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Ultraviolet radiation accelerates BRAF-driven melanomagenesis by targeting TP53

A Corrigendum to this article was published on 21 January 2015

This article has been updated

Abstract

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear1,2. The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event3. To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a BRAF(V600E) mouse model. In mice expressing BRAF(V600E) in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden. Here we show that sunscreen (UVA superior, UVB sun protection factor (SPF) 50) delayed the onset of UVR-driven melanoma, but only provided partial protection. The UVR-exposed tumours showed increased numbers of single nucleotide variants and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in approximately 40% of cases. TP53 is an accepted UVR target in human non-melanoma skin cancer, but is not thought to have a major role in melanoma4. However, we show that, in mice, mutant Trp53 accelerated BRAF(V600E)-driven melanomagenesis, and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans5. Furthermore, we identify TP53/Trp53 as a UVR-target gene that cooperates with BRAF(V600E) to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis. Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

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Figure 1: UVR accelerates BRAF(V600E)-driven naevogenesis.
Figure 2: UVR accelerates BRAF(V600E)-driven melanomagenesis.
Figure 3: Genome analysis of UVR-driven melanomas.
Figure 4: Mutant Trp53 accelerates BRAF(V600E)-driven melanomagenesis.

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Primary accessions

European Nucleotide Archive

Data deposits

Exome sequence and aCGH data have been deposited in the European Nucleotide Archive under study accession number PRJEB6330.

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Acknowledgements

This work was supported by Cancer Research UK (C107/A10433; C5759/A12328; A13540; A17240), the Wenner-Gren Foundations, Stockholm, Teggerstiftelsen (M.P.) and a FEBS Long-Term Fellowship (B.S.-L.). We thank G. Ashton for technical assistance and A. Young for helpful discussions. We would like to acknowledge the contribution of the melanoma specimen donors and research groups to The Cancer Genome Atlas.

Author information

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Authors

Contributions

A.V., B.S.-L. and R.M. designed the study, analysed the data and wrote the paper. M.P. designed and performed experiments and analysed data. S.J.F. designed and performed bioinformatics analysis and analysed data. K.H., J.R., M.R.G., M.C. and N.D. performed experiments. S.E. validated WES SNVs.

Corresponding author

Correspondence to Richard Marais.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Methodology and UVR spectrum.

a, Schematic representation of experimental schedule, showing induction of BRAF(V600E) by tamoxifen at 8 weeks of age followed by weekly exposure to UVR starting 4 weeks later, and for up to 6 months. b, Graph showing spectral radiation distribution for the Waldmann UV6 lamp used in these studies. The UVA and UVB regions are indicated. c, Photomicrograph of Trp53 staining in interfollicular and follicular keratinocytes (arrows) 24 h after UVR exposure. Asterisks indicate hair follicles. Scale bar, 50 μm.

Extended Data Figure 2 UVR induced melanocytic proliferation in BRAF(V600E) mice.

Photomicrographs showing 4′,6-diamidino-2-phenylindole (DAPI), S100 and Ki-67 immunofluorescence staining, together with a merged image in cloth-protected and UVR-exposed areas of the mid-dermis from BRAF(V600E) mice 72 h after UVR exposure. The experiment was repeated in 3 different skin sections of 5 animals. Scale bar, 30 μm.

Extended Data Figure 3 UVR induces naevogenesis in BRAF(V600E) mice.

a, Schematic representation of experimental processing of mouse skin. Three skin sections (long rectangular boxes) were cut perpendicular to the longitudinal axis of the mice to span the UVR-exposed and protected areas (as marked by the dotted line) of the tamoxifen-treated shaved area (pink). b, Photomicrograph of H&E-stained representative skin from the protected and UVR-exposed skin of a tamoxifen-treated BRAF(V600E) mouse subjected to UVR exposure and examined at 7 days (n = 5). Dotted line: UVR treatment line demarcation. Scale bar, 0.4 mm. c, Photomicrograph of H&E-stained skin from the boxed black area in b, showing the cloth-protected skin of a tamoxifen-treated BRAF(V600E) mouse subjected to UVR exposure and examined at 7 days. Black boxes show individual naevi. Double-headed arrow shows example of maximum diameter of a single naevus. Scale bar, 300 µm. d, Photomicrograph of H&E-stained skin from the dashed boxed black area in b, showing the UVR-exposed skin of a tamoxifen-treated BRAF(V600E) mouse subjected to UVR exposure and examined at 7 days. Black boxes show individual naevi. Scale bar, 300 µm. e, Photomicrograph of H&E-stained skin from the cloth-protected and UVR-exposed skin of a tamoxifen-treated CreERT2 control mouse subjected to weekly UVR exposure for 6 months. Scale bar, 300 µm. f, Photograph showing macroscopic appearance of protected and UVR-exposed skin from the tamoxifen-treated CreERT2 control mouse shown in a. Original magnification, ×2.

Extended Data Figure 4 UVR-accelerated BRAF(V600E)-driven tumours have similar histology to BRAF(V600E)-driven tumours from non-UVR-exposed animals.

a, Photomicrograph of an H&E-stained tumour from a UVR-treated BRAF(V600E) mouse highlighting the presence of atypical heterogeneous spindle dendritic and pigment-producing cells (black asterisks and arrow respectively, left panel) and atypical plump spindle cells and mitotic figures (white asterisks and black arrowhead respectively, right panel). Scale bar, 25 µm. b, Photomicrograph of an H&E-stained tumour from a UVR-treated BRAF(V600E) mouse highlighting a region of dermal ulceration (black arrow). Scale bar, 0.5 mm. c, Photomicrograph of H&E-stained tumours from non-UVR exposed (left panel) and UVR-exposed (right panel) BRAF(V600E) mice, showing the presence of nuclear pleomorphism (asterisks) in both tumours. Scale bar, 10 µm. d, Photomicrograph of an S100-stained tumour from a UVR-exposed BRAF(V600E) mouse. Scale bar, 100 µm. e, Photomicrograph of an HMB-45/MelanA-stained tumour from a UVR-exposed BRAF(V600E) mouse. Scale bar, 50 µm. f, Photomicrograph of a Ki-67-stained tumour from a UVR-exposed BRAF(V600E) mouse. Scale bar, 50 µm.

Extended Data Figure 5 Sunscreen blocks the short-term effects of UVR exposure.

a, Photomicrograph showing lack of Trp53 staining in the cloth-protected (Protected) and UVR-exposed, sunscreen-protected (UVR+SS) skin of a BRAF(V600E) mouse after 24 h of UVR exposure (n = 5). Scale bar, 40 µm. b, Photomicrograph of H&E-stained skin from the cloth protected (Protected) and UVR-exposed, sunscreen-protected (UVR+SS) regions of a BRAF(V600E) mouse after 24 h of UVR exposure. Scale bar, 300 µm. c, High-magnification photomicrograph of H&E-stained skin from the cloth protected (Protected) and UVR-exposed sunscreen-protected (UVR+SS) regions of a BRAF(V600E) mouse after 24 h of UVR exposure showing absence of apoptotic keratinocytes. d, Photomicrograph of H&E-stained skin from a cloth-protected (Protected; left), UVA-exposed (UVA; centre), and UVA-exposed, sunscreen-protected (UVA+SS; right) skin of a mouse after 72 h of UVR exposure. Scale bar, 300 µm (n = 5). The photomicrographs below the main images show areas of higher magnification of the images above. Asterisk indicates epidermal hypertrophy; D indicates plump collagen bundles densely located in the dermis. Scale bar, 150 µm.

Extended Data Figure 6 UVA-induced epidermal and dermal thickening after UVA radiation is abrogated by the use of sunscreen.

a, Photomicrographs of H&E-stained epidermis from the cloth-protected (Protected) and UVR-exposed, sunscreen-protected (UVR+SS) skin of a BRAF(V600E) mouse 7 days (d) after UVR exposure showing the similarity in the number and size of naevi in the two regions (n = 5). Scale bar, 300 µm. b, Photographs showing the macroscopic appearance of the cloth-protected (Protected) and UVR-exposed, sunscreen-protected (UVR+SS) skin from a BRAF(V600E) mouse 5 months after UVR. c, Photomicrographs of S100, HMB-45/MelanA- and Ki-67-stained tumours from UVR-exposed, sunscreen-protected (UVR+SS) mice. Scale bar, 50 µm.

Extended Data Figure 7 Tumours with a Trp53/TP53 mutation.

a, Boxplot graph showing median number of SNVs per Mb (SNVs per Mb) in UVR-exposed mutant Trp53, or UVR-exposed wild-type Trp53 (WT-Trp53) tumours. NS, not significant (Mann–Whitney test). b, Photomicrographs of S100, HMB-45/MelanA- and Ki-67-stained tumour sections from a BRAF(V600E)/Trp53(R172H) mouse. Scale bar, 50 µm. c, Kaplan–Meier plot showing melanoma-free survival in control mice (CreERT2; black line); BRAF(V600E) mice (BRAF(V600E) blue line); and BRAF(V600E) mice crossed with PTENflox/+ mice (BRAF(V600E)/ PTENflox/+; orange line). d, Boxplot graphs showing proportion of C-to-T (G-to-A) transitions in human primary melanomas from the TCGA data set (left panel), human metastatic melanomas from the TCGA data set (middle) and human metastatic melanomas from the Yale University data set (right) harbouring wild-type TP53 (TP53 WT) or mutations in TP53 (TP53 mut); WSRT.

Extended Data Table 1 UVR induces Trp53 and apoptosis in epidermal keratinocytes
Extended Data Table 2 Quantification of naevi
Extended Data Table 3 Summary of Trp53 mutations in UVR-exposed BRAF(V600E) mouse tumours and the TP53 mutation context in humans

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Viros, A., Sanchez-Laorden, B., Pedersen, M. et al. Ultraviolet radiation accelerates BRAF-driven melanomagenesis by targeting TP53. Nature 511, 478–482 (2014). https://doi.org/10.1038/nature13298

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