Adipocytes promote breast cancer resistance to chemotherapy, a process amplified by obesity: role of the major vault protein (MVP)

Abstract

Introduction: Clinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated.

Methods: We used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer.

Results: Adipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases.

Conclusions: Collectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.

Keywords: Adipocytes; Breast cancer; Doxorubicin; Mammary adipose tissue; Multidrug resistance; Obesity.

Fig. 1

Coculture with adipocytes promotes multidrug resistance in various human and murine breast cancer cell lines. a Experimental design: breast tumor cells were cocultivated (pink cells) (or not) (blue cells) with adipocytes for 3 days (pre-incubation period) and incubated alone with drugs for 4 h. After washing, cells were cultivated again (or not) with adipocytes for 3 days (post-incubation period). At the end of the post-incubation period, the number of viable cells was determined. b Results of viability assays with various breast tumor cells cocultivated (white bar) (or not) (black bar) with adipocytes and treated (or not) with doxorubicin (DOX) at indicated doses. Bar plots represent the percentage of surviving cells relative to the survival of untreated cells (set to 100%). c Similar viability assays were performed with the MDA-MB436 cell line cocultivated (or not) with adipocytes, treated (or not) with paclitaxel, 5-fluorouracil (5-FU), or mafosfamide at the indicated doses. Abbreviations: C cocultivated cells, NC non-cocultivated cells.

Fig. 2

Adipocytes decrease intracellular doxorubicin accumulation in breast cancer cells independently of major ABC transporters. a Representative experiments showing the intracellular accumulation of doxorubicin (DOX) detected by flow cytometry for indicated times and conditions. Histograms represent the quantification of DOX intracellular fluorescence for each cell line at indicated times after drug exposure. b Quantification of intracellular DOX at indicated times after drug exposure, in the presence (or not) of P-gp inhibitors: verapamil (Vp) and tariquidar (Tar) (left panel), detected by flow cytometry in MDA-MB436 and E0771 cells cocultivated (C) or not (NC) with adipocytes as indicated. Similar experiments were performed by using the MRP family inhibitor, MK-571 (MK) (middle panel), and the BCRP inhibitor, Fumitremorgin-C (FTC) (right panel). Abbreviation: a.u. arbitrary units.

Fig. 3

Adipocytes increase the nuclear efflux of doxorubicin (DOX) and its expulsion from tumor cells via extracellular vesicles (EVs). a Left panel: intracellular localization of DOX visualized by confocal microscopy in non-cocultivated (NC) and cocultivated (C) cells at indicated times after DOX exposure in MDA-MB436 and E0771 cells. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (scale bars, 10 μm). Right panel: corresponding quantification of fluorescence intensity (DOX) in the nuclei. b Analysis of the cytoplasmic localization of DOX in live MDA-MB436 cells using time-lapse video microscopy in control cells (NC) and cells cultivated with adipocyte-conditioned medium (AdCM) for both the pre- and post-incubation steps at indicated times. c Left panel: one representative analysis using nanoparticle tracking analysis (NTA) technology of the number of EVs secreted by MDAMB436 or E0771 cells pre-incubated (C) or not (NC) with adipocytes, treated (DOX) or not (NT) and then post-incubated (C) or not (NC) with adipocyte soluble factors. Right panel: quantification of the number of EVs secreted. d Analysis by flow cytometry of the DOX content in EVs secreted by MDA-MB436 or E0771 cells treated as in c. Abbreviation: a.u. arbitrary units.

Fig. 4

Major vault protein (MVP) is implicated in doxorubicin (DOX) efflux and mediates adipocyte-induced chemoresistance. a Immunoblots against MVP in MDA-MB436 (left panel) and E0771 (right panel) cells non-cocultivated (NC) or cocultivated (C) with adipocytes for the indicated times. Tubulin is shown as a control for equal protein loading. b MVP protein levels were analyzed after transfection of E0771 cells with Ctl and MVP small interfering RNAs (siRNA) by immunoblotting. Tubulin is shown as a control for equal protein loading. c Left panel: intracellular localization of DOX after transfection of E0771 cells with Ctl or MVP siRNAs visualized by confocal microscopy. Cells were post-incubated (C) or not (NC) with adipocytes for 24 h after DOX treatment. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (scale bars, 20 μm). Right panel: corresponding quantification of fluorescence intensity (DOX) in the nuclei. d Analysis of DOX content in the same number of extracellular vesicles secreted by E0771 cells transfected with siCtl or siMVP and then post-incubated (or not) with adipocyte-secreted soluble factors (SF) for 20 h. e Analysis of cytotoxicity induced by DOX or 5-FU treatments in E0771 cells transfected by siCtl or with three independent siMVP and post-incubated with adipocyte-conditioned medium (AdCM) or not (NC) for 72 h using IncuCyte technology (expressed as the ratio of surviving cells post-incubated with AdCM over surviving NC cells). Abbreviation: a.u. arbitrary units.

Fig. 5

Chemoresistance is amplified by obesity and major vault protein (MVP) expression is upregulated at the invasive front of human breast tumors. a Neutral lipid content using bodipy staining (in green) and morphological analyses (transmission, TRANS) of mammary adipocytes embedded in a fibrinogen matrix, isolated from normal weight (NW) or obese patients (scale bars, 100 μm, pictures were taken at day 4 (D4) and day 6 (D6) of culture). Each image shows a maximum projection of about 10 pictures of a z-stack with a distance of 5 μm between each scanning position. b Viability assays after doxorubicin (DOX) exposure of MDA-MB436 breast cancer cell line cocultivated (C), or not (NC), with adipocytes from NW or obese women (n = 5 independent samples). Results are expressed as the ratio of surviving cells in C versus NC cells (set at 1). c Quantification of intracellular fluorescence of DOX in MDA-MB436 cells either non-cocultivated or cocultivated with adipocytes from NW or obese women (n = 5 independent samples) by flow cytometry at indicated times after DOX exposure. d Immunoblots against MVP in MDA-MB436 cells either non-cocultivated or cocultivated with adipocytes from NW or obese women. Tubulin is shown as a control for equal protein loading. Quantification of MVP is shown in arbitrary units. e Histologic examination of a human breast tumor (patient #15) after MVP staining (in brown); (Front) zoom of the MVP staining at the invasive front in one representative area; (Center) zoom of the MVP staining in the center of the tumor in one representative area. In situ carcinoma (black stars) and normal mammary glands (arrowheads) are considered positive and negative internal control, respectively. Note that the quantification did not take into account in situ carcinoma areas. f The ratio of MVP expression between the invasive front and the tumor center was calculated after quantification using Fiji software plug-ins. The results obtained in lean and overweight/obese patients are shown, and each point represents a different patient. Note that, for the clarity of the figure, two patients with ratios greater than 3 (one lean and one obese) are not represented in the figure.

Fig. 6

Schematic representation of the role of tumor-surrounding adipocytes in breast cancer resistance to doxorubicin (DOX). DOX enters tumor cells by passive diffusion and accumulates in the nucleus. Upon stimulation with adipocyte soluble factors, the level of major vault protein (MVP) increases, promoting DOX nuclear efflux. This efflux is followed by sequestration of DOX in cytoplasmic vesicles, and extracellular efflux of the drug is mediated through secretion of these vesicles (extracellular vesicles, EV). This adipocyte-mediated mechanism contributes to the occurrence of a resistant phenotype that is amplified by obesity