Journal of Gastroenterology

, Volume 53, Issue 8, pp 932–944 | Cite as

Insulin receptor substrate-4 is overexpressed in colorectal cancer and promotes retinoblastoma–cyclin-dependent kinase activation

  • Patricia Sanmartín-Salinas
  • María del Val Toledo Lobo
  • Fernando Noguerales-Fraguas
  • Miguel Toro Londoño
  • Antonio Jiménez-Ruiz
  • Luis Gonzalez GuijarroEmail author
Original Article—Alimentary Tract



Insulin receptor substrate 4 (IRS-4) is an adaptor protein for which new evidence suggests plays a role in tumour promotion.


We described nuclear IRS-4 in RKO colon cancer cell lines in biopsies of patients with colorectal cancer (CRC) (n = 20) and in matched adjacent normal colorectal (MANC) tissue (n = 20).


Treatment with physiological doses of IGF-1 promoted nuclear influx of IRS-4 from cellular cytosol in RKO cells. When exogenous IRS-4 was overexpressed in RKO cells, there was an increase in cyclin D1, cyclin E, E2F1, pRB Ser 809/811 and pRB Ser 705 levels compared with the empty vector-transfected cells. Some of these changes returned to control values after wortmannin treatment. Subcellular fractionation showed an overexpression of IRS-4 in the cytoplasm, membrane, and nuclei of tumour samples, whereas the levels of the protein were barely detectable in the three compartments of normal samples. Immunohistochemical studies showed positive nuclear IRS-4 staining in over 74% of the tumour cells. IRS-4 was strongly overexpressed in tumoural tissues from CRC patients compared to MANC tissues. The up-regulation of IRS-4 in CRC samples correlated significantly with the increase of several G1 checkpoint proteins including cyclin D1 (r = 0.6662), Rb (r = 0.7779), pRb Serine 809/811 (r = 0.6864), pRb serine 705 (r = 0.6261) and E2F1 (r = 0.8702).


Taken together, our findings suggest that IRS-4 promotes retinoblastoma–cyclin-dependent kinase activation and it may serve as a pharmacological target since its expression is very low in normal tissue, including colonic epithelium.


Nuclear IRS4 Cell proliferation Colorectal cancer Cell cycle IRS4 



This work was partially supported by the Spanish Grant S2010/BMD-2423 from Comunidad de Madrid. We gratefully acknowledge David Cano Martinez and Borja Hernandez Breijo for helpful discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have not competing interest.


  1. 1.
    Fantin VR, Sparling JD, Slot JW, et al. Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells. J Biol Chem. 1998;273(17):10726–32.CrossRefPubMedGoogle Scholar
  2. 2.
    White MF. The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem. 1998;182(1–2):3–11.CrossRefPubMedGoogle Scholar
  3. 3.
    Mardilovich K, Pankratz SL, Shaw LM. Expression and function of the insulin receptor substrate proteins in cancer. Cell Commun Signal. 2009;7:14 (Epub 2009/06/17).CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sun XJ, Wang LM, Zhang Y, et al. Role of IRS-2 in insulin and cytokine signalling. Nature. 1995;377(6545):173–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Dearth RK, Cui X, Kim HJ, et al. Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2. Cell Cycle. 2007;6(6):705–13 (Epub 2007/03/20).CrossRefPubMedGoogle Scholar
  6. 6.
    Ikink GJ, Boer M, Bakker ER, et al. IRS4 induces mammary tumorigenesis and confers resistance to HER2-targeted therapy through constitutive PI3K/AKT-pathway hyperactivation. Nat Commun. 2016;7:13567 (Epub 2016/11/23).CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hoxhaj G, Dissanayake K, MacKintosh C. Effect of IRS4 levels on PI 3-kinase signalling. PLoS One. 2013;8(9):e73327 (Epub 2013/09/10).CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Weischenfeldt J, Dubash T, Drainas AP, et al. Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking. Nat Genet. 2017;49(1):65–74 (Epub 2016/11/21).CrossRefPubMedGoogle Scholar
  9. 9.
    Cuevas EP, Escribano O, Chiloeches A, et al. Role of insulin receptor substrate-4 in IGF-I-stimulated HEPG2 proliferation. J Hepatol. 2007;46(6):1089–98 (Epub 2007/02/27).CrossRefPubMedGoogle Scholar
  10. 10.
    Chang F, Lee JT, Navolanic PM, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia. 2003;17(3):590–603.CrossRefPubMedGoogle Scholar
  11. 11.
    White MF. IRS proteins and the common path to diabetes. Am J Physiol Endocrinol Metab. 2002;283(3):E413–22.CrossRefPubMedGoogle Scholar
  12. 12.
    Fantin VR, Wang Q, Lienhard GE, et al. Mice lacking insulin receptor substrate 4 exhibit mild defects in growth, reproduction, and glucose homeostasis. Am J Physiol Endocrinol Metab. 2000;278(1):E127–33.CrossRefPubMedGoogle Scholar
  13. 13.
    Numan S, Russell DS. Discrete expression of insulin receptor substrate-4 mRNA in adult rat brain. Brain Res Mol Brain Res. 1999;72(1):97–102.CrossRefPubMedGoogle Scholar
  14. 14.
    Escribano O, Fernández-Moreno MD, Zueco JA, et al. Insulin receptor substrate-4 signaling in quiescent rat hepatocytes and in regenerating rat liver. Hepatology. 2003;37(6):1461–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Siegel R, Desantis C, Jemal A. Colorectal cancer statistics, 2014. CA Cancer J Clin. 2014;64(2):104–17 (Epub 2014/03/17).CrossRefPubMedGoogle Scholar
  16. 16.
    Mehine M, Kaasinen E, Heinonen HR, et al. Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers. Proc Natl Acad Sci USA. 2016;113(5):1315–20 (Epub 2016/01/19).CrossRefPubMedGoogle Scholar
  17. 17.
    Mertens F, Möller E, Mandahl N, et al. The t(X;6) in subungual exostosis results in transcriptional deregulation of the gene for insulin receptor substrate 4. Int J Cancer. 2011;128(2):487–91.CrossRefPubMedGoogle Scholar
  18. 18.
    Cantarini MC, de la Monte SM, Pang M, et al. Aspartyl-asparagyl beta hydroxylase over-expression in human hepatoma is linked to activation of insulin-like growth factor and notch signaling mechanisms. Hepatology. 2006;44(2):446–57.CrossRefPubMedGoogle Scholar
  19. 19.
    Karrman K, Kjeldsen E, Lassen C, et al. The t(X;7)(q22;q34) in paediatric T-cell acute lymphoblastic leukaemia results in overexpression of the insulin receptor substrate 4 gene through illegitimate recombination with the T-cell receptor beta locus. Br J Haematol. 2009;144(4):546–51 (Epub 2008/11/13).CrossRefPubMedGoogle Scholar
  20. 20.
    Fiddes RJ, Janes PW, Sivertsen SP, et al. Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells. Oncogene. 1998;16(21):2803–13.CrossRefPubMedGoogle Scholar
  21. 21.
    Román ID, Cano-Martínez D, Lobo MV, et al. Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease. Biomarkers. 2017;22(2):133–44 (Epub 2016/11/08).CrossRefPubMedGoogle Scholar
  22. 22.
    Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal Quant Cytol Histol. 2001;23(4):291–9.PubMedGoogle Scholar
  23. 23.
    Cuevas EP, Escribano O, Monserrat J, et al. RNAi-mediated silencing of insulin receptor substrate-4 enhances actinomycin D- and tumor necrosis factor-alpha-induced cell death in hepatocarcinoma cancer cell lines. J Cell Biochem. 2009;108(6):1292–301.CrossRefPubMedGoogle Scholar
  24. 24.
    Jacobs AR, LeRoith D, Taylor SI. Insulin receptor substrate-1 pleckstrin homology and phosphotyrosine-binding domains are both involved in plasma membrane targeting. J Biol Chem. 2001;276(44):40795–802 (Epub 2001/08/28).CrossRefPubMedGoogle Scholar
  25. 25.
    Razzini G, Ingrosso A, Brancaccio A, et al. Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains. Mol Endocrinol. 2000;14(6):823–36.CrossRefPubMedGoogle Scholar
  26. 26.
    Lassak A, Del Valle L, Peruzzi F, et al. Insulin receptor substrate 1 translocation to the nucleus by the human JC virus T-antigen. J Biol Chem. 2002;277(19):17231–8 (Epub 2002/02/27).CrossRefPubMedGoogle Scholar
  27. 27.
    Prisco M, Santini F, Baffa R, et al. Nuclear translocation of insulin receptor substrate-1 by the simian virus 40 T antigen and the activated type 1 insulin-like growth factor receptor. J Biol Chem. 2002;277(35):32078–85 (Epub 2002/06/12).CrossRefPubMedGoogle Scholar
  28. 28.
    Sun H, Tu X, Prisco M, et al. Insulin-like growth factor I receptor signaling and nuclear translocation of insulin receptor substrates 1 and 2. Mol Endocrinol. 2003;17(3):472–86 (Epub 2002/12/06).CrossRefPubMedGoogle Scholar
  29. 29.
    Kabuta T, Hakuno F, Asano T, et al. Insulin receptor substrate-3 functions as transcriptional activator in the nucleus. J Biol Chem. 2002;277(9):6846–51 (Epub 2001/11/27).CrossRefPubMedGoogle Scholar
  30. 30.
    Shimwell NJ, Martin A, Bruton RK, et al. Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway. Oncogene. 2009;28(5):686–97 (Epub 2008/11/24).CrossRefPubMedGoogle Scholar
  31. 31.
    Kosugi S, Hasebe M, Matsumura N, et al. Six classes of nuclear localization signals specific to different binding grooves of importin alpha. J Biol Chem. 2009;284(1):478–85 (Epub 2008/11/10).CrossRefPubMedGoogle Scholar
  32. 32.
    Guzińska-Ustymowicz K, Pryczynicz A, Kemona A, et al. Correlation between proliferation markers: PCNA, Ki-67, MCM-2 and antiapoptotic protein Bcl-2 in colorectal cancer. Anticancer Res. 2009;29(8):3049–52.PubMedGoogle Scholar
  33. 33.
    Otto T, Sicinski P. Cell cycle proteins as promising targets in cancer therapy. Nat Rev Cancer. 2017;17(2):93–115.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Wu A, Chen J, Baserga R. Nuclear insulin receptor substrate-1 activates promoters of cell cycle progression genes. Oncogene. 2008;27(3):397–403 (Epub 2007/08/13).CrossRefPubMedGoogle Scholar
  35. 35.
    Wu S, Zhou B, Xu L, et al. Interaction between nuclear insulin receptor substrate-2 and NF-κB in IGF-1 induces response in breast cancer cells. Oncol Rep. 2010;24(6):1541–50.PubMedGoogle Scholar
  36. 36.
    Giovannone B, Scaldaferri ML, Federici M, et al. Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential. Diabetes Metab Res Rev. 2000;16(6):434–41.CrossRefPubMedGoogle Scholar
  37. 37.
    Li C, Qi L, Bellail AC, et al. PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis. Oncol Lett. 2014;7(5):1673–8 (Epub 2014/03/10).CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Esposito DL, Aru F, Lattanzio R, et al. The insulin receptor substrate 1 (IRS1) in intestinal epithelial differentiation and in colorectal cancer. PLoS One. 2012;7(4):e36190 (Epub 2012/04/27).CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Japanese Society of Gastroenterology 2018

Authors and Affiliations

  • Patricia Sanmartín-Salinas
    • 1
  • María del Val Toledo Lobo
    • 2
  • Fernando Noguerales-Fraguas
    • 3
    • 4
  • Miguel Toro Londoño
    • 1
  • Antonio Jiménez-Ruiz
    • 1
  • Luis Gonzalez Guijarro
    • 1
    • 5
    Email author
  1. 1.Department of System Biology,Unit of Biochemistry and Molecular BiologyUniversity of AlcaláAlcalá de HenaresSpain
  2. 2.Department of Biomedicine and Biotechnology,Unit of Cell BiologyUniversity of AlcaláAlcalá de HenaresSpain
  3. 3.Department of Surgery and Medical and Social SciencesUniversity of AlcaláAlcalá de HenaresSpain
  4. 4.Department of General SurgeryPríncipe de Asturias HospitalAlcalá de HenaresSpain
  5. 5.Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD)BarcelonaSpain

Personalised recommendations