14, 15-EET induces breast cancer cell EMT and cisplatin resistance by up-regulating integrin αvβ3 and activating FAK/PI3K/AKT signaling

The study protocol was performed according to the Declaration of Helsinki and was approved by the Ethics Committee of Wuhan No.1 Hospital of Tongji Medical College. All breast cancer patients gave their signed informed consent for the use of biological samples. Tumor tissues and noncancerous tissues were collected from 11 patients in the Wuhan No.1 Hospital of Tongji Medical College.

Human breast cancer cell MCF-7 and MDA-MB-231 were purchased from the China Center for Type Culture Collection (Wuhan, China). BALB/c athymic nude (nu/nu) mice (6–8 weeks old) were obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The mice were maintained in the accredited animal facility of Tongji Medical College, and used for studies approved by the Animal Care and Use Committee of Tongji Medical College.

Rabbit anti-human antibodies against integrin αv and β3, FAK, p-FAK, PI3K, p-PI3K, AKT, p-AKT, E-cadherin, N-cadherin, vimentin, snail and LY294002 (PI3K inhibitor) were purchased from Cell Signaling (Danvers, MA, USA). slug and PF562271 were purchased from Abcam (Cambridge, MA, US). The small interfering RNAs (si RNAs) against integrin αv, integrin β3 and control for experiments using targeted siRNA transfection were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The 14, 15-EET and 14, 15-EEZE were purchased from Cayman chemical (Ann 152 Arbor, MI, USA).

MCF-7 and MDA-MB-231 cells were cultured in flasks in DMEM growth medium supplemented with 5% FBS, 100 U/ml of penicillin, and 100 pg/ml of streptomycin. The cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

A stable metabolite of 14, 15-EET, 14, 15-dihydroxyeicosatrienoic acid (14, 15-DHET) in peripheral venous blood from patients with breast cancer and healthy donors or in BC tissues and noncancerous tissues from breast cancer patients was measured with ELISA (14, 15-EET/DHET ELISA kit; Detroit R&D Inc., Detroit, MI, USA) according to the manual.

Cell viability was performed using an MTT assay. Cells were added to 96-well plates (5 × 10³ cells per well) following to 24 h incubation. On the following day the media were removed and the cells were treated with or without 14, 15-EET and/or 14, 15-EEZE following an incubation for 72 h. After incubation of respective time 10% of an MTT solution (2 mg/mL) was added to each well and the cells were incubated for 4 h at 37 °C. The formazan crystals that formed were dissolved in DMSO (100 μ L/well) with constant shaking for 5 min. The absorbance of the plate was then read with a microplate reader at 540 nm. Three replicate wells were evaluated for each analysis.

Tumor cells were added to 6-well plates (5 × 10⁵ cells per well) which were pre-coated with fibronectin (Sigma). After 2 h incubation at 37 °C, non-adherent cells were harvested. Then, adherent cells were harvested by treatment with trypsin. The percentage of adherent cells was calculated. The results were expressed as A570 values. Each assay was tested in triplicate wells in three independent experiments.

Cells (5 × 10⁴) in serum-free media were seeded onto the upper chambers of modified Boyden chambers (Corning, NY, USA) in which the Transwell filter inserts were coated with Matrigel. In the lower chambers, 5% FBS was added as a chemoattractant. After incubation for 24 h, the membrane was washed briefly with PBS and fixed with 4% paraformaldehyde. The upper side of membrane was wiped gently with a cotton ball. The membrane was then stained using hematoxylin and removed. The magnitude of cells migration was evaluated by counting the migrated cells in six random clones under high-power (× 100) microscope fields. The average number of cells per field was calculated.

For silencing specific gene expression, cells were treated with integrin αv siRNA or integrin β3 siRNA. Briefly, 5 × 10⁵ MCF-7 and MDA-MB-231 cells were seeded into 6-well plate with 2 ml antibiotic-free normal growth medium containing FBS. Transfection of integrin αv siRNA, integrin β3 siRNA or control siRNA was performed according to the manufacture’s protocol.

Total RNA was extracted from cells with TRIzol reagent (Invitrogen, Carlsbad, CA). Quantitative real-time PCR analyses were performed by Applied Biosystems using SYBR Premix Ex Taq™ (TaKaRa, Japan). The mRNA of GAPDH was used as internal control. The primers were as follows: integrin αv, sense5’-CTCGGGACTCCTGCTACCTC-3′, antisense 5’-AAGAAACATCCGGGAAGACG-3′ integrin β3, sense 5’-CCGTGACGAGATTGAGTCA-3′ antisense5’-AGGATGGACTTTCCACTAGAA-3′ and GAPDH, sense 5’-TCATTGACCTCAACTACATGGTTT-3′, antisense 5’-GAAGATGGTGATGGGATTTC-3’.The relative expression of αv and β3 was calculated using 2^-ΔΔCt method.

The whole-cell extracts were prepared using RIPA lysis buffer (Beyotime, China) with phenylmethanesulfonyl fluoride and protease inhibitor cocktail (Roche, USA). Cell lysates were separated by 8–12% SDS-PAGE and electro-transferred onto polyvinylidene difluoride membranes (Bio-Rad, USA). After being blocked using 5% non-fat milk for 1 h at room temperature, membranes were incubated with the indicated primary antibodies overnight at 4 °C and probed with horseradish peroxidase-conjugated secondary antibodies (1:1000). The bands were visualized using a ChemicDocXRS system (Bio-Rad, USA).

Mice were inoculated i.m. in the right hind thigh with MDA-MB-231 cells (2 × 10⁶). Tissue sections were prepared and subjected to immunohistochemical analysis. Anti-human Ki67 antibody, anti-human E-cadherin and anti-human vimentin antibody were used as primary antibodies. HRP-conjugated secondary Ab was used as secondary antibody. Images were obtained using an Olympus-IX71 microscope at 40 × 10 magnification.

For H&E staining, the tumor tissues were embedded in paraffin according to standard histological procedures. Sections were stained with hematoxylin and eosin.

To assay tumor cell arrest in lung during blood flow, MDA-MB-231 cells were labeled with CFSE, and injected into mice via tail vein (2 × 10⁶ cells/mouse, n = 8 for each group). Lungs of mice were harvested 5 h or 24 h after the injection. Frozen sections were prepared and analyzed by fluorescence microscopy. Fluorescent spots were counted from 20 randomly chosen fields in sections of each mouse.

Nude mice were inoculated i.m. in the right hind thigh with MDA-MB-231 cells(2 × 10⁶). The transplanted nude mice were randomly divided into 5 groups (n = 6 for each group). The mice were treated or untreated with14, 15-EET and/or 14, 15-EEZE (i.v. injection, 30 μg/kg/2d). The mice were treated with cisplatin (i.p. injection, 3.0 mg/kg/d) or PBS. All mice were examined every 2 days and sacrificed 35 days after tumor inoculation. Tumor volume (V) was monitored by measuring the length (L) and width (W) and calculated with the formula V = (L × W²) × 0.5.

14, 15-EET has been reported to induce migration and invasion of human cancer cells [5, 6]. 14, 15-EET is very unstable metabolites, and it’s rapidly hydrolyzed by sEH to the more stable metabolites 14, 15-DHETs. We detected the 14, 15-DHET level in serum or in cancer and noncancerous tissues from breast cancer patients. The ELISA results showed that the levels of 14, 15-DHET in serum and cancer tissues in BC patients is much higher than that of healthy donors or noncancerous tissues(Fig. 1a, b). Furthermore, we found that 14, 15-EET enhanced the adhesion ability of MCF-7 and MDA-MB-231 cells (Fig. 1c). Invasion assay showed that 14, 15-EET promoted tumor cell invasion(Fig. 1d), whereas 14, 15-EEZE, an antagonist of 14, 15-EET inhibited EET-induced cell adhesion and invasion.

We further investigated the effect of 14, 15-EET on tumor cell invasion in vivo. The fluorescent spots in lung tissues were significantly increased both 5 h and 24 h after i.v. injection of MCF-7 and MDA-MB-231 cells treated with 14, 15-EET, while 14, 15-EEZE abolished the effect of 14, 15-EET on tumor cell adhesion and invasion in vivo (Fig. 1e). These results indicated that 14, 15-EET promotes breast cancer cell adhesion and extravasation.

Fibronectin presents binding sites for a range of different integrins including integrin αvβ3. As 14, 15-EET enhanced adhesion ability of breast cancer cells to fibronectin, we hypothesized that integrin αvβ3 may be involved in 14, 15-EET-induced breast cancer cells adhesion and invasion. We found that 14, 15-EET increased the mRNA and protein expression of αv- and β3-integrin, Whereas 14, 15-EEZE, reduced EET-induced integrin αvβ3 expression (Fig. 2a, b).

It is widely known that FAK is a downstream integrin αvβ3 kinase. Since 14, 15-EET up-regulated integrin αvβ3 expression, we investigated whether 14, 15-EET affected FAK phosphorylation. We found that 14, 15-EET increased breast cancer cells FAK phosphorylation. PI3K/AKT, downstream FAK signaling molecules were also found to be activated by 14, 15-EET, while 14, 15-EEZE inhibited 14, 15-EET-induced FAK/PI3K/AKT activation (Fig. 2c).

14, 15-EET up-regulated αvβ3 integrin expression and activated FAK/PI3K/AKT signaling in breast cancer cells, therefore, we investigated whether integrin αvβ3 mediated the oncogenic effects of 14, 15-EET. Tumor cells were transfected with integrin αv or β3 siRNA, the expressions of integrin αv and β3 were validated by western blot (Fig. 3a). Knocking down of αv and β3 integrin reduced 14, 15-EET-induced tumor cell FAK/PI3K/AKT phosphorylation (Fig. 3b, c) and invasion (Fig. 3d). These results indicated that 14, 15-EET promotes breast cancer cell invasion and activates FAK/PI3K/AKT signaling through up-regulating integrin αvβ3 expression.

The above data indicated that 14, 15-EET promoted breast cancer cell invasion, given that EMT is known to be the initial step of tumor cell migration and invasion, we examined whether 14, 15-EET affect breast cancer cells EMT. When we seeded 14,15-EET-treated MCF-7 and MDA-MB-231 cells in 6-well plate, it was found that cells lost cell-cell contact and formed a scattered phenomenon (Fig. 4a). Moreover, we found that tumor cells expressed reduced E-cadherin, increased N-cadherin, vimentin, snail and slug after treatment of 14,15-EET, while 14,15-EEZE reversed 14, 15-EET-induced EMT (Fig. 4b). We further examined whether integrin αvβ3 involved in EMT induced by 14, 15-EET. As expected, knockdown of αv or β3 integrin inhibited 14, 15-EET-induced tumor cell EMT (Fig. 4c). PI3K signaling is responsible for EMT, to further confirm the role of FAK/PI3K/AKT signaling in 14, 15-EET-induced EMT, FAK inhibitor PF562271 and PI3K inhibitor LY294002 were utilized. We found that tumor cells treated with PF562271 or LY294002 expressed high levels of E-cadherin and low levels of N-cadherin, vimentin, snail and slug compared with control cells (Fig. 4d). These results suggested that 14, 15-EET induces breast cancer cells EMT through αvβ3/FAK/PI3K/AKT signaling.

Tumor cells display EMT and lead to enhanced drug resistance, as 14, 15-EET induced breast cancer cells EMT, we examined the role of 14, 15-EET in tumor cells sensitivity to cisplatin. MTT assay showed that 14, 15-EET significantly reduced tumor cells sensitivity to cisplatin, while 14, 15-EEZE reversed 14, 15-EET-induced cisplatin resistance (Fig. 5a). Knocking down of αv or β3 integrin reversed 14, 15-EET-induced tumor cells cisplatin resistance (Fig. 5b). Moreover, both PF562271 and LY294002 were found to reduce 14, 15-EET-induced tumor cells cisplatin resistance (Fig. 5c). These data indicated that integrin αvβ3 and FAK/PI3K/AKT signaling mediate 14, 15-EET-induced breast cancer cells cisplatin resistance.

We further evaluated the role of 14, 15-EET on MDA-MB-231 cells EMT and cisplatin resistance in xenograft model. In line with our earlier observations, the expression of E-cadherin decreased while the expression of vimentin increased obviously after 14,15-EET treament. (Fig. 6a). The average tumor volume of cisplatin-treated tumors was significantly smaller than that of 14, 15-EET/cisplatin-treated tumors (Fig. 6b, c). Histologic examination of the tumors showed dramatically increased cellularity in the 14, 15-EET/cisplatin-treated compared with cisplatin-treated tumors (Fig. 6d). Immunohistochemical staining of the 14, 15-EET/cisplatin-treated compared with cisplatin-treated tumors showed increased Ki67 levels (Fig. 6d). Whereas 14, 15-EEZE, reversed 14, 15-EET’s effect. These results suggested that 14, 15-EET promotes breast cancer cells EMT and reduces cisplatin sensitivity in vivo.