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The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth

Abstract

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

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References

  1. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–60.

    Article  CAS  PubMed  Google Scholar 

  2. Mongiat M, Andreuzzi E, Tarticchio G, Paulitti A. Extracellular matrix, a hard player in angiogenesis. Int J Mol Sci 2016; 17. pii: E1822.

    Article  CAS  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182–6.

    Article  CAS  Google Scholar 

  5. Chung AS, Lee J, Ferrara N. Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer. 2010;10:505–14.

    Article  CAS  PubMed  Google Scholar 

  6. Ohtsu A, Shah MA, Van CE, Rha SY, Sawaki A, Park SR, et al. Bevacizumab in combination with chemotherapy as first-line therapy in advanced gastric cancer: a randomized, double-blind, placebo-controlled phase III study. J Clin Oncol. 2011;29:3968–76.

    Article  CAS  PubMed  Google Scholar 

  7. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473:298–307.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Allegra CJ, Yothers G, O’Connell MJ, Sharif S, Petrelli NJ, Lopa SH, et al. Bevacizumab in stage II-III colon cancer: 5-year update of the National Surgical Adjuvant Breast and Bowel Project C-08 trial. J Clin Oncol. 2013;31:359–64.

    Article  CAS  PubMed  Google Scholar 

  9. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell. 2009;15:232–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Vinals F, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15:220–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. McIntyre A, Harris AL. Metabolic and hypoxic adaptation to anti-angiogenic therapy: a target for induced essentiality. EMBO Mol Med. 2015;7:368–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jain RK. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell. 2014;26:605–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wong PP, Demircioglu F, Ghazaly E, Alrawashdeh W, Stratford MR, Scudamore CL, et al. Dual-action combination therapy enhances angiogenesis while reducing tumor growth and spread. Cancer Cell. 2015;27:123–37.

    Article  CAS  PubMed  Google Scholar 

  14. Serini G, Tamagnone L. Bad vessels beware! Semaphorins will sort you out! EMBO Mol Med. 2015;7:1251–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Braghetta P, Ferrari A, De GP, Zanetti M, Volpin D, Bonaldo P, et al. Overlapping, complementary and site-specific expression pattern of genes of the EMILIN/Multimerin family. Matrix Biol. 2004;22:549–56.

    Article  CAS  PubMed  Google Scholar 

  16. Colombatti A, Spessotto P, Doliana R, Mongiat M, Bressan GM, Esposito G. The EMILIN/Multimerin family. Front Immunol. 2011;2:93.

    PubMed  Google Scholar 

  17. Doliana R, Canton A, Bucciotti F, Mongiat M, Bonaldo P, Colombatti A. Structure, chromosomal localization, and promoter analysis of the human elastin microfibril interfase located proteIN (EMILIN) gene. J Biol Chem. 2000;275:785–92.

    Article  CAS  PubMed  Google Scholar 

  18. Bot S, Andreuzzi E, Capuano A, Schiavinato A, Colombatti A, Doliana R. Multiple-interactions among EMILIN1 and EMILI. Matrix Biol. 2015;41:44–55.

    Article  CAS  PubMed  Google Scholar 

  19. Mongiat M, Ligresti G, Marastoni S, Lorenzon E, Doliana R, Colombatti A. Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2. Mol Cell Biol. 2007;27:7176–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mongiat M, Marastoni S, Ligresti G, Lorenzon E, Schiappacassi M, Perris R, et al. The extracellular matrix glycoprotein elastin microfibril interface located protein 2: a dual role in the tumor microenvironment. Neoplasia. 2010;12:294–304.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Marastoni S, Andreuzzi E, Paulitti A, Colladel R, Pellicani R, Todaro F, et al. EMILIN2 down-modulates the Wnt signalling pathway and suppresses breast cancer cell growth and migration. J Pathol. 2014;232:391–404.

    Article  CAS  PubMed  Google Scholar 

  22. Bornstein P, Sage EH. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol. 2002;14:608–16.

    Article  CAS  PubMed  Google Scholar 

  23. Hill VK, Hesson LB, Dansranjavin T, Dallol A, Bieche I, Vacher S, et al. Identification of 5 novel genes methylated in breast and other epithelial cancers. Mol Cancer. 2010;9:51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lorenzon E, Colladel R, Andreuzzi E, Marastoni S, Todaro F, Schiappacassi M, et al. MULTIMERIN2 impairs tumor angiogenesis and growth by interfering with VEGF-A/VEGFR2 pathway. Oncogene. 2012;31:3136–47.

    Article  CAS  PubMed  Google Scholar 

  25. Andreuzzi E, Colladel R, Pellicani R, Tarticchio G, Cannizzaro R, Spessotto P, et al. The angiostatic molecule Multimerin 2 is processed by MMP-9 to allow sprouting angiogenesis. Matrix Biol. 2017. https://doi.org/10.1016/j.matbio.2017.04.002.

    Article  CAS  PubMed  Google Scholar 

  26. Colladel R, Pellicani R, Andreuzzi E, Paulitti A, Tarticchio G, Todaro F, et al. MULTIMERIN2 binds VEGF-A primarily via the carbohydrate chains exerting an angiostatic function and impairing tumor growth. Oncotarget. 2016;7:2022–37.

    Article  PubMed  Google Scholar 

  27. Galvagni F, Nardi F, Spiga O, Trezza A, Tarticchio G, Pellicani R, et al. Dissecting the CD93-Multimerin 2 interaction involved in cell adhesion and migration of the activated endothelium. Matrix Biol 2017. https://doi.org/10.1016/j.matbio.2017.08.003.

    Article  CAS  PubMed  Google Scholar 

  28. Doliana R, Veljkovic V, Prljic J, Veljkovic N, De LE, Mongiat M, et al. EMILINs interact with anthrax protective antigen and inhibit toxin action in vitro. Matrix Biol. 2008;27:96–106.

    Article  CAS  PubMed  Google Scholar 

  29. Veljkovic V, Veljkovic N, Este JA, Huther A, Dietrich U. Application of the EIIP/ISM bioinformatics concept in development of new drugs. Curr Med Chem. 2007;14:441–53.

    Article  CAS  PubMed  Google Scholar 

  30. Gharavi NM, Alva JA, Mouillesseaux KP, Lai C, Yeh M, Yeung W, et al. Role of the Jak/STAT pathway in the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo. J Biol Chem. 2007;282:31460–8.

    Article  CAS  PubMed  Google Scholar 

  31. Xia L, Wang L, Chung AS, Ivanov SS, Ling MY, Dragoi AM, et al. Identification of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation. J Biol Chem. 2002;277:30716–23.

    Article  CAS  PubMed  Google Scholar 

  32. Vincenzi B, Schiavon G, Silletta M, Santini D, Tonini G. The biological properties of cetuximab. Crit Rev Oncol Hematol. 2008;68:93–106.

    Article  PubMed  Google Scholar 

  33. Bronisz A, Godlewski J, Wallace JA, Merchant AS, Nowicki MO, Mathsyaraja H, et al. Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320. Nat Cell Biol. 2012;14:159–67.

    Article  CAS  Google Scholar 

  34. Fukui A, Ohta K, Nishi H, Shigeishi H, Tobiume K, Takechi M, et al. Interleukin-8 and CXCL10 expression in oral keratinocytes and fibroblasts via Toll-like receptors. Microbiol Immunol. 2013;57:198–206.

    Article  CAS  PubMed  Google Scholar 

  35. Nguyen-Jackson HT, Li HS, Zhang H, Ohashi E, Watowich SS. G-CSF-activated STAT3 enhances production of the chemokine MIP-2 in bone marrow neutrophils. J Leukoc Biol. 2012;92:1215–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schiavinato A, Keene DR, Wohl AP, Corallo D, Colombatti A, Wagener R, et al. Targeting of EMILIN-1 and EMILIN-2 to fibrillin microfibrils facilitates their incorporation into the extracellular matrix. J Invest Dermatol. 2016;136:1150–60.

    Article  CAS  PubMed  Google Scholar 

  37. Massena S, Christoffersson G, Hjertstrom E, Zcharia E, Vlodavsky I, Ausmees N, et al. A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils. Blood. 2010;116:1924–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhou J, Yi L, Ouyang Q, Xu L, Cui H, Xu M. Neurotensin signaling regulates stem-like traits of glioblastoma stem cells through activation of IL-8/CXCR1/STAT3 pathway. Cell Signal. 2014;26:2896–902.

    Article  CAS  PubMed  Google Scholar 

  39. Newman AC, Nakatsu MN, Chou W, Gershon PD, Hughes CC. The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation. Mol Biol Cell. 2011;22:3791–3800.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lawler J, Sunday M, Thibert V, Duquette M, George EL, Rayburn H, et al. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J Clin Invest. 1998;101:982–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Rossi A, Kontarakis Z, Gerri C, Nolte H, Holper S, Kruger M, et al. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature. 2015;524:230–3.

    Article  CAS  PubMed  Google Scholar 

  42. Scott A, Powner MB, Gandhi P, Clarkin C, Gutmann DH, Johnson RS. et al. Astrocyte-derived vascular endothelial growth factor stabilizes vessels in the developing retinal vasculature. PLoS ONE. 2010;5:e11863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Maes H, Kuchnio A, Peric A, Moens S, Nys K, De-áBock K, et al. Tumor vessel normalization by chloroquine independent of autophagy. Cancer Cell. 2014;26:190–206.

    Article  CAS  PubMed  Google Scholar 

  44. Mazzone M, Dettori D, Leite de OR, Loges S, Schmidt T, Jonckx B, et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell. 2009;136:839–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Park JS, Kim IK, Han S, Park I, Kim C, Bae J, et al. Normalization of tumor vessels by Tie2 activation and Ang2 inhibition enhances drug delivery and produces a favorable tumor microenvironment. Cancer Cell. 2016;30:953–67.

    Article  CAS  PubMed  Google Scholar 

  46. Cantelmo AR, Conradi LC, Brajic A, Goveia J, Kalucka J, Pircher A, et al. Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy. Cancer Cell. 2016;30:968–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bryja V, Bonilla S, Arenas E. Derivation of mouse embryonic stem cells. Nat Protoc. 2006;1:2082–7.

    Article  CAS  PubMed  Google Scholar 

  48. Colombatti A, Poletti A, Carbone A, Volpin D, Bressan GM. Extracellular matrix of lymphoid tissues in the chick. J Histochem Cytochem. 1989;37:757–63.

    Article  CAS  PubMed  Google Scholar 

  49. Danussi C, Spessotto P, Petrucco A, Wassermann B, Sabatelli P, Montesi M, et al. Emilin1 deficiency causes structural and functional defects of lymphatic vasculature. Mol Cell Biol. 2008;28:4026–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Nakatsu MN, Davis J, Hughes CC. Optimized fibrin gel bead assay for the study of angiogenesis. J Vis Exp 2007; 186. ID=186, doi: 10.3791/186.

  51. Aplin AC, Fogel E, Zorzi P, Nicosia RF. The aortic ring model of angiogenesis. Methods Enzymol. 2008;443:119–36.

    Article  CAS  PubMed  Google Scholar 

  52. Bonaldo P, Braghetta P, Zanetti M, Piccolo S, Volpin D, Bressan GM. Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy. Hum Mol Genet. 1998;7:2135–40.

    Article  CAS  PubMed  Google Scholar 

  53. Kupershmidt I, Su QJ, Grewal A, Sundaresh S, Halperin I, Flynn J, et al. Ontology-based meta-analysis of global collections of high-throughput public data. PLoS One 2010; 5. pii: e13066.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Dr. Veljkovic Veljko for the in silico interaction analyses, Dr. Schiappacassi Monica for the help with the adenoviral and lentiviral constructs, Gustavo Baldassarre for the constructive discussions, Mark Frey and Polk D-Brent for the EGFR mutants, and Raimund Wagener for the anti-mouse EMILIN2 antibody. We thank AIRC the Italian Ministry of Health, the Italian Ministry of University and Research, and the University of Padova for the financial support.

Funding

This work was supported by AIRC (grant # IG-2012-12718 to MM, IG-13126 AColombatti), the Italian Ministry of Health (grant# RF-2010-2312580 to MM), the Italian Ministry of University and Research (grants PRIN 2007 and Progetto Strategico 01.00858.ST97 to GB), and the University of Padova (grants CDPA075559/07 to PBraghetta and CPDA148982/14 to PBonaldo).

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Paulitti, A., Andreuzzi, E., Bizzotto, D. et al. The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth. Oncogene 37, 3399–3414 (2018). https://doi.org/10.1038/s41388-017-0107-x

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