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Use of human tissue to assess the oncogenic activity of melanoma-associated mutations

Nature Genetics volume 37, pages 745–749 (2005)Cite this article

Abstract

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence1,2. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification3,4,5. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification6,7,8. Melanomas also commonly show impairment of the p16INK4A-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16INK4A and ARF protein loss5,9,10,11. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification5,12. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations5,13. TERT amplification also occurs in melanoma5. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

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Figure 1: Expression of mutant proteins in human melanocytes.
Figure 2: Invasive human melanocytic neoplasia.
Figure 3: Effect of disrupting function of p53 and Rb pathways and the impact of hTERT.
Figure 4: Capacity of active PI3K and B-Raf to recapitulate Ras-driven human melanocytic neoplasia.

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Acknowledgements

We thank D. Felsher, A. Oro, S. Artandi, H.Y. Chang, S. Swetter, H. Lee, Z. Siprashvili, F. Scholl, J. Reuter, T. Ridky and P. Dumesic for presubmission review and discussions; T. Cai for PI3K reagents; B. Vogelstein and K. Kinzler for p53 reporter constructs; M. Mihm, S. Kohler and J. Harvell for pathologic evaluation; S. Tao for technical support; L. Attardi for PERP antibody; and R. Marais for B-RafV599E and advice. This work was supported by the US Veterans Affairs Office of Research and Development, by the National Institutes of Arthritis and Musculoskeletal and Skin Diseases (US National Institutes of Health). Y.C. is a Howard Hughes Medical Institute predoctoral fellow. A.E.A. was supported by a training grant from the National Institutes of Arthritis and Musculoskeletal and Skin Diseases (US National Institutes of Health) and a Warren-Whitman-Richardson Fellowship.

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Author notes

  1. Yakov Chudnovsky and Amy E Adams: These authors contributed equally to this work.

Authors and Affiliations

  1. Veterans Affairs Palo Alto Healthcare System, Palo Alto, California 94304, USA and Program in Epithelial Biology, Stanford University School of Medicine, 269 Campus Drive, Room 2145, Stanford, 94305, California, USA

    Yakov Chudnovsky, Amy E Adams, Paul B Robbins, Qun Lin & Paul A Khavari

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  1. Yakov Chudnovsky
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  2. Amy E Adams
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Corresponding author

Correspondence to Paul A Khavari.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Proliferation and apoptosis of human melanocytes after gene transfer. (PDF 325 kb)

Supplementary Fig. 2

Melanocyte neoplasms are vascularized. (PDF 207 kb)

Supplementary Fig. 3

Confirmation of expression of transferred genes. (PDF 498 kb)

Supplementary Fig. 4

Mosaic marker gene expression in genetically induced melanocytic neoplasms. (PDF 167 kb)

Supplementary Fig. 5

Local invasion but lack of metastasis with A375 human melanoma cell line. (PDF 267 kb)

Supplementary Fig. 6

Immunoblots of p53 targets and Rb. (PDF 151 kb)

Supplementary Fig. 7

Mitotic activity in vivo as a function of hTERT transduction. (PDF 218 kb)

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Chudnovsky, Y., Adams, A., Robbins, P. et al. Use of human tissue to assess the oncogenic activity of melanoma-associated mutations. Nat Genet 37, 745–749 (2005). https://doi.org/10.1038/ng1586

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