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

Abstract

Epidermal growth factor receptor (EGFR) signaling drives the formation of many types of cancer, including colon cancer. Docosahexaenoic acid (DHA, 22∶6^{Δ4,7,10,13,16,19}), 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.

Keywords: Cancer; Cholesterol; Membranes/Fluidity; Omega-3 fatty acids; Receptors/Plasma membrane; Super-resolution microscopy.

Fig. 1

Exogenous 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 (∗).

Fig. 2

Dietary 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.

Fig. 3

DHA 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.

Fig. 4

DHA 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.

Fig. 5

Influence 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.

Fig. 6

Exogenous 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.