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. 2019 Mar;8(3):252-265.
doi: 10.1530/EC-18-0543.

Oleic acid induces migration through a FFAR1/4, EGFR and AKT-dependent pathway in breast cancer cells

Cleofas Marcial-Medina  1 , Alejandra Ordoñez-Moreno  1 , Christian Gonzalez-Reyes  1 , Pedro Cortes-Reynosa  1 , Eduardo Perez Salazar  1
Affiliations

Oleic acid induces migration through a FFAR1/4, EGFR and AKT-dependent pathway in breast cancer cells

Cleofas Marcial-Medina et al. Endocr Connect. 2019 Mar.

Abstract

Free fatty acids (FFAs) are an energy source, and induce activation of signal transduction pathways that mediate several biological processes. In breast cancer cells, oleic acid (OA) induces proliferation, matrix metalloproteinase-9 (MMP-9) secretion, migration and invasion. However, the signal transduction pathways that mediate migration and invasion induced by OA in breast cancer cells have not been studied in detail. We demonstrate here that FFAR1 and FFAR4 mediate migration induced by OA in MDA-MB-231 and MCF-7 breast cancer cells. Moreover, OA induces migration, invasion, AKT1 and AKT2 activation, 12-LOX secretion and an increase of NFκB-DNA binding activity in breast cancer cells. Cell migration requires FFAR1, FFAR4, EGFR, AKT and PI3K activity, whereas invasion is mediated though a PI3K/Akt-dependent pathway. Furthermore, OA promotes relocalization of paxillin to focal contacts and it requires PI3K and EGFR activity, whereas NFκB-DNA binding activity requires PI3K and AKT activity.

Keywords: 12-LOX; breast cancer; invasion; migration; oleic acid (OA).

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Figures

Figure 1
Figure 1
FFAR1 and FFAR4 mediate migration induced by OA in breast cancer cells. Panel (A), (B) and (D) Cultures of MDA-MB-231 and MCF-7 cells were treated with DC260126 or AH7614, scratch-wounded and stimulated with 100 µM OA for 48 h. Panel (C) MDA-MB-231 cells were transfected with FFAR4 shRNA or scramble shRNA and cell lysates were analyzed by Western blotting with anti-FFAR4 Ab. Cultures of MDA-MB-231 cells transfected with FFAR4 shRNA or scramble shRNA were scratch-wounded and stimulated with 100 μM OA for 48 h. Graphs represent the mean ± s.d. of at least three independent experiments and are expressed as fold of migrated cells above unstimulated cells. **P < 0.01, ***P < 0.001.
Figure 2
Figure 2
OA induces migration through a PI3K/AKT-dependent pathway. Panel (A) and (B) Cultures of MDA-MB-231 and MCF-7 cells were treated with LY294002 or A6730, scratch-wounded and treated with OA. Panel (C) and (D) Lysates from MDA-MB-231 and MCF-7 cells treated with OA for various times were analyzed by immunoprecipitation (IP) with Akt1 Ab or Akt2 Ab followed by Western blotting with anti-p-Akt-Thr Ab and anti-p-Akt-Ser Ab, respectively. Membranes were analyzed further by Western blotting with anti-Akt1 or Akt2 Ab. Graphs represent the mean ± s.d. of at least three independent experiments and are expressed as fold of migrated cells or p-Akt-1 or -2 above unstimulated cells. *P < 0.05, **P < 0.01 ***P < 0.001.
Figure 3
Figure 3
OA induces migration through an AKT2-dependent pathway. Panel (A) and (B) MDA-MB-231 and MCF-7 cells were transfected with Akt2 siRNA or scrambled siRNA and cell lysates were analyzed by Western blotting with anti-Akt2 Ab. Cultures of MDA-MB-231 and MCF-7 cells transfected with Akt2 siRNA or scrambled siRNA were scratch-wounded and stimulated with OA. Graphs represent the mean ± s.d. of at least three independent experiments and are expressed as fold of Akt2 or migrated cells above unstimulated cells. **P < 0.01 ***P < 0.001.
Figure 4
Figure 4
Baicalein inhibits migration and activation of AKT2 and FAK induced by OA. Panel (A) and (B) Cultures of MDA-MB-231 and MCF-7 cells were treated with baicalein (BAI), scratch-wounded and treated with OA. Panel (C) Lysates from MDA-MB-231 cells treated with BAI and stimulated with OA were analyzed by IP with Akt2 Ab or FAK Ab, followed by Western blotting with anti-p-Akt-Thr Ab or p-FAK Ab. Membranes were analyzed further by Western blotting with anti-Akt2 or FAK Ab. Panel (D) Concentration of 12(S)-HETE was analyzed by ELISA in supernatants from MDA-MB-231 cells treated with OA or AA. Graphs represent the mean ± s.d. of at least three independent experiments and are expressed as fold of migrated cells, p-Akt2, p-FAK or 12(S)-HETE above unstimulated cells. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Focal contacts formation induced by OA requires PI3K activity. MDA-MB-231 cells cultured on coverslips were treated with LY294002 (Panel A) or baicalein (Panel B) and stimulated with OA. Cells were fixed and focal contacts were analyzed by staining with anti-paxillin Ab conjugated to FITC. F-actin was stained with TRITC-conjugated phalloidin. F-actin structures are shown in red, and focal adhesions are shown in green. Graphs represent the mean ± s.d. of at least three independent experiments and are expressed as mean fluorescent intensities of paxillin above unstimulated cells. **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
Role of EGFR in migration and focal contacts formation induced by OA. Panel (A) and (B) Cultures of MDA-MB-231 and MCF-7 cells were treated with AG1478, scratch-wounded and treated with OA. Panel (C) MDA-MB-231 cells cultured on coverslips were treated with AG1478 and stimulated with OA. Cells were fixed and focal contacts were analyzed by staining with anti-paxillin Ab conjugated to FITC. F-actin was stained with TRITC-conjugated phalloidin. F-actin structures are shown in red, and focal adhesions are shown in green. Graphs represent the mean ± s.d. of at least three independent experiments and are expressed as fold of migrated cells or mean fluorescent intensities of paxillin above unstimulated cells. *P < 0.05, ***P < 0.001.
Figure 7
Figure 7
OA induces invasion and NFkB-DNA binding activity. Panel (A) Invasion assays were performed by using the Boyden chamber method and MDA-MB-231 cells treated with LY294002 or A6730 and stimulated with OA. Graph represents the mean ± s.d. of three independent experiments and is expressed as fold of invasion above unstimulated cells. Panel (B) MDA-MB-231 cells were treated with OA for various times and nuclear extracts were obtained. Panel (C, D and E) MCF-7 and MDA-MB-231 cells were treated with LY294002 or A6730, stimulated with OA and nuclear extracts were obtained. NFκB-DNA binding activity was analyzed by EMSA. Control included EMSA reactions with 100-fold excess of cold NFκB competitor (Cold probe). Results shown are representative of three independent experiments. ***P < 0.001.
Figure 8
Figure 8
Model of signal transduction pathways that mediate migration induced by OA in breast cancer cells.
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