Linoleic acid induces secretion of extracellular vesicles from MDA-MB-231 breast cancer cells that mediate cellular processes involved with angiogenesis in HUVECs
Graphical abstract
Introduction
Breast cancer is the most frequent malignant tumor and the second main cause of cancer-related mortality among women worldwide [1,2]. Considering the expression of cellular markers, the breast cancers are categorized into three main groups: (a) estrogen receptor (ER) or progesterone (PR) positive; (b) human epidermal growth factor receptor 2 (Her2/neu) positive (amplification of Her2/neu) with or without ER and PR expression; and (c) triple-negative breast cancer (TNBC), which does not express ER/PR, and does not have amplification of Her2 [3]. The TNBC has a highly aggressive clinical course, an earlier age of onset, greater metastatic potential and poorer clinical outcomes compared with ER/PR positive and Her2 positive tumors [[4], [5], [6]].
Epidemiological and experimental studies have demonstrated that overweight and obesity are promoting factors for breast cancer development and progression, especially in post-menopausal women [[7], [8], [9]]. Mature adipocytes, the major cell type of white adipose tissue and the major component of stromal environment of mammary tumors, storage lipids in the form of triacylglycerol and release lipids as free fatty acids (FFAs) in times of demand 9]. Higher amounts of FFAs have been associated with the production of pro-tumorigenic signaling lipids and then the promotion of cancer progression [10]. Linoleic acid (LA) is an omega-6 polyunsaturated fatty acid (PUFA), and represents the most abundant fatty acid in occidental diets [11]. In human breast cancer cells, LA induces activation of signal transduction pathways that mediate several cell processes including migration and invasion [[12], [13], [14]]. Moreover, LA promotes an epithelial-mesenchymal-transition (EMT)-like process in mammary non-tumorigenic epithelial cells MCF10A [15].
Extracellular vesicles (EVs) are a heterogeneous group of vesicles constituted for a lipid bilayer that are released from healthy and cancer cells into extracellular space, culture medium and body fluids including blood, urine, and plasma. The EVs are classified according to their size and biogenesis into three groups: exosomes, microvesicles and apoptotic bodies [[16], [17], [18]]. Microvesicles (100 nm-1 μm) are formed by outward budding and fission of the plasma membrane, which is mediated by contraction of the actin cytoskeleton, while exosomes (50−200 nm) are vesicles preformed inside of multivesicular bodies, which fuse with the plasma membrane to discharge the exosomes into extracellular space [16,17,19]. Composition of EVs is varied and includes proteins, lipids, mRNAs and microRNAs [20,21].
Angiogenesis is the process by which new capillaries arise from the pre-existing blood vessels, and has been involved in growth, development, response to injury to restore a tissue’s blood supply and promotion of wound healing [22]. The angiogenesis process consists of sequential steps including the stimulation of endothelial cells (ECs) with angiogenic factors, degrading of vascular basement membrane, proliferation of ECs, sprouting and migration, lumen formation, vessel maturation and stabilization [22,23]. The new capillaries can be formed by either sprouting or intussusceptive angiogenesis. During sprouting angiogenesis ECs form sprouts that grow towards an angiogenic stimulus, while in intussusceptive angiogenesis the interstitial tissues invade the existing vessels and form transvascular tissue pillars that expand and split the vessel [24,25]. EVs promote proliferation, migration and sprouting of ECs, maturation of ECs progenitors and angiogenesis [26]. Tumor cells release EVs that induce angiogenesis, and then the angiogenesis promotes growth, survival and metastasis 23,27].
In this study, we demonstrate that LA enhances the secretion of EVs in MDA-MB-231 cells. Moreover EVs from MDA-MB-231 cells stimulated with LA induce activation of FAK and Src, proliferation, an increase of metalloproteinase (MMP)-2 secretion, secretion of MMP-9, migration, invasion and the formation of new tubules in human umbilical vein endothelial cells (HUVECs). Moreover, LA induces the release of EVs from MDA-MB-231 cells that mediate cell processes involved in angiogenesis, via activation of FFAR1 and FFAR4, whereas EVs from MDA-MB-231 cells stimulated with LA induces migration via FAK and Src activity in HUVECs.
Section snippets
Materials
LA sodium salt (99 % purity), PF-573228 and DC260126 were from Sigma-Aldrich (Merck KGaA). Anti-CD9 antibody (Ab) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-fibroblast growth factor (FGF) Ab was from Cell Signaling Technology (Camarillo, CA). AH7614 was from TOCRIS (Minneapolis, MN). Anti-flotillin-2 (Flot-2) Ab and BD Matrigel were from BD Biosciences (Bedford, MA). Anti-vascular endothelial growth factor (VEGF) Ab, anti-epidermal growth factor (EGF) Ab, phosphospecific Abs
LA enhances the secretion of EVs in MDA-MB-231 breast cancer cells
EV fractions from conditioned media of MDA-MB-231 cells unstimulated and stimulated with 90 μM LA for 48 h were characterized by TEM, NTA and Western blotting with anti-CD9 Ab and anti-Flot-2 Ab, because these proteins are associated with the different types of EVs [19,34]. EV fractions showed a heterogeneous population of spherical vesicles that express CD9 and flotillin-2 proteins (Fig. 1A and B). NTA showed that MDA-MB-231 cells release EVs with sizes between 80−700 nm, whereas MDA-MB-231
Discussion
Epidemiological and animal studies have demonstrated a correlation between high dietary fat intake and the risk of developing breast cancer [39,40]. Particularly, western diets characterized for a low consumption of omega-3 fatty acids associated with a high consumption of omega-6 fatty acids are related with a higher risk of developing breast cancer [41]. LA is an omega-6 PUFA and an important component of vegetable oils, being estimated an intake in Western diets of around 15−20 g per day per
Author contributions
Conception and design of the study: A. G-H., J. R-R. and E.P.S. Acquisition of data: A. G-H. and P. C-R. Analysis and interpretation of data: A. G-H., E. L-O. and E.P.S. Technical support: P. C-R. and E. L-O. Funding acquisition: R. T-B and E.P.S. Writing, review and/or revision of the manuscript: A. G-H., J. R-R., P C-R. and E.P.S.
Funding
This work was supported by a grant from Consejo Nacional de Ciencia y Tecnologia-Fondo Sectorial de Investigación en Salud y Seguridad Social of Mexico (Grant number 261637) and Consejo Nacional de Ciencia y Tecnologia of Mexico (Grant number 255429). A. G-H and J R-R were supported for fellowships from Consejo Nacional de Ciencia y Tecnologia of Mexico.
Declaration of Competing Interest
The authors declare no conflict of interest
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