Advertisement

Histochemistry and Cell Biology

, Volume 134, Issue 3, pp 265–276 | Cite as

Overexpression of YAP1 induces immortalization of normal human keratinocytes by blocking clonal evolution

  • Irene D’Addario
  • Claudia Abbruzzese
  • Marco Lo Iacono
  • Massimo Teson
  • Osvaldo Golisano
  • Virginia BaroneEmail author
Original Paper

Abstract

YAP1 is a transcriptional co-activator able to bind several transcription factors. YAP1 was termed a candidate oncogene after it was shown to be in human chromosome 11q22 amplicon; besides the genomic amplification, several experiments indicated that it has oncogenic function. However, YAP1 was also reported to be a tumor suppressor as its gene locus is deleted in some breast cancers. To clarify the role of this protein in the physiology of rapidly renewal cells, we investigated YAP1 in human keratinocytes. Here, we show that YAP1 overexpression in primary human keratinocytes blocks clonal evolution and induces cell immortalization, but not malignant transformation. YAP1 overexpression led to an increase in cell proliferation, colony forming efficiency and holoclone percentage. Cells escaped from senescence, immortalized but still remained unable to grow in soft agar or express mesenchymal markers, suggesting that YAP1 overexpression is not sufficient to promote a complete epithelial–mesenchymal transition and tumorigenic transformation. Protein analysis showed an increase in epithelial proliferation markers and a decrease in epithelial differentiation markers. The expression of LEKTI, a late differentiation marker, dramatically dropped to undetectable levels. Taken together, these data suggest that YAP1-overexpressing keratinocytes are maintained in the proliferative compartment.

Keywords

YAP1 Yes-associated protein Keratinocyte clonal evolution Epidermis Epithelia 

Notes

Acknowledgments

This work was supported by the Italian Ministero della Sanità.

Conflict of interest

None.

References

  1. Barrandon Y, Green H (1985) Cell size as a determinant of the clone forming ability of human keratinocytes. Proc Natl Acad Sci USA 82:5390–5394CrossRefPubMedGoogle Scholar
  2. Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84:2302–2306CrossRefPubMedGoogle Scholar
  3. Basu S, Totty NF, Irwin MS, Sudol M, Downward J (2003) Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14–3-3 and attenuation of p73-mediated apoptosis. Mol Cell 11:11–23CrossRefPubMedGoogle Scholar
  4. Bitoun E, Micheloni A, Lamant L, Bonnart C, Tartaglia-Polcini A, Cobbold C et al (2003) LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome. Hum Mol Genet 12:2417–2430CrossRefPubMedGoogle Scholar
  5. Bravo R, Frank R, Blundell PA, MacDonald-Bravo H (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase δ. Nature 326:515–517CrossRefPubMedGoogle Scholar
  6. Camargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R et al (2007) YAP1 increases organ size and expands undifferentiated progenitor cells. Curr Biol 17:2054–2060CrossRefPubMedGoogle Scholar
  7. Chatterjee A, Sen T, Chang X, Sidransky D (2010) Yes-associated protein 1 regulates the stability of ΔNp63α. Cell Cycle 9:162–167PubMedGoogle Scholar
  8. Chen HI, Sudol M (1995) The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc Natl Acad Sci USA 92:7819–7823CrossRefPubMedGoogle Scholar
  9. Chen HI, Einbond A, Kwak SJ, Linn H, Koepf E, Peterson S et al (1997) Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands. J Biol Chem 272:17070–17077CrossRefPubMedGoogle Scholar
  10. D’Anna F, De Luca M, Cancedda R, Zicca A, Franzi AT (1988) Elutriation of human keratinocytes and melanocytes from in vitro cultured epithelium. Histochem J 20:674–678CrossRefGoogle Scholar
  11. Danovi SA, Rossi M, Gudmundsdottir K, Yuan M, Melino G, Basu S (2008) Yes-associated protein (YAP) is a critical mediator of c-Jun-dependent apoptosis. Cell Death Differ 15:217–219CrossRefPubMedGoogle Scholar
  12. Dellambra E, Vailly J, Pellegrini G, Bondanza S, Golisano O, Macchia C et al (1998) Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa. Hum Gene Ther 9:1359–1370CrossRefPubMedGoogle Scholar
  13. Dellambra E, Golisano O, Bondanza S, Siviero S, Siviero E, Lacal P, Molinari M et al (2000) Downregulation of 14-3-3 sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes. J Cell Biol 149:1117–1129CrossRefPubMedGoogle Scholar
  14. Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA et al (2007) Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130:1120–1133CrossRefPubMedGoogle Scholar
  15. Hanahan D, Weinberg RA (2000) Review: The hallmarks of cancer. Cell 100:57–70CrossRefPubMedGoogle Scholar
  16. Hao Y, Chun A, Cheung K, Rashidi B, Yang X (2008) Tumor suppressor LATS1 is a negative regulator of oncogene YAP. J Biol Chem 283:5496–5509CrossRefPubMedGoogle Scholar
  17. Huang J, Wu S, Barrera J, Matthews K, Pan D (2005) The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell 122:421–434CrossRefPubMedGoogle Scholar
  18. Kang Y, Massagué J (2004) Epithelial–mesenchymal transitions: twist in development and metastasis. Cell 118:277–279 ReviewCrossRefPubMedGoogle Scholar
  19. King KE, Ponnamperuma RM, Yamashita T, Tokino T, Lee LA, Young MF et al (2003) DeltaNp63alpha functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes. Oncogene 22:3635–3644CrossRefPubMedGoogle Scholar
  20. Komuro A, Nagai M, Navin NE, Sudol M (2003) WW domain-containing protein YAP associates with ErbB-4 and acts as a co-transcriptional activator for the carboxyl-terminal fragment of ErbB-4 that translocates to the nucleus. J Biol Chem 278:33334–33341CrossRefPubMedGoogle Scholar
  21. Koster MI, Roop DR (2004) Review: Genetic pathways required for epidermal morphogenesis. Eur J Cell Biol 83:625–629CrossRefPubMedGoogle Scholar
  22. Lee JM, Dedhar S, Kalluri R, Thompson EW (2006) The epithelial–mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172:973–981CrossRefPubMedGoogle Scholar
  23. Lehrer MS, Sun TT, Lavker RM (1998) Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation. J Cell Sci 111:2867–2875PubMedGoogle Scholar
  24. Levy D, Adamovich Y, Reuven N, Shaul Y (2007) The Yes-associated protein 1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73. Cell Death Differ 14:743–751CrossRefPubMedGoogle Scholar
  25. Livak K-J, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 4:402–408CrossRefGoogle Scholar
  26. Mathor MB, Ferrari G, Dellambra E, Cilli M, Mavilio F, Cancedda R et al (1996) Clonal analysis of stably transduced human epidermal stem cells in culture. Proc Natl Acad Sci USA 93:10371–10376CrossRefPubMedGoogle Scholar
  27. Overholtzer M, Zhang J, Smolen GA, Muir B, Li W, Sgroi DC et al (2006) Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc Natl Acad Sci USA 103:12405–12410CrossRefPubMedGoogle Scholar
  28. Panacchia L, Dellambra E, Bondanza S, Paterna P, Maurelli R, Paionni E, Guerra L (2010) Nonirradiated human fibroblasts and irradiated 3T3–J2 murine fibroblasts as a feeder layer for keratinocyte growth and differentiation in vitro on a fibrin substrate. Cells Tissues Organs 191:21–35CrossRefPubMedGoogle Scholar
  29. Parsa R, Yang A, McKeon F, Green H (1999) Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113:1099–1105CrossRefPubMedGoogle Scholar
  30. Pellegrini G, Golisano O, Paterna P, Lambiase A, Bonini S, Rama P et al (1999) Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 145:769–782CrossRefPubMedGoogle Scholar
  31. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza E et al (2001) p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156–3161CrossRefPubMedGoogle Scholar
  32. Steinhardt AA, Gayyed MF, Klein AP, Dong J, Maitra A, Pan D et al (2008) Expression of Yes-associated protein in common solid tumors. Hum Pathol 39:1582–1589CrossRefPubMedGoogle Scholar
  33. Strano S, Munarriz E, Rossi M, Castagnoli L, Shaul Y, Sacchi A et al (2001) Physical interaction with Yes-associated protein enhances p73 transcriptional activity. J Biol Chem 276:15164–15173CrossRefPubMedGoogle Scholar
  34. Strano S, Monti O, Pediconi N, Baccarini A, Fontemaggi G, Lapi E et al (2005) The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA Damage. Mol Cell 18:447–459CrossRefPubMedGoogle Scholar
  35. Sudol M (1994) Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product. Oncogene 9:2145–2152PubMedGoogle Scholar
  36. Sudol M, Bork P, Einbond A, Kastury K, Druck T, Negrini M et al (1995) Characterization of the mammalian YAP (Yes-associated protein) gene and its role in defining a novel protein module, the WW domain. J Biol Chem 270:14733–14741CrossRefPubMedGoogle Scholar
  37. Tomlinson V, Gudmundsdottir K, Luong P, Leung K-Y, Knebel A, Basu S (2010) JNK phosphorilates Yes-associated protein (YAP) to regulate apoptosis. Cell Death Dis 1:e29CrossRefGoogle Scholar
  38. Watt FM, Green H (1981) Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Biol 90:738–742CrossRefPubMedGoogle Scholar
  39. Yagi R, Chen LF, Shigesada K, Murakami Y, Ito Y (1999) A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator. EMBO J 18:2551–2562CrossRefPubMedGoogle Scholar
  40. Yuan M, Tomlinson V, Lara R, Holliday D, Chelala C, Harada T et al (2008) Yes-associated protein (YAP) functions as a tumor suppressor in breast. Cell Death Differ 15:1752–1759CrossRefPubMedGoogle Scholar
  41. Yuan M, Luong P, Hudson C, Gudmundsdottir K, Basu S (2010) c-Abl phosphorylation of Dnp63a is critical for cell viability. Cell Death Dis 1:e16CrossRefGoogle Scholar
  42. Zender L, Spector MS, Xue W, Flemming P, Cordon-Cardo C, Silke J et al (2006) Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125:1253–1267CrossRefPubMedGoogle Scholar
  43. Zhang J, Smolen GA, Haber DA (2008) Negative regulation of YAP by LATS1 underscores evolutionary conservation of the Drosophila Hippo pathway. Cancer Res 68:2789–2794CrossRefPubMedGoogle Scholar
  44. Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J et al (2007) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev 21:2747–2761CrossRefPubMedGoogle Scholar
  45. Zhao B, Ye X, Yu J, Li L, Li W, Li S et al (2008) TEAD mediates YAP-dependent gene induction and growth control. Genes Dev 22:1962–1971CrossRefPubMedGoogle Scholar
  46. Zhao B, Kim J, Ye X, Lai ZC, Guan KL (2009) Both TEAD-binding and WW domains are required for the growth stimulation and oncogenic transformation activity of yes-associated protein. Cancer Res 69:1089–1098CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Irene D’Addario
    • 1
  • Claudia Abbruzzese
    • 1
  • Marco Lo Iacono
    • 2
  • Massimo Teson
    • 3
  • Osvaldo Golisano
    • 4
  • Virginia Barone
    • 1
    • 5
    Email author
  1. 1.Laboratory of Tissue Engineering and Cutaneous PhysiopathologyIstituto Dermopatico dell’Immacolata, IRCCSRomeItaly
  2. 2.Department of Biological and Clinical SciencesUniversity of TurinTurinItaly
  3. 3.Laboratory of Molecular and Cell BiologyIstituto Dermopatico dell’Immacolata, IRCCSRomeItaly
  4. 4.IDI Farmaceutici SPAPomeziaItaly
  5. 5.Molecular Medicine Section, Department of NeuroscienceUniversity of SienaSienaItaly

Personalised recommendations