Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor.


Introduction
Our group recently demonstrated that the inhibitory effects of ω-3 fatty acids on prostate cancer cell proliferation are mediated by FFA4, a G protein-coupled receptor in the free fatty acid receptor (FFAR) family [1]. The purpose of the current study was to determine whether FFARs mediate similar inhibitory effects in human breast cancer cells.
The prevailing mechanistic paradigm has been that n-3 FAs exert anti-inflammatory and potentially anti-cancer effects by competitively reducing production of eicosanoids, and/or more directly by generating metabolites with anti-inflammatory activity (e.g., "resolvins") [11,12]. However, the direct effects of n-3 metabolites on cancer cells, as compared to their anti-inflammatory effects, are under-studied [13]. In one report, resolvin D2, a DHA metabolite, unexpectedly increased proliferation of MCF-7 breast cancer cells [14].
Alternatively, it was shown in the last decade that n-3 FAs are agonist ligands for free fatty acid receptors (FFARs) that were formerly "orphan receptors" [15]. Two G-protein-coupled receptors (GPCRs), FFA1 and FFA4, bind long-chain polyunsaturated fatty acids that include n-3 FAs. The "de-orphanization" discovery has led to the ongoing characterization of the roles of the FFARs in cellular regulation, and to the rapid development of selective FFAR agonists with therapeutic potential [16][17][18][19].
Several studies have specifically explored the mechanism of the inhibitory action of n-3 FAs on breast cancer cells. The pathways implicated in the response include decreased Akt activation [5], increased neutral sphingomyelinase activity [20], increased BRCA levels [21], and increased PTEN levels [22]. GPCR-independent mechanisms have been reviewed [23]. To date, there is little information available concerning the roles of FFARs in breast cancer. It has however been shown that FFA1 is expressed in MCF-7 cells [24], and that MCF-7 and MDA-MB-231 cells express both FFA1 and FFA4 [25,26].
One study investigated the role of FFA4 in breast cancer in a mouse model, focusing on the role of FFA4 in inhibiting inflammation [27]. In this study, n-3 FAs reduced tumor burden even when FFA4 was knocked out in the host mouse. The authors suggest that anti-inflammatory effects of n-3 FAs, mediated by FFA4, are not important for their anti-tumor effects. Using cultured cells derived from their mouse model, the investigators showed that DHA induced apoptosis in either wild-type or FFA4 knockdown cells when applied at high doses (40-100 µM). The role of the alternative n-3 FA receptor, FFA1, has not been examined in breast cancer cell, to our knowledge.
In this study, we utilized two commonly used human breast cancer cell lines: MCF-7 and MDA-MB-231, as experimental models. MCF-7 is a luminal A estrogen receptor positive cell line, while MDA-MB-231 is a highly metastatic triple negative cell line. These two cell lines were used to explore the role of FFARs in the mechanism of action of n-3 FAs in breast cancer.

Cell Culture
MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in RPMI medium supplemented with 10% FBS (Hyclone/Thermo-Fisher Scientific, Waltham, MA, USA). Both cell lines were grown in an incubator at 37˝C in 5% CO 2 on standard tissue culture plastic.

Cell Proliferation Assays
Cells were seeded in 6-well plates at 3ˆ10 5 cells/well in serum-containing medium. After 1 day, the medium was changed to RPMI 1640 without serum. On the next day, the medium was changed to RPMI 1640 with 10% FBS, 10 µM LPA, or 10 nM EGF, in the absence or presence of 100 nM AM966, 10 µM Ki16425, 20 µM EPA or 1 µM TUG-891. Control cells were incubated with the appropriate vehicle (0.03% ethanol, v/v; 0.01% DMSO, v/v; and/or 4 µg/mL BSA). Duplicate wells were prepared for each experimental condition. Cell numbers were evaluated after 24, 48, and 72 h by removing medium, incubating cells with trypsin/EDTA for 5 min, adding trypan blue, and counting the suspended live cells (excluding trypan blue) using a hemacytometer.

Cell Migration Assays
MCF-7 cell migration was assessed using a modified Boyden chamber method, as previously described [28]. Cells were serum starved for 24 h and then seeded in serum-free medium at 2.5ˆ10 4 cells per insert in the upper chambers of 8-µm transwell inserts (BD Biosciences, San Jose, CA, USA). Cells were then treated with 10% FBS, 100 nM AM966, 20 µM EPA, 1 µM TUG-891, 10 µM LPA, or 10 nM EGF, either alone or in combination, with appropriate vehicle controls as described above. Serum-free medium was added to the lower wells. Following a 6-h migration, the insert membranes were fixed and stained using methanol and crystal violet. Cells that invaded the lower chambers were counted by microscopy.

Immunoblotting
Whole-cell extracts containing equal amounts of protein (30 µg) were separated by SDS-PAGE on 10% Laemmli gels, transferred to nitrocellulose, and incubated with primary (overnight at 4˝C) and then secondary (one to two hours at room temperature) antibodies. Blots were developed using enhanced chemiluminescence (GE Healthcare, Pittsburgh, PA, USA), and imaged using a Gel Doc system (BioRad, Hercules, CA, USA). Protein expression was quantified by densitometry using Quantity One software (Bio-Rad). Results were normalized to the actin loading control, and then to the value obtained for untreated control cells.

Statistical Analysis
Data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. The only exceptions were assays in which there was only one time point (e.g., migration assays); these data were analyzed by one-way ANOVA followed by Tukey's mutliple comparisons test. All analyses were done using Prism software (Graphpad, San Diego, CA, USA).

Effects of Lysophosphatidic Acid (LPA) and Epidermal Growth Factor (EGF) on Breast Cancer Cell Proliferation
Before testing for effects of FFAR agonists on breast cancer cells, we first established conditions for using growth factors to stimulate proliferation. Cells were serum-starved before treatments in order to remove confounding effects of LPA contained in serum, and to provide a baseline for testing effects of growth factors. The effects of serum, LPA, and EGF on proliferation of serum-starved MCF-7 and MDA-MB-231 cells are shown in Figure 1. All growth factors significantly increased cell number as compared to control. Serum was significantly more effective in inducing proliferation than LPA or EGF at all time points tested, in both cell lines; this result was expected since serum contains multiple mitogens including LPA. There was no significant difference between responses to LPA versus EGF at any time point.

Immunoblotting
Whole-cell extracts containing equal amounts of protein (30 μg) were separated by SDS-PAGE on 10% Laemmli gels, transferred to nitrocellulose, and incubated with primary (overnight at 4 °C) and then secondary (one to two hours at room temperature) antibodies. Blots were developed using enhanced chemiluminescence (GE Healthcare, Pittsburgh, PA, USA), and imaged using a Gel Doc system (BioRad, Hercules, CA, USA). Protein expression was quantified by densitometry using Quantity One software (Bio-Rad). Results were normalized to the actin loading control, and then to the value obtained for untreated control cells.

Statistical Analysis
Data were analyzed by two-way ANOVA followed by Tukeyʹs multiple comparisons test. The only exceptions were assays in which there was only one time point (e.g., migration assays); these data were analyzed by one-way ANOVA followed by Tukeyʹs mutliple comparisons test. All analyses were done using Prism software (Graphpad, San Diego, CA, USA).

Effects of Lysophosphatidic Acid (LPA) and Epidermal Growth Factor (EGF) on Breast Cancer Cell Proliferation
Before testing for effects of FFAR agonists on breast cancer cells, we first established conditions for using growth factors to stimulate proliferation. Cells were serum-starved before treatments in order to remove confounding effects of LPA contained in serum, and to provide a baseline for testing effects of growth factors. The effects of serum, LPA, and EGF on proliferation of serum-starved MCF-7 and MDA-MB-231 cells are shown in Figure 1. All growth factors significantly increased cell number as compared to control. Serum was significantly more effective in inducing proliferation than LPA or EGF at all time points tested, in both cell lines; this result was expected since serum contains multiple mitogens including LPA. There was no significant difference between responses to LPA versus EGF at any time point.

Signal Transduction Responses to LPA and EGF in Breast Cancer Cells
To further characterize responses to LPA and EGF in the breast cancer cell lines, we performed immunoblotting experiments to test for Erk and Akt activation ( Figure 2). Both LPA and EGF increase activating phosphorylation of Erk and Akt in both MCF-7 and MDA-MB-231 cells ( Figure 2A). These results are consistent with previous reports concerning the mitogenic activity of LPA and EGF in breast cancer cells [29,30]. Wholecell extracts were immunoblotted using antibodies recognizing phosphorylated active Erk and Akt. An immunoblot for total actin was performed as a loading control; (B) Serum-starved MCF-7 cells were incubated for the indicated times with 10 μM LPA or 10 nM EGF. Whole-cell extracts were immunoblotted using antibody recognizing total CCN1. An immunoblot for total actin was performed as a loading control. Each experiment is representative of at least three separate experiments.

Signal Transduction Responses to LPA and EGF in Breast Cancer Cells
To further characterize responses to LPA and EGF in the breast cancer cell lines, we performed immunoblotting experiments to test for Erk and Akt activation ( Figure 2). Both LPA and EGF increase activating phosphorylation of Erk and Akt in both MCF-7 and MDA-MB-231 cells ( Figure 2A). These results are consistent with previous reports concerning the mitogenic activity of LPA and EGF in breast cancer cells [29,30].

Signal Transduction Responses to LPA and EGF in Breast Cancer Cells
To further characterize responses to LPA and EGF in the breast cancer cell lines, we performed immunoblotting experiments to test for Erk and Akt activation ( Figure 2). Both LPA and EGF increase activating phosphorylation of Erk and Akt in both MCF-7 and MDA-MB-231 cells (Figure 2A). These results are consistent with previous reports concerning the mitogenic activity of LPA and EGF in breast cancer cells [29,30].  Whole-cell extracts were immunoblotted using antibodies recognizing phosphorylated active Erk and Akt. An immunoblot for total actin was performed as a loading control; (B) Serum-starved MCF-7 cells were incubated for the indicated times with 10 µM LPA or 10 nM EGF. Whole-cell extracts were immunoblotted using antibody recognizing total CCN1. An immunoblot for total actin was performed as a loading control. Each experiment is representative of at least three separate experiments.
We also examined a more novel response to growth factors, CCN1 induction, in MCF-7 cells. CCN1 is an inducible matricellular protein whose expression is positively correlated with breast cancer progression [31,32]. As shown in Figure 2B, both LPA and EGF stimulate expression of CCN1 in MCF-7 cells. An increase in CCN1 protein levels was seen after only 30 min of treatment with either growth factor. Taken together, the results presented in Figure 2A,B confirm that both LPA and EGF activate pro-mitogenic signaling pathways in human breast cancer cell lines.

Effects of LPA Antagonists on Breast Cancer Cell Proliferation
Previous studies have shown that LPA receptors are expressed in breast cancer cell lines. One group performed a comprehensive analysis of LPA receptor expression in commonly-used breast cancer cell lines [33]. Their data show that MDA-MB-231 and MCF-7 cells, among other cell lines, express LPA 1 , LPA 2 , and LPA 3 . LPA 1 has been determined to mediate many of the actions of LPA in breast cancer cells [34][35][36][37]. RT-PCR experiments conducted in our lab confirmed that LPA 1 was expressed in both MCF-7 and MDA-MB-231 cells under the conditions used herein ( Figure 3A).
We also examined a more novel response to growth factors, CCN1 induction, in MCF-7 cells. CCN1 is an inducible matricellular protein whose expression is positively correlated with breast cancer progression [31,32]. As shown in Figure 2B, both LPA and EGF stimulate expression of CCN1 in MCF-7 cells. An increase in CCN1 protein levels was seen after only 30 min of treatment with either growth factor. Taken together, the results presented in Figure 2A,B confirm that both LPA and EGF activate pro-mitogenic signaling pathways in human breast cancer cell lines.

Effects of LPA Antagonists on Breast Cancer Cell Proliferation
Previous studies have shown that LPA receptors are expressed in breast cancer cell lines. One group performed a comprehensive analysis of LPA receptor expression in commonly-used breast cancer cell lines [33]. Their data show that MDA-MB-231 and MCF-7 cells, among other cell lines, express LPA1, LPA2, and LPA3. LPA1 has been determined to mediate many of the actions of LPA in breast cancer cells [34][35][36][37]. RT-PCR experiments conducted in our lab confirmed that LPA1 was expressed in both MCF-7 and MDA-MB-231 cells under the conditions used herein ( Figure 3A). Two LPA receptor pharmacologic antagonists were used to further study the role of LPA1 in breast cancer cells. Ki16425 is a selective inhibitor of LPA1 and LPA3 [38], while AM966 is an LPA1-selective antagonist [39]. We used a dose-response study ( Figure 3B) to test whether LPA1 inhibitors inhibit LPAinduced proliferation in MDA-MB-231 breast cancer cells. As expected, based on their relative receptor affinities, the LPA1-selective antagonist AM966 was more potent (IC50 = 32 nM) in inhibiting LPA-induced proliferation than was the pan-LPA inhibitor Ki16425 (IC50 = 904 nM). Interestingly, the amount of AM966 needed to completely inhibit MDA-MB-231 cell proliferation was 100-fold higher than in our previously published work using DU145 prostate cancer cells [1], although the amount of Ki16425 required was similar in both MDA-MB-231 and DU145. The dose-response curve for AM966 was very shallow, suggesting the involvement of multiple receptors. These results suggest that LPA1 is not the only LPA receptor that mediates LPA-induced proliferation in these cells.
In Figure 4, we further tested the effects of LPA antagonists. We asked 1) whether the inhibitory effects of LPA antagonists extend to EGF-induced proliferation, and 2) whether LPA antagonists have similar effects in MCF-7 and MDA-MB-231 cells. The results of this series of experiments, which are Two LPA receptor pharmacologic antagonists were used to further study the role of LPA 1 in breast cancer cells. Ki16425 is a selective inhibitor of LPA 1 and LPA 3 [38], while AM966 is an LPA 1 -selective antagonist [39]. We used a dose-response study ( Figure 3B) to test whether LPA 1 inhibitors inhibit LPA-induced proliferation in MDA-MB-231 breast cancer cells. As expected, based on their relative receptor affinities, the LPA 1 -selective antagonist AM966 was more potent (IC 50 = 32 nM) in inhibiting LPA-induced proliferation than was the pan-LPA inhibitor Ki16425 (IC 50 = 904 nM). Interestingly, the amount of AM966 needed to completely inhibit MDA-MB-231 cell proliferation was 100-fold higher than in our previously published work using DU145 prostate cancer cells [1], although the amount of Ki16425 required was similar in both MDA-MB-231 and DU145. The dose-response curve for AM966 was very shallow, suggesting the involvement of multiple receptors. These results suggest that LPA 1 is not the only LPA receptor that mediates LPA-induced proliferation in these cells.
In Figure 4, we further tested the effects of LPA antagonists. We asked 1) whether the inhibitory effects of LPA antagonists extend to EGF-induced proliferation, and 2) whether LPA antagonists have similar effects in MCF-7 and MDA-MB-231 cells. The results of this series of experiments, which are presented in Figure 4, clearly demonstrate that the LPA receptor antagonists block proliferation in response to both LPA and EGF. This response was observed in both MCF-7 and MDA-MB-231 cells.
Taken together, these results are consistent with a crucial role for LPARs in EGF response, as has been noted in other cell types [1,40]. presented in Figure 4, clearly demonstrate that the LPA receptor antagonists block proliferation in response to both LPA and EGF. This response was observed in both MCF-7 and MDA-MB-231 cells.
Taken together, these results are consistent with a crucial role for LPARs in EGF response, as has been noted in other cell types [1,40].

Expression of Free Fatty Acid Receptors (FFARs) in Breast Cancer Cells
We next turned to the effects of n-3 FAs. We examined whether FFARs are expressed in MCF-7 and MDA-MB-231 cells. Both receptors have previously been reported to be present in MCF-7 cells, based on flow cytometry [25]. Using RT-PCR, we assessed mRNA levels for FFA4 and FFA1, the two receptors for long chain free fatty acid ( Figure 5A). Both PCR products were detected, although the PCR product for FFA4 was present at such low levels that it was difficult to visualize. To confirm that FFA4 was expressed, we performed immunoblotting using an antibody previously validated in our laboratory [1], and did detect FFA4 protein ( Figure 5B). We were unable to validate an appropriate antibody for FFA1, but our PCR results suggest that FFA1 is likely expressed at higher levels than FFA4 in the two breast cancer cell lines. These results indicate that the roles of both FFA1 and FFA4 need to be considered in breast cancer cells.

Expression of Free Fatty Acid Receptors (FFARs) in Breast Cancer Cells
We next turned to the effects of n-3 FAs. We examined whether FFARs are expressed in MCF-7 and MDA-MB-231 cells. Both receptors have previously been reported to be present in MCF-7 cells, based on flow cytometry [25]. Using RT-PCR, we assessed mRNA levels for FFA4 and FFA1, the two receptors for long chain free fatty acid ( Figure 5A). Both PCR products were detected, although the PCR product for FFA4 was present at such low levels that it was difficult to visualize. To confirm that FFA4 was expressed, we performed immunoblotting using an antibody previously validated in our laboratory [1], and did detect FFA4 protein ( Figure 5B). We were unable to validate an appropriate antibody for FFA1, but our PCR results suggest that FFA1 is likely expressed at higher levels than FFA4 in the two breast cancer cell lines. These results indicate that the roles of both FFA1 and FFA4 need to be considered in breast cancer cells. presented in Figure 4, clearly demonstrate that the LPA receptor antagonists block proliferation in response to both LPA and EGF. This response was observed in both MCF-7 and MDA-MB-231 cells. Taken together, these results are consistent with a crucial role for LPARs in EGF response, as has been noted in other cell types [1,40].

Expression of Free Fatty Acid Receptors (FFARs) in Breast Cancer Cells
We next turned to the effects of n-3 FAs. We examined whether FFARs are expressed in MCF-7 and MDA-MB-231 cells. Both receptors have previously been reported to be present in MCF-7 cells, based on flow cytometry [25]. Using RT-PCR, we assessed mRNA levels for FFA4 and FFA1, the two receptors for long chain free fatty acid ( Figure 5A). Both PCR products were detected, although the PCR product for FFA4 was present at such low levels that it was difficult to visualize. To confirm that FFA4 was expressed, we performed immunoblotting using an antibody previously validated in our laboratory [1], and did detect FFA4 protein ( Figure 5B). We were unable to validate an appropriate antibody for FFA1, but our PCR results suggest that FFA1 is likely expressed at higher levels than FFA4 in the two breast cancer cell lines. These results indicate that the roles of both FFA1 and FFA4 need to be considered in breast cancer cells.

Effects of FFAR Agonists on Breast Cancer Cell Proliferation
We next performed a dose-response study ( Figure 6) to test whether FFAR agonists inhibit proliferation of MDA-MB-231 cells. EPA, TUG-891, and GW9503 all inhibited proliferation of these cells. EPA was the least potent compound (IC 50 403 nM), which was expected based on results with other cell lines [1]. The IC 50 for the FFA4-selective agonist, TUG-891, was 24 nM. However, a 100-fold higher dose of TUG-891 was required to completely inhibit LPA-induced proliferation in the breast cancer cell line ( Figure 6) as compared to our previously published results with Du145 prostate cancer cells [1]. In addition, the dose-response curve for TUG-891 was very shallow, suggesting the involvement of more than one receptor in the inhibitory response. While TUG-891 is selective for FFA4 over FFA1, neither TUG-891 nor GW9508 is specific for a single receptor. The FFA1-specific agonist GW9508 was 100-fold more potent (IC 50 = 16 nm) in inhibiting LPA-induced proliferation in MDA-MB-231 ( Figure 6) as compared to previous results in DU145 cells where FFA4 response predominates [1]. The published EC 50 for GW9508 at FFA1 is~50 nM. Thus, our results are consistent with a role for FFA1, and possibly also FFA4, in inhibiting proliferation of MDA-MB-231 cells.
We next tested the effects of LPA 1 inhibitors on both LPA-and EGF-induced breast cancer cell proliferation (Figure 7). Neither the LPA antagonists AM966 and Ki16425, nor the FFAR agonists EPA, GW9508, and TUG891, had any significant effects on cell numbers on their own. However, all agents completely inhibited LPA-and EGF-induced proliferation in MCF-7 and MDA-MB-231 cells, when added individually, consistent with the results in Figures 4 and 6. Although EPA appeared to decrease cell numbers slightly below control values in the presence of LPA or EGF, the effect was not significant. There was no additional effect (e.g., cytotoxicity) on cell numbers when LPA antagonists were combined with FFAR agonists GW9508 or TUG891 (Figure 7). Thus, both LPA antagonists and FFAR agonists can block the effects of LPA and EGF on breast cancer cell proliferation.

Effects of FFAR Agonists on Breast Cancer Cell Proliferation
We next performed a dose-response study ( Figure 6) to test whether FFAR agonists inhibit proliferation of MDA-MB-231 cells. EPA, TUG-891, and GW9503 all inhibited proliferation of these cells. EPA was the least potent compound (IC50 403 nM), which was expected based on results with other cell lines [1]. The IC50 for the FFA4-selective agonist, TUG-891, was 24 nM. However, a 100-fold higher dose of TUG-891 was required to completely inhibit LPA-induced proliferation in the breast cancer cell line ( Figure 6) as compared to our previously published results with Du145 prostate cancer cells [1]. In addition, the dose-response curve for TUG-891 was very shallow, suggesting the involvement of more than one receptor in the inhibitory response. While TUG-891 is selective for FFA4 over FFA1, neither TUG-891 nor GW9508 is specific for a single receptor. The FFA1-specific agonist GW9508 was 100-fold more potent (IC50 = 16 nm) in inhibiting LPA-induced proliferation in MDA-MB-231 ( Figure 6) as compared to previous results in DU145 cells where FFA4 response predominates [1]. The published EC50 for GW9508 at FFA1 is ~50 nM. Thus, our results are consistent with a role for FFA1, and possibly also FFA4, in inhibiting proliferation of MDA-MB-231 cells.
We next tested the effects of LPA1 inhibitors on both LPA-and EGF-induced breast cancer cell proliferation (Figure 7). Neither the LPA antagonists AM966 and Ki16425, nor the FFAR agonists EPA, GW9508, and TUG891, had any significant effects on cell numbers on their own. However, all agents completely inhibited LPA-and EGF-induced proliferation in MCF-7 and MDA-MB-231 cells, when added individually, consistent with the results in Figures 4 and 6. Although EPA appeared to decrease cell numbers slightly below control values in the presence of LPA or EGF, the effect was not significant. There was no additional effect (e.g., cytotoxicity) on cell numbers when LPA antagonists were combined with FFAR agonists GW9508 or TUG891 (Figure 7). Thus, both LPA antagonists and FFAR agonists can block the effects of LPA and EGF on breast cancer cell proliferation.    (Panels A and B), or (Panels C and D), 10 μM LPA or 10 nM EGF in the absence and presence of 10 μM TUG891, 10 μM Ki16425, and/or 10 μM AM966. In addition, Panels C and D show the effects of TUG-891 as compared to that of 25 μM EPA, and controls were included for the LPA antagonists alone. Some of the data points from Panels C and D were presented earlier in Figure 4, which was derived from the same series of experiments; panels (A) and (B) are from separate sets of experiments. Although only the 48-h time point is shown, similar inhibitory effects were observed at 24 and 72 h. Each value represents the mean ± SEM (n = 4) of values (number of live cells per well) obtained from two separate experiments, each of which used two separate replicate wells of cells for each experimental condition (**** p < 0.0001 compared to control). Data were analyzed done using two-way ANOVA, followed by Tukey's multiple comparisons test.

Effects of FFAR Agonists on Breast Cancer Cell Migration
Finally, we asked whether FFAR agonists inhibit migration of breast cancer cells. As shown in Figure 8, EPA and GW9508 both block LPA-and EGF-induced migration of MCF-7 cells. The LPAR antagonist Ki16425 has a similar effect. The combination of GW9508 and Ki16425 also yields complete inhibition; i.e., there is no additional effect of the FFAR agonist in the presence of an LPA antagonist. We conclude that either activation of FFARs, or antagonism of LPARs, can inhibit migration in response to either LPA or EGF.  (Panels A and B), or (Panels C and D), 10 µM LPA or 10 nM EGF in the absence and presence of 10 µM TUG891, 10 µM Ki16425, and/or 10 µM AM966. In addition, Panels C and D show the effects of TUG-891 as compared to that of 25 µM EPA, and controls were included for the LPA antagonists alone. Some of the data points from Panels C and D were presented earlier in Figure 4, which was derived from the same series of experiments; panels (A) and (B) are from separate sets of experiments. Although only the 48-h time point is shown, similar inhibitory effects were observed at 24 and 72 h. Each value represents the mean˘SEM (n = 4) of values (number of live cells per well) obtained from two separate experiments, each of which used two separate replicate wells of cells for each experimental condition (**** p < 0.0001 compared to control). Data were analyzed done using two-way ANOVA, followed by Tukey's multiple comparisons test.

Effects of FFAR Agonists on Breast Cancer Cell Migration
Finally, we asked whether FFAR agonists inhibit migration of breast cancer cells. As shown in Figure 8, EPA and GW9508 both block LPA-and EGF-induced migration of MCF-7 cells. The LPAR antagonist Ki16425 has a similar effect. The combination of GW9508 and Ki16425 also yields complete inhibition; i.e., there is no additional effect of the FFAR agonist in the presence of an LPA antagonist. We conclude that either activation of FFARs, or antagonism of LPARs, can inhibit migration in response to either LPA or EGF. Figure 8. Effects of LPAR agonists on MCF-7 cell migration. Serum-starved MCF-7 cells were treated with 10 μM Ki16425, 20 μM EPA, 1 μM GW9508, 10 μM LPA, or 10 nM EGF, either alone or in combination. After a 6-h migration period, cells were analyzed as described in Methods. Each bar represents the mean ± SEM (n = 4) of values (total number of migrated cells/well) obtained from two separate experiments, each of which used two separate replicate wells of cells for each experimental condition (**** p < 0.0001 vs. control). Data were analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test.

Conclusions
In extending our ongoing studies of FFAR activation to breast cancer cells, the intention was to expand our knowledge of the roles of FFARs in cancer. While others have examined the effects of n-3 FAs on breast cancer, to our knowledge no other studies have tested the roles of FFARs in human breast cancer cells.
The current study used LPA as one of the growth factors, based on our previous work implicating LPARs in the mechanism of FFAR-mediated inhibition of cancer cell proliferation. LPA has multiple actions that support breast cancer growth, migration, invasion, and survival [37,[41][42][43][44]]. An LPA antagonist was shown to induce regression of breast tumors in a mouse model [45]. Others reported that either an LPA1 antagonist or LPA1 knockdown decreases MDA-MB-231 cell metastasis, but not primary tumor size, in mice [46]. Together, these data have established a potential role for the LPA axis as a therapeutic target in breast cancer cells, as reviewed by Panupinthu and colleagues [29]. However, the overall roles of LPA and its receptors in breast cancer cell proliferation have not been fully delineated. FFA1/GPR40 was first reported to be present in MCF-7 cells in 2004 [24]. This was later confirmed by another group using flow cytometry [25] and also in the current study by PCR ( Figure 5). Soto-Guzman and colleagues also detected FFA4 expression in MCF-7 cells [25]. Our data demonstrate expression of FFA1 and FFA4 mRNA, and FFAR protein, in both MCF-7 and MDA-MB-231 cells. Interestingly, the results of dose-response studies using two selective FFAR agonists were consistent with a major role for FFA1 in MDA-MB-231 cells ( Figure 6). While these results do not exclude a role for FFA4, this is the first demonstration in our lab that FFA1 and FFA4 may similarly mediate inhibition of cancer cell proliferation. Figures 4 and 7 demonstrate that LPAR inhibition not only impedes LPA-induced proliferation, but also EGF-induced proliferation. This result suggests that, in MCF-7 and MDA-MB-231 cells, EGFR is reliant on one or more LPARs. This result is similar to those obtained with prostate cancer cells [1,40]. LPARs have been described as "masters" of EGFR signaling [47], as confirmed by studies in various cell types [1,[48][49][50][51][52][53]. Further studies will be required to determine whether FFA1 or FFA4 act directly on both LPARs and EGFR to result in inhibition, or indirectly inhibit EGFR via effects on LPARs.

Conclusions
In extending our ongoing studies of FFAR activation to breast cancer cells, the intention was to expand our knowledge of the roles of FFARs in cancer. While others have examined the effects of n-3 FAs on breast cancer, to our knowledge no other studies have tested the roles of FFARs in human breast cancer cells.
The current study used LPA as one of the growth factors, based on our previous work implicating LPARs in the mechanism of FFAR-mediated inhibition of cancer cell proliferation. LPA has multiple actions that support breast cancer growth, migration, invasion, and survival [37,[41][42][43][44]]. An LPA antagonist was shown to induce regression of breast tumors in a mouse model [45]. Others reported that either an LPA 1 antagonist or LPA 1 knockdown decreases MDA-MB-231 cell metastasis, but not primary tumor size, in mice [46]. Together, these data have established a potential role for the LPA axis as a therapeutic target in breast cancer cells, as reviewed by Panupinthu and colleagues [29]. However, the overall roles of LPA and its receptors in breast cancer cell proliferation have not been fully delineated. FFA1/GPR40 was first reported to be present in MCF-7 cells in 2004 [24]. This was later confirmed by another group using flow cytometry [25] and also in the current study by PCR ( Figure 5). Soto-Guzman and colleagues also detected FFA4 expression in MCF-7 cells [25]. Our data demonstrate expression of FFA1 and FFA4 mRNA, and FFAR protein, in both MCF-7 and MDA-MB-231 cells. Interestingly, the results of dose-response studies using two selective FFAR agonists were consistent with a major role for FFA1 in MDA-MB-231 cells ( Figure 6). While these results do not exclude a role for FFA4, this is the first demonstration in our lab that FFA1 and FFA4 may similarly mediate inhibition of cancer cell proliferation. Figures 4 and 7 demonstrate that LPAR inhibition not only impedes LPA-induced proliferation, but also EGF-induced proliferation. This result suggests that, in MCF-7 and MDA-MB-231 cells, EGFR is reliant on one or more LPARs. This result is similar to those obtained with prostate cancer cells [1,40]. LPARs have been described as "masters" of EGFR signaling [47], as confirmed by studies in various cell types [1,[48][49][50][51][52][53]. Further studies will be required to determine whether FFA1 or FFA4 act directly on both LPARs and EGFR to result in inhibition, or indirectly inhibit EGFR via effects on LPARs.
FFAR activation through EPA, GW9508, or TUG-891 abolishes LPA-and EGF-induced proliferation and migration in MCF-7 and MDA-MB-231 cells (Figures 6-8). GW9508 yields a classical dose-response curve with an IC 50 consistent with action at FFA1. At the higher dose of TUG-891 used to achieve complete inhibition in breast cancer cells (10 µM), it is plausible that it is activating FFA1 in addition to, FFA4. Our results are consistent with other reports of inhibitory effects of n-3 FAs in breast cancer cells. In one study, low doses of n-3 FAs were shown to inhibit proliferation of MCF-7 cells, while high doses induced apoptosis [20]. This is consistent with our dose-response results for MDA-MB-231 cells, where inhibition of proliferation was achieved without cytotoxicity ( Figure 6). The EPA concentration (25 µM) used in subsequent experiments was chosen as the dose that maximally inhibited proliferation but did not decrease viability, since our focus was on inhibition of growth factor response rather than toxic responses that may occur at high doses.
Although our results suggest that both FFAR agonists and LPAR antagonists inhibit proliferation in MCF-7 and MDA-MB-231 cells, more work is needed to fully elucidate the mechanism of action. It remains to be definitively determined which FFAR is responsible for the inhibition in breast cancer cells. It is also unclear at this point whether FFAR activation directly influences EGF-mediated signaling, or whether FFARs act indirectly via interference with LPAR activity as appears to be the case in prostate cancer cells [40]. Since the synthetic FFAR agonists used in the current study are not metabolized to resolvins, and are designed to act selectively at GPCRs, utilization of these agonist drugs is helpful in distinguishing receptor-mediated effects from effects of n-3 FAs on lipid metabolism. In addition, the emerging small-molecule FFAR agonists have therapeutic potential for the prevention and/or treatment of breast cancer. In summary, the results presented herein demonstrate for the first time that FFAR activation results in inhibition of breast cancer cell proliferation and migration. The inhibitory response mediated by FFARs needs to be taken into account when considering effects of n-3 fatty acids on breast cancer cells.