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Extracellular vesicles of multiple myeloma cells utilize the proteasome inhibitor mechanism to moderate endothelial angiogenesis

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Abstract

Bone marrow microenvironment is known to support angiogenesis, thus contributing to progression of multiple myeloma (MM). Bortezomib, a proteasome inhibitor (PI) widely used in MM treatment, has anti-angiogenic activity. Extracellular vesicles (EVs), shedding from cell surface, serve as mediators in cell-to-cell communication. We have hypothesized that MM cells (MMCs) treated with bortezomib generate EVs that could diminish angiogenesis, thus limiting MM progression. In the present study, EVs were obtained from MMCs (RPMI-8226), untreated (naïve) or pre-treated with bortezomib. EVs were outlined using NanoSight, FACS, protein arrays and proteasome activity assays. The impact of MMC-EVs on endothelial cell (EC) functions was assessed, employing XTT assay, Boyden chamber and Western blot. A high apoptosis level (annexin V binding 70.25 ± 16.37%) was observed in MMCs following exposure to bortezomib. Compared to naïve EVs, a large proportion of bortezomib-induced EVs (Bi-EVs) were bigger in size (> 300 nm), with higher levels of annexin V binding (p = 0.0043).They also differed in content, presenting with increased levels of pro-inflammatory proteins, reduced levels of pro-angiogenic growth factors (VEGFA, PDGF-BB, angiogenin), and displayed lower proteasome activity. Naïve EVs were found to promote EC migration and proliferation via ERK1/2 and JNK1/2/3 phosphorylation, whereas Bi-EVs inhibited these functions. Moreover, Bi-EVs appeared to reduce EC proteasome activity. EVs released from apoptotic MMCs following treatment with bortezomib can promote angiogenesis suppression by decreasing proliferation and migration of EC. These activities are found to be mediated by specific signal transduction pathways.

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Acknowledgements

We would like to thank Mrs. Sonia Kamenetsky for assistance in the preparation of this manuscript.

Funding

This study was funded by the Israel Cancer Association (Grant Number 20120100) and by the Israel myeloma association (AMEN).

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  1. Tami Katz and Anat Aharon equally contributed to this study.

Authors and Affiliations

  1. Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel

    Moran Zarfati, Irit Avivi, Benjamin Brenner, Tami Katz & Anat Aharon

  2. Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, 7, Ha’Aliya St., Haifa, 3109601, Israel

    Irit Avivi, Benjamin Brenner, Tami Katz & Anat Aharon

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  1. Moran Zarfati
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Correspondence to Anat Aharon.

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10456_2018_9649_MOESM1_ESM.pptx

Supplementary material 1 Effects of MMC-EVs on EC signaling and proteasome activity. HUVEC were incubated in the serum-free medium for 16 hours followed by addition of inhibitors U0126, PD98059, SP600125, and SB203580 for 1 hour. EVs generated from the RPMI 8226 cell line (MMC-EVs: naïve-EVs and Bi-EVs) were added to the culture for 15 minutes. Cellular lysates were separated by SDS-PAGE. Phosphorylation and protein levels of (a) ERK1/2, (b) c-Jun, (c) MAPKAPK-2 were assessed using Western blot (equal protein loading was confirmed with β-actin). Phosphorylation levels were normalized to the total protein expression and displayed in gel pictures S1 (a–c) (PPTX 354 KB)

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Zarfati, M., Avivi, I., Brenner, B. et al. Extracellular vesicles of multiple myeloma cells utilize the proteasome inhibitor mechanism to moderate endothelial angiogenesis. Angiogenesis 22, 185–196 (2019). https://doi.org/10.1007/s10456-018-9649-y

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