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Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells: correlation with ErbB2 and p66 Shc expression

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Abstract

Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r=0.91, p < 0.0002) and PY-ErbB2 levels (r=0.89, p=0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r=− 0.86, p=0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.

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References

  1. Flick MB, Sapi E, Perrotta PL, Maher MG, Halaban R, Carter D, Kacinski BM: Recognition of activated CSF-1 receptor in breast carcinomas by a tyrosine 723 phosphospecific antibody. Oncogene 14: 2553–2561, 1997

    Google Scholar 

  2. Kacinski BM, Scata KA, Carter D, Lee LD, Sapi E, King BL, Chambers SK, Jones MA, Pirro MH, Stanley ER et al.: FMS (CSF-1 receptor) and CSF-1 transcripts and protein are expressed by human breast carcinomas in vivo and in vitro. Oncogene 6: 941–952, 1991

    Google Scholar 

  3. Luqmani YA, Graham M, Coombes RC: Expression of basic fibroblast growth factor, FGFR1 and FGFR2 in normal and malignant human breast, and comparison with other normal tissues. Br J Cancer 66: 273–280, 1992

    Google Scholar 

  4. Tuck AB, Park M, Sterns EE, Boag A, Elliott BE: Coexpression of hepatocyte growth factor and receptor (Met) in human breast carcinoma. Am J Pathol 148: 225–232, 1996

    Google Scholar 

  5. Kannan S, De Santis M, Lohmeyer M et al.: Cripto enhances the tyrosine phosphorylation of Shc and activates mitogenactivated protein kinase (MAPK) in mammary epithelial cells. J Biol Chem 272: 3330–3335, 1997

    Google Scholar 

  6. Dickson RB, Lippman ME: Growth factors in breast cancer. Endocr Rev 16: 559–589, 1995

    Google Scholar 

  7. Hines SJ, Organ C, Kornstein MJ, Krystal GW: Coexpression of the c-kit and stem cell factor genes in breast carcinomas. Cell Growth Differ 6: 769–779, 1995

    Google Scholar 

  8. Alimandi M, Romano A, Curia MC, Muraro R, Fedi P, Aaronson SA, Di FP, Kraus MH: Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 10: 1813–1821, 1995

    Google Scholar 

  9. Carraway KL, Cantley LC: A neu acquaintance for ErbB3 and ErbB4: a role for receptor heterodimerization in growth signaling. Cell 78: 5–8, 1994

    Google Scholar 

  10. Burke CL, Lemmon MA, Coren BA, Engelman DM, Stern DF: Dimerization of the p185neu transmembrane domain is necessary but not sufficient for transformation. Oncogene 14: 687–696, 1997

    Google Scholar 

  11. Earp HS, Dawson TL, Li X, Yu H: Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research. Breast Cancer Res Treat 35: 115–132, 1995

    Google Scholar 

  12. Karunagaran D, Tzahar E, Beerli RR, Chen X, Graus-Porta D, Ratzkin BJ, Seger R, Hynes NE, Yarden Y: ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 15: 254–264, 1996

    Google Scholar 

  13. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177–182, 1987

    Google Scholar 

  14. Hynes N: Amplification and overexpression of the ErbB-2 gene in human tumors: its involvement in tumor development, significance as a prognostic factor, and potential as a target for cancer therapy. Sem Cancer Biol 4: 19–26, 1993

    Google Scholar 

  15. McGuirc W, Tandon A, Allred D, Chamness G, Clark G: How to use prognostic factors in axillary node-negative breast cancer patients. J Natl Cancer Inst 82: 1006–1012, 1990

    Google Scholar 

  16. Bradbury J, Arno J, Edwards P: Induction of epithelial abnormalities that resemble human breast lesions by the expression of the neu/erbB-2 oncogene in reconstituted mouse mammary gland. Oncogene 8: 1551–1558, 1993

    Google Scholar 

  17. Hudziak R, Schlessinger J, Ullrich A: Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH3T3 cells. Proc Natl Acad Sci 84: 7159–7163, 1987

    Google Scholar 

  18. Guy C, Webster M, Schaller M, Parsons T, Cardiff R, Muller W: Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci 89: 10578–10582, 1992

    Google Scholar 

  19. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, Nicoletti I, Grignani F, Pawson T, Pelicci PG: A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 70: 93–104, 1992

    Google Scholar 

  20. Segatto O, Pelicci G, Giuli S, Digiesi G, DiFiore PP, McGlade J, Pawson T, Pelicci PG: She products are substrates of erbB-2 kinase. Oncogene 8: 2105–2112, 1993

    Google Scholar 

  21. Janes PW, Daly RJ, deFazio A, Sutherland RL: Activation of the Ras signalling pathway in human breast cancer cells overexpressing erbB-2. Oncogene 9: 3601–3608, 1994

    Google Scholar 

  22. Xie Y, Pendergast AM, Hung MC: Dominant-negative mutants of Grb2 induced reversal of the transformed phenotypes caused by the point mutation-activated rat HER-2/Neu. J Biol Chem 270: 30717–30724, 1995

    Google Scholar 

  23. Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG et al., Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature 360: 689–692, 1992

    Google Scholar 

  24. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA: Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation [see comments]. Nature 363: 45–51, 1993

    Google Scholar 

  25. Li N, Batzer A, Daly R, Yajnik V, Skolnik E, Chardin P, BarSagi D, Margolis B, Schlessinger J: Guanine-nucleotide releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature 363: 45–51, 1993

    Google Scholar 

  26. Migliaccio E, Mele S, Salcini AE, Pelicci G, Lai KM, Superti-Furga G, Pawson T, Di Fiore PP, Lanfrancone L, Pelicci PG: Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. Embo J 16: 706–716, 1997

    Google Scholar 

  27. Pawson T: Protein modules and signalling networks. Nature 373: 573–580, 1995

    Google Scholar 

  28. Blaikie P, Immanuel D, Wu J, Li N, Yajnik V, Margolis B: A region in She distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors. J Biol Chem 269: 32031–32034, 1994

    Google Scholar 

  29. Kavanaugh WM, Williams LT: An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science 266: 1862–1865, 1994

    Google Scholar 

  30. Batzer AG, Rotin D, Urena JM, Skolnik EY, Schlessinger J: Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol 14: 5192–5201, 1994

    Google Scholar 

  31. Van der Geer P, Pawson T: The PTB domain: a new protein module implicated in signal transduction. Trends Biochem Sci 20: 277–280, 1995

    Google Scholar 

  32. Bork P, Margolis B: A phosphotyrosine interaction domain. Cell 80: 693–694, 1995

    Google Scholar 

  33. Salcini AE, McGlade J, Pelicci G, Nicoletti I, Pawson T, Pelicci PG: Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins. Oncogene 9: 2827–2836, 1994

    Google Scholar 

  34. Ravichandran KS, Lorenz U, Shoelson SE, Burakoff SJ: Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol Cell Biol 15: 593–600, 1995

    Google Scholar 

  35. Clark S, Stern M, Horovitz H: C. elegans cell signalling gene Sem-5 encodes a protein with SH2 and SH3 domains. Nature 356: 593–600, 1992

    Google Scholar 

  36. Maignan S, Guilloteau JP, Fromage N, Arnoux B, Becquart J, Ducruix A: Crystal structure of the mammalian Grb2 adaptor. Science 268: 291–293, 1995

    Google Scholar 

  37. Buday L, Downward J: Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adaptor protein, and Sos nucleotide exchange factor. Cell 73: 611–620, 1993

    Google Scholar 

  38. Skolnik EY, Lee CH, Batzer A, Vicentini LM, Zhou M, Daly R, Myers MJ Jr, Backer JM, Ullrich A, White MF et al.: The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc, implications for insulin control of ras signalling. EMBO J 12: 1929–1936, 1993

    Google Scholar 

  39. Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D: The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1 [see comments]. Nature 363: 83–85, 1993

    Google Scholar 

  40. Sasaoka T, Langlois WJ, Leitner JW, Draznin B, Olefsky JM: The signaling pathway coupling epidermal growth factor receptors to activation of p21ras. J Biol Chem 269: 32621–32625, 1994

    Google Scholar 

  41. Bacus SS, Chin D, Yarden Y, Zelnick CR, Stern DF: Type 1 receptor tyrosine kinases are differentially phosphorylated in mammary carcinoma and differentially associated with steroid receptors. Am J Pathol 148: 549–558, 1996

    Google Scholar 

  42. Kraus M, Fedi P, Starks V, Muraro R, Aaronson S: Demonstration of ligand-dependent signalling by the erbB-3 tyrosine kinase and its constitutive activation in human breast tumor cells. Proc Natl Acad Sci 90: 2900–2904, 1993

    Google Scholar 

  43. Dougall WC, Qian X, Peterson NC, Miller MJ, Samanta A, Greene MI: The neu-oncogene: signal transduction path-ways, transformation mechanisms and evolving therapies. Oncogene 9: 2109–2123, 1994

    Google Scholar 

  44. Hynes NE, Stern DF: The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1198: 165–184, 1994

    Google Scholar 

  45. Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203–212, 1990

    Google Scholar 

  46. Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, Shepard HM: Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother 37: 255–263, 1993

    Google Scholar 

  47. Watanabe T, Shintani A, Nakata M, Shing Y, Folkman J, Igarashi K, Sasada R: Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction. J Biol Chem 269: 9966–9973, 1994

    Google Scholar 

  48. Arteaga CL, Hurd SD, Dugger TC, Winnier AR, Robertson JB: Epidermal growth factor receptors in human breast carcinoma cells: a potential selective target for transforming growth factor alpha-Pseudomonas exotoxin 40 fusion protein. Cancer Res 54: 4703–4709, 1994

    Google Scholar 

  49. Xie Y, Li K, Hung MC: Tyrosine phosphorylation of Shc proteins and formation of Shc/Grb2 complex correlate to the transformation of NIH3T3 cells mediated by the point-mutation activated neu. Oncogene 10: 2409–2413, 1995

    Google Scholar 

  50. Bonfini L, Migliaccio E, Pelicci G, Lanfrancone L, Pelicci PG: Not all Shc's roads lead to Ras. Trends Biochem Sci 21: 257–261, 1996

    Google Scholar 

  51. Pelicci G, Lanfrancone L, Salcini AE, Romano A, Mele S, Grazia Borrello M, Segatto O, Di Fiore PP, Pelicci PG: Constitutive phosphorylation of Shc proteins in human tumors. Oncogene 11: 899–907, 1995

    Google Scholar 

  52. Levitzki A, Gazit A: Tyrosine kinase inhibition: an approach to drug development. Science 267: 1782–1788, 1995

    Google Scholar 

  53. Clark JW, Santos-Moore A, Stevenson LE, Frackelton AR Jr: Effects of tyrosine kinase inhibitors on the proliferation of human breast cancer cell lines and proteins important in the Ras signaling pathway. Int J Cancer 65: 186–191, 1996

    Google Scholar 

  54. DiGiovanna MP, Stern DF: Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor. Cancer Res 55: 1946–1955, 1995

    Google Scholar 

  55. Wallasch C, Weiss FU, Niederfellner G, Jallal B, Issing W, Ullrich A: Heregulin-dependent regulation of HER2/neu oncogenic signalling by heterodimerization with HER3. EMBO J 14: 4267–4275, 1995

    Google Scholar 

  56. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F: Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 15: 1292–1300, 1996

    Google Scholar 

  57. Souttou B, Hamelin R, Crepin M: FGF2 as an autocrine growth factor for immortal human breast epithelial cells. Cell Growth Differ 5: 615–623, 1994

    Google Scholar 

  58. Sivaraman V, Wang H, Nuovo G, Malbon C: Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest 99: 1478–1483, 1997

    Google Scholar 

  59. Daly RJ, Binder MD, Sutherland RL: Overexpression of the Grb2 gene in human breast cancer cell lines. Oncogene 9: 2723–2727, 1994

    Google Scholar 

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Brown University, ???

    Lisa E. Stevenson

  2. Department of Medicine, Brown University and Roger Williams Medical Center, Providence, Rhode Island, USA

    A. Raymond Frackelton Jr.

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Stevenson, L.E., Frackelton, A.R. Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells: correlation with ErbB2 and p66 Shc expression. Breast Cancer Res Treat 49, 119–128 (1998). https://doi.org/10.1023/A:1006007227747

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