Leptin induces cell migration and invasion in a FAK-Src-dependent manner in breast cancer cells

Breast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src- and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 showed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src-dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.


Introduction
Leptin is a hormone with a molecular weight of ~16 kDa, encoded by the LEP gene located on human chromosome 7 (6). It is synthesized and secreted mainly by adipocytes, and in a smaller proportion, by the placenta, stomach, fibroblasts, skeletal muscle, and normal or tumorigenic epithelial mammary tissue (7). One of the primary functions of leptin is the regulation of food intake and energy expenditure, acting primarily through the hypothalamus (8). Leptin also regulates reproductive, immunological and metabolic functions (9). Additionally, leptin is involved in the progression of breast cancer, through the activation of mitogenic, anti-apoptotic and metastatic pathways (2). Leptin exerts these effects through the binding to the ObR receptor, activating various cellular signaling cascades such as JAK-STAT, MAPK and PI3K-Akt (7). Recent evidence showed that leptin levels in the plasma are higher in breast cancer patients compared with healthy individuals (2,10). Furthermore, leptin and its ObR receptor are overexpressed in primary and metastatic mammary tumor tissues, suggesting an autocrine signaling mechanism developed by tumor cells (11).
Importantly, leptin seems to be related to breast cancer risk in premenopausal obese women, however, controversy exists (12). For instance, epidemiological analyses performed by the World Cancer Research Fund and the American Institute for Cancer Research from data up to 2017 showed that being overweight or obese decreased the risk of premenopausal breast cancer (12). Meta-analysis of pre-menopausal patients showed a reduced risk per 5 kg/m 2 increase in the BMI (13). Thus, it was proposed that the pathophysiology between obesity and reduced breast cancer risk in pre-menopause women may be associated on their systemic high levels of estrogens, which in turn reduce gonadotrophin release, and decreased progesterone levels, thus reducing cell proliferation in mammary glands (14). Contradictory studies in this regard have proposed that progesterone may be protective against breast cancer (14). Studies in various populations have shown modest relationships between BMI, obesity and potential to develop breast cancer (15). On the other hand, studies in post-menopausal women showed that obese postmenopausal women presented increased risk for breast cancer compared to non-obese patients; furthermore and the degree of obesity has been correlated to larger tumors and metastasis (16). These patients are characterized by presenting with estrogen (ER-) and progesterone receptor (PR)-positive breast cancers, and not to ER-negative and triple-negative tumors (16). Thus, the effect of increased weight and BMI, as well as the role of leptin and the potential molecular mechanisms by which it contributes to breast cancer progression still remains to be elucidated.
The focal adhesion kinase (FAK) participates in the formation of focal adhesions and activates signaling pathways related to proliferation, survival, cell migration, and angiogenesis (17). Classically, FAK is activated during the formation of focal adhesions, and it is mediated by the interaction between ECM with β-integrins, triggering conformational changes in these receptors (18). This effect is followed by the autophosphorylation of FAK at Y397, which creates a high-affinity binding site for the Src-homology 2 (SH2) domain of Src, a non-receptor tyrosine kinase (19). Active Src phosphorylates the Y576 and Y577 located at the kinase domain of FAK, leading to maximum catalytic activity of FAK, and the formation of a transient FAK-Src signaling complex (17). Cell migration is a key step in metastasis of tumor cells and occurs via two mechanisms: (1) amoeboid, (2) mesenchymal patterns (20). While the amoeboid type of migration has been reported to be independent of integrins and proteases (21), the mesenchymal migration is dependent on integrins, proteases and activation of the FAK signaling pathway (22). In addition, FAK and Src have been associated with migration events such as MMPs expression, secretion and activation which in turn correlates with a highly invasive capacity of tumor cells (23). In particular, MMP-2 and MMP-9 degrade type IV collagen and promote the rupture of basal membranes in colorectal, prostate, lung and breast cancers (24,25). Importantly, serum samples from breast cancer patients have shown that high levels of MMP-2 and MMP-9 are directly associated with metastasis, and further provide evidence of the participation of these MMPs in breast cancer progression. Considering these evidence, we hypothesized that leptin promotes FAK and Src activation, as well as metastasis-associated events such as cell migration, metalloproteases secretion and invasion.
In this study, we evaluated the role of leptin in the activation of FAK and Src kinases, and their roles in cell migration, metalloproteases secretion, and invasion in a cultured cell model of breast cancer. We utilized the breast cancer cell lines MCF7 and MDA-MB-231 and found that leptin activates FAK and Src. Using a combination of inhibitors for these kinases we found a decrease in metastasis-associated events such as cell migration, metalloproteases secretion, and invasion in breast cancer cells. The data represented here contributes to the molecular characterization of the signaling events associated with leptin contributions to cell migration and invasion in breast cancer cell lines.

Materials
Recombinant human leptin, FAK (PF-573228) and Src (PP2) inhibitors were obtained from Sigma-Aldrich. The Src (SU6656) and the STAT3 inhibitor (S31201) were from Merck. Mouse anti-actin, rabbit anti-FAK and anti-Src antibodies were purchased from Santa Cruz Biotechnology. The phospho-specific antibody against FAK, rabbit anti-pY397, was obtained from Invitrogen. The phospho-specific antibody against Src, rabbit anti-pY418, was obtained from MyBiosource (San Diego, CA, USA), species-specific secondary HRP-conjugated antibodies were from Millipore and the secondary antibody antirabbit conjugated with Alexa Fluor 488 was from Invitrogen. Phalloidin coupled to TRITC was purchased from Cytoskeleton (Denver, CO, USA).

Cell culture
The breast cancer cell lines MCF7 and MDA-MB-231 (ATCC) were cultured in DMEM/F12 media (50:50, V:V; Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS) and 1% antibiotics (penicillin G/Streptomycin, Gibco) in a humidified atmosphere containing 5% CO 2 at 37°C. For experimental purposes, cell cultures were serum-starved for 24 h before treatment with either FAK or Src inhibitors and/or leptin. For experimental purposes, cell cultures were used between passages 3 and 15.

Western blot
Whole cell lysates (20 µg) were resolved on 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad). The antiactin, anti-pY397, anti-FAK, anti-pY418 and anti-Src primary antibodies were incubated overnight at 4°C, in agitation at a 1:1000 dilution. Species-specific secondary HRP-conjugated antibodies (1:5000) were incubated for 2 h at room temperature. Membranes were developed using an enhanced chemiluminescence detection system from Bio-Rad.

Immunofluorescence
MCF7 and MDA-MB-231 cells were seeded on glass coverslips, grown to 70% confluence, and stimulated with or without leptin for 0, 15, 60 and 120 min. Cells were fixed for 5 min with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton-X100 in PBS at room temperature. For immunofluorescence (IF) assays, the cells were blocked with 3% albumin in PBS for 1 h at room temperature. Then, the cells were incubated 2 h at room temperature with the rabbit anti-pY397 antibody (1:250 dilution), followed by a 2-h incubation at room temperature with an anti-rabbit conjugated to Alexa Fluor 488 secondary antibody (1:800 dilution). For F-actin staining, cells were incubated with TRITC-Phalloidin (1:500 dilution) for 30 min at room temperature. Cells were counterstained with 4′6-diamidino-2-phenylindole (DAPI), mounted with Fluoroshield/DAPI media (Sigma-Aldrich), and imaged with an Olympus BX43 microscope, using the 100× immersion objective.

Wound healing assays
MCF7 and MDA-MB-231 cells were grown until confluence on 60-mm culture dishes supplemented with DMEM/F12 as described earlier. Cells were starved for 24 h in DMEM/F12 without FBS and treated for 2 h with Cytosine β-D-Arabinofuranoside (AraC) to inhibit cell proliferation during the experiment. After starvation, cells were scratch-wounded using a sterile 200 μL pipette tip, suspended cells were removed by washing with PBS twice, and the cultures were re-fed with DMEM/F12 in the presence or absence of the FAK and Src inhibitors, and/or leptin as indicated in the figures. The progress of cell migration into the wound was monitored at 0 and 48 h using an Olympus BX43 microscope with a 10× objective. The bottom of the plate was marked for reference, and the same field of the monolayers were photographed immediately after performing the wound (time = 0 h) and 48 h after treatments (time = 48 h), five images per plate were analyzed. The distance between the edges of the wound was measured at time 0 and 48 h, and the reported migrated distance corresponds to the difference between these two. The migration area was determined by measuring the total area of the wound using the ImageJ software and the MRI wound-healing tool (26).

Gelatin zymography
Cells were stimulated with leptin and the kinases inhibitors as described earlier, the conditioned medium was collected and concentrated using a 30 kDa cut-off ultra-centrifugal filter units (Amicon, Merck-Millipore). Protein was determined by Bradford (27), and 20 µg of protein from concentrated supernatant were assayed for proteolytic activity on native gelatin-substrate gels (28). Briefly, samples were mixed with non-reducing buffer containing 2.5% SDS, 1% sucrose and 4 mg/mL phenol red, and separated in 8% acrylamide gels co-polymerized with 1 mg/mL gelatin, as previously described (29). After electrophoresis at 72 V for 2.5 h, the gels were rinsed twice in 2.5% Triton X-100, and then incubated in 50 mM Tris-HCl pH 7.4 and 5 mM CaCl 2 assay buffer at 37°C for 24 h. Gels were fixed and stained with 0.25% Coomassie Brilliant Blue G-250 in 10% acetic acid and 30% methanol. Proteolytic activity was detected as clear bands against the background stain of undigested substrate in the gel. Quantification was performed using ImageJ software (26).

Cell invasion assays
Matrigel invasion assays were performed following the Transwell chamber method (30), using 24 well plates containing inserts of 8 mm pore size (Corning Inc, Kennebunk, ME, USA). Briefly, 30 µL of Matrigel (Corning) was added into the inserts and kept at 37°C for 30 min to form a semisolid matrix. Control and cells treated with the FAK or Src inhibitors were plated at 1 × 10 5 cells per insert in serum-free medium on the top chamber. The lower chamber of the Transwell contained 600 µL DMEM supplemented or not with 100 ng/mL leptin. Cells were incubated for 48 h at 37°C in a 5% CO 2 atmosphere. Following incubation, cells and Matrigel on the upper surface of the Transwell membrane were gently removed with cotton swabs. Invading cells on the lower surface of the membrane were washed and fixed with methanol for 5 min, and stained with 0.1% crystal violet in PBS. Cell quantification was performed using a hemocytometer and an Olympus BX43 microscope with the 100× objective.

Statistical analysis
Results are expressed as mean ± s.d. Data were statistically analyzed using one-way ANOVA and the pairwise comparisons were performed using Newman-Keuls and Dunnett's multiple comparison test. Statistical probability of P < 0.05 was considered significant.

Leptin induces FAK activation through the kinase Src in breast cancer cells
FAK activation is triggered by the autophosphorylation of Y397, which is an indicative of its catalytic activity (22,31). This process has been associated with cancer progression and metastasis-related events (31). First, we performed time-course assays to establish whether leptin induces FAK phosphorylation and activation in MDA-MB-231 and MCF7 breast cancer cell lines. We found that MDA-MB-231 cells treated with 100 ng/mL of leptin present a significant increase in FAK phosphorylation on Y397, after 10 min of treatment and peaked after 15 min compared to the non-treated control cells ( Fig. 1A and B). Leptin also activates FAK in MCF7 cells, as Y397 phosphorylation is triggered after 15 min of treatment, reaching a maximum phosphorylation peak at 30 min ( Fig. 1A and C). These data indicate that leptin-induced phosphorylation at Y397 occurs shortly after the stimulus, but is not sustained for long periods in both breast cancer cell lines.
FAK activation is associated to the formation of focal adhesions and cell migration, as it contributes to the weakening of cell-cell adhesion and the cyclic assembly-disassembly of focal adhesions (32). Therefore, we analyzed the effect of leptin in the subcellular localization of phosphorylated FAK at Y397, and whether its activation would promote the formation of stress fibers in MDA-MB-231 and MCF7 cells. Cells were incubated with leptin for 15, 30 and 60 min and formation of stress fibers was monitored by confocal microscopy. Optical planes taken in proximity of the cells with the substrate showed that under normal conditions, the MDA-MB-231 cells expressed activated FAK at the periphery of the cells, which increased upon leptin stimulus (Fig. 1D, in green). In these cells, the cytosolic actin filaments were also abundant, and also increased upon the treatment of leptin (Fig. 1D, in red). Interestingly, the effect of leptin in FAK activation, and formation of stress fibers was more evident in MCF7 cells. Fluorescense microscopy analyses of MCF7 cells showed low levels of activated FAK at the periphery of untreated cells (Fig. 1D, in green), which increased throughout the treatment with leptin. Importantly, these cells also had increased formation of stress fibers upon leptin stimulation (Fig. 1D, in red). Quantification of the fluorescent signal for FAK localization at focal adhesions confirmed the increase of this kinase at these regions (Fig. 1E). These results suggest that leptin promotes the phosphorylation of FAK at Y397 and induces the formation of focal adhesions enriched with stress fibers.
The classic mechanism for FAK activation occurs through autophosphorylation at the Y397 creates a highaffinity binding site for Src (22,33). The interaction between Src and FAK leads to the phosphorylation and activation of Src at Y419, which further result in the phosphorylation of FAK at Y576 and Y577 (22). Src is a tyrosine kinase involved in cell migration and invasion in different triple-negative cancer cell lines (34). Therefore, we asked whether leptin also played a role in the activation of Src, using the highly invasive triple-negative cell line MDA-MB-231 as an in vitro model. Figure 1F shows that leptin induces Src phosphorylation at Y418 in a time-dependent manner, beginning after 10 min of stimulation and is sustained for at least 1 h. Quantification of phosphorylated Src at Y418 further confirmed the activation of this kinase by leptin (Fig. 1G). Then, we asked whether Src was involved on the leptin-induced activation of FAK in both cancer cell lines. To explore this possibility we inhibited Src with the specific inhibitors PP2 ( Fig. 2A, B, C, D and E) and SU5566 (Fig. 2F, G, H, I and J). Western blot analysis and quantification showed that Src inhibition in fact decreases FAK activation dependent on leptin in both cancer cell lines. Western blot analyses of phosphorylated Src at Y418 showed that the kinase was efficiently inactivated by the chemical inhibitors ( Fig.  2A, D and E). The data are in agreement with previous reports of FAK requirement to promote cell migration in MDA-MB-231 and MDA-MB-468 in breast cancer cell lines (35), and with the effect of FAK autophosphorylation at Y397 in cell migration, invasion, and proliferation of gastric carcinomas (36).

Leptin promotes cell migration in a FAK/Src-dependent manner and the STAT3 canonical signaling pathway of the leptin receptor in triple-negative breast cancer cell line
The increased expression of phosphorylated Y397 FAK at focal adhesion points of MDA-MB-231 and MCF7 cells (Fig.  1) is consistent with the requirement for the cycling of focal adhesions during cell migration (37). These observations prompted us to investigate whether FAK was part of the effect of leptin in the migration of these cancer cell lines.   Fig. 3E) presented an epithelial phenotype. Quantification of cell migration for both cell lines showed that cells treated with leptin migrated significantly faster than non-treated control cells (Fig. 3B, D and F). However, in the leptin-stimulated MDA-MB-231 cell line ( Fig.  2A and B) presented enhanced migration capabilities, compared to MCF7 cells ( Fig. 3A and C). Moreover, the MDA-MB-231 cells pre-treated with either FAK or Src inhibitors plus leptin showed a significant decrease in cell migration compared to the leptin-treated cells (Fig. 3A, B, E and F). A similar phenotype was observed in MCF7 cells treated with the FAK inhibitor PF-573228; however, these cells migrated less compared to MDA-MB-231 ( Fig.  3C and D). MCF7 cells presented approximately a 30% decrease in the leptin-induced migration properties when treated with the chemical inhibitor of FAK, compared to the leptin-treated cells (Fig. 3A, B, C and D). These results suggest that leptin-induced migration is dependent on the activation of FAK and Src signaling pathways in both cancer cell lines.
Then, we investigated whether the effects of leptin on FAK and Src activation and the enhanced migratory capabilities of cultured cancer cells were mediated by the canonical leptin-receptor signaling pathway. To this end, we focused on the signal transducer and activator of transcription 3 (STAT3), a transcription factor which is activated upon leptin binding to its receptor (7). STAT3 is overexpressed and constitutively activated in triple-negative breast cancer cells and contributes to a wide variety of cellular processes involved in cancer progression (38). Experiments using S31201, the specific inhibitor for STAT3, showed that the leptin-dependent phosphorylation of FAK at Y397 is different between the two cell lines. MDA-MB-231 cells presented higher levels of FAK phosphorylated at the Y437 residue ( Fig.  4A and B), while in MCF7 cells this phosphorylation was significantly reduced compared to control cells ( Fig. 4A  and C). Surprisingly, the higher level of FAK activation upon STAT3 inhibition did not correlated with the enhanced migratory capabilities of these cancer cells observed before (Fig. 3). In this case, the MDA-MB-231 cells treated with leptin and the STAT3 inhibitor, S3I201, presented a significant decrease wound closure compared to leptin-treated cells (Fig. 4D and E). These data suggest that FAK activation is not related to STAT3 signaling pathway, supporting the hypothesis that two independent mechanisms for signal transduction are activated upon leptin stimulation.

Leptin increased metalloprotease-2 and -9 secretion and activation in breast cancer cells
During migration, the cells need to modify the composition of the ECM by secreting MMPs. MMPs are secreted in response to growth factors, hormones, and cytokines (39). To be active, the MMPs bind to divalent cations, such as Zn 2+ (40). MMPs are required for cell migration, wound healing, tissue remodeling, and angiogenesis among other processes, features by which MMPs are highly relevant in diseases such as arthritis and cancer (41). Metalloproteases secretion, in particular MMP-2 and MMP-9, is linked to invasive and metastatic properties of cancers cells (42). Therefore, we asked whether leptin induces the secretion and activation of MMP-2 and MMP-9. To test this hypothesis, MDA-MB-231 (Fig. 5A, B and C) and MCF7 cells (Fig. 5G, H and I) were stimulated with increasing concentrations of leptin for 24 h. The supernatants were collected, concentrated and analyzed by gelatin zymograms to determine the activity of both MMPs. Figure 5 shows that leptin induced a gradual increase in MMP-2 and MMP-9 secretion and activation, which co-related with the amount of leptin supplemented into the culture media. In both cancer cell lines, a significant increase in the activity of MMP-2 and MMP-9 was observed. In the case of MDA-MB-231 cells, the major change in activation of MMP-2 occurred at 100 ng/mL of leptin, while the activity of MMP-9 was significantly higher at 50 ng/mL compared with the non-treated controls (Fig. 5D, E and F). In the case of MCF7 cells, the activity of MMP-2 was significantly higher at 200 ng/mL of leptin, and for MMP-9 was at 100 ng/mL of the hormone (Fig. 5G, H and I). Both MMPs reached a maximum peak of activation at 400 ng/mL in both cell lines.
To determine the time frame for secretion and activation of the metalloproteases induced by leptin, we collected supernatants of MDA-MB-231 cells stimulated with 100 ng/mL of leptin for 6, 12, 24 and 48 h and assayed for gelatinase activity. Figure 5G, H and I show that leptin induces an increase in both, MMP-2 and MMP-9 secretion after 6 h, reaching a maximum activity peak at 48 h. Together, our data suggest that MMP-2 and MMP-9, two relevant proteins for cell migration and metastatic processes, may be induced and activated by the exposure of chronic and increasing doses of leptin. Secretion and activated of these two MMPs may have a direct co-relation with the enhanced mobility capabilities of these cells observed in our wound-healing assays (Fig. 3).

Leptin promotes invasion and secretion of metalloproteases in a FAK/Src-dependent manner in MDA-MB-231 cells
Metalloproteases secretion is directly related with invasive capacity of tumor cells (43), and might be one of the multiple mechanisms by which FAK positively regulates cell migration (22). If MMP-2 and MMP-9 participate in leptin-dependent migration via FAK activation, their secretion must be controlled by a similar signaling pathway that controls migration of cancer cells. To test this hypothesis, we used the PF-573228 to inhibit FAK signaling pathway and evaluated the effect FAK inactivation on the leptin-induced secretion and activation of MMP-2 and MMP-9. MDA-MB-231 cells were pre-treated with the FAK inhibitor. Then the cells were stimulated with 50 ng/mL of leptin, the supernatants were collected, and concentrated for zymogram analyses. We found that pre-treatment with PF-573228 significantly inhibited MMP-2 ( Fig. 6A and B), and MMP-9 ( Fig. 6A and C) secretion and activation, when compared to the leptintreated cells.
Further, we tested the role of FAK in the invasiveness capabilities of MDA-MB-231 cells stimulated with leptin. Cell invasion assay allows the evaluation of the intrusive potential of tumor cells through the extracellular matrix, which is directly related to processes related to tumor progression and metastasis (44). Therefore, we sought to investigate whether the cancer cells were able to penetrate a model barrier consisting of components of the basement membrane in response to leptin and FAK inhibition. To this end, the cells were seeded in matrigelcoated Transwell chambers in the presence or absence of 100 ng/ml leptin for 48 h. Figure 7A shows representative light microscopy images of MDA-MB-231 cultured in the presence and absence of leptin, where the hormone promoted an increase in cell invasion, as shown by the presence of darker cells characteristic of cell invasion. Also, we observed that invasiveness is a FAK-dependent process, as the cells treated with 5 μM of PF-573228, showed a decrease in invasiveness properties, compared to cells treated with leptin ( Fig. 6D and E). These data is consistent with the contribution of FAK to the highly invasive phenotype of the triple-negative MDA-MB-231 and BT549 cancer cells (35,45).
Finally, we evaluated the role of Src in the secretion and activation of metalloproteases in MDA-MB-231 cells. We observed that leptin promotes the secretion of MMP-2 ( Fig. 8A and B) and MMP-9 ( Fig. 8A and C) through the activation of Src, since culturing the cells with the specific inhibitor of this kinase, PP2, prevented this process. This phenotype was accompanied by a decrease in the leptin-induced invasive capabilities of the MDA-MB-231 cell line treated with PP2  Data represents the mean ± s.d., and it is expressed as fold increase over the un-treated control. Statistical significance was established at *P < 0.05, **P < 0.01, by one-way ANOVA (Newman-Keuls test).
( Fig. 8D and E), supporting the role for Src in cell migration and invasion of cancer cells.
Taken together, our data suggest that leptin activates FAK and Src signaling pathways in a mechanism that is independent to the classic leptin receptor pathway. FAK and Src contribute to the activation and secretion of MMP-2 and MMP-9, which in turn drive the cell migration and invasion phenotypes observed in in vitro models of mammary cancer cells. This leptin-dependent phenotype associated to the activation of FAK and Src is consistent with the aggressive phenotype of the tumorigenic and metastatic cancer cells.

Discussion
Obesity is considered one of the risk factors associated with the development and progression of breast cancer (5,46). Adipose tissue is characterized by an increased synthesis of different adipokines such as leptin (7). Leptin regulates several physiological functions such as food intake and energy expenditure (8). However, in vitro studies demonstrated that leptin also induces the epithelial-mesenchymal transition (EMT), cell migration and MMPs are secreted in the tumor microenvironment in breast epithelial cells (47,48). Thus, leptin is associated with breast cancer progression (7).
In this study, we used the non-invasive MCF7 breast cancer cell line, and the triple-negative highly invasive MDA-MB-231 to investigate the signaling pathways underlying cell migration and invasion. Our results showed that leptin promotes the phosphorylation and consequent activation of FAK at Y397, in both cell lines; however, the phenotypes observed in the triple negative MDA-MB-231 were greater than those observed in the MCF7 cells. We observed that in both cell lines, leptin activates FAK and Src within 10-15 min of stimulation.
Data from the colon cancer cell line SW480 reported by Ratke et al. 2010 showed that leptin also induces FAK activation in a time-specific manner with a maximal activation at 15 min, which is consistent with our studies. Although the exact mechanism of leptin-induced FAK activation has not been described, experimental evidence showed that its canonical activation pathway is through the autophosphorylation of Y397 (49), which is in agreement with our observations. This process is a response to the binding of integrins to ECM during the formation of focal adhesions (17). However, this might not be the only pathway for FAK activation. It has been reported that receptors with tyrosine kinase activity may promote FAK activation in NIH 3T3 fibroblastic cell lines (50). Consistently, in our study FAK phosphorylated at Y397 is localized primarily in focal adhesions bound to stress fibers in both breast cancer cell lines, which suggest that this subcellular localization is regulating cell migration. Interestingly, the activation of FAK/Src seems to be independent of the canonical leptin signaling pathway, as inhibition of STAT3, a downstream effector of the ObR, did not prevent the activation of these kinases, but reduced the migratory capabilities of the cells.
We evaluated the effect of FAK and Src on leptininduced cell migration using the chemical inhibitor PF-573228, and PP2 and SU6656. We found that leptin promotes migration of breast cancer cells in a FAK/Srcdependent manner. Focal adhesions are specialized structures where integrin receptors interact with the ECM, and with the actin cytoskeleton to promote cell migration (51). In addition to regulating the formation of focal adhesions, FAK signaling also affects the remodeling of the actin cytoskeleton by inducing the activation of small GTPases such as Rho, Rac and Cdc42 (52). These are associated to the reorganization of the actin cytoskeleton by generating structures involved in cell migration, such as lamellipodia, filopodia and stress fibers (53).

Figure 8
Representative model of the MMP-2 and MMP-9 secretion induced by leptin in a cultured model for breast cancer. Leptin-dependent activation of FAK occurs via Src and an independent mechanism from the canonical ObR pathway. These kinases promote the secretion of MMPs, cell migration and invasion in cultured models for breast cancer cells. 8:11 An important feature of tumor progression in cancer cells is the increased secretion and activation of metalloproteases (54). We focused our studies in MMP-2 and MMP-9 because they are related to invasive and metastatic processes, and a highly invasive capacity of tumor cells (23). Our data showed that in two breast cancer cellular models, leptin induces an increase in MMPs secretion and activation in a time and dose responsedependent manner. These findings are in agreement with evidence collected from serum samples from breast cancer patients, where high levels of MMP-2 and MMP-9 is directly associated with metastasis, and further provide evidence of the signaling mechanisms (55). MMP-2 and MMP-9 expression is connected with different phases of metastasis, such as the pre-metastatic niche formation, formation of new blood vessels and local invasion (56). Furthermore, leptin promoted MMP-2 and MMP-9 and cell invasion of the triple-negative MDA-MB-231 cell line in a FAK/Src-dependent manner. FAK-dependent induction of MMPs is not limited to cancer; in tenocytes the mechanogrowth factor promotes MMP-2 secretion and cell invasion in a manner dependent on FAK and ERK kinases (57). MMP-9-dependent degradation of fibronectin also stimulates cell migration, and invasion of MCF-7 cells in a FAK/Src-dependent manner (58). The relevance of FAK/Src in cell migration and invasive processes extends to other cellular systems. For instance, the cell adhesion protein Spondin 1 also promotes cell migration and invasion through FAK and Src activation in human osteosarcoma cell lines (59), and Src is well known to be increase cell permeability, EMT, migration, invasion and metastasis of tumor cells (60). Our data strongly suggest that leptin is associated with the establishment of a more aggressive phenotype of the tumor cells promoting local invasion and eventually metastasis of tumor cells (Fig. 8).

Conclusion
Taken together, our results demonstrate that leptin promotes cell migration, metalloprotease secretion and invasion in a FAK and Src-dependent manner, which is independent of the classic leptin route. These events partially explain the association between obesity and the development and progression of breast cancer and suggest that kinases FAK and Src are central molecules, regulating events that favor the metastasis of tumor cells stimulated with leptin, promoting changes to a more aggressive phenotype in breast cancer cells.

Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.