Article | Published:

Identification of a novel EphB4 phosphodegron regulated by the autocrine IGFII/IRA axis in malignant mesothelioma


Malignant mesothelioma is a deadly disease with limited therapeutic options. EphB4 is an oncogenic tyrosine kinase receptor expressed in malignant mesothelioma as well as in a variety of cancers. It is involved in tumor microenvironment mediating angiogenesis and invasive cellular effects via both EphrinB2 ligand-dependent and independent mechanisms. The molecular network underlying EphB4 oncogenic effects is still unclear. Here we show that EphB4 expression in malignant mesothelioma cells is markedly decreased upon neutralization of cancer-secreted IGF-II. In particular, we demonstrate that EphB4 protein expression in malignant mesothelioma cells depend upon a degradation rescue mechanism controlled by the autocrine IGF-II-insulin receptor-A specific signaling axis. We show that the regulation of EphB4 expression is linked to a competing post-translational modification of its carboxy-terminal tail via phosphorylation of its tyrosine 987 by the Insulin receptor isoform-A kinase-associated activity in response to the autocrine IGF-II stimuli. Neutralization of this autocrine-induced EphB4-phosphorylation by IGF-II associates with the increased ubiquitination of EphB4 carboxy-terminal tail and with its rapid degradation. We also describe a novel Ubiquitin binding motif in the targeted region as part of the identified EphB4 phosphodegron and provide 3D modeling data supporting a possible model for the acute EphB4 PTM-driven regulation by IGF-II. Altogether, these findings disclose a novel molecular mechanism for the maintenance of EphB4-expression in malignant mesothelioma cells and other IGF-II-secreting cancers (IGF2omas).

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998;93:741–53.

  2. 2.

    Erber R, Eichelsbacher U, Powajbo V, Korn T, Djonov V, Lin J, et al. EphB4 controls blood vascular morphogenesis during postnatal angiogenesis. EMBO J. 2006;25:628–41.

  3. 3.

    Adams RH, Wilkinson GA, Weiss C, Diella F, Gale NW, Deutsch U, et al. Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 1999;13:295–306.

  4. 4.

    Sinha UK, Kundra A, Scalia P, Smith DL, Parsa B, Masood R, et al. Expression of EphB4 in head and neck squamous cell carcinoma. Ear Nose Throat J. 2003;82:866. 869–70, 887

  5. 5.

    Xia G, Kumar SR, Masood R, Zhu S, Reddy R, Krasnoperov V, et al. EphB4 expression and biological significance in prostate cancer. Cancer Res. 2005;65:4623–32.

  6. 6.

    Huang X, Yamada Y, Kidoya H, Naito H, Nagahama Y, Kong L, et al. EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis. Cancer Res. 2007;67:9800–8.

  7. 7.

    Kumar SR, Scehnet JS, Ley EJ, Singh J, Krasnoperov V, Liu R, et al. Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression. Cancer Res. 2009;69:3736–45.

  8. 8.

    Becerikli M, Merwart B, Lam MC, Suppelna P, Rittig A, Mirmohammedsadegh A, et al. EPHB4 tyrosine-kinase receptor expression and biological significance in soft tissue sarcoma. Int J Cancer. 2015;136:1781–91.

  9. 9.

    Liu R, Ferguson BD, Zhou Y, Naga K, Salgia R, Gill PS, et al. EphB4 as a therapeutic target in mesothelioma. BMC Cancer. 2013;13:269.

  10. 10.

    Martiny-Baron G, Holzer P, Billy E, Schnell C, Brueggen J, Ferretti M, et al. The small molecule specific EphB4 kinase inhibitor NVP-BHG712 inhibits VEGF driven angiogenesis. Angiogenesis. 2010;13:259–67.

  11. 11.

    Steinle JJ, Meininger CJ, Forough R, Wu G, Wu MH, Granger HJ. Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway. J Biol Chem. 2002;277:43830–5.

  12. 12.

    Noren NK, Foos G, Hauser CA, Pasquale EB. The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway. Nat Cell Biol. 2006;8:815–25.

  13. 13.

    Yang NY, Pasquale EB, Owen LB, Ethell IM. The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization. J Biol Chem. 2006;281:32574–86.

  14. 14.

    Rutkowski R, Mertens-Walker I, Lisle JE, Herington AC, Stephenson SA. Evidence for a dual function of EphB4 as tumor promoter and suppressor regulated by the absence or presence of the ephrin-B2 ligand. Int J Cancer. 2012;131:E614–24.

  15. 15.

    Atapattu L, Lackmann M, Janes PW. The role of proteases in regulating Eph/ephrin signaling. Cell Adh Migr. 2014;8:294–307.

  16. 16.

    Dynkevich Y, Rother KI, Whitford I, Qureshi S, Galiveeti S, Szulc AL, et al. Tumors, IGF-2, and hypoglycemia: insights from the clinic, the laboratory, and the historical archive. Endocr Rev. 2013;34:798–826.

  17. 17.

    Louvi A, Accili D, Efstratiadis A. Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev Biol. 1997;189:33–48.

  18. 18.

    Sciacca L, Costantino A, Pandini G, Mineo R, Frasca F, Scalia P, et al. Insulin receptor activation by IGF-II in breast cancers: evidence for a new autocrine/paracrine mechanism. Oncogene. 1999;18:2471–9.

  19. 19.

    Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, et al. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells. Mol Cell Biol. 1999;19:3278–88.

  20. 20.

    Christofori G, Naik P, Hanahan D. A second signal supplied by insulin-like growth factor II in oncogene-induced tumorigenesis. Nature. 1994;369:414–8.

  21. 21.

    Nussbaum T, Samarin J, Ehemann V, Bissinger M, Ryschich E, Khamidjanov A, et al. Autocrine insulin-like growth factor-II stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis. Hepatology. 2008;48:146–56.

  22. 22.

    El-Badry OM, Minniti C, Kohn EC, Houghton PJ, Daughaday WH, Helman LJ. Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma tumors. Cell Growth Differ. 1990;1:325–31.

  23. 23.

    Rutten AA, Bermudez E, Stewart W, Everitt JI, Walker CL. Expression of insulin-like growth factor II in spontaneously immortalized rat mesothelial and spontaneous mesothelioma cells: a potential autocrine role of insulin-like growth factor II. Cancer Res. 1995;55:3634–9.

  24. 24.

    Rogler CE, Yang D, Rossetti L, Donohoe J, Alt E, Chang CJ, et al. Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice. J Biol Chem. 1994;269:13779–84.

  25. 25.

    Scalia P, Heart E, Comai L, Vigneri R, Sung CK. Regulation of the Akt/Glycogen synthase kinase-3 axis by insulin-like growth factor-II via activation of the human insulin receptor isoform-A. J Cell Biochem. 2001;82:610–8.

  26. 26.

    Xu WW, Li B, Guan XY, Chung SK, Wang Y, Yip YL, et al. Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression. Nat Commun. 2017;8:14399.

  27. 27.

    Ritter MR, Dorrell MI, Edmonds J, Friedlander SF, Friedlander M. Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis. Proc Natl Acad Sci. USA. 2002;99:7455–60.

  28. 28.

    Daughaday WH, Trivedi B, Baxter RC. Serum “big insulin-like growth factor II” from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing. Proc Natl Acad Sci. USA. 1993;90:5823–7.

  29. 29.

    Marks AG, Carroll JM, Purnell JQ, Roberts CT Jr. Plasma distribution and signaling activities of IGF-II precursors. Endocrinology. 2011;152:922–30.

  30. 30.

    Kim KW, Bae SK, Lee OH, Bae MH, Lee MJ, Park BC. Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma. Cancer Res. 1998;58:348–51.

  31. 31.

    Morcavallo A, Gaspari M, Pandini G, Palummo A, Cuda G, Larsen MR, et al. Research resource: New and diverse substrates for the insulin receptor isoform A revealed by quantitative proteomics after stimulation with IGF-II or insulin. Mol Endocrinol. 2011;25:1456–68.

  32. 32.

    Pundir S, Martin MJ, O’Donovan C. UniProt Protein Knowledgebase. Methods Mol Biol. 2017;1558:41–55.

  33. 33.

    Stapleton D, Balan I, Pawson T, Sicheri F. The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization. Nat Struct Biol. 1999;6:44–9.

  34. 34.

    Swatek KN, Komander D. Ubiquitin modifications. Cell Res. 2016;26:399–422.

  35. 35.

    Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 2015;43:D512–20.

  36. 36.

    Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 2007;28:730–8.

  37. 37.

    Bennett BD, Wang Z, Kuang WJ, Wang A, Groopman JE, Goeddel DV, et al. Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily. J Biol Chem. 1994;269:14211–8.

  38. 38.

    Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer. 2010;10:165–80.

  39. 39.

    Xia G, Kumar SR, Masood R, Koss M, Templeman C, Quinn D, et al. Up-regulation of EphB4 in mesothelioma and its biological significance. Clin Cancer Res. 2005;11:4305–15.

  40. 40.

    Salgia R, Kulkarni P, Gill PS. EphB4: A promising target for upper aerodigestive malignancies. Biochim Biophys Acta. 2018;1869:128–37.

  41. 41.

    Ferguson BD, Tan YH, Kanteti RS, Liu R, Gayed MJ, Vokes EE, et al. Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer. Sci Rep. 2015;5:10641.

  42. 42.

    Noren NK, Yang NY, Silldorff M, Mutyala R, Pasquale EB. Ephrin-independent regulation of cell substrate adhesion by the EphB4 receptor. Biochem J. 2009;422:433–42.

  43. 43.

    Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, et al. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell. 2005;123:291–304.

  44. 44.

    Chen T, Liu X, Yi S, Zhang J, Ge J, Liu Z. EphB4 is overexpressed in gliomas and promotes the growth of glioma cells. Tumour Biol. 2013;34:379–85.

  45. 45.

    Zhao S, Ulrich HD. Distinct consequences of posttranslational modification by linear versus K63-linked polyubiquitin chains. Proc Natl Acad Sci. USA. 2010;107:7704–9.

  46. 46.

    Pandini G, Vigneri R, Costantino A, Frasca F, Ippolito A, Fujita-Yamaguchi Y, et al. Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling. Clin Cancer Res. 1999;5:1935–44.

  47. 47.

    Baserga R. The decline and fall of the IGF-I receptor. J Cell Physiol. 2013;228:675–9.

  48. 48.

    Tawo R, Pokrzywa W, Kevei E, Akyuz ME, Balaji V, Adrian S, et al. The ubiquitin ligase CHIP integrates proteostasis and aging by regulation of insulin receptor turnover. Cell. 2017;169:470–82 e413.

  49. 49.

    Vihanto MM, Plock J, Erni D, Frey BM, Frey FJ, Huynh-Do U. Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin. FASEB J. 2005;19:1689–91.

  50. 50.

    Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–64.

  51. 51.

    Sciacca L, Mineo R, Pandini G, Murabito A, Vigneri R, Belfiore A. In IGF-I receptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor isoform A. Oncogene. 2002;21:8240–50.

  52. 52.

    Pandini G, Frasca F, Mineo R, Sciacca L, Vigneri R, Belfiore A. Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved. J Biol Chem. 2002;277:39684–95.

  53. 53.

    Greenall SA, Donoghue J, Johns TG, Adams TE. Differential sensitivity of human hepatocellular carcinoma xenografts to an IGF-II neutralizing antibody may involve activated STAT3. Transl Oncol. 2018;11:971–8.

  54. 54.

    Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins. 2006;65:712–25.

  55. 55.

    Bussi G, Donadio D, Parrinello M. Canonical sampling through velocity rescaling. J Chem Phys. 2007;126:014101.

Download references


We thank Dr. Steven Albelda (University of Pennsylvania, USA) for providing the MSTO211H cell line and his constructive criticism during the manuscript preparation; Dr Veronica Vella (University of Catania, Italy) for IR system expression data in colorectal cancer cell lines; Dr Parkash S Gill (USC, Los Angeles, CA) for critical insights in manuscript revision. We also thank ASEM srl (Treviso, Italy) and the GianAmico Alessandrini Family for supporting PS research.

Authors contributions

PS conceptualized the study, designed and performed experiments, performed sequence comparison, conserved domains search and prepared manuscript; GP prepared cDNAs from cancer cells, provided unpublished data used for Table 1 and contributed to the manuscript preparation; EC performed 3D modeling analysis, AG provided conceptual insight and logistic support; SJW performed experiments, contributed to manuscript preparation and managed technical and administrative tasks.

Author information

Correspondence to Pierluigi Scalia.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7