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BRAFE600-associated senescence-like cell cycle arrest of human naevi

Abstract

Most normal mammalian cells have a finite lifespan1, thought to constitute a protective mechanism against unlimited proliferation2,3,4. This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16INK4a (ref. 5). In cultured cells, senescence can be elicited prematurely by oncogenes6; however, whether such oncogene-induced senescence represents a physiological process has long been debated. Human naevi (moles) are benign tumours of melanocytes that frequently harbour oncogenic mutations (predominantly V600E, where valine is substituted for glutamic acid) in BRAF7, a protein kinase and downstream effector of Ras. Nonetheless, naevi typically remain in a growth-arrested state for decades and only rarely progress into malignancy (melanoma)8,9,10. This raises the question of whether naevi undergo BRAFV600E-induced senescence. Here we show that sustained BRAFV600E expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16INK4a and senescence-associated acidic β-galactosidase (SA-β-Gal) activity, a commonly used senescence marker. Validating these results in vivo, congenital naevi are invariably positive for SA-β-Gal, demonstrating the presence of this classical senescence-associated marker in a largely growth-arrested, neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ, we observed a marked mosaic induction of p16INK4a, suggesting that factors other than p16INK4a contribute to protection against BRAFV600E-driven proliferation. Naevi do not appear to suffer from telomere attrition, arguing in favour of an active oncogene-driven senescence process, rather than a loss of replicative potential. Thus, both in vitro and in vivo, BRAFV600E-expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.

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Figure 1: Sustained expression of BRAF E600 induces a senescence-like arrest in normal human melanocytes.
Figure 2: Physiological levels of BRAF E600 induce senescence-like arrest in normal human fibroblasts in a p16 INK4a -independent manner.
Figure 3: Human melanocytic naevi display the hallmarks of senescent cells.
Figure 4: No apparent telomere loss in naevi.

References

  1. Hayflick, L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37, 614–636 (1965)

    Article  CAS  Google Scholar 

  2. Mathon, N. F. & Lloyd, A. C. Cell senescence and cancer. Nature Rev. Cancer 1, 203–213 (2001)

    Article  CAS  Google Scholar 

  3. Lowe, S. W., Cepero, E. & Evan, G. Intrinsic tumour suppression. Nature 432, 307–315 (2004)

    Article  ADS  CAS  Google Scholar 

  4. Campisi, J. Senescent cells, tumour suppression, and organismal aging: good citizens, bad neighbors. Cell 120, 513–522 (2005)

    Article  CAS  Google Scholar 

  5. Shay, J. W. & Roninson, I. B. Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene 23, 2919–2933 (2004)

    Article  CAS  Google Scholar 

  6. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602 (1997)

    Article  CAS  Google Scholar 

  7. Pollock, P. M. et al. High frequency of BRAF mutations in nevi. Nature Genet. 33, 19–20 (2003)

    Article  CAS  Google Scholar 

  8. Kuwata, T., Kitagawa, M. & Kasuga, T. Proliferative activity of primary cutaneous melanocytic tumours. Virchows Arch. A Pathol. Anat. Histopathol. 423, 359–364 (1993)

    Article  CAS  Google Scholar 

  9. Bennett, D. C. Human melanocyte senescence and melanoma susceptibility genes. Oncogene 22, 3063–3069 (2003)

    Article  CAS  Google Scholar 

  10. Chin, L., Merlino, G. & DePinho, R. A. Malignant melanoma: modern black plague and genetic black box. Genes Dev. 12, 3467–3481 (1998)

    Article  CAS  Google Scholar 

  11. Robinson, W. A. et al. Human acquired naevi are clonal. Melanoma Res. 8, 499–503 (1998)

    Article  CAS  Google Scholar 

  12. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954

  13. Wellbrock, C. et al. V599EB-RAF is an oncogene in melanocytes. Cancer Res. 64, 2338–2342 (2004)

    Article  CAS  Google Scholar 

  14. Mooi, W. J. & Krausz, T. Biopsy Pathology of Melanocytic Disorders 56–105 (Chapman & Hall Medical, London, 1992)

    Google Scholar 

  15. Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995)

    Article  ADS  CAS  Google Scholar 

  16. Sharpless, E. & Chin, L. The INK4a/ARF locus and melanoma. Oncogene 22, 3092–3098 (2003)

    Article  CAS  Google Scholar 

  17. Zhu, J., Woods, D., McMahon, M. & Bishop, J. M. Senescence of human fibroblasts induced by oncogenic Raf. Genes Dev. 12, 2997–3007 (1998)

    Article  CAS  Google Scholar 

  18. Lin, A. W. et al. Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signalling. Genes Dev. 12, 3008–3019 (1998)

    Article  CAS  Google Scholar 

  19. Narita, M. et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113, 703–716 (2003)

    Article  CAS  Google Scholar 

  20. Gruis, N. A. et al. Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds. Nature Genet. 10, 351–353 (1995)

    Article  CAS  Google Scholar 

  21. Bandyopadhyay, D. et al. The human melanocyte: a model system to study the complexity of cellular aging and transformation in non-fibroblastic cells. Exp. Gerontol. 36, 1265–1275 (2001)

    Article  CAS  Google Scholar 

  22. Wang, Y. L., Uhara, H., Yamazaki, Y., Nikaido, T. & Saida, T. Immunohistochemical detection of CDK4 and p16INK4 proteins in cutaneous malignant melanoma. Br. J. Dermatol. 134, 269–275 (1996)

    Article  CAS  Google Scholar 

  23. Kamb, A. et al. A cell cycle regulator potentially involved in genesis of many tumour types. Science 264, 436–440 (1994)

    Article  ADS  CAS  Google Scholar 

  24. Beausejour, C. M. et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 22, 4212–4222 (2003)

    Article  CAS  Google Scholar 

  25. Bastian, B. C. Understanding the progression of melanocytic neoplasia using genomic analysis: from fields to cancer. Oncogene 22, 3081–3086 (2003)

    Article  CAS  Google Scholar 

  26. Miracco, C. et al. Quantitative in situ evaluation of telomeres in fluorescence in situ hybridization-processed sections of cutaneous melanocytic lesions and correlation with telomerase activity. Br. J. Dermatol. 146, 399–408 (2002)

    Article  CAS  Google Scholar 

  27. Peeper, D. S. & Mooi, W. J. Pathogenesis of melanocytic naevi: growth arrest linked with cellular senescence? Histopathology 41, S139–S143 (2002)

    Google Scholar 

  28. Patton, E. E. et al. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr. Biol. 15, 249–254 (2005)

    Article  CAS  Google Scholar 

  29. Hingorani, S. R., Jacobetz, M. A., Robertson, G. P., Herlyn, M. & Tuveson, D. A. Suppression of BRAF(V599E) in human melanoma abrogates transformation. Cancer Res. 63, 5198–5202 (2003)

    CAS  PubMed  Google Scholar 

  30. Peeper, D. S. et al. A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence. Nature Cell Biol. 4, 148–153 (2002)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Atsma, E. Mesman and J. Zevenhoven for help with immunohistochemistry; S. Douma for analytical support; L. Oomen, L. Brocks and J. van Rheenen for help with microscopy; N. Gruis and C. Out for p16INK4a-deficient fibroblasts; L. Zaal and A. van der Wal for help with obtaining congenital naevus specimens; M. Voorhoeve and R. Agami for pRetroSuper, pRetroSuper-Blasticidin and pRetroSuper-GFP; S. Gryaznov for the telomere probe; R. Beijersbergen and M. van Lohuizen for reagents; G. Abou-Rjaily for help with lentiviral infections; P. Krimpenfort and colleagues in the Peeper laboratory for discussions; R. Bernards for support; and M. van Lohuizen and A. Berns for suggestions and reading of the manuscript. M.S.S is supported by an NIH grant. M.S.S. is a V Foundation for Cancer Research Scholar. L.C.W.V., T.K. and D.S.P. were supported by the Netherlands Organization for Scientific Research (NWO).

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Correspondence to Wolter J. Mooi or Daniel S. Peeper.

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Supplementary information

Supplmentary Figure S1

Ectopic overexpression of BRAFE600 causes a senescence-like cell cycle arrest. (PDF 236 kb)

Supplmentary Figure S2a-f

Physiologic levels of BRAFE600 induce senescence in a p16INK4a-independent manner. (PDF 818 kb)

Supplementary Figure 2g-l

Physiologic levels of BRAFE600 induce senescence in a p16INK4a-independent manner. (PDF 436 kb)

Supplmentary Figure S3

BRAFE600 fails to induce p14ARF. (PDF 23 kb)

Supplementary Figure S4

Sustained knockdown of p16INK4a cause an increase in cellular proliferation rate (PDF 48 kb)

Supplementary Figure S5

BRAFE600 mutational analysis in human congenital nevi. (PDF 39 kb)

Supplementary Figure S6

Mosaic pattern of p16INK4a-positivity in nevi. (PDF 152 kb)

Supplementary Figure S7

No apparent telomere loss in nevi. (PDF 648 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (RTF 14 kb)

Supplementary Methods

Additional descriptions of methods used in this study, to accompany those detailed in the main text. (RTF 9 kb)

Supplmentary Table

Tumorigenicity assay. (PDF 42 kb)

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Michaloglou, C., Vredeveld, L., Soengas, M. et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005). https://doi.org/10.1038/nature03890

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