attenuate oncogenic kras-driven proliferation by altering plasma membrane nanoscale proteolipid composition.
      ,
      • Levental K.R.
      • Surma M.A.
      • Skinkle A.D.
      • Lorent J.H.
      • Zhou Y.
      • Klose C.
      • Chang J.T.
      • Hancock J.F.
      • Levental I.
      ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis.
      ,
      • Hou T.Y.
      • Barhoumi R.
      • Fan Y.-Y.
      • Rivera G.M.
      • Hannoush R.N.
      • McMurray D.N.
      • Chapkin R.S.
      n-3 polyunsaturated fatty acids suppress CD4+ T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization.
      ,
      • Chapkin R.S.
      • Wang N.
      • Fan Y.-Y.Y.
      • Lupton J.R.
      • Prior I.A.
      Docosahexaenoic acid alters the size and distribution of cell surface microdomains.
      ). Therefore, it is possible that the functionality of other membrane receptors may be impacted. Although assessment of membrane rigidity provides information on the structure of the membrane itself, at present, it is difficult to predict its association with protein signaling. This is because the biophysical properties of the plasma membrane such as rigidity are the result of a myriad of variables, including lipid saturation, cholesterol content, lipid-protein interactions, and cytoskeletal dynamics (
      • Salinas M.L.
      • Fuentes N.R.
      • Choate R.
      • Wright R.C.
      • McMurray D.N.
      • Chapkin R.S.
      AdipoRon attenuates wnt signaling by reducing cholesterol-dependent plasma membrane rigidity.
      ,
      • Dinic J.
      • Ashrafzadeh P.
      • Parmryd I.
      Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains.
      ,
      • Alvarez-Guaita A.
      • Vilà de Muga S.
      • Owen D.M.
      • Williamson D.
      • Magenau A.
      • García-Melero A.
      • Reverter M.
      • Hoque M.
      • Cairns R.
      • Cornely R.
      • Tebar F.
      • Grewal T.
      • Gaus K.
      • Ayala-Sanmartín J.
      • Enrich C.
      • et al.
      Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains.
      ). Similarly, we cannot rule out the contribution of a DHA bioactive metabolite, although to date there is no evidence that secreted lipid metabolites (in general) have membrane-altering properties. Therefore, additional work is needed to determine if DHA impacts the nanoscale architecture of other EGFR mutants that arise from anti-EGFR therapies (
      • Yu H.A.
      • Arcila M.E.
      • Rekhtman N.
      • Sima C.S.
      • Zakowski M.F.
      • Pao W.
      • Kris M.G.
      • Miller V.A.
      • Ladanyi M.
      • Riely G.J.
      Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers.
      ), including the clinically relevant EGFR family member HER2 (
      • Tobin S.J.
      • Wakefield D.L.
      • Jones V.
      • Liu X.
      • Schmolze D.
      • Jovanović-Talisman T.
      Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2.
      ,
      • Bertotti A.
      • Migliardi G.
      • Galimi F.
      • Sassi F.
      • Torti D.
      • Isella C.
      • Cora D.
      • Di Nicolantonio F.
      • Buscarino M.
      • Petti C.
      • Ribero D.
      • Russolillo N.
      • Muratore A.
      • Massucco P.
      • Pisacane A.
      • et al.
      A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.
      ).
      Although experimental, epidemiological, preclinical, and clinical evidence support a protective benefit of DHA against colon and breast tumorigenesis (
      • Cockbain A.J.
      • Volpato M.
      • Race A.D.
      • Munarini A.
      • Fazio C.
      • Belluzzi A.
      • Loadman P.M.
      • Toogood G.J.
      • Hull M.A.
      Anticolorectal cancer activity of the omega-3 polyunsaturated fatty acid eicosapentaenoic acid.
      ,
      • Fabian C.J.
      • Kimler B.F.
      • Hursting S.D.
      Omega-3 fatty acids for breast cancer prevention and survivorship.
      ,
      • Serini S.
      • Calviello G.
      Modulation of Ras/ERK and phosphoinositide signaling by long-chain n-3 PUFA in breast cancer and their potential complementary role in combination with targeted drugs.
      ), this may not be true for all cancers. Recently, it was reported that ELOVL2, an elongase that functions in the synthesis of the long-chain n-3 and n-6 PUFAs, e.g., DHA and docosapentaenoic acid (DPA, 22∶5Δ4,7,10,13,16), respectively, is required for the maintenance of glioblastoma stem cells and that dual targeting of PUFA synthesis and EGFR signaling had a combinatorial cytotoxic effect of glioblastoma stem cells (
      • Gimple R.C.
      • Kidwell R.L.
      • Kim L.J.Y.
      • Sun T.
      • Gromovsky A.D.
      • Wu Q.
      • Wolf M.
      • Lv D.
      • Bhargava S.
      • Jiang L.
      • Prager B.C.
      • Wang X.
      • Ye Q.
      • Zhu Z.
      • Zhang G.
      • et al.
      Glioma stem cell–specific superenhancer promotes polyunsaturated fatty-acid synthesis to support EGFR signaling.
      ). The mechanism underlying the unique effects of DHA in these two tissues is not understood but may involve how EGFR nanocluster proteolipid composition is maintained in each instance. Although in most cases, DHA is relatively nontoxic to nontransformed cells, the previous example highlights the benefit of selective targeting of DHA. This is consistent with previous reports that DHA specifically targets tumor subtypes (
      • Reynolds L.
      • Mulik R.S.
      • Wen X.
      • Dilip A.
      • Corbin I.R.
      Low-density lipoprotein-mediated delivery of docosahexaenoic acid selectively kills murine liver cancer cells.
      ,
      • Wen X.
      • Reynolds L.
      • Mulik R.S.
      • Kim S.Y.
      • Van Treuren T.
      • Nguyen L.H.
      • Zhu H.
      • Corbin I.R.
      Hepatic arterial infusion of low-density lipoprotein docosahexaenoic acid nanoparticles selectively disrupts redox balance in hepatoma cells and reduces growth of orthotopic liver tumors in rats.
      ), attenuating EGFR nanoclustering.
      In conclusion, our novel findings suggest that the ability of DHA to reduce EGFR nanocluster formation plays a crucial role in the attenuation of hyperactive EGFR-driven signaling and phenotypes. These findings support the feasibility of using dietary strategies that target plasma membrane nanoscale architecture in order to reduce oncogenic signaling and cancer risk.

      Data availability

      All data reported in this study are located within the article.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      The authors thank Dr Michiyuki Matsuda (Kyoto University, Kyoto, Japan) for providing the H- and K-Ras Raichu plasmids, Dr Guangwei Du (UT Health Science Center Houston, TX) for providing the Spo20-GFP plasmid, Dr Gregory Fairn (University of Toronto, Canada) for providing the D4H-mCherry plasmid, and Dr Jing Kang (Harvard University) for the Fat-1 breeder mice. This work was supported by the Allen Endowed Chair in Nutrition & Chronic Disease Prevention, and the National Institutes of Health (R35-CA197707, RO1-CA244359, and P30-ES029067). The use of the Texas A&M Microscopy and Imaging Center is acknowledged.

      Author contribution

      N. R. F., M. M., I. R. C., J. K., and R. S. C. designed the studies and wrote the manuscript. N. R. F., M. M., X. W., G. W., S. C.-A., and M. L. S. performed the experimental work.

      Author ORCIDs

      Funding and additional information

      N. R. F. was supported by the Texas A&M University Regulatory Science in Environmental Health and Toxicology Training Grant (T32-ES026568) and is a former recipient of a Predoctoral Fellowship in Pharmacology/Toxicology from the PhRMA Foundation. G. W. was supported by the National Science Foundation REU Site Summer Undergraduate Research Program in Biochemistry (NSF DBI-1358941).

      Supplemental data

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