[CANCER RESEARCH 62, 6724 – 6730, November 15, 2002] Ink4a/Arf Deficiency Promotes Ultraviolet Radiation-induced Melanomagenesis 1 Juan A. Recio, Frances P. Noonan, Hisashi Takayama, 2 Miriam R. Anver, Paul Duray, Walter L. Rush, Gerd Lindner, Edward C. De Fabo, Ronald A. DePinho, and Glenn Merlino 3 Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892-4264 [J. A. R., H. T., G. M.]; Laboratory of Photobiology and Photoimmunology, Departments of Immunology and Dermatology, George Washington University Medical School, Washington, DC 20037 [F. P. N., E. C. D. F.]; Pathology/Histotechnology Laboratory, Science Applications International Corporation, National Cancer Institute at Frederick, Maryland 21702-1201 [M. R. A.]; Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892 [P. D.]; Department of Dermatopathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000 [W. L. R.]; Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany D-20246 [G. L.]; and Department of Adult Oncology, Medicine and Genetics, Dana- Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [R. A. D.] ABSTRACT Cutaneous malignant melanoma (CMM), already known for its highly aggressive behavior and resistance to conventional therapy, has evolved into a health crisis by virtue of a dramatic elevation in incidence. The underlying genetic basis for CMM, as well as the fundamental role for UV radiation in its etiology, is now widely accepted. However, the only bona fide genetic locus to emerge from extensive analysis of CMM suppressor candidates is INK4a/ARF at 9p21, which is lost frequently in familial and occasionally in somatic CMM. The functional relationship between INK4a/ARF and UV radiation in the pathogenesis of CMM is largely unknown. Recently, we reported that hepatocyte growth factor/scatter factor (HGF/SF)-transgenic mice develop melanomas after a single ery- themal dose of neonatal UV radiation, supporting epidemiological data implicating childhood sunburn in CMM. Here we show that neonatal UV irradiation induces a full spectrum of melanocyte pathology from early premalignant lesions through distant metastases. Cutaneous melanomas arise with histopathological and molecular pathogenetic features remark- ably similar to CMM, including loss of ink4a/arf. A role for ink4a/arf in UV-induced melanomagenesis was directly assessed by placing the HGF/SF transgene on a genetic background devoid of ink4a/arf. Median time to melanoma development induced by UV radiation was only 50 days in HGF/SF ink4a/arf / mice, compared with 152 and 238 days in HGF/SF ink4a/arf / and HGF/SF ink4a/arf / mice, respectively. These studies provide experimental evidence that ink4a/arf plays a critical role in UV-induced melanomagenesis and strongly suggest that sunburn is a highly significant risk factor, particularly in families harboring germ-line mutations in INK4a/ARF. INTRODUCTION CMM 4 is a highly aggressive, potentially fatal malignancy often resistant to currently available therapy. The recent alarming elevation in incidence of CMM (1, 2) has emphasized the need to understand the molecular mechanisms by which melanoma develops in humans and the risks that influence that process. It is well appreciated that such risks include both genetic and environmental factors (3, 4). Documentation of germ-line and somatic mutations in familial and sporadic CMM has unambiguously identified INK4a/ARF at 9p21 as a melanoma susceptibility locus, whereas a variety of other candidates remain to be discovered or confirmed (reviewed in Refs. 4 – 6). The INK4a/ARF locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19arf in mice), encoding two unre- lated proteins in alternative reading frames (7). Acting through pRB and p53, respectively, these factors help regulate transit through the cell cycle, as well as cellular senescence and apoptosis (reviewed in Ref. 8). Data obtained from human tumors have implicated loss of p16INK4a, and with it pRB function, as the most significant muta- tional event at this locus in melanomagenesis (reviewed in Ref. 4). In sporadic CMM, where INK4a/ARF mutations are less frequent, func- tional loss of p16INK4a can also occur through epigenetic mecha- nisms such as promoter hypermethylation (9, 10). Germ-line muta- tions in cyclin-dependent kinase 4 that prevent binding to p16INK4a also abolish normal pRB-mediated cell cycle control, which could explain susceptibility to CMM in those families (11). Transgenic mouse models have been used to test these hypotheses. Interestingly, although ink4a/arf -/- mice did not exhibit melanoma susceptibility (12), expression of activated H-ras on an Ink4a/Arf-deficient back- ground produced a high incidence of melanoma with a relatively short latency (13). p16ink4a-specific gene targeting can also facilitate mela- nomagenesis, particularly after exposure to 7,12-dimethylbenz(a)an- thracene (14, 15). Mice expressing the cyclin-dependent kinase 4 R24C allele identified in melanoma-prone kindreds are susceptible to spontaneous and 7,12-dimethylbenz(a)anthracene-initiated melanoma (16, 17). Receptor tyrosine kinases play an important role in melanocyte function and melanoma development, including the HGF/SF receptor c-Met (reviewed in Refs. 4, 18 –20). HGF/SF can serve as a multi- functional regulator of proliferation, motility, and morphogenesis in cells expressing c-MET (reviewed in Refs. 21, 22) and is required for the development of liver, placenta, and skeletal muscle (23–25). HGF/SF has been shown to stimulate growth and invasiveness and associated angiogenesis in tumor cells (reviewed in Refs. 26, 27). HGF/SF-Met autocrine loops have been detected in numerous human tumor types, including melanoma (21, 27, 28). The c-MET proto- oncogene resides at chromosome 7q33, a region amplified in human melanoma (29, 30), where it has been implicated in metastatic pro- gression (31, 32). Transgenic mice broadly expressing HGF/SF de- velop a number of tumor types, demonstrating its potential as an oncogenic agent (33). At a mean onset age of 21 months, HGF/SF- transgenic mice develop melanoma, 15% of which acquire a met- astatic phenotype (33, 34). With respect to environmental factors, exposure to the UV spec- trum of solar radiation is thought to be a causal agent in at least 80% of CMM (3, 35, 36). Retrospective epidemiological data currently suggest that unlike other skin cancers that are associated with cumu- lative lifetime UV exposure, CMM is provoked by intense intermittent exposure to UV, particularly during childhood (37, 38). However, the functional relationship between genes and the environment in mela- noma pathogenesis is not well understood. These circumstances are fueled, at least in part, by the lack of a suitable genetically tractable, UV-dependent mouse melanoma model. Melanoma-prone transgenic mouse models have been previously described (reviewed in Ref. 39; see above), and some have demonstrated sensitivity to UV irradiation (40, 41); however, responses have been relatively inefficient. More- Received 6/17/02; accepted 10/3/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported, in part, by NIH Award CA-92258 (to F. P. N.) and the National Cancer Institute under Contract NOI-CO-56000. 2 Present address: Department of Internal Medicine, Fujioka General Hospital, 942-1 Fujioka, Gunma 375-8503, Japan. 3 To whom requests for reprints should be addressed, at Laboratory of Molecular Biology, National Cancer Institute, Building 37, Room 5002, Bethesda, MD 20892-4264. Phone: (301) 496-4270; Fax: (301) 480-7618; E-mail: gmerlino@helix.nih.gov. 4 The abbreviations used are: CMM, cutaneous malignant melanoma; HGF/SF, hepa- tocyte growth factor/scatter factor; RB, retinoblastoma; SED, standard erythemal dose; TRP, tyrosinase-related protein. 6724 Research. on September 5, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from