AIM: 1) To study the participation of Focal Adhesion Kinase (FAK) in regulation of Breast Cancer cell migration in relation with MMP-9 and other signaling proteins. 2) To study the effect of some natural products on FAK. METHODS: Cell culture, Western Blot, Immunoprecipitation, Immunocytochemistry, Zymography, SiRNA transfection, RT-PCR, Real-Time PCR. RESULTS: For our study on FAK, we selected invasive Breast Cancer cell line MDA-MB-231 and treated the cells with Fibronectin (FN). Treatment of FN was found to increase FAK expression, phosphorylation (Tyr 397). FAK was found to be involved in re- gulation of breast cancer cell migration and MMP-9 expression, activity. Fi-bronectin increases association of FAK with integrin α5β1, Paxillin, Actin, ERK, PI3K and localization at Focal Adhesion sites. FAK was found to be involved in modulation of ERK and PI3K phosphorylation. Moreover, FAK signal was found to be transduced through ERK and PI3K, which modulate MMP-9 and thereby cell migration. CONCLUSION: FAK expression, phosphorylation and processing are induced in response to Cell-ECM interactions. Integrin α5β1 is involved in FN induced FAK phosphorylation. FAK is a potent regulator of MMP-9 expression and activity. FAK is involved in regulation of ERK and PI3K phosphorylation. ERK and PI3K are involved in FAK regulated MMP-9 expression & activity. FAK regulates MMP-9 expression and activity and thereby migration of human breast cancer cell. By the regulation of FAK, cell attachment and migration may be regulated by Curcumin, ATRA or EGCG treatment. It may be concluded that invasive potential of breast cancer cells may be modulated by regulation of FAK.
One of the common crucial events for cell migration, survival, apoptosis or cell proliferation is cell attachment with Extra Cellular Matrix (ECM). Integrins are the receptors through which cells attach with ECM. Focal Adhesion Kinase (FAK) transmits the signal, produced as a result of Integrin-ECM binding, to cell-interior. FAK is a non-receptor protein tyrosine kinase of 125 kD which directly interacts with β-subunit of integrin molecule, resides within the Focal Adhesion complex and initiates Integrin mediated signaling. FAK is situated at the signaling junctions where information comes from mainly four sources-ECM-integrin interaction, growth factor signaling, G-protein coupled receptor signaling and mechanical forces imposed on the cell [1,2]. After FAK activation, it interacts with a number of signaling molecules which situate FAK at the crossroad of different signaling pathway [
Matrix Metalloproteinases (MMP) are a family of zinc dependent enzymes responsible for degradation of ECM including basement membrane collagen, interstitial collagens, Fibronectin (FN), and various proteoglycans both in physiological and pathological conditions [
Fibronectin (FN) is one of the important ECM-glycolproteins which binds with Integrin. FN is also overexpressed in breast cancer [
In this communication we report a role of FAK in FNmediated human breast cancer cell migration involving regulation of MMP-9 expression and activity. We have shown involvement of Integrin α5β1, PI3K and ERK in this signaling pathway. Moreover, we have shown that some natural compounds may be used to regulate FAK expression and thereby MMP-9 expression-activity and cell migration.
Leibovitz’s L-15 Medium, fetal bovine serum (FBS) were purchased from GIBCO™—Invitrogen, Carlsbad, CA. Fibronectin (440 kDa), Protease Inhibitor Cocktail Tablets (complete, mini, EDTA-free, Cat No. 11836170 001), Protein G agarose were purchased from Roche, Germany. Gelatin Sepharose 4B beads was purchased from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. All primary antibodies (monoclonal and polyclonal), seconddary antibodies (FITC and HRP), FAK SiRNA and negative control SiRNA were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. USA. SYBR Green JumpStartTM Taq Readymix TM was purchased from SigmaAldrich, St. Louis, MO, USA. Primers (FAK, MMP-9, TIMP-1 and G3PDH) were synthesized by Operon, Germany. RNAqueous 4 PCR (Total RNA isolation kit) and Retroscript (RT-PCR Kit) were purchased from Ambion, Austin, TX, USA. ERK inhibitor (PD 98059), PI-3K inhibitor (LY 294002) were purchased from Promega, Madison, WI. LipofectamineTM 2000 was purchased from Invitrogen, Life Technologies (USA). SuperSignal West Pico Chemiluminescent Substrate kit was purchased from Pierce, Thermo Fisher Scientific Inc. Rockford, USA). Alltrans Retinoic Acid (ATRA), Curcumin and EGCG was obtained from Sigma-Aldrich, USA.
MDA-MB-231 (human breast cancer cell line) was obtained from National Centre for Cell Sciences (NCCS), Pune, India. The cells were grown and maintained in Leibovitz’s L-15 Medium containing 10% FBS in a 5% CO2 incubator at 37˚C. All experiments with MDA-MB-231 were performed at early passages of the cell line.
MDA-MB-231 (300,000 cells/ml) cells were grown in SFCM with treatment as required in the experimental condition. Then the cells were washed with ice cold PBS and were scraped into lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% Deoxycholate, Protease inhibitor cocktail tablets (following manufacturer protocol), 1 mM sodium orthovanadate and 1 mM Sodium fluoride) on ice and clarified by centrifugation. Protein concentrations were determined using a Lowry method. The samples were then subjected to electrophoresis on SDS-PAGE and the proteins were transferred onto PVDF membrane (Millipore) by Western blot. Nonspecific binding sites on the membrane were pre-blocked in 4% BSA. Blots were incubated with anti-FAK, anti-pFAK (Tyr397), anti-PI3K, anti-pPI3K, anti-ERK, anti-pERK, anti-MMP-9 or anti-Paxillin (Santa Cruz) antibodies. Following incubation in horseradish peroxidase (HRP)-conjugated secondary, detection was performed with the Super Signal West Pico Chemiluminescent Substrate kit (Pierce, USA) following the manufacturer’s protocol. All blots were reprobed with anti β-tubulin antibody as internal loading control.
MDA-MB-231 (300,000 cells/ml) cells were allowed to grow without or with Fibronectin (20 μg/ml) for 2 hr. in SFCM. For Immunoprecipitation cells were lysed with NP40 buffer (50 mM Tris, 150 mM NaCl, 1% NP40, pH 8, Protease inhibitor cocktail tablets, 1 mM sodium Orthovanadate and 1 mM Sodium fluoride). Preclearing of lysate was done by incubation with Protein G agarose and anti-human IgG at 4˚C using for 1 hr, followed by protein estimation by Lowry method. Immunoprecipitation was performed from 100 μg of protein with 1 μg of anti-integrin α5 (H-104) sc10729 or anti FAK (A-17): sc 557 antibody (Santa Cruz Biotechnology, USA) followed by precipitation with Protein-G-Agarose for 2 hr at 4˚C, then the beads were washed with NP40 buffer (without Protease inhibitor cocktail, sodium orthovanadate and sodium fluoride). The samples were prepared for SDS-PAGE with the addition of Laemmli buffer and elution at 100˚C for 4 min. Western blotting was performed as described.
MDA-MB-231 cells (300,000 cells/ml) were grown in absence and presence of FAK SiRNA in required concentrations and time periods in serum free culture medium (SFCM). MDA-MB-231 cells (300,000 cells/ml) were grown in presence of FN (20μg/ml) for 2 hours and then were kept with or without α5 antibody for an additional 1 hour. After either of these treatments, Gelatin Zymography was performed using a 7.5% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) co-polymerized with 0.1% gelatin as described in earlier report [
MDA-MB-231 cells (300,000 cells/ml) were grown in absence (control) and presence of FN (20 μg/ml) for 2 hr after treatment with ERK inhibitor (PD 98059) (50 μM), PI-3K inhibitor (LY 294002) (20 μM) for 1 hour in SFCM. The SFCM was collected and Zymography was performed as described.
MDA-MB-231 (300,000 cells/ml) cells were allowed to grow on surface coated or not coated (Control) with Fibronectin (20 μg/ml SFCM) upto 2 hr at 37˚C in a CO2 incubator. The coverslips were then washed in PBS, fixed with 3.5% formaldehyde, treated with 0.5% Triton-X 100 and nonspecific sites were blocked with 1% BSA. The cells were then incubated with anti-FAK primary antibody (1:1000 dilution) for overnight at 4˚C followed by wash and incubation with FITC-coupled second anti-body (1:1000 dilution) at 37˚C for 1.5 hr in a humidified chamber. After washing with PBS, the coverslips were mounted with glycerol on glass slides and observed under a fluorescence microscope (Leica).
RNA was extracted from MDA-MB-231 cells (1 × 106 cells/ml) either grown in absence (Control) or in presence of Fibronectin (20 μg/ml) for 1 hr and 2 hr; or grown in absence or presence of FAK SiRNA and control SiRNA prior to treatment with Fibronectin (20 μg/ml) for 2 hr. Cells were washed in PBS and total RNA was extracted (RNAqueous, Ambion, USA) as previously discussed [
hFAK: 5’-GCGCTGGCTGGAAAAAGAGGAA-3’ (forward) 5’-TCGGTGGGTGCTGGCTGGTAGG-3’(rever-se);
hMMP-9: 5’-CGCTACCACCTCGAACTTTG-3’- (forward) 5’-GCCATTCACGTCGTCCTTAT-3’-(reverse);
hTIMP-1: 5’-CACCCACAGACGGCCTTCTGC-3’- (forward) 5’-AGTGTAGGTCTTGGTGAAGCC-3’-(rever-se);
GAPDH: 5’-CGGAGTCAACGGATTTGGTCGTAT- 3’(forward) 5’-AGCCTTCTCCATGGTGGTGAAGAC3’(reverse).
RT-PCR was carried out using Two Step RT-PCR kit (Retroscript Ambion, USA). Conditions used for PCR consisted of 40 cycles for FAK, MMP-9, and TIMP-1 at 94˚C for 30 sec, 58˚C for 30 sec and 72˚C for 30 sec with a final incubation at 72˚C for 7 min in DNA thermal cycler.
Human breast cancer cell line MDA-MB-231 were seeded in 35 mm dishes and grown to 60% confluence. For the transfection process, FAK SiRNA and negative control SiRNA were transfected using LipofectamineTM 2000 following the manufacturer’s protocol. The transfection agent (LipofectamineTM 2000) was incubated with serum-free culture medium for 10 mins at room temperature. Subsequently, respective SiRNA (FAK SiRNA (h), sense-GCAUGUGGCCUGCUAUGG; antisense-CCAUAGCAGGCCACAUGC) mixed with serum-free culture medium was added to it and incubated at room temperature for additional 15 min. The mixtures were then diluted in serum free culture medium and added to each dish so that the final concentration of the SiRNA in each plate was 100 nM. The mixture was overlaid on the cells in full media without serum and without antibiotic and incubated at 37˚C in the presence of 5% CO2. After 24 hr, the transfection mixture was replaced with fresh media supplemented with 10% FBS and antibiotic and the transfected cells allowed growing for another 48 hr. Cells were exposed to FN for 2 hr and later collected for western blot, gelatin Zymography and real-time RT-PCR assays.
Cells were kept in SFCM for 1 h with thrice change and were exposed or not exposed to FN (20 μg/ml) in culture dish and grown for 2 hr. The monolayer was scratched with sterile tip, washing 3× with SFCM. The cells were maintained in fresh SFCM and cell migration was observed under microscope and photographed at different time points (0, 12, and 24 hr) [
MDA-MB-231 cells (300,000 cells/ml) were treated with 20 µM of ATRA, Curcumin and EGCG separately for 24 hr [29-31]. Control cells were treated with equal concentration of DMSO. In case of experiments with fibronectin treatment, cell were grown in absence or presence of 20 μg/ml fibronectin for 2 hr. after the treatment of the natural products.
Bands of Zymography, Western blots and RT-PCR were quantitated using Image J Launcher (version 1.4.3.67).
Western Blot analysis showed increased expression, phosphorylation and processing of FAK in time dependent manner when cells were exposed to FN for upto 2 hr, compared to the FN-untreated control cells. The expression, phosphorylation and cleavage of FAK were found to be increased from 30 min to 2 hr (
MDA-MB-231 treated with FN for 1 hr and 2 hr clearly shows that FAK and MMP-9 expression was up-regulated 4 fold whereas TIMP-1 message decreased 2 folds. GAPDH gene expression was used as standard internal control to normalize RNA integrity and equal loading (
When MDA-MB-231 cells were grown on Fibronectin coated surface for 30 min, 1 hr and 2 hr, FAK (upper panel) and p-FAK (lower panel) were found to be localized at adhesion plaques. It became increasingly distinct from 30 min to 2 hr. However these punctuate fashion was not observed in cells without FN treatment (
α5β1 integrin was immunoprecipitated with α5 monoclonal antibody from cell extract. With this immunoprecipitate a blot was developed with anti-FAK antibody and it was observed that FAK was associated with α5β1. In another set of experiment Paxillin (upper panel) & Actin (lower panel) was found to be immunoprecipitated with FAK, upon immunoprecipitation with anti-FAK antibody. The associations were increased several folds when cells were exposed to FN than the untreated control cells (
Western Blot analysis indicates when α5 integrin was masked by treating cells with anti-α5-integrin antibody for 1 hr prior to FN treatment, the amount of FAK expression and phosphorylation became as good as FN untreated cells (
Appreciable decrease in FAK and MMP-9 mRNA expression was observed by Real-Time PCR (
downregulated in response to FAK inhibition. Observations revealed expression of FAK, MMP-9 and MMP-9 activity were increased upon exposure of cells to FN. Whereas, treatment of cells with FAK SiRNA prior to FN-exposure appreciably downregulated FAK, MMP-9 expression and MMP-9 activity. Cells treated with control SiRNA did not show any notable change of FAK, MMP-9 expression or MMP-9 activity.
From the Western Blot it was found that PI3K and ERK co-precipitate with FAK when it is immunoprecipitated with anti-FAK antibody. The association increases several folds in FN-treated cells (
Western blot analysis shows, phosphorylation of PI3K and ERK is increased a several folds when cell are exposed to FN. But the phosphorylation of PI3K and ERK was appreciably down regulated when cell were treated with FAK SiRNA prior to FN treatment. In contrary, the activity remain unaltered when cell were treated with Control SiRNA prior to FN treatment (
When MDA-MB-231 cells were treated with ERK or PI3K inhibitor prior to FN treatment, MMP-9 activity was found to be downregulated a several folds, compared to only FN treated cells (
Effect of FAK silencing on cell migration was observed in FN treated and untreated conditions. Depletion of
FAK was found to retard cell migration even after 24 hr of making wound compared to the control cells (
When MDA-MB-231 cells were treated with Curcumin (20 µM), ATRA (20 µM) and EGCG (20 µM), the cell were found to become constricted, spindle shaped and/or roundish with stretched attachment after 24 hr, compared to the untreated cells (
Western blot analysis showed that expression of FAK was downregulated upon treatment of MDA-MB-231 cells with Curcumin (20 µM), ATRA (20 µM) and EGCG (20 µM) for 24hr. Upon treatment with Curcumin, ATRA and EGCG, FAK expression was to found to be downregulated by 2.14, 1.68, 1.81 folds respectively. β-Tubulin was taken as loading control (
When MDA-MB-231 cells were treated with Curcumin
(20 µM), ATRA (20 µM) and EGCG (20 µM) for 24 hr, MMP-9 activity was found to be downregulated by 49.32, 4.81, 7.33 folds (
Cell migration assay demonstrated that the migration of MDA-MB-231 cells was accelerated when the cells were exposed to Fibronectin (FN) for 2 hr. But when the cells were treated with Curcumin (20 µM), ATRA (20 µM) and EGCG (20 µM) for 24 hr prior to exposure to FN, cell migration were drastically decreased (
Overexpression and increased phosphorylation of Focal Adhesion Kinase (FAK) in human breast cancer tissue have been reported by several groups. Studies have shown that increase of FAK have relation with progression of several cancers including breast cancer [32-35]. In invitro studies, depletion of FAK shows its importance in a number of cellular activities like cell migration and cytoskeletal organization, as well as cell survival and cell cycle progression [14,36-38]. An important ECM glycoprotein Fibronectin (FN) plays crucial role in regulating activity of FAK both in physiological and pathological conditions. A number of groups have reported FN-mediated FAK activation and its participation in transducing the signal from ECM to cell interior [10,39]. Involvement FAK in regulation of MMP-9 has also been reported in different cell type [16,22]. Our lab has shown induction of MMP-9 expression and activity in FN induced MDA-MB-231 [
used FN-treated MDA-MB-231 model to study the participation of FAK in regulation of MMP-9 and thereby cell migration.
Our current findings clearly indicate FAK plays the pivotal role in regulation of cell migration in FN induced MDA-MB-231 breast cancer cells. Consistent with earlier reports we found an increased expression and phosphorylation (Tyr 397) of FAK, increased expression and activity of MMP-9, and reduced expression of TIMP-1 in this model [
Induction of FAK and signaling events at downstream of it that affect cell migration is complex. For example, after binding of FN-integrin interaction FAK is activated by autophosphorylation at Tyr 397. This event initiates the signaling, which leads to interaction of FAK with several proteins and its phosphorylation at other sites. The SH2 domain of p85 subunit of PI3K interacts with FAK at Tyr397 [18,41]. In addition, activation of FAK induces ERK activation [1,42]. Again reports show in different systems that FAK regulates MMP-9 activity which is an important modulator of cell migration by degradation of ECM [
To better understand the mechanism by which FAK regulates Fibronectin-induced cell migration, we examined the expression and activity of MMP-9 which is the downstream target of integrin receptors, responsible for ECM degradation. Upon treatment of MDA-MB-231 cells with the FAK SiRNA; we found reduced activation of FAK as evidenced by reduced phosphorylation at its Tyr397 residues. This inhibition showed the reduction of MMP-9 expression and activity. Thus, it seems that FAK promotes MDA-MB-231 breast cancer cell migration through modulation of MMP-9.
We examined signaling involved in FN induced FAK activation. Consistent with earlier reports in other cell types [
of FAK at Focal Adhesion sites as well as its association with Paxillin and Actin. So, in response to FN treatment FAK recruited in Focal Adhesion points where it is phosphorylated after interaction with integrin α5β1. Paxillin is a focal adhesion protein. FAK-Paxillin association and their interaction with integrin β1 was reported as positive modulator of cancer cell migration [
Consistent with previous report [
In this model, reduction of MMP-9 activity after inhibition of ERK indicates the involvement of ERK in FN induced MMP-9 regulation. Studies in ovarian carcinoma cells have shown that FAK and ERK are important for Fibronectin stimulated invasiveness and MMP9 secretion by these cells [
Chinese hamster ovary (CHO) cells, Heinz et al. have shown involvement of PI3K in FAK-promoted cell migration [
Recently, several groups are trying to develop therapeutic strategies making FAK as a target molecule. These include inhibitory-RNA, small molecular inhibitory proteins or natural products. Different groups including our laboratory have demonstrated inhibitory role of some natural and/or synthetic compounds on Integrin mediated signaling [29,30,46]. But studies are required in detail to confirm their effects on FAK. We have tried to search whether these products can regulate FAK/MMP-9 signaling and thereby cell migration. In a part of this study we have focused on the effect of some natural products on FAK.
Interestingly, treatment of the natural products like ATRA, Curcumin and EGCG were found to decrease cell attachments. Results suggested ATRA, Curcumin and EGCG as inhibitor of FAK expression and MMP-9 activity in MDA-MB-231 breast cancer cell line.
We have also shown that FAK is the regulator of MMP- 9 activity. So here we may say that ATRA, Curcumin and EGCG downregulate MMP-9 activity probably by regulating FAK expression.
Cell migration is an important event which depends on efficient coordination between cell attachment and detachment on extracellular matrix [
In summary, we propose that Fibronectin induced MDAMB-231 breast cancer cell migration is regulated by FAK. FAK is potent regulator of MMP-9 expression and activity. FN induces FAK through integrin α5β1. Again FAK transduces signal through ERK and PI3K by regulating their phosphorylation which regulate MMP-9 activity. Moreover, we have shown that Curcumin, ATRA and EGCG downregulate FAK expression MMP-9 activity and thereby cell migation. These signaling pathways may have crucial roles in modulating migration of breast cancer cells. The findings in this communication strengthen the role of FAK in regulation of cell migration and it’s potential therapeutic implication in breast cancer.
The authors wish to express their thanks to Dr. Jaydip Biswas, Director, Chittaranjan National Cancer Institute, for continuous inspiration and financial support and to Defence Research & Development Organisation (DRDO) (Grant no: DLS/81/48222/LSRB-145/ID/2008) for funding this project.
ECM: Extracellular matrix,
ERK: Extracellular regulated kinase,
FAK: Focal adhesion kinase,
FBS: Fetal bovine serum,
FITC: Fluorecin isothiocyanate,
FN: Fibronectin,
GAPDH: Glyceraldehyde phosphate dehydrogenase,
HRP: Horseradish peroxidase,
MMP: Matrix metalloproteinase,
PI-3K: Phosphatidyl inositol 3 kinase,
SFCM: Serum free culture media,
SiRNA: Small interfering RNA,
TIMP: Tissue inhibitor of Metalloproteinases,
Tyr: Tyrosine.