Laetitia Delort

Leptin Induces a Proliferative Response in Breast Cancer Cells but Not in Normal Breast Cells

Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, a hormone excessively produced during obesity, is suggested to be involved in breast cancer. The aim of the study was to investigate procarcinogenic potential of leptin by evaluating influence of leptin on cell proliferation, cell cycle, apoptosis, and signaling on numerous breast cells lines, including 184B5 normal cells, MCF10A fibrocystic cells and MCF-7, MDA-MB-231, and T47D cancer cells. Expressions of leptin and Ob-R were analyzed using qRT-PCR and immunohistochemistry, proliferation using fluorimetric resazurin reduction test and xCELLigence system, apoptosis and cell cycle by flow cytometry, and effect of leptin on different signalling pathways using qRT-PCR and Western blot. Cells were exposed to increasing concentrations of leptin. All cell lines expressed mRNA and protein of leptin and Ob-R. Leptin stimulated proliferation of all cell lines except for 184B5 and MDA-MB-231 cells. Leptin inhibited apoptosis but didnt alter proportion of cells within cell cycle in MCF7 cells. Leptin induced overexpression of leptin, Ob-R, estrogen receptor, and aromatase mRNA in MCF-7 and T47D cells. Autoregulation induced by leptin, relationship with estrogen pathway, and proliferative and antiapoptic activity in breast cancer cells may explain that obesity-associated hyperleptinemia may be a breast cancer risk factor.

Leptin, adipocytes and breast cancer: Focus on inflammation and anti-tumor immunity

More than one million new cases of breast cancer are diagnosed worldwide each year and more than 400,000 deaths are caused by the disease. The origin of this pathology is multifactorial and involved genetic, hormonal, environmental and nutritional factors including obesity in postmenopausal women. The role played by the adipose tissue and their secretions, ie adipokines, is beginning to be recognized. Plasma adipokine levels, which are modulated during obesity, could have “remote” effects on mammary carcinogenesis. Breast cancer cells are surrounded and locally influenced by an adipocyte microenvironment, which is probably more extensive in obese people. Hence, leptin appears to be strongly involved in mammary carcinogenesis and may contribute to the local pro-inflammatory mechanisms, especially in obese patients, who have increased metastatic potential and greater risk of mortality. This review presents the multifaceted role of leptin in breast cancer development and the different molecular pathways involved such as inflammation, oxidative stress and antitumor immunity.

Breast Cancer and Obesity: In Vitro Interferences between Adipokines and Proangiogenic Features and/or Antitumor Therapies?

Obesity is now considered as a risk factor for breast cancer in postmenopausal women. Adipokine levels are modulated in obesity, and may play a role in carcinogenesis. Moreover, obesity increases risk of cancer mortality. Here, we hypothesized that this increase could be due to a modification in angiogenesis, capital event in the development of metastases, and/or in effectiveness of cancer treatments. To test these assumptions, following a same experimental design and simultaneously the effects of leptin and adiponectin on angiogenesis were investigated, and the impact of hyperleptinemia on anticancer drug effectiveness was measured in physiological and obesity situations. Focusing on angiogenesis, the proliferation of endothelial cells (HUVEC), which expressed leptin and adiponectin receptors, was stimulated by leptin and inhibited by adiponectin. Both adipokines globally reduced apoptosis and caspase activity. Leptin increased migration whereas adiponectin decreased migration, and leptin enhanced the area of the tubes formed by HUVEC cells while adiponectin inhibited their formation. MCF7 and MDA-MB-231 cells treated with leptin secreted more VEGF than untreated cells, whereas adiponectin treatment inhibited VEGF secretion. Finally, MCF7 cells pre-treated with leptin were more invasive than untreated cells. This effect was not reproduced in MDA-MB-231 cells. In the MCF7 breast cancer cell line, leptin could induce cell proliferation and reduced the efficacy of all breast cancer therapies (tamoxifen, 5-fluorouracil, taxol and vinblastin). These results suggest that, in obesity situation, leptin– in contrast to adiponectin – may promote tumor invasion and angiogenesis, leading to metastases ‘apparition, and reduce treatment efficacy, which could explain the increased risk of cancer mortality in cases of overweight. The evidence suggests adipokines influence breast cancer issue and could play a significant role, especially in obese patients for which hyperleptinemia, hypoadiponectinemia and increased metastatic potential are described.

Reciprocal Interactions between Breast Tumor and Its Adipose Microenvironment Based on a 3D Adipose Equivalent Model

Breast cancer has become the most common cancer among women in industrialized countries. Obesity is well established as a risk factor, in particular owing to the attendant secretion of the entities called adipokines; there is growing evidence for a role of cells and factors present in the mammary tumor microenvironment such as fibroblasts, preadipocytes, adipocytes and their secretions. To study how the microenvironment influences breast cancer growth, we developed a novel tridimensional adipose model epithelialized with normal human keratinocytes or with breast cancer cell lines. These mimicked a breast tumor in contact with an adipose microenvironment and allowed monitoring of the interactions between the cells. Leptin and adiponectin, two major adipokines, and their respective receptors, ObRt and AdipoR1, were expressed in the model, but not the second adiponectin receptor, AdipoR2. The differentiation of preadipocytes into adipocytes was greater when they were in contact with the breast cancer cell lines. The contact of breast cancer cell lines with the microenvironment completely modified their transcriptional programs by increasing the expression of genes involved in cell proliferation (cyclinD1, MAPK), angiogenesis (MMP9, VEGF) and hormonal pathways (ESR1, IL6). This tridimensional adipose model provides new insights into the interactions between breast cancer cells and their adipose microenvironment, and provides a tool to develop new drugs for the treatment of both cancer and obesity.

New insights into anticarcinogenic properties of adiponectin: a potential therapeutic approach in breast cancer?

Obesity is a recognized breast cancer risk factor in postmenopausal women. A recent hypothesis suggests a major role for adipose tissue in carcinogenesis. During many years, the adipose tissue was only considered as a fat storage of energy. This tissue is now described as an endocrine organ secreting a large range of molecules called adipokines. Among these adipokines, adiponectin may play a major role in breast cancer. Plasma adiponectin levels were found to be decreased in cases of breast cancer and in obese patients. Adiponectin may act directly on breast cancer cells by inhibiting proliferation and angiogenesis or by stimulating apoptosis. Increasing adiponectin levels may be of major importance in the prevention and/or the treatment of breast cancer. This therapeutic approach may be of particular significance for obese patients. The beneficial effects of adiponectin and its possible therapeutic applications will be discussed in this review.

Supernatants of Adipocytes From Obese Versus Normal Weight Women and Breast Cancer Cells: In Vitro Impact on Angiogenesis.

Breast cancer is correlated with a higher risk of metastasis in obese postmenopausal women. Adipokines, whose plasma concentrations are modulated in obese subjects and adipocytes surround mammary cells, suggesting that adipocyte secretome affect mammary tumorogenesis. We hypothesize that mature adipocyte secretions from obese women conditioned or not by breast neoplasic cells, increase changes on the angiogenesis stages. Supernatants of human mature adipocytes, differentiated from stem cells of either adipose tissue of normal weight (MA20) or obese (MA30) women or obtained from co‐cultures between MA20 and MA30 and breast cancer cell line MCF‐7, were collected. The impact of these supernatants was investigated on proliferation, migration, and tube formation by endothelial cells (HUVEC). MA20 and MA30 showed a preservation of their “metabolic memory” (increase of Leptin, ObR, VEGF, CYP19A1, and a decrease of Adiponectin expression in MA30 compared to MA20). Supernatants from obese‐adipocytes increased HUVEC proliferation, migration, and sprouting like with supernatants obtained from co‐cultures of MA/MCF‐7 regardless the womens BMI. Additional analyses such as the use of neutralizing antibodies, analysis of supernatants (Milliplex®) and variations in gene expression (qRT‐PCR), strongly suggest an implication of IL‐6, or a synergistic action among adipokines, probably associated with that of VEGF or IL‐6. As a conclusion, supernatants from co‐cultures of MA30 and MCF‐7 cells increase proliferation, migration, and sprouting of HUVEC cells. These results provide insights into the interaction between adipocytes and epithelial cancer cells, particularly in case of obesity. The identification of synergistic action of adipokines would therefore be a great interest in developing preventive strategies. J. Cell. Physiol. 232: 1808–1816, 2017.

P168: Les sécrétions adipocytaires induisent-elles une diminution de l’efficacité des traitements d’hormonothérapie en situation d’obésité ?

Introduction et but de l’etude L’obesite, facteur de risque de cancer du sein chez les femmes menopausees, est associee a un pronostic defavorable avec survenue de recidives. Au cours de la chimiotherapie, les femmes obeses ou ayant une prise de poids >5% presentent une moindre reponse therapeutique associee a un risque accru de rechutes et de mortalite. Ceci pourrait impliquer une interaction entre secretions adipocytaires et therapie. Dans cette etude, l’objectif est de caracteriser, in vitro, i/ l’impact d’une secretion adipocytaire specifique, la leptine (LEP), sur l’efficacite de l’hormonotherapie : deux anti-œstrogenes (Tamoxifene (Tx) et Fulvestrant (Fv)) et un anti-aromatase (Letrozole (Lz)) ; ii/ l’influence du secretât adipocytaire global de la femme mince et obese sur la reponse au Tx. Materiel et methodes Pour cela, i/ la proliferation des cellules mammaires neoplasiques (MCF-7) ou issues de dystrophie fibro-kystique (MCF10a) en presence de Tx, Fv ou Lz et de LEP (10-100-1000ng/mL) a ete evaluee (fluorescence, rezasurine). ii/ Un modele original (1) de culture tridimensionnelle entre « MCF-7-fibro-blastes-adipocytes matures issus de la differenciation de cellules souches adipocytaires de femmes minces (AM20) ou obeses (AM30) » a ete utilise en presence ou non de Tx. Des marquages immunohistologiques (marqueur de proliferation, Ki67), un suivi de variations d’expressions geniques et une analyse en composante principale (ACP) ont ete realises dans ce modele 3D. Resultats et Analyse statistique L’inhibition de proliferation des MCF-7 induite par le Tx et le Lz (−15 %, −12 % respectivement, pxa0 Conclusion Ces resultats montrent que la LEP amoindrit l’efficacite de l’hormonotherapie (Tx et Lz), et dans une moindre proportion, celle du Fv. Le modele 3D met en evidence que les secretions adipocytaires, notamment celles des AM30, contrecarrent l’effet du Tx. Cette etude permet de suggerer des liens etroits entre secretions adipocytaires et resistance a l’hormonotherapie chez les femmes en surpoids.

P164: Interaction entre cellules mammaires et adipocytaires en situation d’obésité : implication dans le cancer du sein

Introduction et but de l’etude L’obesite est un facteur de risque d’apparition du cancer du sein chez les femmes menopau-sees. Une influence du microenvironnement adipocytaire est aujourd’hui demontree dans le developpement de cette pathologie. Ainsi, l’objectif de notre etude est de caracteriser, dans un contexte d’obesite, les interactions entre les cellules mammaires et leur microenvironnement adipeux et plus particulierement les adipocytes matures (AM) et leurs progeniteurs (cellules souches adipocytaires [ASC] et preadipocytes [PA]). Materiel et methodes Afin d’evaluer l’influence de l’obesite, des ASC extraites a partir de tissu adipeux sous-cutane abdominal de femmes minces et obeses (n =6) ont ete mises a differencier en pre-adipocytes (PA20 et PA30) puis en adipocytes matures (AM20 et AM30) et des analyses par qRT-PCR effectuees. Ces deux types de cellules adipeuses ont ensuite ete co-cultivees en presence de cellules mammaires cancereuses (MCF-7 et MDA-MB-231) ou non cancereuses (MCF10a) et leur proliferation a ete mesuree (fluorescence a la resazurine, Fluoroskan Ascent FL®, n =6). Resultats et Analyse statistique Les resultats montrent que les AM provenant de femmes obeses compares a ceux issus de femmes de poids normal expriment plus fortement la leptine (x8,6, p =0,03), son recepteur (x3,5, p =0,01), l’aromatase (x13,06, p =0,01) et le VEGF (x2, p =0,01) et plus faiblement l’adiponectine (x0,3, p =0,04), ce qui suggere que les AM gardent en memoire leur environnement metabolique d’origine. Les capacites de proliferation des cellules cancereuses mammaires MCF-7 positives pour le recepteur aux œstrogenes (RE +) sont augmentees lorsqu’elles sont co-cultivees en presence des PA30, AM20 et AM30 (+125%, p =0,03 ; +150%, p=0,01; +170%, pxa0 Conclusion Ainsi, ces resultats montrent l’existence d’interactions reciproques entre cellules mammaires et adipeuses, se traduisant par des modifications du comportement des differents types cellulaires presents dans le microenvironnement et pouvant jouer un role cle dans la progression de la cancerogenese mammaire.