Obese adipose tissue extracellular vesicles raise breast cancer cell malignancy

in Endocrine-Related Cancer
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  • 1 Laboratory of Celular and Molecular Pharmacology, Departamento de Biologia Celular, IBRAG, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brasil
  • | 2 Redox Biology Laboratory Programa de Pesquisa em Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
  • | 3 Serviço de Cirurgia Plástica, Departamento de Cirurgia Geral, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
  • | 4 Laboratory of Inflammation and Metabolism, Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, Brasil

Correspondence should be addressed to M Renovato-Martins: marianarenovato@id.uff.br

*(M Renovato-Martins and C Barja-Fidalgo contributed equally to this work)

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Obesity is a chronic low-grade inflammatory condition that strongly impacts breast cancer. Aside from inflammatory mediators, obese adipose tissue (AT) secretes high amounts of extracellular vesicles (EVs), which are capable of transferring molecules to target cells and promoting cell-to-cell communication. Here, we investigated how soluble mediators and EVs secreted by human obese AT influence MCF-7 and MDA-MB-231 mammary adenocarcinoma cell lines by modulating cell proliferation, migration, invasion, and signaling pathways. Both cell lineages were stimulated with conditioned media (CM) or EVs obtained from cultures of AT explants collected from lean or obese individuals who underwent plastic or bariatric surgeries, respectively. EVs derived from obese AT increased the proliferative potential of both cell lines and further potentiated the migratory and invasive properties of MDA-MB-231 cells. The proliferative effects of CM and EVs on MCF-7 cells were dependent on ERK/MAPK pathway activation, while the migration and invasiveness of MDA-MB-231 cells were dependent on PI3K/AKT pathway activation. Furthermore, CM derived from obese AT potentiated the pro-angiogenic effect of MDA-MB-231 on endothelial cells. We also detected that EVs derived from obese AT were enriched in leptin and bioactive matrix metallopeptidase 9 (MMP9), and stimulation of MDA-MD-231 cells with those EVs or CM derived from obese AT potentiated the release of MMP9 by those cells. Our data indicate that obese AT secretes molecules and EVs with pro-tumoral activities capable of increasing breast cancer cell malignancy and provide strong evidence of the key role of AT-derived EV signaling in the tumor microenvironment.

Abstract

Obesity is a chronic low-grade inflammatory condition that strongly impacts breast cancer. Aside from inflammatory mediators, obese adipose tissue (AT) secretes high amounts of extracellular vesicles (EVs), which are capable of transferring molecules to target cells and promoting cell-to-cell communication. Here, we investigated how soluble mediators and EVs secreted by human obese AT influence MCF-7 and MDA-MB-231 mammary adenocarcinoma cell lines by modulating cell proliferation, migration, invasion, and signaling pathways. Both cell lineages were stimulated with conditioned media (CM) or EVs obtained from cultures of AT explants collected from lean or obese individuals who underwent plastic or bariatric surgeries, respectively. EVs derived from obese AT increased the proliferative potential of both cell lines and further potentiated the migratory and invasive properties of MDA-MB-231 cells. The proliferative effects of CM and EVs on MCF-7 cells were dependent on ERK/MAPK pathway activation, while the migration and invasiveness of MDA-MB-231 cells were dependent on PI3K/AKT pathway activation. Furthermore, CM derived from obese AT potentiated the pro-angiogenic effect of MDA-MB-231 on endothelial cells. We also detected that EVs derived from obese AT were enriched in leptin and bioactive matrix metallopeptidase 9 (MMP9), and stimulation of MDA-MD-231 cells with those EVs or CM derived from obese AT potentiated the release of MMP9 by those cells. Our data indicate that obese AT secretes molecules and EVs with pro-tumoral activities capable of increasing breast cancer cell malignancy and provide strong evidence of the key role of AT-derived EV signaling in the tumor microenvironment.

Introduction

Obesity is a major public health problem with rapidly increasing rates worldwide. It is characterized by a chronic low-grade inflammatory condition in adipose tissue (AT) (Ouchi et al. 2011). In obesity, the AT expansion caused by hyperplasia and hypertrophy of adipocytes leads to the release of pro-inflammatory adipokines, such as TNF-α, IL-6, IL-1β, and leptin (Spiegelman & Flier 2001, Ouchi et al. 2011). These mediators promote the infiltration of pro-inflammatory M1 macrophages, which potentiate the inflammatory profile of obese AT (Weisberg et al. 2003, Olefsky & Glass 2010). This condition contributes to the development of obesity-associated comorbidities, such as diabetes, hypertension, cardiovascular diseases, dyslipidemia, and several types of cancer (Bray 2004, Dandona et al. 2004, Rahmouni et al. 2005, Van Gaal et al. 2006, Hursting & Berger 2010, Wolin et al. 2010, Klop et al. 2013).

Breast cancer is the most common type of cancer among women (Bray et al. 2018). Compared to lean women, obese women are prone to a diagnosis of larger tumors, higher incidence of metastasis, and higher risk of recurrence (Calle & Kaaks 2004, Ford et al. 2013). Recent data show a significant association between being overweight and increased breast cancer risk with worse prognosis, recurrence, and mortality in both postmenopausal and premenopausal women (Renehan et al. 2008, Chan et al. 2014, Bray et al. 2018).

Along with the AT release of adipokines that may contribute to tumor progression, extracellular vesicles (EVs) derived from obese AT also play a role in this scenario (Robado de Lope et al. 2018). EVs are small membrane vesicles coated with a lipid bilayer membrane and actively secreted by different tissues during physiological and pathological processes (Minciacchi et al. 2015, Gopal et al. 2017). The population of EVs include exosomes (30 to 100 nm) and larger vesicles called microparticles (0.1 to 1 µm) (Colombo et al. 2014). EV composition depends on cellular origin and may contain enzymes, transcription factors, receptors, mRNA, microRNA, lipids, and other bioactive molecules, which can be transferred to target cells in order to play a key role in cell-to-cell communication (Colombo et al. 2014, Yoon et al. 2014, van Niel et al. 2018).

Several works have demonstrated that tumor-derived EVs can promote cancer progression and modulate the tumor microenvironment (Minciacchi et al. 2015, Webber et al. 2015, Gopal et al. 2017). The EVs released by tumor cells can influence the stroma (Webber et al. 2015), and stroma-derived EVs can promote protrusive activity, motility, and metastasis of breast cancer cells via the activation of the autocrine Wnt signaling pathway (Luga et al. 2012).

During obesity, levels of circulating EVs are significantly increased (Stepanian et al. 2013, Campello et al. 2016, Renovato-Martins et al. 2017). We recently demonstrated that the AT of obese subjects releases large amounts of EVs mainly derived from leukocytes and preadipocytes. These EVs carry toll-like receptor 8 (TLR8), which can be transferred to human blood monocytes and polarize these cells in favor of a pro-inflammatory profile (CD16+), subsequently enabling them to migrate toward the inflamed obese AT (Renovato-Martins et al. 2017). Concerning cancer, it has been shown that adipocyte-derived exosomes can drive melanoma cancer cells to a more aggressive phenotype by transferring enzymes implicated in fatty acid oxidation, which suggests crosstalk between adipocytes and cancer cells (Lazar et al. 2016). Furthermore, the in vitro treatment of breast cancer cells with EVs derived from AT mesenchymal stem cells enhanced cell migration and growth, triggering signaling pathways associated with tumor progression (Lin et al. 2013).

However, despite the often-described association between breast cancer and obesity, few studies have evaluated the role of EVs derived from AT in breast cancer cellular functions. In this study, we investigated how obesity affects crosstalk between AT-derived EVs and mammary adenocarcinoma cells, analyzed the consequences of these interactions on cell functions, and determined the molecular mechanisms involved.

Materials and methods

Participants

Two groups of subjects were chosen for this study. The obese group (OB) was comprised of obese patients who clinically elected for bariatric surgery and were selected by Antônio A Peixoto de Souza (General Surgery Department HUCFF-UFRJ). The control group (CTL) was comprised of lean individuals who submitted to mammary plastic surgery and were selected by César Cláudio da Silva (General Surgery Department HUCFF-UFRJ). All individuals provided written informed consent. The experiments were approved by and in accordance with the Ethics Committee of Pedro Ernesto University Hospital, Rio de Janeiro State University, Rio de Janeiro, Brazil (UERJ/CAAE 36880914.0.0000.5259).

Reagents

The following culture reagents were purchased from Sigma-Aldrich: BSA, penicillin, streptomycin, trypsin, trypan blue, ribonuclease A (RNase A), protease inhibitor phenylmethylsulfonyl fluoride (PMSF), leupeptin, MCDB-131 medium, Dulbecco’s modified Eagle’s medium (DMEM), Medium 199 (M199), 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenol tetrazolium bromide (MTT), Mitomycin C, Tween 20, phenol red, propidium iodide (P4170), gelatin type A from porcine skin, and Coomassie brilliant blue R-250. Fetal bovine serum (FBS) was purchased from Cutilab (Campinas, SP, Brazil). The bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific. For electrophoresis, the nitrocellulose membranes and Rainbow TM molecular weight markers were obtained from GE Healthcare, and the enhanced chemiluminescence (ECL) system (Super Signal West Pico chemiluminescent substrate kit) was purchased from Pierce Biotechnology. The panoptic staining kit was obtained from LB Laborclin (PR, Brazil), and the Matrigel® was from BD Biosciences Labware (Bedford, MA, USA).

Antibodies and inhibitors

The anti-ERK 1/2 (sc154; rabbit) antibody was from Santa Cruz Biotechnology, the anti-AKT/PKB (#05-591; mouse) was from Millipore, the anti-phospho-ERK 1/2 (#9106; mouse) and anti-phospho-Akt S473 (#4058; mouse) were from Cell Signaling, and the anti-MMP2 (ab3158; goat) and anti-MMP9 (ab119906; mouse) were from Abcam. MAPK inhibitor PD98059 (US1513000) and PI3K inhibitor LY294002 (US1440202) were from Calbiochem.

Culture of AT explants and procurement of CM

Fragments of s.c. AT from lean individuals (IMC < 25) were collected during plastic surgeries (control group (CTL)), and fragments of omental AT were obtained from obese individuals (IMC ≥ 30) during gastric sleeve surgeries (obese group (OB)). After the surgeries, the excised ATs were rinsed and cleaned with PBS. Explants of 100 mg of AT were cut into small pieces and incubated in 1 mL of 199 medium (M199) supplemented with 1% FBS at 37°C for 24 h. After that, the supernatants from the cultures, herein referred to as conditioned media (CM), were aspirated (onto ice) and centrifuged at 350 g for 10 min at 4°C to remove small fragments and cells before being stored at −80°C for further experiments.

Isolation of EVs

The EVs released by the AT explants within the supernatants were obtained as previously described (Renovato-Martins et al. 2017). Briefly, the CM were first centrifuged at 2000 g for 10 min at 4°C to clear the cells and cell debris, then further centrifuged at 20,000 g for 70 min at 4°C (Le Bihan et al. 2012). The EV-enriched pellets were resuspended in M199 (1% FBS). Using this protocol, we obtained mostly microparticles, which are the largest EVs.

Cell lines and cultures

The breast cancer cell lines MCF-7 (a luminal non-invasive type) and MDA-MB-231 (a basal invasive type) as well as HMEC-1, a human microvasculature endothelial cell line, were obtained from the American Type Culture Collection Cell Bank (ATCC). The tumor cells were maintained in DMEM supplemented with 10% FBS, penicillin (0.5 U/mL), streptomycin (0.5 mg/mL), HEPES (15 mM), NaHCO3 (14.04 mM), and L-glutamine (2 mM). HMEC-1 cells were maintained in MCDB-131 medium supplemented with 10% FBS, epidermal cell growth factor (EGF) (10 mg/mL), hydrocortisone (10 mg/mL), penicillin (0.5 U/mL), streptomycin (0.5 mg/mL), HEPES (15 mM), NaHCO3 (14.04 mM), and L-glutamine (2 mM). All cells were cultured at 5% CO2, pH 7.2, and 37°C. The cells were allowed to grow until confluency was reached, then detached for use after a brief treatment with trypsin (0.1%) and ethylenediaminetetraacetic acid (EDTA, 0.01%). Both the MDA-MB-231 and MCF-7 cells were stimulated with 20% (v/v) of CM or EVs derived from lean or obese AT, which is in accordance with our previous study protocol (Renovato-Martins et al. 2017).

MTT assay

The MTT assay was performed through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenol tetrazolium bromide to formazan crystals method. MCF-7 and MDA-MB-231 cells were seeded in 96-well plates. Both cell types were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h. The stimuli were diluted in DMEM supplemented with 1% FBS (in the presence or absence of 20 µM ERK MAPK inhibitor PD98059). Next, MTT (2 mg/mL) was added to the wells. Four hours later, the formazan crystals were dissolved in isopropyl alcohol. The plates were read on an EnVision™ plate reader (PerkinElmer) at an absorbance of 570 nm.

Proliferation assay with tritiated thymidine

MCF-7 and MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT in 96-well plates for 24 h. For the final 6 h, [3H]-thymidine (10 µCi/mL) was added to the wells. After that, the cells were lysed in RIPA lysis buffer for 30 min at room temperature. The radioactivity was counted with a Packard Tri-Carb 1600 TR liquid scintillation analyzer (PerkinElmer).

Cell migration assay

The cell migration assay was performed by the wound healing method. Briefly, MCF-7 and MDA-MB-231 cells were plated in 6-well plates until 100% confluence was reached. Next, the cells were pre-stimulated with CM (20% v/v) or EVs (20 µg/mL) derived from lean or obese AT for 24 h. Both stimuli were diluted in DMEM supplemented with 1% FBS (in the presence or absence of 20 µM PI3K/Akt inhibitor LY294002). The supernatants were aspired, and the cells were incubated with mitomycin (5 µg/mL) for 2 h before being washed. Then, a risk on the plate was performed with the aid of a tip. Representative images were captured at 0 h and 24 h after scraping at 10× magnification (Olympus IX71; Olympus Corporation). Cell migration was quantified as a percentage of wound closure compared to the cells at 0 h in each group using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).

Invasion assay

The invasive ability of MDA-MB-231 cells was measured using a transwell chamber containing membranes with 8 µm pores, which were initially coated with 1% gelatin. Previously plated MDA-MB-231 cells were stimulated with CM (20% v/v) or EVs (20 µg/mL) derived from lean or obese AT for 24 h. Both stimuli were diluted in DMEM supplemented with 1% FBS (in the presence or absence of 20 µM PI3K/AKT inhibitor LY294002). Next, the cells were detached, resuspended in serum-free medium, and seeded into the transwell upper chamber. DMEM supplemented with 10% FBS was added to the lower chamber as a chemotactic agent. After 24 h of incubation at 37°C, the cells that migrated through gelatin to the lower chamber were fixed and stained with a panoptic kit. The invasive cells were quantified by manual counting under an inverted microscope at 10× magnification (Olympus IX71; Olympus Corporation). For each experimental group, ten randomly selected fields were analyzed.

Immunoblotting

MCF-7 and MDA-MB-231 cells were stimulated with CM (20% v/v) or EVs (20 µg/mL) derived from lean or obese AT (DMEM supplemented with 1% FBS for 24 h) and lysed in RIPA lysis buffer. Pelleted EVs from the centrifugation at 20,000 g for 70 min were further lysed in RIPA lysis buffer. The samples and electrophoresis were prepared according the protocol of Helal-Neto et al. (2016). Briefly, 30 µg of proteins from cells and 20 µg of proteins from EVs were loaded onto electrophoresis gels. Next, the proteins were transferred from the gels to nitrocellulose membranes and blocked with 5% BSA containing 0.1% Tween 20 in TBS. The blocked membranes were incubated overnight at 4°C with the following primary antibodies: anti-pAKT (1:1000), anti-AKT (1:1000), anti-pERK (1:1000), anti-ERK (1:1000), anti-MMP9 (1:500), and anti-MMP2 (1:500). After washing in Tween-TBS, the membranes were incubated for 2 h with peroxidase-conjugated secondary antibody. The bands were visualized using ECL and quantified by densitometry using ImageJ software (NIH). The results were expressed as arbitrary units (AU).

Matrigel™ tube formation assay

HMEC-1 cells were stimulated for 24 h with the supernatants of MDA-MB-231 cells, which were previously stimulated with CM (20% v/v) or EVs (20 µg/mL) derived from lean or obese AT and diluted in DMEM supplemented with 5% FBS. The number of tubules formed was determined through a Matrigel tube formation assay as described by Helal-Neto et al. (2016). Four phase-contrast images were taken for each individual well from different locations using an Olympus BX40 microscope at 10× magnification (Olympus Corporation). The extent of tube formation was quantified by counting the number and measuring the total length of the formed tubes using Adobe Photoshop CS5 software (Adobe Inc). We defined a tubule as having a length greater than 30 px. Fragments of structures cut out by the photos were disregarded.

Quantification of leptin

The amount of leptin present in the EVs derived from lean and obese AT were quantified by an ELISA Development Kit from Peprotech (Rocky Hill, NJ, USA) according to the manufacturer’s instructions.

Zymography assay

Gelatin zymography was performed for both EVs derived from lean or obese AT and the supernatants of MDA-MB-231 cells previously stimulated with CM (20% v/v) or EVs (20 μg/mL). MDA-MB-231 cell supernatants were concentrated in a SpeedVac concentrator (Savant SPD111; Thermo Fisher Scientific) for 2 h. Both the EVs and the resulting pellet of MDA-MB-231 supernatants were resuspended in Triton lysis buffer (50 mM HEPES buffer, pH 7.5 containing 250 mM NaCl, 1% Triton X-100, and 10% glycerin) supplemented with protease inhibitors. The samples were then subjected to SDS-PAGE as described by Quirico-Santos et al. (2013). Briefly, the gels (SDS-PAGE, 7.5%) were copolymerized with 2 mg/mL gelatin. For each sample, 15 μg of total serum protein was loaded. The gels were further washed in renaturation buffer (2.5% Triton X-100 in 50 mM Tris–HCl, pH 7.5) and incubated at 37°C for 24 h in substrate buffer (10 mM Tris–HCl buffer, pH 7.5; 5 mM CaCl2; and 1 mM ZnCl2). The gels were then stained with a 30% methanol/10% acetic acid solution containing 0.5% Coomassie blue and discolored using the same solution without dye. Areas of enzymatic activity appeared as clear bands over the dark background. The image was digitally inverted so that the integration of the bands was reported as positive values. Matrix metallopeptidase (MMP) production was measured by scoring the intensity of bands as arbitrary OD units via image analysis performed with ImageJ software (NIH).

Statistical analyses

The data are expressed as means ± s.e. The quantitative variables between normal and log-normal transformed groups were performed using a one-way ANOVA with Sidak’s multiple comparisons or the Student’s t-test. For all analyses, a P value of <0.05 was considered statistically significant. Statistical evaluation was performed using GraphPad Prism software version 7.0 (GraphPad Software Inc).

Results

CM and EVs derived from obese AT act differently on MCF-7 and MDA-MB-231 cell proliferation

To evaluate the effect of AT on the proliferative behavior of MCF-7 and MDA-MB-231 cells, both were incubated with CM or EVs derived from lean or obese AT for 24 h. The proliferative capacity was analyzed by MTT and tritiated thymidine incorporation assays. As demonstrated in Fig. 1A and B, both CM and EVs derived from obese AT increased the proliferative capacity of MCF-7 cells by approximately 30% compared to those from lean AT. No differences in MDA-MB-231 cell proliferation rates were found when these cells were incubated with CM or EVs derived from obese AT (Fig. 1C and D). In order to investigate the molecular mechanism underlying the effects of CM and EVs derived from obese AT on MCF-7 and MDA-MB-231 cells, we evaluated the ERK/MAPK signaling pathways, which are involved in cellular proliferation (Chang et al. 2003). In Fig. 1E, we show that stimulation of MCF-7 cells with CM and EVs derived from obese AT resulted in an approximately two-fold increase in ERK phosphorylation relative to those from to lean AT. However, no differences were observed in MDA-MB-231 cells (Fig. 1F).

Figure 1
Figure 1

Obese AT secretion increases MCF-7 and MDA-MB-231 cell proliferation. MCF-7 and MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h. Proliferation assays were evaluated by MTT in (A) MCF-7 and (C) MDA-MB-231 cells and by thymidine incorporation in (B) MCF-7 and (D) MDA-MB-231 cells. Lysate samples of both cells were subjected to immunoblotting for detection of pERK/ERK levels in (E) MCF-7 and (F) MDA-MB-231 cells. The results were quantified by densitometry through ImageJ software (NIH). The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

Citation: Endocrine-Related Cancer 27, 10; 10.1530/ERC-19-0507

In order to better understand the role of the ERK pathway in MCF-7 cell proliferation triggered by obese AT, we performed the same functional assay in the presence of PD98059, a specific inhibitor of this signaling pathway. Figure 2 highlights that inhibiting the ERK/MAPK pathway impaired the effect, underlying the importance of this cellular pathway in tumoral cell proliferation triggered by obese AT.

Figure 2
Figure 2

Increase in MCF-7 cell proliferation triggered by obese AT is dependent on ERK phosphorylation. MCF-7 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h (concomitantly in the presence or absence of 20 µM ERK signaling pathway inhibitor PD98059). Proliferation was evaluated by the MTT method. The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

Citation: Endocrine-Related Cancer 27, 10; 10.1530/ERC-19-0507

CM and EVs derived from obese AT enhance migration and invasiveness of MDA-MB-231 cells via AKT pathway

We evaluated the influence of secretions by obese AT on the migratory activity of MCF-7 and MDA-MB-231 cells. Figure 3A illustrates that treatment with CM or EVs derived from obese AT did not interfere significantly with the typically weak migratory activity of MCF-7 cells. On the other hand, CM and EVs from obese AT led to a two-fold increase in the already high rates of MDA-MB-231 cell migration (Fig. 3B) when compared to both stimuli derived from lean AT.

Figure 3
Figure 3

Obese AT secretion increases MDA-MB-231 cell migration and invasiveness. MCF-7 and MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT. The cell migration assay was evaluated by the wound healing method for 24 h (compared to 0 h) in (A) MCF-7 and (B) MDA-MB-231 cells. The photos were taken with an inverted microscope at 10× magnification (two photos per field). Lysate samples of both cells were subjected to immunoblotting for the detection of pAkt/AKT levels in (C) MCF-7 and (D) MDA-MB-231 cells. The results were quantified by densitometry from the calculation of the ratio between pAKT/AKT levels using ImageJ software (NIH). (E) MDA-MB-231 cell invasion was evaluated by migration through a transwell system coated with gelatin over 24 h. The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05, **P < 0.01, ***P < 0.005. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

Citation: Endocrine-Related Cancer 27, 10; 10.1530/ERC-19-0507

In order to assess which signaling pathway could be underlying this effect, we evaluated the PI3K/AKT pathway, which is primarily involved in cellular migration (Yuan & Cantley 2008). Neither CM nor AT-derived EVs altered Akt phosphorylation of MCF-7 cells (Fig. 3C). On the other hand, EVs but not CM derived from obese AT induced a four-fold higher increase in AKT phosphorylation of MDA-MB-231 cells (Fig. 3D). It is well established that motility facilitates the process of invasion, which is characterized by the ability of tumor cells to cross the epithelial basement membrane of adjacent tissues, thereby invading the interstitial space and initiating metastasis (Aznavoorian et al. 1993). In addition to the increase in migratory capacity, CM and EVs derived from obese AT increased the invasive capacity of MDA-MB-231 cells through gelatin-coated transwell chambers by 50% (Fig. 3E). In contrast, CM and EVs derived from lean or obese AT did not alter the invasive capacity of MCF-7 cells, which was almost absent (data not shown).

To better identify the involvement of the PI3K/Akt pathway in MDA-MB-231 cell migration and invasiveness, we performed the experiments in the presence of the PI3K/AKT inhibitor LY294002. Figure 4 demonstrates that inhibition of the PI3K/AKT pathway impaired the effects induced by CM or EVs derived from obese AT on migration (Fig. 4A) and invasion (Fig. 4B) compared to those from lean AT, thus revealing that this pathway contributes to the motility of MDA-MB-231 cells triggered by obese AT.

Figure 4
Figure 4

Increases in MDA-MB-231 cell migration and invasiveness triggered by obese AT are dependent on Akt phosphorylation. MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT in the presence or absence of 20 µM PI3K/AKT signaling pathway inhibitor LY294002. (A) After 24 h, cell migration was evaluated by the wound healing method (compared to 0 h). The photos were taken with an inverted microscope at 10× magnification (two photos per field). (B) The cell invasion assay was evaluated by migration through a transwell system coated with gelatin over 24 h. The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

Citation: Endocrine-Related Cancer 27, 10; 10.1530/ERC-19-0507

Obese AT secretion potentiates MDA-MB-231 cell capacity for inducing tubulogenic properties in endothelial cells

It is known that migration and invasiveness are key properties of metastatic tumor cells and that the angiogenic process is necessary to support tumor cell growth and the invasion process (Bergers & Benjamin 2003). Based on these concepts, we investigated if, in addition to potentiating the migratory and invasive capacities of MDA-MB-231 cells, the obese AT environment could affect the pro-angiogenic profile of this tumoral lineage. HMEC-1 cells were stimulated with the supernatants released from MDA-MB-231 cells, which were previously stimulated with CM or EVs derived from obese AT. Next, a tubulogenesis assay was performed on the cells.

Figure 5A shows that the supernatants of MDA-MB-231 cells previously treated with the CM from obese AT but not EVs boosted tubule formation by HMEC-1 cells. Quantification of the number and size of formed tubules is represented in Fig. 5B and C, respectively.

Figure 5
Figure 5

Obese AT secretion increases MDA-MB-231 angiogenic properties upon endothelial cell tubule formation. HMEC-1 cells were treated for 24 h with the supernatants of MDA-MB-231 cells previously stimulated with CM (20% v/v) or EVs (20 µg/mL) derived from lean or obese AT. Tubulogenesis was performed using Matrigel for 6 h. Photos were obtained under an inverted microscope. The size (in pixels) and number of structures formed were measured. (A) Photos taken with an inverted microscope demonstrate tubule formation. The bar graphs are representative of (B) number and (C) size quantification of structures formed. The results are representative of four different experiments. The Student's t-test was used to evaluate the differences between groups. Results represent means ± s.e. **P < 0.01, ***P < 0.005. HMEC-1, a human microvasculature endothelial cell line; CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

Citation: Endocrine-Related Cancer 27, 10; 10.1530/ERC-19-0507

EVs derived from obese AT present increased content of leptin and bioactive MMP9

In cancer, EVs can modulate the tumor microenvironment by carrying hormones, such as leptin, as well as proteases, such as MMPs, which may contribute to tumoral cell invasiveness (Aoki et al. 2010, dos Anjos Pultz et al. 2017, Shimoda 2019). Since it is known that AT releases leptin (Ouchi et al. 2011), MMP2, and MMP9 (Bouloumié et al. 2001), we investigated whether EVs released from obese AT carry different levels of leptin and MMPs. Figure 6A shows that EVs derived from obese AT present a 6.7-fold increase in leptin levels compared to EVs derived from lean AT. Figure 6B shows that EVs released by obese AT presented higher MMP9 expression, while no differences in MMP2 expression were detected among EVs derived from lean and obese AT. Corroborating these data, the zymography results presented herein revealed high amounts of mature MMP9 with degrading activity within EVs derived from obese AT, while no differences were detected concerning MMP2 activity (Fig. 6C). Thus, our results demonstrated that the stimulation of MDA-MB-231 cells with either CM or EVs from obese AT potentiated the capacity of this cell lineage to secrete mature MMP9 with degrading activity, thereby contributing to its enhanced invasiveness (Fig. 6D).

Figure 6
Figure 6

EVs from obese AT are enriched in leptin and MMP9. (A) Leptin content in EVs derived from lean and obese AT was measured by ELISA, and the results are presented as picograms per milliliter (pg/mL). (B) EVs derived from lean and obese AT were resuspended in RIPA lysis buffer and then subjected to immunoblotting for the detection of MMP9 and MMP2. (C) EV lysates were subjected to a zymography assay for the detection of MMP9 and MMP2 activity. (D) MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h. Next, both stimuli were changed to a medium without serum for another 24 h and the supernatants were collected. The supernatants were concentrated, lysed, and subjected to a zymography assay for the detection of MMP9 activity. The results are representative of four different experiments. The Student's t-test or a one-way ANOVA with Sidak’s post-test were used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05, ***P < 0.005. MMP, matrix metallopeptidase; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients.

Citation: Endocrine-Related Cancer 27, 10; 10.1530/ERC-19-0507

Discussion

The focus of this investigation was to better understand the role of AT in breast cancer cells in the obesity context, highlighting the contribution of AT-derived EVs in this scenario. Here, we obtained the secretions derived from AT biopsies from lean and obese individuals, which allowed us to mimic the interaction between AT and breast cancer cells in vitro. Thus, we investigated the effects of AT secretions (CM and EVs) on the cellular functions that sustain cancer development in two different breast cancer cell lines, a luminal non-invasive cell type MCF-7 and a basal invasive cell type MDA-MB-231 (Kenny et al. 2007).

Our results demonstrated that EVs from obese AT enhanced the proliferation of MCF- 7 cells types and potentiated the migratory and invasive capacities of MDA-MB-231 cells. It is noteworthy that our findings showed that EVs have a pivotal role in these processes. However, stimuli from obese AT did not induce migratory and invasive profiles in MCF-7 cells.

The correlation of high BMI with the risk for hormone-responsive postmenopausal breast tumors is well established (Suzuki et al. 2009). It has also been reported that obese women are 20% more likely to develop triple-negative breast cancer than non-obese women (Pierobon & Frankefeld 2013). Furthermore, through insulin resistance and secretion of pro-inflammatory adipokines by AT, obesity can contribute to the progression of triple-negative breast cancers (Phipps et al. 2008). In agreement with these findings, we demonstrated that the secretions derived from obese AT increased the migratory and invasive capacities of triple-negative MDA-MB-231 cells. In line with our data, a recent study showed an increase in the aggressiveness of colon cancer cells co-cultured with adipocytes or stimulated with CM obtained from obese patient adipocytes (Nimri et al. 2019).

ERK/MAPK and PI3K/AKT, which are classic signaling pathways usually modified in cancer, have previously been associated with increased proliferation and invasiveness, respectively (Chang et al. 2003, Yuan & Cantley 2008). Similarly, our results indicated that, during obesity, ERK/MAPK plays an important role in communication between the AT and non-invasive tumor cells (MCF-7) to increase proliferation, which could lead to a larger tumor mass and consequently a worse prognosis. It is known that both visceral and s.c. AT can be a source of estrogens (Hetemäki et al. 2017) and that, during obesity, adipose tissue functions as major tissue site of conversion of androstenedione to estrone (Siiteri 1987) due to higher levels of aromatase (Baglietto et al. 2009, Zahid et al. 2016). This could explain, at least in part, the increased phosphorylated ERK levels observed in the triple-positive MCF-7 cells, once this hormone can regulate MAPK/ERK activation in cancer cells (Sun et al. 2017).

Furthermore, the PI3K/AKT pathway was shown to play a pivotal role in MDA-MB-231 cell migration and invasiveness by CM and EVs released by obese AT. Inhibition of PI3K decreased the invasive potential of MDA-MB-231 cells even after treatment with CM and EVs released by lean AT. However, the decrease was much more pronounced in cells that were stimulated with CM and EVs derived from obese AT (2.3 and 1.2-fold decreases, respectively), suggesting a pivotal function of the PI3K/AKT pathway in the invasive process under obese conditions. These data indicate that obese AT secretions influence distinct cell functions in the two breast cancer cell lines through different signaling pathways. Additionally, we observed that the P38/MAPK pathway did not play a significant role in AT secretion effects on both cell lines (data not shown).

We have previously demonstrated that the CM derived from AT of obese individuals contains higher levels of pro-inflammatory adipokines, such as TNFA, leptin, MIP1A, and VEGF (Renovato-Martins et al. 2017). It is well established that obesity leads to an increase in circulating levels of pro-inflammatory adipokines (Vendrell et al. 2004) like TNFA and IL-6, both of which are associated with the development and progression of breast cancer (Vozarova et al. 2001, Bachelot et al. 2003). Moreover, VEGF and TNFA are known as main contributors to angiogenesis (Bergers & Benjamin 2003). Accordingly, we showed here that stimulating MDA-MB-231 cells with CM but not EVs derived from obese AT altered their secretory profile, which further increased endothelial cell differentiation into capillary-like structures. We hypothesize that these adipokines present in the CM could be promoting alterations in MDA-MB-231 cells, which potentiated tubulogenesis in endothelial cells.

Saxena et al. (2007) demonstrated that leptin induces phosphorylation of AKT in hepatocellular carcinoma cells and potently induces invasion and migration. Along this line, we suggest that the AKT phosphorylation observed in MDA-MB-231 cells could be induced by leptin. Our results demonstrated that EVs released by obese AT contained a 6.7-fold increase in leptin levels compared to EVs derived from lean AT. Likewise, Aoki et al. (2010) showed that EVs derived from 3T3-L1 adipocytes contain adipokines like TNFA and leptin, which may be transported to endothelial cells and mediate intercellular communication.

More recently, studies have emphasized the importance of EVs modulating the tumor microenvironment through the transfer of their contents (dos Anjos Pultz et al. 2017, Shimoda 2019). We demonstrated that EVs derived from obese AT have a high content of active MMP9. It is noteworthy that, despite the heterogeneity in MMP9 content assayed among the EV-samples derived from AT from distinct patients, we detected increased MMP9 activity in MDA-MB-231 cells stimulated with those EVs. This enzyme, with its potent proteolytic activity, may prepare pre-metastatic niches and thereby contribute to the metastasis process (Chaffer & Weinberg 2011). EVs from obese and lean patients presented no differences in MMP2 activity, suggesting that this MMP may have a role in AT physiology and that obesity does not interfere with its action.

Interestingly, our data showed that MDA-MB-231 cells stimulated with EVs derived from obese AT secreted increased levels of active MMP9. We believe that, in this scenario, EVs may transfer bioactive MMP9 directly to MDA-MB-231 cells, triggering an increase in their malignancy. Another possibility is that the PI3K/AKT pathway activation driven by the stimulation of MDA-MB-231 cells with EVs derived from obese AT could be upregulating MMP9 proteolytic activity (Dilly et al. 2013). This occurrence would make the pathway at least a partial mechanism for the increased MMP9 activity of MDA-MB-231 cells modulated by EVs. In addition, other proteins that are normally increased in secretions from obese AT, such as cathepsin K (Lafarge et al. 2011), TNFA, IL1B (Ouchi et al. 2011), and IL-17 (Chehimi et al. 2017), may be involved in MMP9 activation as described by other studies (Yoo et al. 2002, Christensen & Shastri 2015, Ohta et al. 2017, Ren et al. 2017), thus potentiating MDA-MB-231 cell invasiveness triggered by EVs.

Taken together, our data clearly indicate that inflammatory mediators and EVs released by AT from obese individuals present pro-tumoral activities capable of upregulating the ERK pathway in MCF-7 cells, which triggers an increase in proliferation, and the AKT pathway in MDA-MB-231 cells, which promotes increases in migration, invasiveness, and pro-angiogenic effects. EVs released from obese AT may also contribute to the formation of pre-metastatic niches followed by development of the tumor.

It is well established that EVs can promote communication between the stroma and cancer cells, modulating the tumor microenvironment and contributing to cancer progression (Greening et al. 2015, Saleem & Abdel-Mageed 2015, Webber et al. 2015, Dos Anjos Pultz et al. 2017, Shimoda 2019). Nevertheless, it is important to mention that most studies correlating AT and cancer cells are performed with in vitro differentiated cell lineages of preadipocytes into adipocytes.

The difference in our work is that we obtained EVs and CM from the AT of obese patients, which allowed us to more accurately mimic what happens in the obese microenvironment and determine real effects on breast cancer cell functions. Therefore, we propose that, in obesity, EVs released from AT could be considered an important mode of communication between AT and cancer cells capable of interfering in tumor progression. Obese AT-derived EVs may be a possible therapeutic target for somehow preventing this communication and improving cancer prognosis. Furthermore, the ERK and AKT pathways could serve as additional tools for complementing the treatments of luminal MCF-7 and basal triple-negative MDA-MB-231 cells, respectively. However, it is still necessary to further investigate the EV contents, including other adipokines, signaling pathways, and cellular functions that may be involved in worse breast cancer prognoses, in order to develop effective therapeutic strategies.

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.

Funding

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Acknowledgements

The authors thank Genilson Rodrigues da Silva and Gabriele Estevão Muniz dos Santos for their technical assistance. The authors also thank Dr Marco Antonio Leite and Dr Luiz Gustavo Oliveira (Coordinator of the Bariatric and Metabolic Surgery Program of the Federal Hospital of Ipanema) for their involvement in patient recruitment.

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    Obese AT secretion increases MCF-7 and MDA-MB-231 cell proliferation. MCF-7 and MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h. Proliferation assays were evaluated by MTT in (A) MCF-7 and (C) MDA-MB-231 cells and by thymidine incorporation in (B) MCF-7 and (D) MDA-MB-231 cells. Lysate samples of both cells were subjected to immunoblotting for detection of pERK/ERK levels in (E) MCF-7 and (F) MDA-MB-231 cells. The results were quantified by densitometry through ImageJ software (NIH). The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

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    Increase in MCF-7 cell proliferation triggered by obese AT is dependent on ERK phosphorylation. MCF-7 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h (concomitantly in the presence or absence of 20 µM ERK signaling pathway inhibitor PD98059). Proliferation was evaluated by the MTT method. The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

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    Obese AT secretion increases MDA-MB-231 cell migration and invasiveness. MCF-7 and MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT. The cell migration assay was evaluated by the wound healing method for 24 h (compared to 0 h) in (A) MCF-7 and (B) MDA-MB-231 cells. The photos were taken with an inverted microscope at 10× magnification (two photos per field). Lysate samples of both cells were subjected to immunoblotting for the detection of pAkt/AKT levels in (C) MCF-7 and (D) MDA-MB-231 cells. The results were quantified by densitometry from the calculation of the ratio between pAKT/AKT levels using ImageJ software (NIH). (E) MDA-MB-231 cell invasion was evaluated by migration through a transwell system coated with gelatin over 24 h. The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05, **P < 0.01, ***P < 0.005. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

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    Increases in MDA-MB-231 cell migration and invasiveness triggered by obese AT are dependent on Akt phosphorylation. MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT in the presence or absence of 20 µM PI3K/AKT signaling pathway inhibitor LY294002. (A) After 24 h, cell migration was evaluated by the wound healing method (compared to 0 h). The photos were taken with an inverted microscope at 10× magnification (two photos per field). (B) The cell invasion assay was evaluated by migration through a transwell system coated with gelatin over 24 h. The data were normalized by cells without stimulus (represented by the dashed line). The results are representative of four different experiments. A one-way ANOVA with Sidak’s post-test was used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

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    Obese AT secretion increases MDA-MB-231 angiogenic properties upon endothelial cell tubule formation. HMEC-1 cells were treated for 24 h with the supernatants of MDA-MB-231 cells previously stimulated with CM (20% v/v) or EVs (20 µg/mL) derived from lean or obese AT. Tubulogenesis was performed using Matrigel for 6 h. Photos were obtained under an inverted microscope. The size (in pixels) and number of structures formed were measured. (A) Photos taken with an inverted microscope demonstrate tubule formation. The bar graphs are representative of (B) number and (C) size quantification of structures formed. The results are representative of four different experiments. The Student's t-test was used to evaluate the differences between groups. Results represent means ± s.e. **P < 0.01, ***P < 0.005. HMEC-1, a human microvasculature endothelial cell line; CM, conditioned media; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients; s.e., standard error.

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    EVs from obese AT are enriched in leptin and MMP9. (A) Leptin content in EVs derived from lean and obese AT was measured by ELISA, and the results are presented as picograms per milliliter (pg/mL). (B) EVs derived from lean and obese AT were resuspended in RIPA lysis buffer and then subjected to immunoblotting for the detection of MMP9 and MMP2. (C) EV lysates were subjected to a zymography assay for the detection of MMP9 and MMP2 activity. (D) MDA-MB-231 cells were stimulated with 20% (v/v) of CM or EVs (20 µg/mL) derived from lean or obese AT for 24 h. Next, both stimuli were changed to a medium without serum for another 24 h and the supernatants were collected. The supernatants were concentrated, lysed, and subjected to a zymography assay for the detection of MMP9 activity. The results are representative of four different experiments. The Student's t-test or a one-way ANOVA with Sidak’s post-test were used to evaluate the differences between groups. The bar graphs represent means ± s.e. *P < 0.05, ***P < 0.005. MMP, matrix metallopeptidase; EVs, extracellular vesicles; AT, adipose tissue; CTL, lean (control) patients; OB, obese patients.

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