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p63 steps into the limelight: crucial roles in the suppression of tumorigenesis and metastasis

Abstract

The role of p63 in cancer has been an area of intense debate and controversy. Is TP63 (which encodes p63) a tumour suppressor gene or an oncogene? This debate is partly due to the complexity of the gene. There are several p63 isoforms — some with tumour suppressive functions and others with oncogenic functions. In this Opinion article, we focus on the recent advances in understanding p63 biology and its roles in cancer. In this regard, we discuss the role of p63 in multiple stem cell compartments, ageing, in the response to DNA damage and in DNA repair. Finally, we highlight the importance of understanding the interactions between all three p53 family members and the potential impact of this knowledge on cancer therapy and regenerative medicine.

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Figure 1: p53, p63 and p73 sequence and structural similarity.
Figure 2: Extensive interaction between p53 and TAp63 in response to DNA damage.
Figure 3: Mechanisms used by TAp63 to suppress metastasis.
Figure 4: TAp63 and ΔNp63 are expressed in discrete areas of the dermis and epidermis.

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References

  1. Lane, D. P. Cancer p53, guardian of the genome. Nature 358, 15–16 (1992).

    CAS  PubMed  Google Scholar 

  2. Levine, A. J., Hu, W. & Feng, Z. The P53 pathway: what questions remain to be explored? Cell Death Differ. 13, 1027–1036 (2006).

    CAS  PubMed  Google Scholar 

  3. Kaghad, M. et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 90, 809–819 (1997).

    CAS  PubMed  Google Scholar 

  4. Osada, M. et al. Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nature Med. 4, 839–843 (1998).

    CAS  PubMed  Google Scholar 

  5. Schmale, H. & Bamberger, C. A novel protein with strong homology to the tumor suppressor p53. Oncogene 15, 1363–1367 (1997).

    CAS  PubMed  Google Scholar 

  6. Trink, B. et al. A new human p53 homologue. Nature Med. 4, 747–748 (1998).

    PubMed  Google Scholar 

  7. Flores, E. R. et al. Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell 7, 363–373 (2005).

    CAS  PubMed  Google Scholar 

  8. Guo, X. et al. TAp63 induces senescence and suppresses tumorigenesis in vivo. Nature Cell Biol. 11, 1451–1457 (2009).

    CAS  PubMed  Google Scholar 

  9. Su, X. et al. TAp63 suppresses metastasis through coordinate regulation of Dicer and miRNAs. Nature 467, 986–990 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Tomasini, R. et al. TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev. 22, 2677–2691 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Nemajerova, A., Petrenko, O., Trumper, L., Palacios, G. & Moll, U. M. Loss of p73 promotes dissemination of Myc-induced B cell lymphomas in mice. J. Clin. Invest. 120, 2070–2080 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Yang, A. et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol. Cell 2, 305–316 (1998).

    CAS  PubMed  Google Scholar 

  13. Mills, A. A. et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713 (1999).

    CAS  PubMed  Google Scholar 

  14. Yang, A. et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718 (1999).

    CAS  PubMed  Google Scholar 

  15. Flores, E. R. et al. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 416, 560–564 (2002).

    CAS  PubMed  Google Scholar 

  16. Su, X. et al. TAp63 prevents premature aging by promoting adult stem cell maintenance. Cell Stem Cell 5, 64–75 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Yang, A. et al. Relationships between p63 binding, DNA sequence, transcription activity, and biological function in human cells. Mol. Cell 24, 593–602 (2006).

    CAS  PubMed  Google Scholar 

  18. Brady, C. A. et al. Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell 145, 571–583 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, T. et al. Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence. Cell 149, 1269–1283 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Donehower, L. A. et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221 (1992).

    CAS  PubMed  Google Scholar 

  21. Jacks, T. et al. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4, 1–7 (1994).

    CAS  PubMed  Google Scholar 

  22. Davison, T. S. et al. p73 and p63 are homotetramers capable of weak heterotypic interactions with each other but not with p53. J. Biol. Chem. 274, 18709–18714 (1999).

    CAS  PubMed  Google Scholar 

  23. Yang, A. et al. Genome-wide mapping indicates that p73 and p63 co-occupy target sites and have similar dna-binding profiles in vivo. PLoS ONE 5, e11572 (2010).

    PubMed  PubMed Central  Google Scholar 

  24. Helton, E. S., Zhu, J. & Chen, X. The unique NH2-terminally deleted (ΔN) residues, the PXXP motif, and the PPXY motif are required for the transcriptional activity of the ΔN variant of p63. J. Biol. Chem. 281, 2533–2542 (2006).

    CAS  PubMed  Google Scholar 

  25. el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W. & Vogelstein, B. Definition of a consensus binding site for p53. Nature Genet. 1, 45–49 (1992).

    CAS  PubMed  Google Scholar 

  26. Cho, M. S., Chan, I. L. & Flores, E. R. ΔNp63 transcriptionally regulates brachyury, a gene with diverse roles in limb development, tumorigenesis and metastasis. Cell Cycle 9, 2434–2441 (2010).

    CAS  PubMed  Google Scholar 

  27. Lin, Y. L. et al. p63 and p73 transcriptionally regulate genes involved in DNA repair. PLoS Genet. 5, e1000680 (2009).

    PubMed  PubMed Central  Google Scholar 

  28. Vigano, M. A. et al. New p63 targets in keratinocytes identified by a genome-wide approach. EMBO J. 25, 5105–5116 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Koster, M. I., Kim, S., Huang, J., Williams, T. & Roop, D. R. TAp63α induces AP-2γ as an early event in epidermal morphogenesis. Dev. Biol. 289, 253–261 (2006).

    CAS  PubMed  Google Scholar 

  30. Koster, M. I. et al. p63 induces key target genes required for epidermal morphogenesis. Proc. Natl Acad. Sci. USA 104, 3255–3260 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Belloni, L. et al. DNp73α protects myogenic cells from apoptosis. Oncogene 25, 3606–3612 (2006).

    CAS  PubMed  Google Scholar 

  32. Jost, C. A., Marin, M. C. & Kaelin, W. G. Jr. p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature 389, 191–194 (1997).

    CAS  PubMed  Google Scholar 

  33. Di Como, C. J., Gaiddon, C. & Prives, C. p73 function is inhibited by tumor-derived p53 mutants in mammalian cells. Mol. Cell. Biol. 19, 1438–1449 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Gaiddon, C., Lokshin, M., Ahn, J., Zhang, T. & Prives, C. A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain. Mol. Cell. Biol. 21, 1874–1887 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Chen, C., Gorlatova, N., Kelman, Z. & Herzberg, O. Structures of p63 DNA binding domain in complexes with half-site and with spacer-containing full response elements. Proc. Natl Acad. Sci. USA 108, 6456–6461 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Deutsch, G. B. et al. DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer. Cell 144, 566–576 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lill, N. L., Grossman, S. R., Ginsberg, D., DeCaprio, J. & Livingston, D. M. Binding and modulation of p53 by p300/CBP coactivators. Nature 387, 823–827 (1997).

    CAS  PubMed  Google Scholar 

  38. MacPartlin, M. et al. p300 regulates p63 transcriptional activity. J. Biol. Chem. 280, 30604–30610 (2005).

    CAS  PubMed  Google Scholar 

  39. Olsson, A., Manzl, C., Strasser, A. & Villunger, A. How important are post-translational modifications in p53 for selectivity in target-gene transcription and tumour suppression? Cell Death Differ. 14, 1561–1575 (2007).

    CAS  PubMed  Google Scholar 

  40. Zdzalik, M. et al. Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73. Cell Cycle 9, 4584–4591 (2010).

    CAS  PubMed  Google Scholar 

  41. Zeng, X. et al. MDM2 suppresses p73 function without promoting p73 degradation. Mol. Cell. Biol. 19, 3257–3266 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kadakia, M., Slader, C. & Berberich, S. J. Regulation of p63 function by Mdm2 and MdmX. DNA Cell Biol. 20, 321–330 (2001).

    CAS  PubMed  Google Scholar 

  43. Agami, R., Blandino, G., Oren, M. & Shaul, Y. Interaction of c-Abl and p73α and their collaboration to induce apoptosis. Nature 399, 809–813 (1999).

    CAS  PubMed  Google Scholar 

  44. Gong, J. G. et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806–809 (1999).

    CAS  PubMed  Google Scholar 

  45. MacPartlin, M., Zeng, S. X. & Lu, H. Phosphorylation and stabilization of TAp63γ by IκB kinase-β. J. Biol. Chem. 283, 15754–15761 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. White, E. & Prives, C. DNA damage enables p73. Nature 399, 734–737 (1999).

    CAS  PubMed  Google Scholar 

  47. Yuan, Z. M. et al. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 399, 814–817 (1999).

    CAS  PubMed  Google Scholar 

  48. Rossi, M. et al. The E3 ubiquitin ligase Itch controls the protein stability of p63. Proc. Natl Acad. Sci. USA 103, 12753–12758 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Rossi, M. et al. The ubiquitin-protein ligase Itch regulates p73 stability. EMBO J. 24, 836–848 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Li, Y., Peart, M. J. & Prives, C. Stxbp4 regulates ΔNp63 stability by suppression of RACK1-dependent degradation. Mol. Cell. Biol. 29, 3953–3963 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Senoo, M. Manis, J. P., Alt, F. W. & McKeon, F. p63 and p73 are not required for the development and p53-dependent apoptosis of T cells. Cancer Cell 6, 85–89 (2004).

    CAS  PubMed  Google Scholar 

  52. Jacobs, W. B. et al. p63 is an essential proapoptotic protein during neural development. Neuron 48, 743–756 (2005).

    CAS  PubMed  Google Scholar 

  53. Beyer, U., Moll-Rocek, J., Moll, U. M. & Dobbelstein, M. Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes. Proc. Natl Acad. Sci. USA 108, 3624–3629 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Suh, E. K. et al. p63 protects the female germ line during meiotic arrest. Nature 444, 624–628 (2006).

    CAS  PubMed  Google Scholar 

  55. Holembowski, L. et al. While p73 is essential, p63 is completely dispensable for the development of the central nervous system. Cell Cycle 10, 680–689 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Su, X. et al. Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf. EMBO J. 28, 1904–1915 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Flores, E. R. p73 is critical for the persistence of memory. Cell Death Differ. 18, 381–382 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Yang, A. et al. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404, 99–103 (2000).

    CAS  PubMed  Google Scholar 

  59. Wetzel, M. K. et al. p73 regulates neurodegeneration and phospho-tau accumulation during aging and Alzheimer's disease. Neuron 59, 708–721 (2008).

    CAS  PubMed  Google Scholar 

  60. Agostini, M. et al. p73 regulates maintenance of neural stem cell. Biochem. Biophys. Res. Commun. 403, 13–17 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Fujitani, M. et al. TAp73 acts via the bHLH Hey2 to promote long-term maintenance of neural precursors. Curr. Biol. 20, 2058–2065 (2010).

    CAS  PubMed  Google Scholar 

  62. Gonzalez-Cano, L. et al. p73 deficiency results in impaired self renewal and premature neuronal differentiation of mouse neural progenitors independently of p53. Cell Death Dis. 1, e109 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Talos, F. et al. p73 is an essential regulator of neural stem cell maintenance in embryonal and adult CNS neurogenesis. Cell Death Differ. 17, 1816–1829 (2010).

    CAS  PubMed  Google Scholar 

  64. Keyes, W. M. et al. ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis. Cell Stem Cell 8, 164–176 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Di Como, C. J. et al. p63 expression profiles in human normal and tumor tissues. Clin. Cancer Res. 8, 494–501 (2002).

    CAS  PubMed  Google Scholar 

  66. Romano, R. A. et al. ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation. Development 139, 772–782 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Iwakuma, T., Lozano, G. & Flores, E. R. Li-Fraumeni syndrome: a p53 family affair. Cell Cycle 4, 865–867 (2005).

    CAS  PubMed  Google Scholar 

  68. Lang, G. A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861–872 (2004).

    CAS  PubMed  Google Scholar 

  69. Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119, 847–860 (2004).

    CAS  PubMed  Google Scholar 

  70. Adorno, M. et al. A Mutant-p53/Smad complex opposes p63 to empower TGFβ-induced metastasis. Cell 137, 87–98 (2009).

    CAS  PubMed  Google Scholar 

  71. Muller, P. A. et al. Mutant p53 drives invasion by promoting integrin recycling. Cell 139, 1327–1341 (2009).

    PubMed  Google Scholar 

  72. Keyes, W. M. & Mills, A. A. p63: a new link between senescence and aging. Cell Cycle 5, 260–265 (2006).

    CAS  PubMed  Google Scholar 

  73. Keyes, W. M. et al. p63 heterozygous mutant mice are not prone to spontaneous or chemically induced tumors. Proc. Natl Acad. Sci. USA 103, 8435–8440 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Talos, F., Wolff, S., Beyer, U., Dobbelstein, M. & Moll, U. M. Brdm2 - an aberrant hypomorphic p63 allele. Cell Death Differ. 17, 184–186 (2010).

    CAS  PubMed  Google Scholar 

  75. Senoo, M. Pinto, F., Crum, C. P. & McKeon, F. p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell 129, 523–536 (2007).

    CAS  PubMed  Google Scholar 

  76. Ihrie, R. A. et al. Perp is a p63-regulated gene essential for epithelial integrity. Cell 120, 843–856 (2005).

    CAS  PubMed  Google Scholar 

  77. Nylander, K. et al. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J. Pathol. 198, 417–427 (2002).

    CAS  PubMed  Google Scholar 

  78. Rocco, J. W., Leong, C. O., Kuperwasser, N., DeYoung, M. P. & Ellisen, L. W. p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9, 45–56 (2006).

    CAS  PubMed  Google Scholar 

  79. Sniezek, J. C., Matheny, K. E., Westfall, M. D. & Pietenpol, J. A. Dominant negative p63 isoform expression in head and neck squamous cell carcinoma. Laryngoscope 114, 2063–2072 (2004).

    CAS  PubMed  Google Scholar 

  80. Hedvat, C. V. et al. Expression of p63 in diffuse large B-cell lymphoma. Appl. Immunohistochem. Mol. Morphol. 13, 237–242 (2005).

    CAS  PubMed  Google Scholar 

  81. Buza, N., Cohen, P. J., Pei, H. & Parkash, V. Inverse p16 and p63 expression in small cell carcinoma and high-grade urothelial cell carcinoma of the urinary bladder. Int. J. Surg. Pathol. 18, 94–102 (2010).

    PubMed  Google Scholar 

  82. Castillo-Martin, M., Domingo-Domenech, J., Karni-Schmidt, O., Matos, T. & Cordon-Cardo, C. Molecular pathways of urothelial development and bladder tumorigenesis. Urol. Oncol. 28, 401–408 (2010).

    CAS  PubMed  Google Scholar 

  83. Comperat, E. et al. p63 gene expression study and early bladder carcinogenesis. Urology 70, 459–462 (2007).

    PubMed  Google Scholar 

  84. Comperat, E. et al. Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma. A tissue microarray study of 158 cases. Virchows Arch. 448, 319–324 (2006).

    CAS  Google Scholar 

  85. Karni-Schmidt, O. et al. Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression. Am. J. Pathol. 178, 1350–1360 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Stepan, A., Margaritescu, C., Simionescu, C. & Ciurea, R. E-cadherin and p63 immunoexpression in dysplastic lesions and urothelial carcinomas of the bladder. Rom. J. Morphol. Embryol. 50, 461–465 (2009).

    CAS  PubMed  Google Scholar 

  87. Dhillon, P. K. et al. Aberrant cytoplasmic expression of p63 and prostate cancer mortality. Cancer Epidemiol. Biomarkers Prev. 18, 595–600 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Green, D. R. & Kroemer, G. Cytoplasmic functions of the tumour suppressor p53. Nature 458, 1127–1130 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Hanker, L. et al. Clinical relevance of the putative stem cell marker p63 in breast cancer. Breast Cancer Res. Treat. 122, 765–775 (2010).

    CAS  Google Scholar 

  90. Crook, T., Nicholls, J. M., Brooks, L., O'Nions, J. & Allday, M. J. High level expression of ΔN-p63: a mechanism for the inactivation of p53 in undifferentiated nasopharyngeal carcinoma (NPC)? Oncogene 19, 3439–3444 (2000).

    CAS  PubMed  Google Scholar 

  91. Rinne, T., Brunner, H. G. & van Bokhoven, H. p63-associated disorders. Cell Cycle 6, 262–268 (2007).

    CAS  PubMed  Google Scholar 

  92. Cabanillas, M. et al. A novel heterozygous point mutation in the p63 gene in a patient with ectodermal dysplasia associated with B-cell leukemia. Pediatr. Dermatol. 28, 707–710 (2011).

    PubMed  Google Scholar 

  93. Flores, E. R. & Lozano, G. The p53 family grows old. Genes Dev. 26, 1997–2000 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Rufini, A. et al. TAp73 depletion accelerates aging through metabolic dysregulation. Genes Dev. 26, 2009–2014 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Burgess, D. J. Metabolism: TAp63 tips the energy balance? Nature Rev. Cancer 12, 736–737 (2012).

    CAS  Google Scholar 

  96. Su, X. et al. TAp63 is a master transcriptional regulator of lipid and glucose metabolism. Cell Metab. 16, 511–525 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Mani, S. A. et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Beaudry, V. G. et al. Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis. PLoS Genet. 6, e1001168 (2010).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

E.R.F. is a scholar of the Leukemia and Lymphoma Society of America, the Rita Allen Foundation and the V Foundation for Cancer Research. E.R.F. is supported by grants from NCI (R01CA160394), NCI (R01CA134796), CPRIT (RP120124), the Mel Klein Foundation, and the Hildegardo E. and Olga M. Flores Foundation. D.C. is a CPRIT Scholar (RP101502).

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Su, X., Chakravarti, D. & Flores, E. p63 steps into the limelight: crucial roles in the suppression of tumorigenesis and metastasis. Nat Rev Cancer 13, 136–143 (2013). https://doi.org/10.1038/nrc3446

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