Bisphenol A Induces Migration through a GPER-, FAK-, Src-, and

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Bisphenol A induces migration through a GPER, FAK, Src and ERK2-dependent pathway in MDA-MB-231 breast cancer cells Rocio Castillo-Sanchez, Rocio Gomez-Ortega, and Eduardo Perez-Salazar Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00457 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on February 29, 2016

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Chemical Research in Toxicology

Bisphenol A induces migration through a GPER, FAK, Src and ERK2dependent pathway in MDA-MB-231 breast cancer cells

Rocio Castillo Sanchez#, Rocio Gomez-Ortega# and Eduardo Perez Salazar&

#

Departamento de Toxicologia, &Departamento de Biologia Celular, Cinvestav-IPN, Mexico D.F., Mexico.

Corresponding Author: Eduardo Perez Salazar, PhD Departamento de Biologia Celular Cinvestav-IPN Av. IPN # 2508 Ciudad de Mexico 07360 Mexico Telephone:

52 –55- 5747-3991

Fax number: 52 -55- 5747-3393 E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

Bisphenol A (BPA) is an industrial synthetic chemical utilized in the production of numerous products including food and beverage containers. Humans are exposed to BPA during ingestion of contaminated water and food, because it can leach from polycarbonate containers, beverage cans and epoxy resins. BPA has been related with the development of several diseases including breast cancer. However, the signal transduction pathways mediated by BPA and its role like promoter of migration and invasion in breast cancer cells remain to be investigated. Here we demonstrate that BPA promotes migration, invasion and an increase in the number of focal contacts in MDA-MB-231 breast cancer cells. Moreover, MDA-MB-231 cells express GPER and BPA promotes migration through a GPER-dependent pathway. BPA also induces activation of FAK, Src and ERK2; whereas migration induced by BPA requires the activity of these kinases. In addition, BPA induces an increase on AP-1- and NFκB-DNA binding activity through a Src and ERK2dependent pathway. In conclusion, our findings demonstrate, that BPA induces the activation of signal transduction pathways, which mediate migration, AP-1/NFκBDNA binding activity, as well as an invasion process in MDA-MB-231 breast cancer cells.

Keywords: Breast cancer; BPA; GPER; FAK; Src; migration, invasion



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INTRODUCTION

Bisphenol A (BPA) is an industrial synthetic chemical that is utilized in the production of many products including polycarbonate plastics and epoxy resins. Therefore, BPA is found in numerous food and beverage containers, as well as it is a component of lining of metal food cans and dental materials (resins).1-3 Humans are exposed to BPA during ingestion of contaminated water and food, because BPA can leach from polycarbonate plastic containers, beverage cans, epoxy resins and other products in contact with water and food.1,4-6

BPA has been related with development of several diseases, such as breast cancer. Particularly, prepubertal BPA exposition of rats increases cell proliferation in mammary gland, whereas perinatal exposition of BPA to mice promotes intraductal hyperplasias in adult mice.7,8 BPA is able to bind the estrogen receptor α and β, as well as it is a ligand for the G-protein-coupled receptor (GPCR) namely G protein-coupled estrogen receptor (GPER).9,10 In breast cancer cells, BPA induces proliferation and migration through a GPER-dependent pathway.11,12 Moreover, it has been reported the presence of BPA in several human fluids and tissues, including urine, plasma, saliva, blood, maternal breast milk and adipose tissue.2,13,14 Particularly, several reports demonstrate the presence of BPA in blood, serum and plasma of nonpregnant adults with mean concentrations in the range of 1 ng/ml.2 Another study demonstrate that postmenopausal women with medium concentration of BPA in serum of 0.55 ng/ml and with a maximum detectable value of 8.77 ng/ml have elevated breast density; whereas BPA is also


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present in human adipose tissue with a GM of 3.95 and a maximum value of 20.9 ng/g.13,15

Cell migration plays an important role in normal and pathological processes including embryonic development, wound healing and metastasis. Focal adhesions are structures where integrin receptors mediate adhesion between actin cytoskeleton and extracellular matrix (ECM), and their components are diverse including scaffolding proteins, GTPases, kinases and phosphatases.16,17 A protein tyrosine kinase localized in focal adhesions is the 125 kDa focal adhesion kinase (FAK), which has been implicated in several biological processes including spreading, differentiation, proliferation, apoptosis, migration, invasion, survival and angiogenesis.18-20 Cell adhesion promotes FAK activation that is recruited to focal contacts and is autophosphorylated at tyrosine (Tyr)-397, which creates a highaffinity binding site for SH2 domain-containing proteins, including Src family kinases, PI3K, PLC and Grb7.18,19 FAK-Src complex formation promotes that Src phosphorylates FAK at additional Tyr residues, such as Tyr-407, Tyr-576, Tyr-577 and Tyr-925. Particularly, Tyr-576 and Tyr-557 are present in the activation domain and their phosphorylation is required for maximal FAK activation. In addition, phosphorylation at Tyr-925 creates a binding site for Grb2 and then activation of Ras/MEK/ERK pathway.20-22

In the present study, we demonstrate that BPA induces migration, invasion and an increase in the number of focal contacts in MDA-MB-231 breast cancer cells. BPA also induces activation of FAK, Src and ERK2; whereas migration induced by BPA requires activity of these kinases and GPER. In addition, BPA induces an increase



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on AP-1- and NFκB-DNA binding activity through a Src and ERK2-dependent pathway.

MATERIALS AND METHODS

Chemicals

BPA, epidermal growth factor (EGF) and phalloidin-tetramethylrhodamine B isothiocyanate (TRITC-conjugated phalloidin) were obtained from Sigma-Aldrich Co.

(St.

Louis,

MO).

PP2

(4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo

pyrimidine) , PP3 (4-Amino-1-phenylpyrazolo pyrimidine) and ERK inhibitor (3-(2Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4

thiazolidinedione

hydrochloride,

HCl) were obtained from Merck Millipore (Rockland, MA). FAK antibody (Ab) C-20, Src family Ab SRC-2, retinoblastoma (Rb) Ab C-15, NFκB p50 Ab 4D1, IκBα (FL) Ab and GPR30 (GPER) Ab (K-19)-R were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Paxillin Ab and phospho-specific Abs to Tyr-397 of FAK (anti-pFAK) and Tyr-418 of Src (anti-p-Src) were obtained from Invitrogen (Caramillo, CA). ERK1/2 Ab and phosphospecific Ab E10 to threonine (Thr)-202 and Tyr-204 of ERK1/2 (anti-p-ERK1/2) were obtained from Cell Signaling Technology (Camarillo, CA). Actin Ab was kindly provided by Dr. Jose Manuel-Hernandez (Cinvestav-IPN). Live/Dead Cellular Viability/Cytotoxicity kit was obtained from Molecular Probes (Eugene, OR). [γ−32P]ATP was obtained from Perkin-Elmer (Boston, MA).


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Cell Culture

The human MDA-MB-231 breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 3.7 g/l sodium bicarbonate and antibiotics in a humidified atmosphere containing 5% CO2 and 95% air at 37 ºC.

The non-tumorigenic epithelial cells MCF10A and MCF12A were cultured in DMEM/F12 (3:1) medium supplemented with 5% FBS, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 20 ng/ml EGF and antibiotics in a humidified atmosphere containing 5% CO2 and 95% air at 37 ºC. For experimental purposes, MDA-MB-231 cells were starved for 24 h in DMEM without FBS; whereas MCF10A and MCF12A cells were starved for 4 h in DMEM without FBS and supplements (insulin, hydrocortisone and EGF) before treatment.

Cell viability assays

After starvation, MDA-MB-231, MCF10A and MCF12A cells were washed twice with phosphate buffered saline (PBS). Cells were equilibrated in DMEM without FBS and/or supplements for at least 30 min at 37 ºC. Cultures were treated for 2 h with 12 µM mitomycin C, BPA was added to cell cultures to the indicated concentrations for 48 h, and then cell viability assays were performed by using the Live/Dead Cellular Viability/Cytotoxicity kit. The kit quickly discriminates live from dead cells by simultaneously staining with green-fluorescent calcein-AM to indicate



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intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity.

Cell Stimulation

After starvation, cultures were washed twice with PBS. Cells were equilibrated in DME M without FBS and supplements for at least 30 min at 37 ºC. BPA was added to cell cultures to the indicated concentrations and stimulation was terminated by aspirating the medium. Cells were solubilized in 0.5 ml of ice-cold RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 1.5 mM MgCl2, 0.1% SDS and 1 mM phenylmethylsulfonyl fluoride).

Western blotting

Equal amounts of protein (200 µg) were separated by SDS-PAGE using 8 and 10% separating gels followed by transfer to nitrocellulose membranes. After transfer, membranes were blocked using 5% non-fat dried milk in PBS pH 7.2, and incubated overnight at 4 ºC with the primary Ab. The membranes were washed three times with PBS/0.1% Tween 20, and then incubated with secondary Ab (1:5,000) for 2 h at room temperature. After washing three times with PBS/0.1% Tween 20, the immunoreactive bands were visualized using the western blotting


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luminol reagent (Santa Cruz Biotechnology). Autoradiograms were scanned and the labeled bands were quantified using the Image J software (NIH, USA).

Scratch-Wound assay Migration assays were performed as described previously.24 Cells were grown to confluence on 60-mm culture dishes and starved. After starvation, cultures were washed twice with PBS. Cells were equilibrated in DMEM without FBS and supplements for at least 30 min at 37 ºC. Cultures were treated for 2 h with 12 µM mitomycin C to inhibit proliferation, and they were scratch-wounded using a sterile 200 µl pipette tip and washed twice with PBS. Cells were re-fed with DMEM without FBS and/or supplements. Next, cells were treated without or with inhibitors and/or BPA. After 48 h of incubation at 37 °C, the progress of cell migration into the wound was photographed using an inverted microscope coupled to a camera. Cell migration was evaluated by using the Image J software.

Chemotactic migration assay (Boyden chamber method) Migration assays were performed as described previously.24 Cell migration assays were performed in 24-well plates containing 12 cell culture inserts with 8 µm pore size (Costar, Corning, Inc). Cultures were washed twice with PBS, and cells were equilibrated in DMEM without FBS for at least 30 min at 37 ºC. Cells were pretreated for 2 h with 12 µM mitomycin C and they were seeded into the upper



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chamber of Boyden chamber at 1×105 cells/well. Vehicle and/or BPA were added to the lower chamber. Upper and lower chambers contained FBS free-DMEM. After 48 h of incubation at 37 °C, nonmigrated cells were removed from the upper side of the membrane with cotton swabs, and the cells on the lower surface of the membrane were fixed in methanol for 5 min. The membrane was stained with 0.1% crystal violet in PBS. The dye was eluted with 200 µl of 10% acetic acid, and the absorbance at 600 nm was measured. The background value was obtained from wells without cells.

Interference RNA

GPER expression was silenced in MDA-MB-231 cells by using the silencer siRNA kit from Santa Cruz Biotechnology, according the manufacturer´s guidelines. One control of scramble siRNAs was included. The MDA-MB-231 cells transfected with GPER siRNAs and scramble siRNA were used to perform scratch-wound assays.

Invasion assays Invasion assays were performed as described previously.24 Matrigel invasion assays were performed by the modified Boyden chamber method in 24-well plates containing 12 cell culture inserts with 8 µm pore size (Costar, Corning, Inc). Matrigel BD (30 µl) was added into culture inserts and kept overnight at 37 ºC to form a semisolid matrix. After starvation, cultures were washed twice with PBS,


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and cells were equilibrated in DMEM without FBS for at least 30 min at 37 ºC. Cells were pretreated for 2 h with 12 µM mitomycin C and then they were seeded into the upper chamber of Boyden chamber at 1×105 cells/well. DMEM with or without BPA was added to the lower chamber at the indicated concentrations. Both the upper and lower chambers contained FBS free-DMEM. After 48 h of incubation at 37 °C, nonmigrated cells were removed from the upper side of the membrane with cotton swabs, and the cells on the lower surface of the membrane were fixed in methanol for 5 min. The membrane was stained with 0.1% crystal violet in PBS. The dye was eluted with 200 µl of 10% acetic acid, and the absorbance at 600 nm was measured. The background value was obtained from wells without cells.

Preparation of nuclear extracts Nuclear extracts were prepared as described previously.25 Cultures of MDA-MB231 cells (1.2 x 106 cells) were stimulated with BPA at different times. Cells were lysed with 0.1% non-ionic detergent Nonidet P40 in Buffer A (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 6 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF), lysates were pelleted at 2,600 rpm for 15 min and resuspended in Buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 20% glicerol 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 1 mM DTT, 0.2 mM PMSF). Nuclear extracts were recovered by centrifugation at 14,000 rpm for 15 min at 4 °C and the protein level of each sample was determined by the micro Bradford protein assay.



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Electrophoretic mobility shift assay (EMSA)

Double-strand oligonucleotides containing specific binding sites for AP-1, 5’GATCCTTCGTGACTCAGCGGGATCCTTCGTGACTCAGCGG-3’ and NF-kB, 5’AGCTAAGGGACTTTCCGCTGGGGACTTTCCAGG-3’ were used as probes.26,27 An amount of 20 pmol of annealed oligonucleotide was labeled with [γ-32P] ATP using T4 polynucleotide kinase. The

32

P-labeled oligonucleotide probe (~1 ng) was

incubated with 5 µg nuclear extracts in a reaction mixture containing 3 µg poly (dIdC), 0.25 M HEPES pH 7.5, 0.6 M KCl, 50 mM MgCl2, 1 mM EDTA, 7.5 mM DTT and 9% glycerol for 20 min at 4 ºC. One hundred-fold excess of unlabeled specific and non-specific probes were used as specific and non-specific competitors respectively. The samples were fractionated on a 6% polyacrylamide gel in 0.5X Tris borate-EDTA buffer. Gels were dried and analyzed by autoradiography.

Overexpression of FRNK

In order to exogenously express FRNK (pFLAG-CMV-2-FRNK) in MDA-MB-231 cells. Cell cultures were transfected with 8 µg of plasmid using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. Following transfection, cells were incubated with complete medium for 24 h, and cells were serum-starved for 12 h, washed with PBS and equilibrated in DMEM without FBS. Cells were pretreated for 2 h with 12 µM mitomycin C and migration assays were performed.


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Immunofluorescence confocal microscopy

Cells were grown on coverslips, washed with PBS, equilibrated in DMEM without FBS and treated with BPA. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked for 1 h with 3% BSA. Cells were stained with TRITC-conjugated phalloidin to reveal Factin. For staining of focal contacts, cells were incubated for 12 h with anti-paxillin Ab, followed by FITC- labelled anti-mouse secondary Ab for 2 h at room temperature.

Number of focal contacts was determined by using a Leica confocal microscope (Model TCS SP2; Leica Microsystems, Wetzlar, Germany). Serial optical sections of 0.8-0.9 µm thick were taken in both xyz and xzy. To prevent interference from the fluorescent probes, images of the same optical section were taken as separate channels, and they were analyzed by using the Image J software.

Statistical analysis

Results are expressed as mean ± S.D. Data were statistically analyzed using oneway ANOVA and Dunnett´s multiple comparison test. Statistical probability of P

Bisphenol A Induces Migration through a GPER-, FAK-, Src-, and

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