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Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells

Cancer Gene Therapy volume 29, pages 1056–1069 (2022)Cite this article

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Abstract

Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand–receptor interactions, which may have important implications in cancer.

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Fig. 1: Characterization of small extracellular vesicles collected from MDA-MB-231 and MCF-7 cells in recollection medium.
Fig. 2: Notch components are present in proteins extracted from MDA-MB-231 and MCF-7 cells and associated small extracellular vesicles.
Fig. 3: Notch components packaged into MDA-MB-231 small extracellular vesicles trigger expression changes in Notch target genes and epithelial-to-mesenchymal transition marker genes in MCF-7 cells.
Fig. 4: MDA-MB-231 small extracellular vesicles induce a more aggressive phenotype in MCF-7 cells that is Notch signaling-dependent.
Fig. 5: N1ICD loaded into MDA-231-SEVs induces expression changes in Notch target genes and epithelial-to-mesenchymal transition marker genes in MCF-7 cells.
Fig. 6: N1ICD loaded into MDA-231-SEVs enhances cell motility and proliferation in recipient MCF-7 cells.
Fig. 7: N1ICD loaded into MDA-231-SEVs suppresses E-cadherin expression and increases Ki67 expression in MCF-7 tumors in vivo.
Fig. 8: Model of regulation of breast cancer cell progression by non-classical Notch signaling via small extracellular vesicles.

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Data are available upon request to the authors by email.

References

  1. Andersen P, Uosaki H, Shenje LT, Kwon C. Non-canonical Notch signaling: emerging role and mechanism. Trends Cell Biol. 2012;22:257–65.

    Article  CAS  Google Scholar 

  2. MacGrogan D, Münch J, de la Pompa JL. Notch and interacting signalling pathways in cardiac development, disease, and regeneration. Nat Rev Cardiol. 2018;15:685–704.

    Article  Google Scholar 

  3. Bray SJ. Notch signalling in context. Nat Rev Mol Cell Biol. 2016;17:722–35.

    Article  CAS  Google Scholar 

  4. Diévart A, Beaulieu N, Jolicoeur P. Involvement of Notch1 in the development of mouse mammary tumors. Oncogene. 1999;18:5973–81.

    Article  Google Scholar 

  5. Hu C, Diévart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Clin Pathol. 2006;168:973–90.

    Article  CAS  Google Scholar 

  6. Zhong Y, Shen S, Zhou Y, Mao F, Lin Y, Guan J, et al. NOTCH1 is a poor prognostic factor for breast cancer and is associated with breast cancer stem cells. Onco Targets Ther. 2016;9:6865–71.

    Article  CAS  Google Scholar 

  7. Amthor H, Macharia R, Navarrete R, Brown SC, Otto A, Voit T, et al. NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proc Natl Acad Sci USA. 2007;104:18261–6.

    Article  Google Scholar 

  8. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

    Article  CAS  Google Scholar 

  9. Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019;20:69–84.

    Article  CAS  Google Scholar 

  10. Meurette O, Mehlen P. Notch signaling in the tumor microenvironment. Cancer Cell. 2018;34:536–48.

    Article  CAS  Google Scholar 

  11. Tao J, Jiang MM, Jiang L, Salvo JS, Zeng HC, Dawson B, et al. Notch activation as a driver of osteogenic sarcoma. Cancer Cell. 2014;26:390–401.

    Article  CAS  Google Scholar 

  12. Radtke F, Raj K. The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer. 2003;3:756–67.

    Article  CAS  Google Scholar 

  13. Kovall RA, Blacklow SC. Mechanistic insights into Notch receptor signaling from structural and biochemical studies. Curr Top Dev Biol. 2010;92:31–71.

    Article  CAS  Google Scholar 

  14. Sharghi-Namini S, Tan E, Ong L-LS, Ge R, Asada HH. Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment. Sci Rep. 2014;4:4031.

    Article  Google Scholar 

  15. Sheldon H, Heikamp E, Turley H, Dragovic R, Thomas P, Oon CE, et al. New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood. 2010;116:2385–94.

    Article  CAS  Google Scholar 

  16. Gonzalez-King H, García NA, Ontoria-Oviedo I, Ciria M, Montero JA, Sepúlveda P. Hypoxia inducible factor-1α potentiates jagged 1-mediated angiogenesis by mesenchymal stem cell-derived exosomes. Stem Cells. 2017;35:1747–59.

    Article  CAS  Google Scholar 

  17. Cooks T, Pateras IS, Jenkins LM, Patel KM, Robles AI, Morris J, et al. Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246. Nat Commun. 2018;9:771.

    Article  Google Scholar 

  18. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;7:367(6478).

  19. Meldolesi J. Exosomes and ectosomes in intercellular communication. Curr Biol. 2018;28:R435–R444.

    Article  CAS  Google Scholar 

  20. Mathieu M, Martin-Jaular L, Lavieu G, Théry C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol. 2019;21:9–17.

    Article  CAS  Google Scholar 

  21. Syn N, Wang L, Sethi G, Thiery JP, Goh BC. Exosome-mediated metastasis: from epithelial–mesenchymal transition to escape from immunosurveillance. Trends Pharm Sci. 2016;37:606–17.

    Article  CAS  Google Scholar 

  22. Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009;9:581–93.

    Article  Google Scholar 

  23. Harris DA, Patel SH, Gucek M, Hendrix A, Westbroek W, Taraska JW. Exosomes released from breast cancer carcinomas stimulate cell movement. PLoS ONE. 2015;10:e0117495.

    Article  Google Scholar 

  24. Bolos V, Mira E, Martinez-Poveda B, Luxan G, Canamero M, Martinez AC, et al. Notch activation stimulates migration of breast cancer cells and promotes tumor growth. J Breast Cancer. 2013;15:R54.

    Google Scholar 

  25. Ivascu A, Kubbies M. Diversity of cell-mediated adhesions in breast cancer spheroids. Int J Oncol. 2007;31:1403–13.

    CAS  PubMed  Google Scholar 

  26. Manuel Iglesias J, Beloqui I, Garcia-Garcia F, Leis O, Vazquez-Martin A, Eguiara A, et al. Mammosphere Formation in Breast Carcinoma Cell Lines Depends upon Expression of E-cadherin. PLoS ONE. 2013;8:e77281.

    Article  Google Scholar 

  27. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J Clin 2021. 2021;71:209–49.

  28. Bonadeo N, Becu-Villalobos D, Cristina C, Lacau-Mengido IM. The Notch system during pubertal development of the bovine mammary gland. Sci Rep. 2019;9:8899.

    Article  Google Scholar 

  29. Callahan R, Egan SE. Notch signaling in mammary development and oncogenesis. J Mammary Gland Biol Neopla. 2004;9:145–63.

    Article  Google Scholar 

  30. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. J Breast Cancer. 2004;6:R605–R615.

    CAS  Google Scholar 

  31. Sethi N, Dai X, Winter CG, Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells. Cancer Cell. 2011;19:192–205.

    Article  CAS  Google Scholar 

  32. Miao K, Lei JH, Valecha MV, Zhang A, Xu J, Wang L, et al. NOTCH1 activation compensates BRCA1 deficiency and promotes triple-negative breast cancer formation. Nat Commun. 2020;11:3256.

    Article  CAS  Google Scholar 

  33. Lobry C, Oh P, Mansour MR, Look AT, Aifantis I. Notch signaling: switching an oncogene to a tumor suppressor. Blood. 2014;123:2451–9.

    Article  CAS  Google Scholar 

  34. Wang Z, Li Y, Kong D, Ahmad A, Banerjee S, Sarkar FH. Crosstalk between miRNA and Notch signaling pathways in tumor development and progression. Cancer Lett. 2010;292:141–8.

    Article  CAS  Google Scholar 

  35. Xu F, Zhu Y, He Q, Wu L-Y, Zhang Z, Shi W-H, et al. Identification of microRNA-regulated pathways using an integration of microRNA-mRNA microarray and bioinformatics analysis in CD34+ cells of myelodysplastic syndromes. Sci Rep. 2016;6:32232.

    Article  CAS  Google Scholar 

  36. Wu X, Zhou Z, Xu S, Liao C, Chen X, Li B, et al. Extracellular vesicle packaged LMP1-activated fibroblasts promote tumor progression via autophagy and stroma–tumor metabolism coupling. Cancer Lett. 2020;478:93–106.

    Article  CAS  Google Scholar 

  37. Yan W, Wu X, Zhou W, Fong MY, Cao M, Liu J, et al. Cancer-cell-secreted exosomal miR-105 promotes tumour growth through the MYC-dependent metabolic reprogramming of stromal cells. Nat Cell Biol. 2018;20:597–609.

    Article  CAS  Google Scholar 

  38. Wieland E, Rodriguez-Vita J, Liebler SS, Mogler C, Moll I, Herberich SE, et al. Endothelial Notch1 activity facilitates metastasis. Cancer Cell. 2017;31:355–67.

    Article  CAS  Google Scholar 

  39. Sahlgren C, Gustafsson MV, Jin S, Poellinger L, Lendahl U. Notch signaling mediates hypoxia-induced tumor cell migration and invasion. Proc Natl Acad Sci USA. 2008;105:6392–7.

    Article  CAS  Google Scholar 

  40. Cho OH, Shin HM, Miele L, Golde TE, Fauq A, Minter LM, et al. Notch regulates cytolytic effector function in CD8 T cells. J Immunol Res. 2009;182:3380.

    CAS  Google Scholar 

  41. O’Neil J, Grim J, Strack P, Rao S, Tibbitts D, Winter C, et al. FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors. J Exp Med. 2007;204:1813–24.

    Article  Google Scholar 

  42. Qiu H, Zmina PM, Huang AY, Askew D, Bedogni B. Inhibiting Notch1 enhances immunotherapy efficacy in melanoma by preventing Notch1 dependent immune suppressive properties. Cancer Lett. 2018;434:144–51.

    Article  CAS  Google Scholar 

  43. Thery C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006; Chapter 3: Unit 3 22.

  44. Melo SA, Sugimoto H, O’Connell JT, Kato N, Villanueva A, Vidal A, et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell. 2014;26:707–21.

  45. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain bysystemic injection of targeted exosomes. Nat Biotechnol 2011;29:341–5.

  46. Lamichhane TN, Raiker RS, Jay SM. Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery. Mol Pharm. 2015;12:3650–7.

  47. Yousefnia S, Ghaedi K, Forootan FS, Esfahani MHN. Characterization of the stemness potency of mammospheres isolated from the breast cancer cell lines. Tumour Biol. 2019;41.

  48. Ciria M, García NA, Ontoria-Oviedo I, González-King H, Carrero R, De La Pompa JL, et al. Mesenchymal stem cel migration and proliferation are mediated by hypoxia-inducible factor-1ɑ upstream of Notch and SUMO pathways. Stem Cells Dev. 2017;26:973–85.

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Acknowledgements

We thank Dr. Kenneth McCreath for manuscript editing and helpful comments. The data presented herein were obtained using core facilities at IIS La Fe (Cell Culture Unit, Animal Housing Facility and Microscopy Unit) and the Cytometry Unit at Centro de Investigación Principe Felipe. This work was supported by grants from the Instituto de Salud Carlos III (grants: PI16/00107, PI19/00245, RD16/0011/0004, cofunded by the European Regional Development Fund (FEDER “una manera de hacer Europa”), Conselleria de Sanitat Universal i Salut Pública, fellowships ACIF/2018/259 and ACIF/2017/318 co-financed by the European Union through the Operational Program of FEDER of the Valencian Community 2014–2020.

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Authors and Affiliations

  1. Regenerative Medicine and Heart Transplantation Unit, Instituto de Investigación Sanitaria la Fe, Valencia, Spain

    Hernán González-King, Sandra Tejedor, María Ciria, Marta Gil-Barrachina, Rafael Sánchez-Sánchez, Pilar Sepúlveda & Nahuel A. García

  2. Electron Microscopy Core Facility, Centro de Investigación Príncipe Felipe, Valencia, Spain

    Mario Soriano-Navarro

  3. Gecorp, Buenos Aires, Argentina

    Nahuel A. García

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  1. Hernán González-King
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Contributions

Conceptualization: HGK, NAG. Methodology: HGK, STG, MCC, MSN, MG. Investigation: HGK, NAG. Visualization: HGK, STG. Funding acquisition: PSS. Project administration: PS. Supervision: NAG. Writing—original draft: HGK, NAG. Writing—review & editing: NAG, PS, HGK, STG, RSS.

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Correspondence to Pilar Sepúlveda or Nahuel A. García.

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González-King, H., Tejedor, S., Ciria, M. et al. Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells. Cancer Gene Ther 29, 1056–1069 (2022). https://doi.org/10.1038/s41417-021-00411-8

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