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Cell and Tissue Research

, Volume 351, Issue 1, pp 59–72 | Cite as

Epidermal growth factor-induced modulation of cytokeratin expression levels influences the morphological phenotype of head and neck squamous cell carcinoma cells

  • Galina Makarova
  • Michael Bette
  • Ansgar Schmidt
  • Ralf Jacob
  • Chengzhong Cai
  • Fiona Rodepeter
  • Thomas Betz
  • Johannes Sitterberg
  • Udo Bakowsky
  • Roland Moll
  • Andreas Neff
  • Andreas Sesterhenn
  • Afshin Teymoortash
  • Matthias Ocker
  • Jochen A. Werner
  • Robert MandicEmail author
Regular Article

Abstract

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Keywords

Head and neck squamous cell carcinoma Epidermal growth factor receptor Cytokeratin Morphology Human 

Notes

Acknowledgments

Technical assistance of Ms. Roswitha Peldszus and Ms. Grazyna Sadowski (both Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Marburg, Germany) and Ms. Viktoria Morokina (Institute of Pathology, University Hospital Giessen and Marburg GmbH, Campus Marburg, Marburg, Germany) was greatly appreciated.

Supplementary material

441_2012_1500_Fig10_ESM.jpg (8 mb)

(JPEG 8229 kb)

References

  1. Astanina K, Jacob R (2010) KIF5C, a kinesin motor involved in apical trafficking of MDCK cells. Cell Mol Life Sci 67:1331–1342PubMedCrossRefGoogle Scholar
  2. Beil M, Micoulet A, von Wichert G, Paschke S, Walther P, Omary MB, Van Veldhoven PP, Gern U, Wolff-Hieber E, Eggermann J, Waltenberger J, Adler G, Spatz J, Seufferlein T (2003) Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells. Nat Cell Biol 5:803–811PubMedCrossRefGoogle Scholar
  3. Bowden PE, Haley JL, Kansky A, Rothnagel JA, Jones DO, Turner RJ (1995) Mutation of a type II keratin gene (K6a) in pachyonychia congenita. Nat Genet 10:363–365PubMedCrossRefGoogle Scholar
  4. Cameron BR, Berean KW (2003) Cytokeratin subtypes in thyroid tumours: immunohistochemical study with emphasis on the follicular variant of papillary carcinoma. J Otolaryngol 32:319–322PubMedCrossRefGoogle Scholar
  5. Chao SC, Tsai YM, Yang MH, Lee JY (2003) A novel mutation in the keratin 4 gene causing white sponge naevus. Br J Dermatol 148:1125–1128PubMedCrossRefGoogle Scholar
  6. Chung CH, Parker JS, Karaca G, Wu J, Funkhouser WK, Moore D, Butterfoss D, Xiang D, Zanation A, Yin X, Shockley WW, Weissler MC, Dressler LG, Shores CG, Yarbrough WG, Perou CM (2004) Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression. Cancer Cell 5:489–500PubMedCrossRefGoogle Scholar
  7. Chung CH, Ely K, McGavran L, Varella-Garcia M, Parker J, Parker N, Jarrett C, Carter J, Murphy BA, Netterville J, Burkey BB, Sinard R, Cmelak A, Levy S, Yarbrough WG, Slebos RJ, Hirsch FR (2006) Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas. J Clin Oncol 24:4170–4176PubMedCrossRefGoogle Scholar
  8. Dobrossy L (2005) Epidemiology of head and neck cancer: magnitude of the problem. Cancer Metastasis Rev 24:9–17PubMedCrossRefGoogle Scholar
  9. Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3:362–374PubMedCrossRefGoogle Scholar
  10. Fuchs E (1996) The cytoskeleton and disease: genetic disorders of intermediate filaments. Annu Rev Genet 30:197–231PubMedCrossRefGoogle Scholar
  11. Garrod DR (1995) Desmosomes and cancer. Cancer Surv 24:97–111PubMedGoogle Scholar
  12. Gigi O, Geiger B, Eshhar Z, Moll R, Schmid E, Winter S, Schiller DL, Franke WW (1982) Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody. EMBO J 1:1429–1437PubMedGoogle Scholar
  13. Hammerl P, Hartl A, Thalhamer J (1993) Particulate nitrocellulose as a solid phase for protein immobilization in immuno-affinity chromatography. J Immunol Methods 165:59–66PubMedCrossRefGoogle Scholar
  14. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674PubMedCrossRefGoogle Scholar
  15. Hatzfeld M, Franke WW (1985) Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol 101:1826–1841PubMedCrossRefGoogle Scholar
  16. Herrmann H, Aebi U (2004) Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular scaffolds. Annu Rev Biochem 73:749–789PubMedCrossRefGoogle Scholar
  17. Hesse M, Zimek A, Weber K, Magin TM (2004) Comprehensive analysis of keratin gene clusters in humans and rodents. Eur J Cell Biol 83:19–26PubMedCrossRefGoogle Scholar
  18. Jiang CK, Magnaldo T, Ohtsuki M, Freedberg IM, Bernerd F, Blumenberg M (1993) Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16. Proc Natl Acad Sci U S A 90:6786–6790PubMedCrossRefGoogle Scholar
  19. Kolsch A, Windoffer R, Leube RE (2009) Actin-dependent dynamics of keratin filament precursors. Cell Motil Cytoskeleton 66:976–985PubMedCrossRefGoogle Scholar
  20. Krohn V, Wiegand S, Werner JA, Mandic R (2011) EGFR codon 497 polymorphism—implications for receptor sensitivity to inhibitors in HNSCC cell lines. Anticancer Res 31:59–65PubMedGoogle Scholar
  21. Ku NO, Liao J, Omary MB (1998) Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins. EMBO J 17:1892–1906PubMedCrossRefGoogle Scholar
  22. Ku NO, Strnad P, Zhong BH, Tao GZ, Omary MB (2007) Keratins let liver live: mutations predispose to liver disease and crosslinking generates Mallory–Denk bodies. Hepatology 46:1639–1649PubMedCrossRefGoogle Scholar
  23. Kuropkat C, Lippert BM, Werner JA (2002) Follow-up with serum Cyfra 21-1 in patients with squamous cell carcinomas of the head and neck. Oncology 63:280–285PubMedCrossRefGoogle Scholar
  24. Lansford C, Grenman R, Bier H, Somers KD, Kim S-Y, Whiteside TL, Clayman G, Carey TE (1999) Head and neck cancers. In: Masters J (ed) Human cell culture vol 2, cancer cell lines part 2. Kluwer Academic Press, Dordrecht, pp 185–255Google Scholar
  25. Lee CH, Coulombe PA (2009) Self-organization of keratin intermediate filaments into cross-linked networks. J Cell Biol 186:409–421PubMedCrossRefGoogle Scholar
  26. Liao J, Lowthert LA, Ku NO, Fernandez R, Omary MB (1995) Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues. J Cell Biol 131:1291–1301PubMedCrossRefGoogle Scholar
  27. Mandic R, Eikelkamp N, Peldszus R, Sadowski M, Sesterhenn AM, Dunne AA, Werner JA (2001) Variations of EGF-R surface expression in squamous cell carcinomas of the head and neck region. Anticancer Res 21:3413–3418PubMedGoogle Scholar
  28. Mandic R, Schamberger CJ, Muller JF, Geyer M, Zhu L, Carey TE, Grenman R, Dunne AA, Werner JA (2005) Reduced cisplatin sensitivity of head and neck squamous cell carcinoma cell lines correlates with mutations affecting the COOH-terminal nuclear localization signal of p53. Clin Cancer Res 11:6845–6852PubMedCrossRefGoogle Scholar
  29. Mandic R, Rodgarkia-Dara CJ, Zhu L, Folz BJ, Bette M, Weihe E, Neubauer A, Werner JA (2006) Treatment of HNSCC cell lines with the EGFR-specific inhibitor cetuximab (Erbitux) results in paradox phosphorylation of tyrosine 1173 in the receptor. FEBS Lett 580:4793–4800PubMedCrossRefGoogle Scholar
  30. Mandic R, Rodgarkia-Dara CJ, Krohn V, Wiegand S, Grenman R, Werner JA (2009) Cisplatin resistance of the HNSCC cell line UT-SCC-26A can be overcome by stimulation of the EGF-receptor. Anticancer Res 29:1181–1187PubMedGoogle Scholar
  31. McLean WH, Rugg EL, Lunny DP, Morley SM, Lane EB, Swensson O, Dopping-Hepenstal PJ, Griffiths WA, Eady RA, Higgins C (1995) Keratin 16 and keratin 17 mutations cause pachyonychia congenita. Nat Genet 9:273–278PubMedCrossRefGoogle Scholar
  32. Mertens C, Hofmann I, Wang Z, Teichmann M, Sepehri CS, Schnolzer M, Franke WW (2001) Nuclear particles containing RNA polymerase III complexes associated with the junctional plaque protein plakophilin 2. Proc Natl Acad Sci U S A 98:7795–7800PubMedCrossRefGoogle Scholar
  33. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24PubMedCrossRefGoogle Scholar
  34. Moll R, Holzhausen HJ, Mennel HD, Kuhn C, Baumann R, Taege C, Franke WW (2006) The cardiac isoform of alpha-actin in regenerating and atrophic skeletal muscle, myopathies and rhabdomyomatous tumors: an immunohistochemical study using monoclonal antibodies. Virchows Arch 449:175–191PubMedCrossRefGoogle Scholar
  35. Moll R, Divo M, Langbein L (2008) The human keratins: biology and pathology. Histochem Cell Biol 129:705–733PubMedCrossRefGoogle Scholar
  36. Omary MB, Coulombe PA, McLean WH (2004) Intermediate filament proteins and their associated diseases. N Engl J Med 351:2087–2100PubMedCrossRefGoogle Scholar
  37. Oshima RG, Baribault H, Caulin C (1996) Oncogenic regulation and function of keratins 8 and 18. Cancer Metastasis Rev 15:445–471PubMedCrossRefGoogle Scholar
  38. Prieto VG, Lugo J, McNutt NS (1996) Intermediate- and low-molecular-weight keratin detection with the monoclonal antibody MNF116. An immunohistochemical study on 232 paraffin-embedded cutaneous lesions. J Cutan Pathol 23:234–241PubMedCrossRefGoogle Scholar
  39. Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE (2007) Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 104:973–978PubMedCrossRefGoogle Scholar
  40. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302:1704–1709PubMedCrossRefGoogle Scholar
  41. Rogers MA, Edler L, Winter H, Langbein L, Beckmann I, Schweizer J (2005) Characterization of new members of the human type II keratin gene family and a general evaluation of the keratin gene domain on chromosome 12q13.13. J Investig Dermatol 124:536–544PubMedCrossRefGoogle Scholar
  42. Schmidt A, Jager S (2005) Plakophilins—hard work in the desmosome, recreation in the nucleus? Eur J Cell Biol 84:189–204PubMedCrossRefGoogle Scholar
  43. Schmidt A, Langbein L, Rode M, Pratzel S, Zimbelmann R, Franke WW (1997) Plakophilins 1a and 1b: widespread nuclear proteins recruited in specific epithelial cells as desmosomal plaque components. Cell Tissue Res 290:481–499PubMedCrossRefGoogle Scholar
  44. Schwarz J, Ayim A, Schmidt A, Jager S, Koch S, Baumann R, Dunne AA, Moll R (2006) Differential expression of desmosomal plakophilins in various types of carcinomas: correlation with cell type and differentiation. Hum Pathol 37:613–622PubMedCrossRefGoogle Scholar
  45. Schweizer J, Bowden PE, Coulombe PA, Langbein L, Lane EB, Magin TM, Maltais L, Omary MB, Parry DA, Rogers MA, Wright MW (2006) New consensus nomenclature for mammalian keratins. J Cell Biol 174:169–174PubMedCrossRefGoogle Scholar
  46. Sesterhenn AM, Mandic R, Dunne AA, Werner JA (2005) Cytokeratins 6 and 16 are frequently expressed in head and neck squamous cell carcinoma cell lines and fresh biopsies. Anticancer Res 25:2675–2680PubMedGoogle Scholar
  47. Smith FJ, Jonkman MF, van Goor H, Coleman CM, Covello SP, Uitto J, McLean WH (1998) A mutation in human keratin K6b produces a phenocopy of the K17 disorder pachyonychia congenita type 2. Hum Mol Genet 7:1143–1148PubMedCrossRefGoogle Scholar
  48. Sterz CM, Kulle C, Dakic B, Makarova G, Bottcher MC, Bette M, Werner JA, Mandic R (2010) A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9. Oral Oncol 46:116–122PubMedCrossRefGoogle Scholar
  49. van der Velden LA, Schaafsma HE, Manni JJ, Link M, Ruiter DJ, Ramaekers FC, Kuijpers W (1996) Cytokeratin and vimentin expression in normal epithelium and benign lesions of the vocal cords. Acta Otolaryngol 116:325–331PubMedCrossRefGoogle Scholar
  50. Weichselbaum RR, Dunphy EJ, Beckett MA, Tybor AG, Moran WJ, Goldman ME, Vokes EE, Panje WR (1989) Epidermal growth factor receptor gene amplification and expression in head and neck cancer cell lines. Head Neck 11:437–442PubMedCrossRefGoogle Scholar
  51. Windoffer R, Beil M, Magin TM, Leube RE (2011) Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia. J Cell Biol 194:669–678PubMedCrossRefGoogle Scholar
  52. Yamashiro Y, Takei K, Umikawa M, Asato T, Oshiro M, Uechi Y, Ishikawa T, Taira K, Uezato H, Kariya K (2010) Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma. Biochem Biophys Res Commun 399:365–372PubMedCrossRefGoogle Scholar
  53. Yanagawa T, Yoshida H, Yamagata K, Onizawa K, Tabuchi K, Koyama Y, Iwasa S, Shimoyamada H, Harada H, Omura K (2007) Loss of cytokeratin 13 expression in squamous cell carcinoma of the tongue is a possible sign for local recurrence. J Exp Clin Cancer Res 26:215–220PubMedGoogle Scholar
  54. Yiasemides E, Trisnowati N, Su J, Dang N, Klingberg S, Marr P, Melbourne W, Tran K, Chow CW, Orchard D, Varigos G, Murrell DF (2008) Clinical heterogeneity in recessive epidermolysis bullosa due to mutations in the keratin 14 gene, KRT14. Clin Exp Dermatol 33:689–697PubMedCrossRefGoogle Scholar
  55. Zhao M, Sano D, Pickering CR, Jasser SA, Henderson YC, Clayman GL, Sturgis EM, Ow TJ, Lotan R, Carey TE, Sacks PG, Grandis JR, Sidransky D, Heldin NE, Myers JN (2011) Assembly and initial characterization of a panel of 85 genomically validated cell lines from diverse head and neck tumor sites. Clin Cancer Res 17:7248–7264PubMedCrossRefGoogle Scholar
  56. Zhu L, Werner JA, Mandic R (2007) Implications of tropomyosin-related kinase B (TrkB) in head and neck cancer. Anticancer Res 27:3121–3126PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Galina Makarova
    • 1
  • Michael Bette
    • 2
  • Ansgar Schmidt
    • 3
  • Ralf Jacob
    • 4
  • Chengzhong Cai
    • 1
  • Fiona Rodepeter
    • 1
  • Thomas Betz
    • 5
  • Johannes Sitterberg
    • 5
  • Udo Bakowsky
    • 5
  • Roland Moll
    • 3
  • Andreas Neff
    • 6
  • Andreas Sesterhenn
    • 1
  • Afshin Teymoortash
    • 1
  • Matthias Ocker
    • 7
  • Jochen A. Werner
    • 1
  • Robert Mandic
    • 1
    Email author
  1. 1.Department of Otorhinolaryngology, Head and Neck SurgeryUniversity Hospital Giessen and MarburgMarburgGermany
  2. 2.Department of Molecular Neuroscience, Institute of Anatomy and Cell BiologyPhilipps UniversityMarburgGermany
  3. 3.Institute of PathologyPhilipps University of MarburgMarburgGermany
  4. 4.Department of Cell Biology and Cell PathologyPhilipps UniversityMarburgGermany
  5. 5.Department of Pharmaceutical Technology and BiopharmacyPhilipps UniversityMarburgGermany
  6. 6.Department of Oral and Maxillofacial Surgery, University Hospital Giessen and MarburgCampus Marburg, Medical Faculty of Philipps UniversityMarburgGermany
  7. 7.Institute for Surgical ResearchPhilipps UniversityMarburgGermany

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