Michael B Mann1,2, Michael A Black3, Devin J Jones1, Jerrold M Ward2,12, Christopher Chin Kuan Yew2,12, Justin Y Newberg1, Adam J Dupuy4, Alistair G Rust5,12, Marcus W Bosenberg6,7, Martin McMahon8,9, Cristin G Print10,11, Neal G Copeland1,2,13, & Nancy A Jenkins1,2,13
1 Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.
2 Institute of Molecular and Cell Biology, Singapore.
3 Department of Biochemistry, University of Otago, Dunedin, New Zealand.
4 Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
5 Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, UK.
6 Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut, USA.
7 Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA.
8 Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco,
San Francisco, California, USA.
9 Department of Cell and Molecular Pharmacology, University of California, San Francisco,
San Francisco, California, USA.
10 Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.
11 New Zealand Bioinformatics Institute, University of Auckland, Auckland, New Zealand.
12 Present addresses: Global VetPathology, Montgomery Village, Maryland, USA (J.M.W.), National Heart Research Institute Singapore, Singapore (C.C.K.Y.) and Institute of Cancer Research, London, UK (A.G.R.).
13 These authors contributed equally to this work.
Correspondence should be addressed to N.A.J. (email@example.com)
Published online in Nature Genetics on 13 April 2015.
Although nearly half of human melanomas harbor oncogenic BrafV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BrafV600E melanoma and discover new genes with potential clinical importance in human melanoma.
Figure Legend: SB-mediated mutagenesis promotes melanoma formation in BrafV600E mutant mice. (a) Pigmentation changes in SB|Braf
mice appear uniform at 25 weeks of age with
all 4-OHT–painted surfaces appearing almost
completely black; this was most obvious on tail skin in comparing sibling mice. Green, yellow
and white asterisks denote Braf, SB|Braf and
SB sibling mice, respectively. (b) Image of the
underside of a dorsal skin specimen after it
was removed during necropsy of an SB|Braf
mouse. In wild-type mice, the dorsal skin was uniformly non-pigmented, but many individual black clones of BrafV600E-positive nevi that
covered 4-OHT–painted surfaces were found
in SB|Braf mice. Scale bar, 5 mm. (c) Kaplan-Meier survival curves comparing experimental SB|Braf and control sibling SB and Braf mice
(log-rank test, P < 0.0001). (d–i) Histology
and tumor classification from sections of skin
masses stained with hematoxylin and eosin (d–f) and undergoing immunohistochemistry (IHC)
analysis (g–i). (d) Melanoma showing both a
loose cell pattern and a more typical melanoma pattern with nerve-like structures (400×).
(e) Melanoma displaying focal schwannomatous features containing pigment granules (400×). (f) Melanoma differentiation focus with tumor cells containing pigment granules (1,000×). (g) S-100 expression in the nucleus of melanoma cells
from an unpigmented melanoma (100×); inset, low magnification depicting robust S-100 expression throughout the tumor and adjacent normal skin lacking S-100 expression. (h) Cytoplasmic staining for the melanocyte-specific protein Tyrp1 using antiserum to Tyrp1 (PEP1) (400×). (i) Robust staining of nuclear SB transposase in SB|Braf melanoma cells (400×). Scale bars, 100 μm (d–l); inset, 3 mm (g).