The Cancer Modeling Section seeks to elucidate the complex molecular/genetic program governing tumor genesis and progression through the development and analysis of genetically engineered mouse models of human cancer. Our efforts in this regard are focused primarily on cutaneous malignant melanoma. Exposure to ultraviolet (UV) radiation is a causal agent in the vast majority of melanoma. Retrospective epidemiological data have suggested that melanoma is provoked by intermittent, intense exposure to UV, particularly during childhood. Previously, we tested this hypothesis in transgenic mice in which the receptor tyrosine kinase MET was deregulated by virtue of ectopic expression of its ligand, hepatocyte growth factor. We discovered that a single neonatal dose of burning UV radiation in these mice was necessary and sufficient to induce tumors reminiscent of human melanoma with shortened latency (Noonan et al., Nature 413: 271-2, 2001). A critical role for the INK4A/ARF locus, which helps regulate the pRb and p53 pathways and is widely regarded as a key melanoma suppressor in human patients, was confirmed in our animal model by demonstrating that UV-induced melanoma was significantly accelerated in Ink4a/Arf-deficient mice (Recio et al., Cancer Res. 62: 6724-30, 2002). These results strongly suggest that sunburn is a highly significant risk factor in kindreds harboring germline mutations in INK4A/ARF (Merlino and Noonan, Trends Mol. Med. 9: 102-8, 2003). Hepatocyte growth factor transgenic mice harboring an oncogenic mutation in CDK4, carried by some melanoma-prone kindreds, also exhibited highly accelerated melanoma genesis (Tormo et al., Am. J. Pathol. 169: 665-72, 2006). There has been controversy surrounding the relative risks associated with UV-B versus UV-A radiation. We used albino hepatocyte growth factor transgenic mouse to show that UV-B, but not UV-A, alone is able to induce the full melanoma phenotype (DeFabo et al., Cancer Res. 64: 6372-6, 2004). Remarkably, our most recent results indicate that in pigmented hepatocyte growth factor transgenic mice, UV-A is highly melanomagenic (unpublished data). The relevance of the p53- and pRb-tumor suppressor pathways to the development of most, if not all types of cancer is unequivocal. However, critical questions remain concerning the relative roles of specific p53- and pRb-pathway members, and how these roles vary among tumors arising from distinct cellular lineages. In particular, functional differences between p53 and its indirect positive regulator ARF in the suppression of tumorigenesis have not been clarified; p53 is degraded by MDM2, an activity that can be blocked by ARF. Enigmatically, although the most commonly mutated gene in human cancer, p53 does not appear to play a significant role in the development of melanoma. In contrast to p53, ARF is frequently inactivated in melanoma through loss at the INK4A/ARF locus, long associated with heritable susceptibility to melanoma, as discussed above. Using a series of animal- and cell culture-based models of melanoma we have found that oncogene-induced senescence, a barrier against early tumor progression, can be overcome by a deficiency in the tumor suppressor ARF, but not p53, facilitating rapid development of melanoma (Ha et al., Proc. Natl. Acad. Sci. 104: 10968-73, 2007). Accordingly, oncogenic NRAS was found to collaborate with a deficiency in ARF, but not p53, to fully transform melanocytes in vitro. We also discovered that ARF helps regulates p53-independent melanocyte senescence by blocking E2F1 function. Taken together, our data help explain in human melanoma the relative abundance and paucity of mutations in ARF and TP53, respectively, and demonstrate that ARF and p53, although linked in a common pathway, suppress tumorigenesis through diverse, lineage-dependent mechanisms. Thus, therapeutics currently being designed to restore wild-type p53 function may be insufficient to counter melanoma and other malignancies in which ARF holds significant p53-independent tumor suppressor activity (Ha et al., Cell Cycle 7:1944-8, 2008). Although an extensive accumulation of epidemiological evidence supports a fundamental role for UV in melanoma, the specific UV-affected molecular pathways and mechanisms remain largely unidentified, limiting the development of successful prevention and treatment strategies. In fact, few specific UV signature mutations such as C to T transitions have been found in genes thought to contribute to melanoma. These uncertainties suggest that mechanisms other than UV-induced DNA mutagenesis may be involved in melanoma initiation. These mechanisms have been difficult to study because UV irradiation of cultured melanocytic cells simply cannot reproduce events in vivo. It is extremely difficult to gauge what levels of UV are comparable to what cells experience in vivo, and more importantly there is no microenvironment in cultured cells, which absolutely must play a role in any response to UV. To help determine the role(s) of UV in melanoma in vivo, we have recently developed an experimental mouse model that allows melanocytes, specifically and inducibly labeled with green fluorescent protein, to be highly purified from disaggregated mouse skin by fluorescence activated cell sorting following UV irradiation in vivo. We have already identified a pattern of UV-B induced gene expression changes in melanocytes isolated from mice that are consistent with inflammatory alterations and may spare melanocytes post-UV destruction. We hypothesize that melanoma may escape immune destruction by co-opting such pathways. We are also subjecting in vivo exposed melanocytes to deep sequencing to try to identify genomic targets of UV radiation. We will use fluorescence activated cell sorting to isolate green fluorescent protein-labeled melanocytes from all stages of melanoma development in an attempt to catalog their precise genomic alterations. We anticipate that this in vivo model will provide novel insights into the nature of UV-induced damage, and the mechanisms by which UV provokes melanoma.
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