Proteomic Profiling of Extracellular Vesicles Released by Leptin-Treated Breast Cancer Cells: A Potential Role in Cancer Metabolism

Abstract

Tumor extracellular vesicles (EVs), as endocytic vesicles able to transport nucleic acids, proteins, and metabolites in recipient cells, have been recognized fundamental mediators of cell-to-cell communication in breast cancer. The biogenesis and release of EVs are highly regulated processes and both the quantity of EVs and their molecular cargo might reflect the metabolic state of the producing cells. We recently demonstrated that the adipokine leptin, whose circulating levels correlate with adipose tissue expansion, is an inducer of EV release from breast cancer cells. Here, we show a specific proteomic signature of EVs released by MCF-7 breast cancer cells grown in the presence of leptin (Lep-EVs), in attempt to find additional molecular effectors linking obesity to breast cancer biology. An analysis of the proteomic profile of Lep-EVs by LC-MS/MS revealed a significant enrichment in biological processes, molecular functions, and cellular components mainly related to mitochondrial machineries and activity, compared to protein content of EVs from untreated breast cancer cells. Metabolic investigations, carried out to assess the autocrine effects of these vesicles on breast cancer cells, revealed that Lep-EVs were able to increase ATP levels in breast cancer cells. This result is associated with increased mitochondrial respiration evaluated by Seahorse analyzer, supporting the concept that Lep-EVs can modulate MCF-7 breast cancer cell oxidative metabolism. Moreover, taking into account the relevance of tumor immune cell crosstalk in the tumor microenvironment (TME), we analyzed the impact of these vesicles on macrophage polarization, the most abundant immune component in the breast TME. We found that tumor-derived Lep-EVs sustain the polarization of M0 macrophages, derived from the human THP-1 monocytic cells, into M2-like tumor-associated macrophages, in terms of metabolic features, phagocytic activity, and increased expression of CD206-positive population. Overall, our results indicate that leptin by inducing the release of EV-enriched in mitochondrial proteins may control the metabolism of MCF-7 breast cancer cells as well as that of macrophages. Characterization of tumor-derived EV protein cargo in an obesity-associated milieu, such as in the presence of elevated leptin levels, might allow identifying unique features and specific metabolic mechanisms useful to develop novel therapeutic approaches for treatment of breast cancer, especially in obese patients.

Regulation of mitochondrial metabolism by autophagy supports leptin-induced cell migration

“…Particularly, the increase in leptin secretion mediated by adipocytes can influence tumor cell metabolism, and also the metabolism of cells adjacent to the tumor 51 . For example, it has been shown that leptin influenced the protein cargo and the biological activity of extracellular vesicles secreted from breast cancer cells, increased mitochondrial metabolism of breast cancer cells, and also regulated macrophage metabolism, which are the most abundant immunological component in the tumor microenvironment 52 . Additionally, it has been demonstrated in estrogen receptor-positive (ER + ) cells, that leptin increased mitochondrial ATP production and enhanced mitochondrial respiration 53 – 55 .…”

“…In this scenario, autophagy is an easily-activated process that the cell uses to speed up nutrient bioavailability and satisfy the energetic demands required to sustain cancer features considered of a high energetic demand. Moreover, metabolic changes regulated by autophagy could influence the metabolism of non-tumor cells adjacent to the tumor, since it was suggested that leptin induced extracellular vesicle secretion with an important role in the metabolic crosstalk within the immune cells in the breast tumor microenvironment in which leptin concentration could be higher than in the blood 52 . This hypothesis needs to be tested in future studies.…”

Leptin: A Heavyweight Player in Obesity-Related Cancers

“…Leptin, through Heat Shock Protein 90, is able to modulate the expression, at the post-transcriptional level, of the tumor susceptibility gene 101, known to be a key component of the endosomal sorting complex required for transport I [174]. Additionally, the same group reported that leptin affects the extracellular vesicle cargo, sustaining the expression of proteins mainly related to mitochondrial machinery and activity, thus modifying the behavior of recipient breast cancer cells and macrophages in terms of metabolism and energy production [175].…”