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.

Epidemiological and animal studies have revealed an association between dietary fatty acids and the incidence of breast cancer (1)(2)(3). In addition, emerging evidence indicates that obesity, which is characterized by hyperlipidemia and elevated circulating free-fatty acids (FFA) 1 (4), is associated with en-hanced cancer risk (5). 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). 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). However, several biological effects appear to be PPAR independent (8,9).
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). 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). In addition, a 1:10 molar ratio of oleate versus palmitate was sufficient to annihilate the proapoptotic action of palmitate (10). 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). 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). 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 (12,13).
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). GPCRs for fatty acid derivatives such as prostaglandins (15), leukotrienes (16), lysophosphatidic acid (LPA) (17), sphingosine 1-phosphate (18), and eicosatetraenoic acid (19) are well characterized. Agonist stimulation of these GPCRs induces a variety of cellular responses, including cell proliferation (18). 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). Recently, three independent groups found that the orphan receptor GPR40 is activated by medium and long chain FFAs (21)(22)(23). Using a ligand-fishing strategy based on measurement of intracellular calcium concentrations ([Ca 2ϩ ] i ), they showed that FFAs in the absence of bovine serum albumin (BSA) increased [Ca 2ϩ ] i in GPR40-overexpressing cells. GPR40 is highly expressed in pancreatic ␤-cells (21,22), but it is also present in other tissues (23). Interestingly, GPR40 is expressed in the human breast cancer cell line MCF-7 in which unsaturated, but not saturated, FFAs bound to BSA increase [Ca 2ϩ ] i (24).
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. 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% CO 2 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 (Ն99% purity) at 37°C with 5% fatty acid-free BSA as described before (25). After being adjusted to pH 7.4, the solution was filtered through a 0.22-m 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%.

Materials-Sodium
Cell Proliferation-For cell growth assay, 5000 cells/well were seeded in 96-well plates and incubated for 24 h in standard medium (10). 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 [ 3 H]thymidine (1 Ci/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). 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 ϫ 10 5 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 NA 3 VO 4 , 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 g) 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).
Cell Transfection-Cells seeded in 6-well plates at 4 ϫ 10 5 cells/well were incubated for 24 h in standard medium. Cells were transiently transfected with 5 g 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 5Ј-AAGGGCATATTGCT-TCAGTTC-3Ј. 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.
Statistics-Data are presented as mean Ϯ S.E. Differences between two conditions were assessed with a Student's t test for related samples. A p value Ͻ0.05 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). Fig. 1A confirms this observation because the PI3-K inhibitor wortmannin (28) 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). Because the EGFR has been reported to be activated by oleate in endothelial cells (14), 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), did not significantly affect oleate-induced [ 3 H]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). These results establish that the EGFR is not implicated in oleate-induced proliferation of MDA-MB-231 cells.

Oleate-induced MDA-MB-231 Cell Proliferation Is Decreased by Specific Inhibitors of
and PKC-Several fatty acid derivatives such as LPA are known to act via GPCRs and to activate PI3-K via G proteins (13). We first studied the effect of pertussis toxin, an inhibitor of G i /G o proteins (31), on oleate-induced cell proliferation. As shown in Fig. 1C, pertussis toxin decreased oleate-induced [ 3 H]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 G i /G o proteins (32). 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 G i /G o is considered highly specific (31).
Agonist stimulation of GPCR caused the activation of a wide variety of signaling pathways, including modulation of phospholipases and protein kinases (20). 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) 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) and of mitogenic-extracellular signal-regulated kinase 1/2 (MEK1/2) (35), respectively, reduced the proliferative effect of oleate by about 40% (Fig. 1, E and F). Moreover, PKC, 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 PKC (36) blocked the oleate-induced proliferation (Fig. 1G). However, this peptide inhibitor also decreased the proliferation in the control situation, suggesting that PKC is important for the growth of MDA-MB-231 cells. Blocking protein kinase A, another downstream effector of GPCR, with H-89 (37) did not affect oleateinduced 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 PKC, 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). 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), 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), 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] i in GPR40-overexpressing cells (21)(22)(23). Because GPR40 is expressed in MDA-MB-231 breast cancer cells at ϳ12,000 copies/g of total RNA as we evaluated by real-time PCR, we measured changes of [Ca 2ϩ ] i in fura-2/AM-loaded cells following oleate or palmitate treatments. In the absence of BSA, oleate and palmitate caused a rapid increase in [Ca 2ϩ ] i by 2and 1.5-fold, respectively (Fig. 4A). Interestingly, in the presence of BSA, a more physiological condition, oleate still caused a rapid increase in [Ca 2ϩ ] i by 2-fold (Fig. 4B). In contrast, palmitate did not affect [Ca 2ϩ ] i in the presence of BSA.

GPR40 Is Involved in Oleate-induced Cell Proliferation-To
determine whether GPR40 is implicated in the proliferative action of oleate in MDA-MB-231 cells, we first attempted to reduce the expression level of GPR40 in MDA-MB-231 cells using RNA interference. Because the efficiency of transfection is about 60% in this cell line (data not shown), cells were co-transfected with a plasmid encoding GFP together with a plasmid encoding an siRNA against GPR40 or with a control scrambled siRNA, followed by selection of cells expressing GFP with a fluorescent-activated cell sorter. At the end of the selection, GPR40 mRNA expression was reduced by 80% in siRNAtransfected cells as determined by real-time PCR (Table I). Using a standard protocol to evaluate cell proliferation, fluorescent-activated cell sorter-selected cells were serum-starved for 24 h and then incubated for an additional 24 h in serum-free medium supplemented with 0.5% BSA (control) or various concentrations of BSA-bound oleate. The increase in [ 3 H]thymidine incorporation following oleate stimulation was signifi-FIG. 3. Oleate stimulates AKT phosphorylation. After 24 h of serum starvation in minimal essential medium in the presence of 0.5% BSA, cells were incubated for 2 h in Dulbecco's PBS containing 5 mM glucose and stimulated with 0.1 mM BSA-bound oleate or 100 ng/ml EGF for the indicated times. A, cell lysates were analyzed by immunoblotting using an AKT phospho-specific antibody that recognizes AKT phosphorylated at Ser-473 (p-AKT). The membrane was stripped and reprobed with an antibody recognizing total AKT. The figure shows a representative experiment that has been repeated three times in triplicate or quadruplicate. B, p-AKT and AKT were quantified by densitometry, and the data was expressed as p-AKT/AKT ratios (percent of the starting time control). Means Ϯ S.E. of three independent experiments performed in triplicate or quadruplicate. cantly reduced at all tested concentrations of the FFA in GFPpositive cells expressing the GPR40-specific siRNA (Fig. 5B). The decreased expression of GPR40 was particularly efficient in reducing the stimulation of DNA synthesis caused by low concentrations (1-5 M) of the FFA.
We next performed experiments to evaluate [ 3 H]thymidine incorporation following overexpression of GPR40 (Table I) in the presence of increasing concentrations of oleate (Fig. 5A). The stimulation of DNA synthesis by oleate was increased about 1.3-fold in MDA-MB-231 cells overexpressing GPR40 at concentrations of 10 M and above when compared with the Renilla luciferase control situation. To obtain strong additional evidence in favor of a role for GPR40, we attempted to identify breast cancer cell lines that would express GPR40 at lower levels. Unfortunately, among the four cell lines tested, GPR40 was expressed at levels relatively similar to the one measured in MDA-MB-231 cells (data not shown). To circumvent this problem, we examined the effect of GPR40 overexpression in the breast cancer cell lines T47D and MCF-7, which, respectively, do not or only slightly proliferate in the presence of oleate (10). Interestingly, in these two cell lines the effect of oleate on [ 3 H]thymidine incorporation was increased by nearly 2-fold when GPR40 was overexpressed (Fig. 5). The reason why oleate, in the absence of GPR40 overexpression, promotes a strong proliferative signal in MDA-MB-231 cells but a poor response in T47D and MCF-7 cells that expressed GPR40 mRNA at approximately the same level is unclear. It may be that GPR40 is coupled more tightly to its associated G proteins in MDA-MB-231 cells than in the two other cell lines. Alternatively, the expression level of the GPR40 protein may be lower in T47D and MCF7 cells even though the three cell lines expressed the GPR40 transcript at similar levels. Altogether, the siRNA and overexpression results indicate that oleate-induced proliferation of MDA-MB-231 cells is mediated at least in part through GPR40.

DISCUSSION
This study was prompted by the intriguing link between the incidence of breast cancer and obesity noted in both animal and human studies (1-3). The mechanism for this potential link, however, is enigmatic. To address this issue, we focused a series of studies on the abundant fatty acid, oleate, because it has been shown to stimulate the proliferation of breast cancer cells (10). Inhibition of individual signaling cascades, at the level of G i /G o proteins, Src, MEK1/2, PI3-K, or PLC resulted in a substantial reduction of oleate-induced DNA synthesis, suggesting that integration or cooperation of signals from multiple pathways is necessary to drive cells to enter S-phase in response to oleate. We also found that a GPCR is implicated in the proliferative signal transduction induced by this monounsaturated FFA. Thus, pertussis toxin, a very specific inhibitor of some G proteins (31), attenuated both LPA and oleate-induced proliferation, suggesting the implication of G i /G o proteins in this process. LPA has previously been shown to induce the proliferation of fibroblasts via GPCRs coupled to G i /G o (32). In addition, the fact that we provide evidence (see below) that the GPCR GPR40 is implicated in the proliferative action of oleate is in itself strong evidence for a role of a GPCR in oleate action.
It is noteworthy that, except for a possible role in insulin secretion, no other physiological action has been attributed to the GPCR GPR40 (21). Several lines of evidence demonstrate that GPR40 is implicated in oleate-induced proliferation of breast cancer cells. First, [Ca 2ϩ ] i is increased in response to oleate treatment in GPR40-expressing MDA-MB-231 cells. Second, overexpression of GPR40 amplified the oleate-induced cell  proliferation in three breast cancer cell lines, suggesting that oleate acts via this FFA receptor in these cells. Third, treatment of MDA-MB-231 cells with an siRNA-targeting GPR40 reduced the proliferative effect of oleate. Activation of GPCR signaling leading to cell growth has previously been documented for different GPCR agonists, including lysophospholipids and thrombin (18). Because only a partial inhibition of the proliferative effect of oleate with siRNA-targeting GPR40 was observed here, we could on one hand speculate that additional receptors for oleate or other signaling pathways participate in oleate-induced cell proliferation. For instance, LPA interacts with several GPCRs to mediate its biological actions (40), and it may also act via a receptor-independent pathway to cause cell growth (41). Interestingly, another FFA-activated GPCR, GPR120, was recently identified (42), and its contribution to oleate-induced proliferation of breast cancer cells remains to be studied. On the other hand, the importance of GPR40 in oleateinduced proliferation is likely to be underestimated because the siRNA did not completely block its expression. Moreover, as we do not have information on GPR40 protein level and stability in these cells, it is difficult to draw quantitative conclusions. The marked decrease in oleate-induced proliferation with the PLC inhibitor suggests a significant role of this phospholipase in this process. PLC is a major contributor of GPCR signaling (43). One might speculate that oleate binding to the receptor activates PLC-mediated hydrolysis of phosphatidylinositol 4,5-biphosphate into diacylglycerol and inositol trisphosphate, which, respectively, activates PKC and mobilizes calcium from the endoplasmic reticulum (44). Both PKC and calcium are effectors that have been reported to modulate processes required for breast cancer cell proliferation (45). Moreover, several studies have shown that unsaturated FFAs, including oleate, stimulated PLC␥ activity (46). In addition, our results show that oleate and palmitate caused a rapid and transient increase in [Ca 2ϩ ] i in MDA-MB-231 cells in the absence of BSA. However, in the presence of BSA, palmitate did not increase [Ca 2ϩ ] i in contrast to oleate, which still elicited a robust Ca 2ϩ response. Similar results have been observed in MCF-7 breast cancer cells (24). The difference observed between these two fatty acids might be explained in part by a better availability of unbound oleate for GPR40. In comparison to palmitate, oleate has a slightly lower affinity for BSA (47) and lower partition coefficient into cellular membranes (48). Thus, oleate may promote breast cancer cell proliferation by binding more efficiently to GPR40 than palmitate. In addition, as shown in our previous studies (10,11), differences in the metabolism of these two fatty acids are involved in their opposite effects on cell fate: the saturated fatty acid palmitate induces apoptosis of breast cancer cells via a mechanism that possibly implicates enhanced cardiolipin turnover and a reduction of this mitochondrial phospholipid, whereas the unsaturated fatty acid oleate, by sustaining cardiolipin synthesis, permits cell proliferation.
The rapid and transient increase in [Ca 2ϩ ] i induced by oleate suggests calcium mobilization from intracellular stores that implicates G q protein activation in response to many receptor agonists (18). Thus, these results favor the view that oleate stimulates, in part, MDA-MB-231 cell proliferation via GPR40 coupled to a G q protein and via the PLC/Ca 2ϩ pathway. GPR40 is mainly coupled to G q in MIN6 cells and Chinese hamster ovary cells overexpressing GPR40 (21,22). However, GPR40 is also coupled to G i /G o and G q in MCF-7 cells (24). Hence, in addition to G q , oleate-induced proliferation of MDA-MB-231 cells via GPR40 is likely to implicate G i /G o , because pertussis toxin reduced but did not abolish the proliferative effect of oleate in these cells. An orphan GPCR coupled to G i /G o proteins has been shown to be activated by the unsaturated FFA arachidonate and to cause a decrease in cAMP levels (19). The subunits of several G i /G o proteins are able to inhibit adenylyl cyclases resulting in a decrease in cAMP levels (49). However, cAMP levels were not affected by oleate in MDA-MB-231 cells when compared with LPA, which elicited a reduction in the cellular cAMP content (data not shown). This is consistent with the observation that H-89, a protein kinase A inhibitor, did not impair oleate-induced DNA synthesis. It is well established that many of the diverse biological effects resulting from the activation of G i /G o proteins are not mediated via the cAMP transduction system (50). For example, members of the G i /G o protein family directly affect Src (50) and Ca 2ϩ channels (51).
The increase in DNA synthesis induced by oleate appears to be mediated in part by the Src and MEK1/2 pathway as supported by the experiments using pharmacological inhibitors. The activity of Src and ERK1/2, the direct downstream effector of MEK1/2, is critical for cell survival and proliferation mediated by diverse growth factors (52,53). Consistent with this view, a previous study reported that oleate induces ERK1/2 activation via GPR40 in the MIN6 pancreatic ␤-cell line (21). AKT is rapidly activated by oleate in MDA-MB-231 cells, but not by palmitate. AKT controls cell cycle progression and is well known to participate in cell proliferation and survival (38). Agonist-induced stimulation of GPCRs may lead to the transactivation of the EGFR via Src and subsequently the activation of a wide variety of signaling pathways, including AKT and ERK1/2 (26,30). However, oleate did not induce EGFR activation in the cellular model we used, and blocking EGFR activity with AG1478 did not affect oleate-induced proliferation. Oleate can activate the EGFR in endothelial cells (14) but had no effect on EGFR tyrosine phosphorylation in Hs578T breast cancer cells (54). Whether stimulation of GPR40 by oleate leads to the transactivation of other growth factor receptors linked to tyrosine kinases and the activation of AKT is currently under investigation. Alternatively, a G␤␥ subunit coupled to GPR40 might directly activate PI3-K as frequently described (reviewed in Ref. 55).
In summary, the molecular, biochemical, and pharmacological results in this study suggest the following cascade of events in response to oleate in MDA-MD-231 cells. The unsaturated FFA binds to GPR40 and possibly other FFA receptor(s) coupled to G i /G o and G q , resulting in the activation of Src proteins, PI3-K, AKT, and Ca 2ϩ signaling, thus promoting cell growth. It cannot be discounted that other non-GPCR receptors are also implicated in oleate-induced activation of the PLC, Src, PI3-K/ AKT, MEK1/2, and PKC pathways that may participate in the proliferative effect. Taken together, these data provide a novel mechanism for the action of oleate in breast cancer cells in relation to cell growth by showing that this monounsaturated FFA acts as an extracellular signaling molecule to regulate breast cancer cell proliferation via the FFA receptor GPR40. Thus, GPR40 is not only a receptor that may participate in the control of insulin secretion by FFA, but it might also play an important role in the control of cell growth/apoptosis by some FFA. Hence, the possibility should be considered that GPR40 provides a link between fat and/or obesity and cancer. In this respect, the emerging evidence suggest an important association between insulin resistance, obesity, type 2 diabetes, and several cancers, in particular colon, prostate, and breast cancer (5). Finally, it could be hypothesized that overexpression of GPR40 in a subset of tumors could render them more susceptible to progression in patients eating a diet rich in unsaturated fatty acids.