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

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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.

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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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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

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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