Abstract P1-05-04: Leptin modulates exosome biogenesis in breast cancer cells through an enhanced Hsp90/Tsg101 interaction

Abstract

Abstract Exosomes, small membrane vesicles secreted by both normal and malignant cells upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane,play an important role in cell-to-cell communication. Several reports have highlighted the involvement of exosomes in many aspects of breast cancer (BC) development and progression, thus mounting interest in the potential exploitation of these vesicles and their cargoes as cancer biomarkers, drug delivery systems, and for the development of novel therapies. It has been extensively demonstrated the involvement of the obesity hormone leptin in all steps of breast tumorigenesis, but up to now its role in modulating breast cancer exosome generation has not been investigated. Here, we studied the effect of leptin on exosome biogenesis and secretion in estrogen receptor a (ERa)-positive MCF-7 and triple negative MDA-MB-231 BC cells. First, we revealed by Transmission Electron Microscopy (TEM) that the number of MVBs in the cytoplasm of leptin-treated MCF-7 and MDA-MB-231 BC cells was significantly increased compared to control untreated cells. Next, we characterized size distribution, particle number and protein cargo of exosomes, isolated by ultracentrifugation method, from conditioned media (CM) of cells treated or not with leptin. Nanoparticle Tracking Analysis revealed that the concentration of exosomes in the leptin treated MCF-7- and MDA-MB-231-CM was significantly higher compared to untreated samples. Furthermore, exosomes quantification by Acetylcholinesterase activity showed that the full leptin receptor antagonist, peptide LDFI, abrogated leptin-induced exosome secretion. Exosomes from leptin treated cells showedan increased expression of the leptin target gene Heat shock protein 90 (Hsp90) and its client protein HER2, along with activated leptin signaling effectors (pJAK2, pSTAT3, and pMAPK42/44) compared to exosomes from untreated cells. Mechanistically, our results demonstrated that, among proteins involved in exosome biogenesis, leptin significantly increased protein expression of the well-known exosomal luminal marker Tumor susceptibility gene 101 (Tsg101), without affecting its mRNA levels. Thus, we aimed to analyse specific Tsg101 protein-protein interaction as a possible mechanism able to modulate its stability or half-life within the cells. To answer to this question, we performed, in MCF-7 BC cells, co-immunoprecipitation studies combined with mass spectrometry. Analysis of the spectra identified Hsp90 as a specific Tsg101 interacting protein, and concomitantly immunoblotting assays revealed a specific interaction between HSP90 and Tsg101 in basal condition that was further increased upon leptin exposure. Accordingly, leptin-induced Tsg101 protein levels were completely abrogated in the presence of specific Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamicin. In conclusion, our results demonstrate for the first time that leptin was able to increase exosomes release in BC cells, through an up-regulation of Tsg101 expression at posttranslational level. These findings, providing additional insights into the molecular mechanism governing exosome generation in BC cells, might open new avenues for therapeutic intervention in BC. Citation Format: Gelsomino L, Giordano C, Barone I, Panza S, Augimeri G, Bonofiglio D, Catalano S, Andò S. Leptin modulates exosome biogenesis in breast cancer cells through an enhanced Hsp90/Tsg101 interaction [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-04.