MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells
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Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis.
Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays.
We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC-derived exosomes on the expression of VEGF in breast cancer-derived cells. In addition, we found that down-regulation of VEGF mediated by MSC-derived exosomes can affect the vascular behavior of endothelial cells in vitro.
Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.
KeywordsMesenchymal stem cells Exosome Angiogenesis Vascular endothelial growth factor Breast cancer
The authors are grateful to the members of the Departments of Genetics and Biochemistry, Tarbiat Modares University, for their excellent technical assistance and advice. This work was supported by research grants from the Tarbiat Modares University and the Council for Development of Stem Cell Sciences and Technologies (Grant No. 11/77227).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- 9.H. Zhong, K. Chiles, D. Feldser, E. Laughner, C. Hanrahan, M.M. Georgescu, J.W. Simons, G.L. Semenza, Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: Implications for tumor angiogenesis and therapeutics. Cancer Res 60, 1541–1545 (2000)PubMedGoogle Scholar
- 16.B.D. Roorda, A. ter Elst, W.A. Kamps, E.S. de Bont, Bone marrow-derived cells and tumor growth: Contribution of bone marrow-derived cells to tumor micro-environments with special focus on mesenchymal stem cells. Crit Rev Oncol Hematol 69, 187–198 (2009). doi: 10.1016/j.critrevonc.2008.06.004 CrossRefPubMedGoogle Scholar
- 21.S.R. Baglio, K. Rooijers, D. Koppers-Lalic, F.J. Verweij, M. Perez Lanzon, N. Zini, B. Naaijkens, F. Perut, H.W. Niessen, N. Baldini, D.M. Pegtel, Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. Stem Cell Res Ther 6, 127 (2015). doi: 10.1186/s13287-015-0116-z CrossRefPubMedPubMedCentralGoogle Scholar
- 26.D. Chen, Y. Sun, Y. Yuan, Z. Han, P. Zhang, J. Zhang, M.J. You, J. Teruya-Feldstein, M. Wang, S. Gupta, M.C. Hung, H. Liang, L. Ma, miR-100 induces epithelial-mesenchymal transition but suppresses tumorigenesis, migration and invasion. PLoS Genet 10, e1004177–e1002014. doi: 10.1371/journal.pgen.1004177
- 28.L. Deng, L. Shang, S. Bai, J. Chen, X. He, R. Martin-Trevino, S. Chen, X.Y. Li, X. Meng, B. Yu, X. Wang, Y. Liu, S.P. McDermott, A.E. Ariazi, C. Ginestier, I. Ibarra, J. Ke, T. Luther, S.G. Clouthier, L. Xu, G. Shan, E. Song, H. Yao, G.J. Hannon, S.J. Weiss, M.S. Wicha, S. Liu, MicroRNA100 inhibits self-renewal of breast cancer stem-like cells and breast tumor development. Cancer Res 74, 6648–6660 (2014). doi: 10.1158/0008-5472.CAN-13-3710 CrossRefPubMedPubMedCentralGoogle Scholar
- 29.A. Petrelli, R. Carollo, M. Cargnelutti, F. Iovino, M. Callari, D. Cimino, M. Todaro, L.R. Mangiapane, A. Giammona, A. Cordova, F. Montemurro, D. Taverna, M.G. Daidone, G. Stassi, S. Giordano, By promoting cell differentiation, miR-100 sensitizes basal-like breast cancer stem cells to hormonal therapy. Oncotarget 6, 2315–2330 (2015). doi: 10.18632/oncotarget.2962 CrossRefPubMedGoogle Scholar
- 32.S. Ohno, M. Takanashi, K. Sudo, S. Ueda, A. Ishikawa, N. Matsuyama, K. Fujita, T. Mizutani, T. Ohgi, T. Ochiya, N. Gotoh, M. Kuroda, Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther 21, 185–191 (2012). doi: 10.1038/mt.2012.180 CrossRefPubMedPubMedCentralGoogle Scholar
- 33.A. Caivano, F. La Rocca, V. Simeon, M. Girasole, S. Dinarelli, I. Laurenzana, A. De Stradis, L. De Luca, S. Trino, A. Traficante, G. D'Arena, G. Mansueto, O. Villani, G. Pietrantuono, L. Laurenti, L. Del Vecchio, P. Musto, MicroRNA-155 in serum-derived extracellular vesicles as a potential biomarker for hematologic malignancies - a short report. Cell Oncol 40, 97–103 (2017). doi: 10.1007/s13402-016-0300-x CrossRefGoogle Scholar
- 35.C. Thery, S. Amigorena, G. Raposo,A. Clayton, Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell. Biol. Chapter 3, Unit 3 22 (2006) doi: 10.1002/0471143030.cb0322s30
- 36.L. Chen, Y. Wang, Y. Pan, L. Zhang, C. Shen, G. Qin, M. Ashraf, N. Weintraub, G. Ma, Y. Tang, Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury. Biochem Biophys Res Commun 431, 566–571 (2013). doi: 10.1016/j.bbrc.2013.01.015 CrossRefPubMedPubMedCentralGoogle Scholar
- 38.F. Collino, M.C. Deregibus, S. Bruno, L. Sterpone, G. Aghemo, L. Viltono, C. Tetta, G. Camussi, Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs. PLoS One 5, e11803 (2010). doi: 10.1371/journal.pone.0011803 CrossRefPubMedPubMedCentralGoogle Scholar
- 42.T.H. Lee, S. Seng, M. Sekine, C. Hinton, Y. Fu, H.K. Avraham, S. Avraham, Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1. PLoS Med 4, e186 (2007). doi: 10.1371/journal.pmed.0040186 CrossRefPubMedPubMedCentralGoogle Scholar
- 46.M.S. Rotundo, T. Galeano, P. Tassone, P. Tagliaferri, mTOR inhibitors, a new era for metastatic luminal HER2-negative breast cancer? A systematic review and a meta-analysis of randomized trials. Oncotarget 7, 27055–27066 (2016). doi: 10.18632/oncotarget.7446 CrossRefPubMedPubMedCentralGoogle Scholar
- 47.S. Grundmann, F.P. Hans, S. Kinniry, J. Heinke, T. Helbing, F. Bluhm, J.P. Sluijter, I. Hoefer, G. Pasterkamp, C. Bode, M. Moser, MicroRNA-100 regulates neovascularization by suppression of mammalian target of rapamycin in endothelial and vascular smooth muscle cells. Circulation 123, 999–1009 (2011). doi: 10.1161/CIRCULATIONAHA.110.000323 CrossRefPubMedGoogle Scholar
- 49.A.K. Nagaraja, C.J. Creighton, Z. Yu, H. Zhu, P.H. Gunaratne, J.G. Reid, E. Olokpa, H. Itamochi, N.T. Ueno, S.M. Hawkins, M.L. Anderson, M.M. Matzuk, A link between mir-100 and FRAP1/mTOR in clear cell ovarian cancer. Mol Endocrinol 24, 447–463 (2010). doi: 10.1210/me.2009-0295 CrossRefPubMedPubMedCentralGoogle Scholar
- 54.M. Galie, G. Konstantinidou, D. Peroni, I. Scambi, C. Marchini, V. Lisi, M. Krampera, P. Magnani, F. Merigo, M. Montani, F. Boschi, P. Marzola, R. Orru, P. Farace, A. Sbarbati, A. Amici, Mesenchymal stem cells share molecular signature with mesenchymal tumor cells and favor early tumor growth in syngeneic mice. Oncogene 27, 2542–2551 (2008). doi: 10.1038/sj.onc.1210920 CrossRefPubMedGoogle Scholar
- 56.X.L. Yan, C.J. Fu, L. Chen, J.H. Qin, Q. Zeng, H.F. Yuan, X. Nan, H.X. Chen, J.N. Zhou, Y.L. Lin, X.M. Zhang, C.Z. Yu, W. Yue, X.T. Pei, Mesenchymal stem cells from primary breast cancer tissue promote cancer proliferation and enhance mammosphere formation partially via EGF/EGFR/Akt pathway. Breast Cancer Res Treat 132, 153–164 (2012). doi: 10.1007/s10549-011-1577-0 CrossRefPubMedGoogle Scholar
- 57.A.Y. Khakoo, S. Pati, S.A. Anderson, W. Reid, M.F. Elshal, A.T. Rovira II, D. Nguyen, C.A. Malide, G. Combs, J. Hall, M. Zhang, T.B. Raffeld, W. Rogers, J.A. Stetler-Stevenson, M. Frank, T.F. Reitz, Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma. J Exp Med 203, 1235–1247 (2006). doi: 10.1084/jem.20051921 CrossRefPubMedPubMedCentralGoogle Scholar
- 58.B. Cousin, E. Ravet, S. Poglio, F. De Toni, M. Bertuzzi, H. Lulka, I. Touil, M. Andre, J.L. Grolleau, J.M. Peron, J.P. Chavoin, P. Bourin, L. Penicaud, L. Casteilla, L. Buscail, P. Cordelier, Adult stromal cells derived from human adipose tissue provoke pancreatic cancer cell death both in vitro and in vivo. PLoS One 4, e6278 (2009). doi: 10.1371/journal.pone.0006278 CrossRefPubMedPubMedCentralGoogle Scholar
- 59.P. Secchiero, S. Zorzet, C. Tripodo, F. Corallini, E. Melloni, L. Caruso, R. Bosco, S. Ingrao, B. Zavan, G. Zauli, Human bone marrow mesenchymal stem cells display anti-cancer activity in SCID mice bearing disseminated non-Hodgkin's lymphoma xenografts. PLoS One 5, e11140 (2010). doi: 10.1371/journal.pone.0011140 CrossRefPubMedPubMedCentralGoogle Scholar
- 62.J.K. Lee, S.R. Park, B.K. Jung, Y.K. Jeon, Y.S. Lee, M.K. Kim, Y.G. Kim, J.Y. Jang, C.W. Kim, Exosomes derived from mesenchymal stem cells suppress angiogenesis by down-regulating VEGF expression in breast cancer cells. PLoS One 8, e84256 (2013). doi: 10.1371/journal.pone.0084256 CrossRefPubMedPubMedCentralGoogle Scholar