Oleate Promotes the Proliferation of Breast Cancer Cells via the G Protein-coupled Receptor GPR40

Evidence from epidemiological studies and animal models suggests a link between high levels of dietary fat intake and risk of breast cancer. In addition, obesity, in which circulating lipids are elevated, is associated with increased risk of various cancers. Relative to this point, we previously showed that oleate stimulates the proliferation of breast cancer cells and that phosphatidylinositol 3-kinase plays a role in this process. Nonetheless, questions remain regarding the precise mechanism(s) by which oleate promotes breast cancer cell growth. Pharmacological inhibitors of the GTP-binding proteins Gi/Go, phospholipase C, Src, and mitogenic-extracellular signal-regulated kinase 1/2 (MEK 1/2) decreased oleate-induced [3H]thymidine incorporation in the breast cancer cell line MDA-MB-231. In addition, oleate caused a rapid and transient rise in cytosolic Ca2+ and an increase in protein kinase B phosphorylation. Overexpressing in these cells the G protein-coupled receptor GPR40, a fatty acid receptor, amplified oleate-induced proliferation, whereas silencing the GPR40 gene using RNA interference decreased it. Overexpressing GPR40 in T47D and MCF-7 breast cancer cells that are poorly responsive to oleate allowed a robust proliferative action of oleate. The data indicate that the phospholipase C, MEK 1/2, Src, and phosphatidylinositol 3-kinase/protein kinase B signaling pathways are implicated in the proliferative signal induced by oleate and that these effects are mediated at least in part via the G protein-coupled receptor GPR40. The results suggest that GPR40 is implicated in the control of breast cancer cell growth by fatty acids and that GPR40 may provide a link between fat and cancer. Evidence from epidemiological studies and animal models suggests a link between high levels of dietary fat intake and risk of breast cancer. In addition, obesity, in which circulating lipids are elevated, is associated with increased risk of various cancers. Relative to this point, we previously showed that oleate stimulates the proliferation of breast cancer cells and that phosphatidylinositol 3-kinase plays a role in this process. Nonetheless, questions remain regarding the precise mechanism(s) by which oleate promotes breast cancer cell growth. Pharmacological inhibitors of the GTP-binding proteins Gi/Go, phospholipase C, Src, and mitogenic-extracellular signal-regulated kinase 1/2 (MEK 1/2) decreased oleate-induced [3H]thymidine incorporation in the breast cancer cell line MDA-MB-231. In addition, oleate caused a rapid and transient rise in cytosolic Ca2+ and an increase in protein kinase B phosphorylation. Overexpressing in these cells the G protein-coupled receptor GPR40, a fatty acid receptor, amplified oleate-induced proliferation, whereas silencing the GPR40 gene using RNA interference decreased it. Overexpressing GPR40 in T47D and MCF-7 breast cancer cells that are poorly responsive to oleate allowed a robust proliferative action of oleate. The data indicate that the phospholipase C, MEK 1/2, Src, and phosphatidylinositol 3-kinase/protein kinase B signaling pathways are implicated in the proliferative signal induced by oleate and that these effects are mediated at least in part via the G protein-coupled receptor GPR40. The results suggest that GPR40 is implicated in the control of breast cancer cell growth by fatty acids and that GPR40 may provide a link between fat and cancer. Epidemiological and animal studies have revealed an association between dietary fatty acids and the incidence of breast cancer (1.Fay M.P. Freedman L.S. Clifford C.K. Midthune D.N. Cancer Res. 1997; 57: 3979-3988PubMed Google Scholar, 2.Lee M.M. Lin S.S. Annu. Rev. Nutr. 2000; 20: 221-248Crossref PubMed Scopus (87) Google Scholar, 3.Rose D.P. Am. J. Clin. 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In addition, emerging evidence indicates that obesity, which is characterized by hyperlipidemia and elevated circulating free-fatty acids (FFA) 1The abbreviations used are: FFA, free-fatty acid; PI3-K, phosphatidylinositol 3-kinase; GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; [Ca2+]i, intracellular calcium concentration; BSA, bovine serum albumin; EGF, epidermal growth factor; EGFR, EGF receptor; PKCζ, protein kinase Cζ; ERK1/2, extracellular signal-regulated kinase 1/2; AKT, protein kinase B; PBS, phosphate-buffered saline; siRNA, small interfering RNA; GFP, green fluorescent protein; PLC, phospholipase C; MEK1/2, mitogenic-extracellular signal-regulated kinase 1/2. 1The abbreviations used are: FFA, free-fatty acid; PI3-K, phosphatidylinositol 3-kinase; GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; [Ca2+]i, intracellular calcium concentration; BSA, bovine serum albumin; EGF, epidermal growth factor; EGFR, EGF receptor; PKCζ, protein kinase Cζ; ERK1/2, extracellular signal-regulated kinase 1/2; AKT, protein kinase B; PBS, phosphate-buffered saline; siRNA, small interfering RNA; GFP, green fluorescent protein; PLC, phospholipase C; MEK1/2, mitogenic-extracellular signal-regulated kinase 1/2. (4.Felber J.P. Golay A. Int. J. Obes. Relat. Metab. Disord. 2002; 26: 39-45Crossref PubMed Scopus (131) Google Scholar), is associated with enhanced cancer risk (5.Calle E.E. Kaaks R. Nat. Rev. Cancer. 2004; 4: 579-591Crossref PubMed Scopus (2631) Google Scholar). However, relative little information exists on the mechanisms by which exogenous FFAs influence breast cancer cell growth. FFAs play pivotal roles in many biological processes. They serve as an abundant source of energy and as precursors of many cellular signaling and structural molecules (6.McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar). As natural ligands for the nuclear receptors peroxisomal proliferated-activated receptors (PPARs), they also control the transcription of several genes involved in lipid and glucose metabolisms (7.Ferre P. Diabetes. 2004; 53: 43-50Crossref PubMed Google Scholar). However, several biological effects appear to be PPAR independent (8.Sauer L.A. Dauchy R.T. Blask D.E. Cancer Res. 2000; 60: 5289-5295PubMed Google Scholar, 9.Louet J.F. Chatelain F. Decaux J.F. Park E.A. Kohl C. Pineau T. Girard J. Pegorier J.P. Biochem. J. 2001; 354: 189-197Crossref PubMed Google Scholar). We have previously reported that the monounsaturated FFA oleate (C18:1) and the saturated FFA palmitate (C16:0), the two most abundant fatty acids in plasma, are not equivalent with respect to their actions on breast cancer cell proliferation and apoptosis (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar). Oleate stimulates the proliferation of breast cancer cells, whereas palmitate induces apoptosis. Moreover, we found as a general principle that saturated FFAs (palmitic, myristic, and stearic) are proapoptotic, whereas unsaturated FFAs (oleic, linoleic, arachidonic, and eicosapentaenoic) increase proliferation of MDA-MB-231 breast cancer cells (11.Hardy S. El-Assaad W. Przybytkowski E. Joly E. Prentki M. Langelier Y. J. Biol. Chem. 2003; 278: 31861-31870Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In addition, a 1:10 molar ratio of oleate versus palmitate was sufficient to annihilate the proapoptotic action of palmitate (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar). Important differences in the metabolism of these two FFAs in MDA-MB-231 cells contribute to their opposite effects on cell fate. An early enhancement of cardiolipin turnover and a decrease in the level of this mitochondrial phospholipid necessary for cytochrome c retention are involved in the proapoptotic effect of palmitate. By contrast oleate, by channeling palmitate to inert triglyceride stores and by permitting sustained cardiolipin synthesis, not only blocks palmitate-induced apoptosis but also permits cell proliferation (11.Hardy S. El-Assaad W. Przybytkowski E. Joly E. Prentki M. Langelier Y. J. Biol. Chem. 2003; 278: 31861-31870Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In addition, oleate but not palmitate appears to act like a growth factor because it stimulates cell proliferation at very low concentrations and rapidly activates phosphatidylinositol 3-kinase (PI3-K) in these cells (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar). These findings suggest the existence of signaling pathways via membrane receptors such as receptor tyrosine kinases or G protein-coupled receptors (GPCR), which are known to activate PI3-K (12.Grant S. Qiao L. Dent P. Front. Biosci. 2002; 7: 376-389Crossref PubMed Scopus (163) Google Scholar, 13.Yart A. Chap H. Raynal P. Biochim. Biophys. Acta. 2002; 1582: 107-111Crossref PubMed Scopus (58) Google Scholar). Unsaturated FFAs including oleate but not saturated FFAs have been shown to trigger tyrosine phosphorylation and epidermal growth factor receptor (EGFR) activation in an endothelial cell line (14.Vacaresse N. Lajoie-Mazenc I. Auge N. Suc I. Frisach M.F. Salvayre R. Negre-Salvayre A. Circ. Res. 1999; 85: 892-899Crossref PubMed Scopus (62) Google Scholar). GPCRs for fatty acid derivatives such as prostaglandins (15.Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar), leukotrienes (16.Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. Herrity N.C. Halsey W. Sathe G. Muir A.I. Nuthulaganti P. Dytko G.M. Buckley P.T. Wilson S. Bergsma D.J. Hay D.W. Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (301) Google Scholar), lysophosphatidic acid (LPA) (17.Mills G.B. Moolenaar W.H. Nat. Rev. Cancer. 2003; 3: 582-591Crossref PubMed Scopus (923) Google Scholar), sphingosine 1-phosphate (18.Radeff-Huang J. Seasholtz T.M. Matteo R.G. Brown J.H. J. Cell. Biochem. 2004; 92: 949-966Crossref PubMed Scopus (171) Google Scholar), and eicosatetraenoic acid (19.Hosoi T. Koguchi Y. Sugikawa E. Chikada A. Ogawa K. Tsuda N. Suto N. Tsunoda S. Taniguchi T. Ohnuki T. J. Biol. Chem. 2002; 277: 31459-31465Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) are well characterized. Agonist stimulation of these GPCRs induces a variety of cellular responses, including cell proliferation (18.Radeff-Huang J. Seasholtz T.M. Matteo R.G. Brown J.H. J. Cell. Biochem. 2004; 92: 949-966Crossref PubMed Scopus (171) Google Scholar). These effects implicate the activation of a wide variety of signaling pathways, including the modulation of adenylyl cyclases, phospholipases, ion channels, and mitogen-activated protein kinases (20.Schulte G. Fredholm B.B. Cell. Signal. 2003; 15: 813-827Crossref PubMed Scopus (391) Google Scholar). Recently, three independent groups found that the orphan receptor GPR40 is activated by medium and long chain FFAs (21.Itoh Y. Kawamata Y. Harada M. Kobayashi M. Fujii R. Fukusumi S. Ogi K. Hosoya M. Tanaka Y. Uejima H. Tanaka H. Maruyama M. Satoh R. Okubo S. Kizawa H. Komatsu H. Matsumura F. Noguchi Y. Shinohara T. Hinuma S. Fujisawa Y. Fujino M. Nature. 2003; 422: 173-176Crossref PubMed Scopus (1213) Google Scholar, 22.Briscoe C.P. Tadayyon M. Andrews J.L. Benson W.G. Chambers J.K. Eilert M.M. Ellis C. Elshourbagy N.A. Goetz A.S. Minnick D.T. Murdock P.R. Sauls Jr., H.R. Shabon U. Spinage L.D. Strum J.C. Szekeres P.G. Tan K.B. Way J.M. Ignar D.M. Wilson S. Muir A.I. J. Biol. Chem. 2003; 278: 11303-11311Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 23.Kotarsky K. Nilsson N.E. Flodgren E. Owman C. Olde B. Biochem. Biophys. Res. Commun. 2003; 301: 406-410Crossref PubMed Scopus (306) Google Scholar). Using a ligand-fishing strategy based on measurement of intracellular calcium concentrations ([Ca2+]i), they showed that FFAs in the absence of bovine serum albumin (BSA) increased [Ca2+]i in GPR40-overexpressing cells. GPR40 is highly expressed in pancreatic β-cells (21.Itoh Y. Kawamata Y. Harada M. Kobayashi M. Fujii R. Fukusumi S. Ogi K. Hosoya M. Tanaka Y. Uejima H. Tanaka H. Maruyama M. Satoh R. Okubo S. Kizawa H. Komatsu H. Matsumura F. Noguchi Y. Shinohara T. Hinuma S. Fujisawa Y. Fujino M. Nature. 2003; 422: 173-176Crossref PubMed Scopus (1213) Google Scholar, 22.Briscoe C.P. Tadayyon M. Andrews J.L. Benson W.G. Chambers J.K. Eilert M.M. Ellis C. Elshourbagy N.A. Goetz A.S. Minnick D.T. Murdock P.R. Sauls Jr., H.R. Shabon U. Spinage L.D. Strum J.C. Szekeres P.G. Tan K.B. Way J.M. Ignar D.M. Wilson S. Muir A.I. J. Biol. Chem. 2003; 278: 11303-11311Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar), but it is also present in other tissues (23.Kotarsky K. Nilsson N.E. Flodgren E. Owman C. Olde B. Biochem. Biophys. Res. Commun. 2003; 301: 406-410Crossref PubMed Scopus (306) Google Scholar). Interestingly, GPR40 is expressed in the human breast cancer cell line MCF-7 in which unsaturated, but not saturated, FFAs bound to BSA increase [Ca2+]i (24.Yonezawa T. Katoh K. Obara Y. Biochem. Biophys. Res. Commun. 2004; 314: 805-809Crossref PubMed Scopus (87) Google Scholar). In the present study, we investigated the mechanisms by which oleate increases the proliferation of the breast cancer cell line MDA-MB-231. The results suggest that multiple pathways are involved in the proliferative action of oleate in these cells and that the oleate effect implicates a GPCR. In addition, evidence is provided that the oleate-induced proliferation of breast cancer cells is mediated at least in part through GPR40. Materials—Sodium salts of fatty acids were purchased from Nu-Check Prep (Elysian, MN). Fatty acid-free BSA (fraction V) was obtained from Sigma. Fura-2/AM was from Molecular Probes Inc. (Eugene, OR). [3H]thymidine (specific activity, 71 Ci/mmol) was obtained from PerkinElmer Life Sciences. Lipofectamine 2000 and FuGENE 6 were purchased from Invitrogen and from Roche Applied Sciences, respectively. U73122, PD98059, PP1, LPA, H-89, wortmannin, protein kinase Cζ (PKCζ) peptide inhibitor were from Biomol (Plymouth Meeting, PA). Pertussis toxin and AG1478 were from Calbiochem (La Jolla, CA). Cell Culture—The human breast cancer cell lines MDA-MB-231, T47D, and MCF-7 were obtained from the American Type Culture Collection. Cells were cultured at 37 °C with 5% CO2 in phenol red-free minimal essential medium containing non-essential amino acids, 2 mm glutamine, 10 mm Hepes (pH 7.4), and 5% heat-inactivated fetal bovine serum (standard medium). BSA-bound fatty acids were prepared by stirring fatty acid sodium salts (≥99% purity) at 37 °C with 5% fatty acid-free BSA as described before (25.Roche E. Buteau J. Aniento I. Reig J.A. Soria B. Prentki M. Diabetes. 1999; 48: 2007-2014Crossref PubMed Scopus (122) Google Scholar). After being adjusted to pH 7.4, the solution was filtered through a 0.22-μm filter, and the fatty acid concentration was measured using a NEFA C kit (GmbH; Wako Chemicals). When BSA-bound fatty acids were added to serum-free culture medium, the final concentration of BSA was adjusted to 0.5%. Cell Proliferation—For cell growth assay, 5000 cells/well were seeded in 96-well plates and incubated for 24 h in standard medium (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar). After a 24-h starvation period in medium without serum, cells were incubated without or with BSA-bound fatty acids for 24 h. DNA synthesis was then assayed with a pulse of [3H]thymidine (1 μCi/well) during the last 4 h of the incubation. Cells were harvested with a PHD cell harvester from Cambridge Technology (Watertown, MA), and the radioactivity retained on the dried glass fiber filters was measured by liquid scintillation. Immunoprecipitation and Immunoblotting Analyses—For measuring EGFR activation, immunoprecipitation and immunoblotting were performed as previously described (26.Buteau J. Foisy S. Joly E. Prentki M. Diabetes. 2003; 52: 124-132Crossref PubMed Scopus (318) Google Scholar). For extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation analyses, cells were seeded in 60-mm Petri dishes at 2 × 105 cells/dish and incubated for 24 h in standard medium. After a 24-h period of serum starvation in medium containing 0.5% BSA, cells were incubated in Dulbecco's phosphate-buffered saline (PBS) containing 5 mm glucose and 0.5% BSA for 2 h and stimulated with oleate or epidermal growth factor (EGF) for the indicated time. This incubation in Dulbecco's PBS was used to reduce basal levels of ERK1/2 and AKT phosphorylation. After stimulation, cells were washed and lysed in protein extraction buffer containing 20 mm Tris (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm NaPi, 1 mm NA3VO4, 10 mg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation, and protein concentrations of the supernatants were determined using the Bio-Rad DC colorimetric assay. Equal amounts of protein (25 μg) were separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblotting was performed according to the manufacturer's instructions using phospho(Thr-202/Tyr-204)-ERK1/2 (phosphorylated-ERK1/2), ERK1/2, phospho(Ser-473)-AKT (phosphorylated AKT), or AKT-specific antibodies (Cell Signaling Technology). Cytosolic Calcium Determination—Cells grown in 150-mm Petri dishes (70% confluent) were trypsinized, centrifuged, and resuspended at 2 × 106 cells/ml in Dulbecco's PBS containing 5 mm glucose, 20 mm Hepes (pH 7.4), 2.5 mm probenecid, and 3 mm fura-2/AM. After 30 min of fura-2/AM loading at 25 °C, cells were washed and resuspended as above but without fura-2/AM and were dispensed at 2 × 105 cells/well into 96-well plates. [Ca2+]i was measured at 37 °C by the ratiometric method (emission fluorescence at 500 nm and excitation wavelengths at 340 and 380 nm) using a FLUOstar OPTIMA microplate reader (BMG Labtechnologies Inc., Durham, NC). Calcium concentrations were calculated as described by Grynkiewicz et al. (27.Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Cell Transfection—Cells seeded in 6-well plates at 4 × 105 cells/well were incubated for 24 h in standard medium. Cells were transiently transfected with 5 μg of the plasmid pIRESpuro-GPR40 expressing the human GPR40 (provided by Bjorn Olde, Wallenberg Neuroscience Center, Lund, Sweden) or a control plasmid expressing Renilla luciferase (CMV-RLuc) using Lipofectamine 2000 (MDA-MB-231) and FuGENE 6 (T47D and MCF-7) according to the manufacturer's instructions. Five hours post-transfection, cells were seeded into 96-well plates at 5000 cells/well and assayed for cell growth as described above. RNA Interference—Vectors that express hairpin small interfering RNAs (siRNA) under the control of the human H1 promoter were constructed by inserting pairs of annealed DNA oligonucleotides into pSilencer H1 3.0 vector (Ambion, Austin, TX) between BamH1 and HindIII restriction sites according to the manufacturer's instructions. The target sequence for human GPR40 was 5′-AAGGGCATATTGCTTCAGTTC-3′. Cells were co-transfected with plasmid encoding the green fluorescent protein (GFP) and siRNA targeting GPR40 gene or a scrambled siRNA control (Ambion). Cells expressing the GFP were enriched by selection on a fluorescent-activated cell sorter (FAC-Star Plus; BD Biosciences). Cells were seeded into 96-well plates at 5000 cells/well and assayed for cell growth as described above. Quantitative Real-time Reverse Transcription-PCR—Total RNA was isolated using TRIzol reagent (Invitrogen). RNA (5 μg) was reverse-transcribed using the Omniscript reverse transcriptase kit (Qiagen Inc., Valencia, CA). Quantitative real-time PCR was performed on a Rotor-Gene (Corbett Research, Sidney, Australia) using a QuantiTect SYBR Green PCR kit (Qiagen Inc.) according to the manufacturer's instructions. The primers used were as follows: GPR40 5′-AGCTCTCCTTCGGCCTCTATG-3′ (forward) and 5′-CAGAGAGACTGTCAGCAGCAG-3′ (reverse), glyceraldehyde-3-phosphate dehydrogenase, 5′-GACCACAGTCCATGCCATCAC-3′ (forward) and 5′-AGGTCCACCACTGACACGTTG-3′ (reverse). Statistics—Data are presented as mean ± S.E. Differences between two conditions were assessed with a Student's t test for related samples. A p value <0.05 was considered significant. Oleate-induced Cell Proliferation Does Not Involve EGFR Activation—We previously showed that the monounsaturated fatty acid oleate stimulates the proliferation of three different breast cancer cell lines and that PI3-K is implicated in this effect (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar). Fig. 1A confirms this observation because the PI3-K inhibitor wortmannin (28.Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar) markedly curtailed oleate-induced proliferation of MDA-MB-231 cells. In addition, we showed that PI3-K is rapidly activated by oleate, suggesting signaling through a receptor (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar). Because the EGFR has been reported to be activated by oleate in endothelial cells (14.Vacaresse N. Lajoie-Mazenc I. Auge N. Suc I. Frisach M.F. Salvayre R. Negre-Salvayre A. Circ. Res. 1999; 85: 892-899Crossref PubMed Scopus (62) Google Scholar), we first examined whether the activation of the EGFR could be involved in oleate-induced proliferation of MDA-MB-231 cells. AG1478, a pharmacological inhibitor of EGFR activity (29.Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1608) Google Scholar), did not significantly affect oleate-induced [3H]thymidine incorporation (Fig. 1B). However, AG1478 efficiently prevented the phosphorylation of EGFR induced by EGF (Fig. 2A). In addition, an examination of the time course of EGFR phosphorylation induced by oleate revealed no activation of EGFR over 240 min of treatment (Fig. 2B). However, EGF induced a strong activation of the EGFR at 2 min that became maximal at 15 min and decreased thereafter. Interestingly, LPA transiently produced a modest increase in EGFR phosphorylation resulting probably from transactivation of the EGFR following LPA binding to its receptor (30.Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (584) Google Scholar). These results establish that the EGFR is not implicated in oleate-induced proliferation of MDA-MB-231 cells.Fig. 2EGFR is not activated by oleate in MDA-MB-231 cells. A, cells were stimulated with BSA alone (Cont), 0.1 mm oleate (Ole), or palmitate (Pal) bound to BSA (0.5%) or 100 ng/ml EGF for 10 min in the absence or presence of 250 nm AG1478. B, cells were stimulated with 0.1 mm oleate or 10 μm LPA bound to BSA or 100 ng/ml EGF for the indicated times. Cells were lysed, and EGFR was immunoprecipitated from total protein extracts to perform immunoblot analysis with an anti-phospho-tyrosine (p-Tyr)-specific antibody. Membranes were stripped and reprobed with an EGFR-specific antibody. The figure shows representative experiments that have been repeated twice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oleate-induced MDA-MB-231 Cell Proliferation Is Decreased by Specific Inhibitors of Gi/Go, Phospholipase C, Src, MEK1/2, and PKCζ—Several fatty acid derivatives such as LPA are known to act via GPCRs and to activate PI3-K via G proteins (13.Yart A. Chap H. Raynal P. Biochim. Biophys. Acta. 2002; 1582: 107-111Crossref PubMed Scopus (58) Google Scholar). We first studied the effect of pertussis toxin, an inhibitor of Gi/Go proteins (31.Moss J. Vaughan M. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 303-379PubMed Google Scholar), on oleate-induced cell proliferation. As shown in Fig. 1C, pertussis toxin decreased oleate-induced [3H]thymidine incorporation by 70%. The same extent of inhibition was observed with LPA, which has been shown to increase cell proliferation through GPCR activation via Gi/Go proteins (32.Fang X. Yu S. LaPushin R. Lu Y. Furui T. Penn L.Z. Stokoe D. Erickson J.R. Bast Jr., R.C. Mills G.B. Biochem. J. 2000; 352: 135-143Crossref PubMed Scopus (103) Google Scholar). This strongly supports that the effect of oleate on breast cancer cell proliferation is mediated at least in part by GPCR(s), because the action of pertussis toxin to inhibit Gi/Go is considered highly specific (31.Moss J. Vaughan M. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 303-379PubMed Google Scholar). Agonist stimulation of GPCR caused the activation of a wide variety of signaling pathways, including modulation of phospholipases and protein kinases (20.Schulte G. Fredholm B.B. Cell. Signal. 2003; 15: 813-827Crossref PubMed Scopus (391) Google Scholar). To assess a possible role of these different signal transduction pathways in the action of oleate on breast cancer cell proliferation, we tested the effect of different classes of specific inhibitors. Treating cells with the phospholipase C (PLC) inhibitor U73122 (33.Smith R.J. Sam L.M. Justen J.M. Bundy G.L. Bala G.A. Bleasdale J.E. J. Pharmacol. Exp. Ther. 1990; 253: 688-697PubMed Google Scholar) resulted in an 80% reduction of oleate- or LPA-induced proliferation (Fig. 1D). PP1 and PD98059, which are pharmacological inhibitors of the Src-like family (34.Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1775) Google Scholar) and of mitogenic-extracellular signal-regulated kinase 1/2 (MEK1/2) (35.Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3242) Google Scholar), respectively, reduced the proliferative effect of oleate by about 40% (Fig. 1, E and F). Moreover, PKCζ, a downstream effector of PI3-K, appears also to be implicated in the proliferative effect of oleate because a specific membrane-permeant peptide inhibitor of PKCζ (36.Zhou G. Seibenhener M.L. Wooten M.W. J. Biol. Chem. 1997; 272: 31130-31137Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) blocked the oleate-induced proliferation (Fig. 1G). However, this peptide inhibitor also decreased the proliferation in the control situation, suggesting that PKCζ is important for the growth of MDA-MB-231 cells. Blocking protein kinase A, another downstream effector of GPCR, with H-89 (37.Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar) did not affect oleate-induced DNA synthesis (Fig. 1H). Taken together, the results are consistent with the view that oleate signals at least in part via GPCR(s) and that many downstream signal transduction pathways, including PLC, Src, MEK1/2, and PKCζ, may participate in its proliferative effect. Oleate Induces a Rapid Increase in AKT Phosphorylation— ERK1/2 and AKT, the direct downstream effectors of MEK1/2 and PI3-K, respectively, are major regulators of cell proliferation (38.Brazil D.P. Yang Z.Z. Hemmings B.A. Trends Biochem. Sci. 2004; 29: 233-242Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). We therefore examined the role of ERK1/2 and AKT in oleate-induced cell proliferation by using antibodies recognizing the activated/phosphorylated forms of Thr-202/Tyr-204-ERK1/2 and Ser-473-AKT. Because basal ERK1/2 activity is very high in MDA-MB-231 cells as previously described (39.Price J.T. Tiganis T. Agarwal A. Djakiew D. Thompson E.W. Cancer Res. 1999; 59: 5475-5478PubMed Google Scholar), we were unable to detect significant increases in ERK1/2 phosphorylation in response to either oleate or the usual positive control EGF (data not shown). In sharp contrast, both oleate and EGF stimulated AKT phosphorylation (Fig. 3, A and B). Oleate-induced AKT phosphorylation, which increased more than 2-fold during the first 5 min of treatment, peaked at 30 min and then slowly declined. The same extent of AKT activation was observed with five different preparations of oleate-BSA. In agreement with our previous data showing that oleate, but not palmitate, activates PI3-K (10.Hardy S. Langelier Y. Prentki M. Cancer Res. 2000; 60: 6353-6358PubMed Google Scholar), AKT phosphorylation was not induced by two different preparations of palmitate-BSA that had been shown to be active in inducing apoptosis (data not shown). Considering the PI3-K inhibitor data described in Fig. 1A, the results indicate that the proliferative signal induced by oleate is mediated at least in part via PI3-K/AKT activation. Oleate Increases [Ca2+]i in the Presence and Absence of BSA—GPR40 was first recognized to be activated by medium and long chain FFA (not bound to BSA) by assays measuring [Ca2+]i in GPR40-overexpressing cells (21.Itoh Y. Kawamata Y. Harada M. Kobayashi M. Fujii R. Fukusumi S. Ogi K. Hosoya M. Tanaka Y. Uejima H. Tanaka H. Maruyama M. Satoh R. Okubo S. Kizawa H. Komatsu H. Matsumura F. Noguchi Y. Shinohara T. Hinuma S. Fujisawa Y. Fujino M. Nature. 2003; 422: 173-176Crossref PubMed Scopus (1213) Google Scholar, 22.Briscoe C.P. Tadayyon M. Andrews J.L. Benson W.G. Chambers J.K. Eilert M.M. Ellis C. Elshourbagy N.A. Goetz A.S. Minnick D.T. Murdock P.R. Sauls Jr., H.R. Shabon U. Spinage L.D. Strum J.C. Szekeres P.G. Tan K.B. Way J.M. Ignar D.M. Wilson S. Muir A.I. J. Biol. Chem. 2003; 278: 11303-11311Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 23.Kotarsky K. Nilsson N.E. Flodgren E. Owman C. Olde B. Biochem. Bioph