Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Yes and Lyn play a role in nuclear translocation of the epidermal growth factor receptor

Oncogene volume 32, pages 759–767 (2013)Cite this article

Subjects

A Correction to this article was published on 05 December 2018

Abstract

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (CtxR) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFKs), and nuclear EGFR had a role in resistance to cetuximab. To better understand SFK-mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated upregulation of the SFK members Yes (v-Yes-1 yamaguchi sarcoma viral oncogene) and Lyn (v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog) in all CtxR clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in CtxR clones, but not in cetuximab-sensitive (CtxS) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR-expressing CtxS parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all CtxR clones exhibited upregulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101), and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101, which influences EGFR nuclear translocation in this model of cetuximab resistance.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

Abbreviations

BCRP:

breast cancer-resistance protein

CRC:

colorectal cancer

CtxR:

cetuximab-resistant

CtxS:

cetuximab-sensitive

DMSO:

dimethyl sulfoxide

EGF:

epidermal growth factor

EGFR:

epidermal growth factor receptor

ER:

endoplasmic reticulum

FBS:

fetal bovine serum

FGFR:

fibroblast growth factor receptor

HNSCC:

head and neck squamous cell carcinoma

iNOS:

inducible nitric oxide synthase

Lyn:

v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog

mAb:

monoclonal antibody

NSCLC:

non-small cell lung cancer

PGFR:

platelet-derived growth factor

PI3K:

phosphatidylinositol 3-kinase

PKC:

protein kinase C

PLCγ:

phospholipase C-gamma

qPCR:

quantitative PCR

RTK:

receptor tyrosine kinase

SFK:

Src-family kinases

STAT:

signal transducers and activators of transcription

Yes:

v-Yes-1 yamaguchi sarcoma viral oncogene

References

  1. Yarden Y, Sliwkowski MX . Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001; 2: 127–137.

    CAS  PubMed  Google Scholar 

  2. Wheeler DL, Dunn EF, Harari PM . Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nat Rev Clin Oncol 2010; 7: 493–507.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Brand TM, Iida M, Wheeler DL . Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab. Cancer Biol Ther 2011; 11: 777–792.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Pao W, Chmielecki J . Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer. Nat Rev Cancer 2010; 10: 760–774.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Gschwind A, Fischer OM, Ullrich A . The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat Rev Cancer 2004; 4: 361–370.

    CAS  PubMed  Google Scholar 

  6. Marmor MD, Skaria KB, Yarden Y . Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys 2004; 58: 903–913.

    CAS  PubMed  Google Scholar 

  7. Wang YN, Yamaguchi H, Hsu JM, Hung MC . Nuclear trafficking of the epidermal growth factor receptor family membrane proteins. Oncogene 2010; 29: 3997–4006.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Brand TM, Iida M, Li C, Wheeler DL . The nuclear epidermal growth factor receptor signaling network and its role in cancer. Discov Med 2011; 12: 419–432.

    PubMed  PubMed Central  Google Scholar 

  9. Carpenter G, Liao HJ . Trafficking of receptor tyrosine kinases to the nucleus. Exp Cell Res 2009; 315: 1556–1566.

    CAS  PubMed  Google Scholar 

  10. Lo HW, Ali-Seyed M, Wu Y, Bartholomeusz G, Hsu SC, Hung MC . Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1. J Cell Biochem 2006; 98: 1570–1583.

    CAS  PubMed  Google Scholar 

  11. Hsu SC, Hung MC . Characterization of a novel tripartite nuclear localization sequence in the EGFR family. J Biol Chem 2007; 282: 10432–10440.

    CAS  PubMed  Google Scholar 

  12. Wang YN, Yamaguchi H, Huo L, Du Y, Lee HJ, Lee HH et al. The translocon Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus. J Biol Chem 2010; 285: 38720–38729.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Liao HJ, Carpenter G . Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression. Mol Biol Cell 2007; 18: 1064–1072.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 2001; 3: 802–808.

    CAS  PubMed  Google Scholar 

  15. Hanada N, Lo HW, Day CP, Pan Y, Nakajima Y, Hung MC . Co-regulation of B-Myb expression by E2F1 and EGF receptor. Mol Carcinog 2006; 45: 10–17.

    CAS  PubMed  Google Scholar 

  16. Lo HW, Hsu SC, Ali-Seyed M, Gunduz M, Xia W, Wei Y et al. Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway. Cancer Cell 2005; 7: 575–589.

    CAS  PubMed  Google Scholar 

  17. Hung LY, Tseng JT, Lee YC, Xia W, Wang YN, Wu ML et al. Nuclear epidermal growth factor receptor (EGFR) interacts with signal transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene expression. Nucleic Acids Res 2008; 36: 4337–4351.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lo HW, Cao X, Zhu H, Ali-Osman F . Cyclooxygenase-2 is a novel transcriptional target of the nuclear EGFR-STAT3 and EGFRvIII-STAT3 signaling axes. Mol Cancer Res 2010; 8: 232–245.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Jaganathan S, Yue P, Paladino DC, Bogdanovic J, Huo Q, Turkson J . A functional nuclear epidermal growth factor receptor, SRC and Stat3 heteromeric complex in pancreatic cancer cells. PLoS One 2011; 6: e19605.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Huang WC, Chen YJ, Li LY, Wei YL, Hsu SC, Tsai SL et al. Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells. J Biol Chem 2011; 286: 20558–20568.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Piccione EC, Lieu TJ, Gentile CF, Williams TR, Connolly AJ, Godwin AK et al. A novel epidermal growth factor receptor variant lacking multiple domains directly activates transcription and is overexpressed in tumors. Oncogene 2012; 31: 2953–2967.

    CAS  PubMed  Google Scholar 

  22. Wang SC, Nakajima Y, Yu YL, Xia W, Chen CT, Yang CC et al. Tyrosine phosphorylation controls PCNA function through protein stability. Nat Cell Biol 2006; 8: 1359–1368.

    CAS  PubMed  Google Scholar 

  23. Dittmann K, Mayer C, Fehrenbacher B, Schaller M, Raju U, Milas L et al. Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase. J Biol Chem 2005; 280: 31182–31189.

    CAS  PubMed  Google Scholar 

  24. Lo HW, Xia W, Wei Y, Ali-Seyed M, Huang SF, Hung MC . Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer. Cancer Res 2005; 65: 338–348.

    CAS  PubMed  Google Scholar 

  25. Hadzisejdic I, Mustac E, Jonjic N, Petkovic M, Grahovac B . Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis. Mod Pathol 2010; 23: 392–403.

    CAS  PubMed  Google Scholar 

  26. Xia W, Wei Y, Du Y, Liu J, Chang B, Yu YL et al. Nuclear expression of epidermal growth factor receptor is a novel prognostic value in patients with ovarian cancer. Mol Carcinog 2009; 48: 610–617.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Psyrri A, Yu Z, Weinberger PM, Sasaki C, Haffty B, Camp R et al. Quantitative determination of nuclear and cytoplasmic epidermal growth factor receptor expression in oropharyngeal squamous cell cancer by using automated quantitative analysis. Clin Cancer Res 2005; 11: 5856–5862.

    CAS  PubMed  Google Scholar 

  28. Li CF, Fang FM, Wang JM, Tzeng CC, Tai HC, Wei YC et al. EGFR nuclear import in gallbladder carcinoma: nuclear phosphorylated EGFR upregulates iNOS expression and confers independent prognostic impact. Ann Surg Oncol 2012; 19: 443–454.

    PubMed  Google Scholar 

  29. Li C, Iida M, Dunn EF, Ghia AJ, Wheeler DL . Nuclear EGFR contributes to acquired resistance to cetuximab. Oncogene 2009; 28: 3801–3813.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Li C, Iida M, Dunn EF, Wheeler DL . Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma. Radiother Oncol 2010; 97: 330–337.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Hsu SC, Miller SA, Wang Y, Hung MC . Nuclear EGFR is required for cisplatin resistance and DNA repair. Am J Transl Res 2009; 1: 249–258.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Liccardi G, Hartley JA, Hochhauser D . EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment. Cancer Res 2011; 71: 1103–1114.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Dittmann K, Mayer C, Kehlbach R, Rodemann HP . Radiation-induced caveolin-1 associated EGFR internalization is linked with nuclear EGFR transport and activation of DNA-PK. Mol Cancer 2008; 7: 69.

    PubMed  PubMed Central  Google Scholar 

  34. Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ . c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J Biol Chem 1999; 274: 8335–8343.

    CAS  PubMed  Google Scholar 

  35. Maa MC, Leu TH, McCarley DJ, Schatzman RC, Parsons SJ . Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers. Proc Natl Acad Sci USA 1995; 92: 6981–6985.

    CAS  PubMed  Google Scholar 

  36. Wheeler DL, Iida M, Dunn EF . The role of Src in solid tumors. Oncologist 2009; 14: 667–678.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim LC, Song L, Haura EB . Src kinases as therapeutic targets for cancer. Nat Rev Clin Oncol 2009; 6: 587–595.

    PubMed  Google Scholar 

  38. Thomas SM, Brugge JS . Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 1997; 13: 513–609.

    CAS  PubMed  Google Scholar 

  39. Bromann PA, Korkaya H, Courtneidge SA . The interplay between Src family kinases and receptor tyrosine kinases. Oncogene 2004; 23: 7957–7968.

    CAS  PubMed  Google Scholar 

  40. Tice DA, Biscardi JS, Nickles AL, Parsons SJ . Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl Acad Sci USA 1999; 96: 1415–1420.

    CAS  PubMed  Google Scholar 

  41. Kloth MT, Laughlin KK, Biscardi JS, Boerner JL, Parsons SJ, Silva CM . STAT5b, a mediator of synergism between c-Src and the epidermal growth factor receptor. J Biol Chem 2003; 278: 1671–1679.

    CAS  PubMed  Google Scholar 

  42. Ottenhoff-Kalff AE, Rijksen G, van Beurden EA, Hennipman A, Michels AA, Staal GE . Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product. Cancer Res 1992; 52: 4773–4778.

    CAS  PubMed  Google Scholar 

  43. Lu KV, Zhu S, Cvrljevic A, Huang TT, Sarkaria S, Ahkavan D et al. Fyn and SRC are effectors of oncogenic epidermal growth factor receptor signaling in glioblastoma patients. Cancer Res 2009; 69: 6889–6898.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Wheeler DL, Iida M, Kruser TJ, Nechrebecki MM, Dunn EF, Armstrong EA et al. Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab. Cancer Biol Ther 2009; 8: 696–703.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang J, Kalyankrishna S, Wislez M, Thilaganathan N, Saigal B, Wei W et al. SRC-family kinases are activated in non-small cell lung cancer and promote the survival of epidermal growth factor receptor-dependent cell lines. Am J Pathol 2007; 170: 366–376.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Fu YN, Yeh CL, Cheng HH, Yang CH, Tsai SF, Huang SF et al. EGFR mutants found in non-small cell lung cancer show different levels of sensitivity to suppression of Src: implications in targeting therapy. Oncogene 2008; 27: 957–965.

    CAS  PubMed  Google Scholar 

  47. Koppikar P, Choi SH, Egloff AM, Cai Q, Suzuki S, Freilino M et al. Combined inhibition of c-Src and epidermal growth factor receptor abrogates growth and invasion of head and neck squamous cell carcinoma. Clin Cancer Res 2008; 14: 4284–4291.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Wheeler DL, Huang S, Kruser TJ, Nechrebecki MM, Armstrong EA, Benavente S et al. Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members. Oncogene 2008; 27: 3944–3956.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Brand TM, Dunn EF, Iida M, Myers RA, Kostopoulos KT, Li C et al. Erlotinib is a viable treatment for tumors with acquired resistance to cetuximab. Cancer Biol Ther 2011; 12: 436–446.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Massie C, Mills IG . The developing role of receptors and adaptors. Nat Rev Cancer 2006; 6: 403–409.

    CAS  PubMed  Google Scholar 

  51. Carpenter G . Nuclear localization and possible functions of receptor tyrosine kinases. Curr Opin Cell Biol 2003; 15: 143–148.

    CAS  PubMed  Google Scholar 

  52. Wang SC, Hung MC . Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors. Clin Cancer Res 2009; 15: 6484–6489.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Lo HW, Hung MC . Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br J Cancer 2006; 94: 184–188.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Zhao Y, He D, Saatian B, Watkins T, Spannhake EW, Pyne NJ et al. Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase Cdelta, Lyn kinase, and matrix metalloproteinases. J Biol Chem 2006; 281: 19501–19511.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Xi S, Zhang Q, Dyer KF, Lerner EC, Smithgall TE, Gooding WE et al. Src kinases mediate STAT growth pathways in squamous cell carcinoma of the head and neck. J Biol Chem 2003; 278: 31574–31583.

    CAS  PubMed  Google Scholar 

  56. Kasai A, Shima T, Okada M . Role of Src family tyrosine kinases in the down-regulation of epidermal growth factor signaling in PC12 cells. Genes Cells 2005; 10: 1175–1187.

    CAS  PubMed  Google Scholar 

  57. Su T, Bryant DM, Luton F, Verges M, Ulrich SM, Hansen KC et al. A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor. Nat Cell Biol 2010; 12: 1143–1153.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Boerner JL, Demory ML, Silva C, Parsons SJ . Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol Cell Biol 2004; 24: 7059–7071.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This project was supported, in part, by grant P30CA014520 from the National Cancer Institute, grant 1UL1RR025011 from the Clinical and Translational Science Award program of the National Center for Research Resources and the National Institutes of Health (DLW) by grant RSG-10-193-01-TBG from the American Cancer Society (DLW), and by NIH grant T32 GM08.1061-01A2 from Graduate Training in Cellular and Molecular Pathogenesis of Human Diseases (TMB).

Author information

Author notes

  1. M Iida and T M Brand: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA

    M Iida, T M Brand, D A Campbell, C Li & D L Wheeler

Authors
  1. M Iida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T M Brand
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D A Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D L Wheeler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D L Wheeler.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Iida, M., Brand, T., Campbell, D. et al. Yes and Lyn play a role in nuclear translocation of the epidermal growth factor receptor. Oncogene 32, 759–767 (2013). https://doi.org/10.1038/onc.2012.90

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.90

Keywords

This article is cited by

Search

Advanced search

Quick links