Malignant progression results from a dynamic cross-talk between stromal and cancer cells. Recent evidence suggests that this cross-talk is mediated to a significant extent by exosomes, nanovesicles secreted by most cell types and which allow the transfer of proteins, lipids, and nucleic acids between cells. Adipocytes are a major component of several tumor microenvironments, including that of invasive melanoma, where cells have migrated to the adipocyte-rich hypodermic layer of the skin. We show that adipocytes secrete exosomes in abundance, which are then taken up by tumor cells, leading to increased migration and invasion. Using mass spectrometry, we analyzed the proteome of adipocyte exosomes. Interestingly, these vesicles carry proteins implicated in fatty acid oxidation (FAO), a feature highly specific to adipocyte exosomes. We further show that, in the presence of adipocyte exosomes, FAO is increased in melanoma cells. Inhibition of this metabolic pathway completely abrogates the exosome-mediated increase in migration. Moreover, in obese mice and humans, both the number of exosomes secreted by adipocytes as well as their effect on FAO-dependent cell migration are amplified. These observations might in part explain why obese melanoma patients have a poorer prognosis than their nonobese counterparts. Cancer Res; 76(14); 4051-7. Ó2016 AACR.

Extracellular vesicles are emerging key actors in adipocyte communication. Notably, small extracellular vesicles shed by adipocytes stimulate fatty acid oxidation and migration in melanoma cells and these effects are enhanced in obesity. However, the vesicular actors and cellular processes involved remain largely unknown. Here, we elucidate the mechanisms linking adipocyte extracellular vesicles to metabolic remodeling and cell migration. We show that adipocyte vesicles stimulate melanoma fatty acid oxidation by providing both enzymes and substrates. In obesity, the heightened effect of extracellular vesicles depends on increased transport of fatty acids, not fatty acid oxidation-related enzymes. These fatty acids, stored within lipid droplets in cancer cells, drive fatty acid oxidation upon being released by lipophagy. This increase in mitochondrial activity redistributes mitochondria to membrane protrusions of migrating cells, which is necessary to increase cell migration in the presence of adipocyte vesicles. Our results provide key insights into the role of extracellular vesicles in the metabolic cooperation that takes place between adipocytes and tumors with particular relevance to obesity.

Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.

Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity-induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri-adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL-6, G-CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro-inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri-adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT-dependent CTGF promoter activity, which depends on β1-integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co-activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT-induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation.

The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M(r) as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.

Prostate gland is surrounded by periprostatic adipose tissue (PPAT), which is increasingly believed to play a paracrine role in prostate cancer progression. Our previous work demonstrates that adipocytes promote homing of prostate cancer cells to PPAT and that this effect is upregulated by obesity. Here, we show that once tumor cells have invaded PPAT (mimicked by an in vitro model of coculture), they establish a bidirectional crosstalk with adipocytes, which promotes tumor cell invasion. Indeed, tumor cells induce adipocyte lipolysis and the free fatty acids (FFA) released are taken up and stored by tumor cells. Incubation with exogenous lipids also stimulates tumor cell invasion, underlining the importance of lipid transfer in prostate cancer aggressiveness. Transferred FFAs (after coculture or exogenous lipid treatment) stimulate the expression of one isoform of the pro-oxidant enzyme NADPH oxidase, NOX5. NOX5 increases intracellular reactive oxygen species (ROS) that, in turn, activate a HIF1/MMP14 pathway, which is responsible for the increased tumor cell invasion. In obesity, tumor-surrounding adipocytes are more prone to activate the depicted signaling pathway and to induce tumor invasion. Finally, the expression of NOX5 and MMP14 is upregulated at the invasive front of human tumors where cancer cells are in close proximity to adipocytes and this process is amplified in obese patients, underlining the clinical relevance of our results. Implications: Our work emphasizes the key role of adjacent PPAT in prostate cancer dissemination and proposes new molecular targets for the treatment of obese patients exhibiting aggressive diseases.

IntroductionClinical 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.MethodsWe 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.ResultsAdipocytes 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.ConclusionsCollectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1088-6) contains supplementary material, which is available to authorized users.

Summary Over the last decade, it has become increasingly clear that adipose tissue, and particularly adipocytes, contributes to tumor progression. Obesity, an ever‐increasing worldwide phenomenon, exacerbates this effect. The influence of obesity on melanoma remains poorly studied, although recent data do underline an association between the two diseases in both humans and murine models. Herein, we review the impact of obesity on melanoma incidence and progression and discuss the underlying mechanisms known to be involved. Adipose tissue favors the proliferation and aggressiveness of melanoma cells through a direct dialog, mediated by soluble factors and by exosomes, and through remodeling of the tumor microenvironment. This knowledge could, in the future, help to design new personalized therapeutic options for obese melanoma patients.