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/scatter factor (HGF/SF). 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). There has been controversy surrounding the relative risks associated with UV-B versus UV-A radiation. We used albino HGF/SF 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 clearly that in pigmented HGF/SF-transgenic mice, UV-A is highly melanomagenic (Noonan et al., submitted), demonstrating the melanin is a double-edged sword with respect to melanoma risk. 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. Using animal- and cell culture-based models of melanoma we found that oncogene-induced senescence, a barrier against early tumor progression, can be overcome by a deficiency in 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. 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. Therapeutics designed to restore wild-type p53 function may be insufficient to counter cancers in which ARF holds significant p53-independent tumor suppressor activity (Ha et al., Cell Cycle 7:1944-8, 2008). The downstream targets of ARF are currently being investigated, along with its role in regulating differentiation. We recently compared the oncogenic roles of the 3 major NRas downstream effectors, Raf, PI3K and Ral guanine exchange factor (RalGEF), using our Arf-deficient immortalized mouse melanocytes as a model system (Mishra et al., Oncogene 29:2249-2256, 2010). Although no single downstream pathway could recapitulate all the consequences of oncogenic NRas, we found a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. We also discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be evaluated as a possible therapeutic target. 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. 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 cannot reproduce the in vivo microenvironment. To determine the role(s) of UV in melanoma in vivo, we have developed an experimental mouse model (iDCT-GFP) that allows melanocytes, specifically and inducibly labeled with green fluorescent protein (GFP), to be highly purified from disaggregated mouse skin by FACS following UV irradiation in vivo. We have 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 remodeling-associated destruction. We have identified an interferon (IFN)-gamma signaling signature arising in melanocytes after neonatal UV irradiation. The source was macrophages recruited to the skin after UV exposure;IFN-gamma in turn activated melanocytes and the expression of genes that could facilitate immunoevasive mechanisms. Transplanted neonatal macrophages were found to significantly enhance melanoma growth in vivo in an IFN-gamma-dependent fashion. This was surprising considering that IFN-alpha has been used to treat melanoma patients, albeit with very limited success. We hypothesize that melanomas may escape immune destruction by co-opting these pathways already hard-wired in melanocytes, and suggest that the IFN-gamma signaling pathway may represent a promising new therapeutic target for melanoma patients. This work was recently published in Nature (Zaidi et al., Nature 469:548-553, 2011). We are also subjecting in vivo exposed melanocytes to RNA sequencing to try to identify genomic targets of UV radiation. We will use fluorescence activated cell sorting to isolate GFP-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. We have also hypothesized that late stage melanoma cells can co-opt pathways hard-wired into normal developing melanoblasts to achieve a more aggressive and metastatic phenotype. Both the embryonic melanoblast and the metastatic melanoma cell must undergo a similar EMT and become invasive, highly migratory, and survive to colonize at a remote sites. We again employed our iDCT-GFP model to isolate melanoblasts from key stages of melanocyte development. RNA sequencing and microarray-based gene expression profiling will be performed representing each developmental stage. Genes will be identified whose expression is characteristically up-regulated (or down-regulated) in both melanoblasts and metastatic melanoma relative to adult melanocytes, which may represent new therapeutic targets against metastasis in melanoma.
| Huang, Liping; Qin, Yifei; Zuo, Qiang et al. (2018) Ezrin mediates both HGF/Met autocrine and non-autocrine signaling-induced metastasis in melanoma. Int J Cancer 142:1652-1663 |
| Yu, Yanlin; Dai, Meng; Lu, Andrew et al. (2018) PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation. Oncogene 37:2225-2236 |
| Zuo, Qiang; Liu, Jing; Huang, Liping et al. (2018) AXL/AKT axis mediated-resistance to BRAF inhibitor depends on PTEN status in melanoma. Oncogene 37:3275-3289 |
| Harris, Melissa L; Fufa, Temesgen D; Palmer, Joseph W et al. (2018) A direct link between MITF, innate immunity, and hair graying. PLoS Biol 16:e2003648 |
| Michael, Helen T; Merlino, Glenn (2017) A Topical Solution to the Sunless Tanning Problem. Trends Mol Med 23:771-773 |
| Leachman, Sancy A; Merlino, Glenn (2017) Medicine: The final frontier in cancer diagnosis. Nature 542:36-38 |
| Mishra, Prasun J; Mishra, Pravin J; Merlino, Glenn (2016) Integrated Genomics Identifies miR-32/MCL-1 Pathway as a Critical Driver of Melanomagenesis: Implications for miR-Replacement and Combination Therapy. PLoS One 11:e0165102 |
| Merlino, Glenn; Herlyn, Meenhard; Fisher, David E et al. (2016) The state of melanoma: challenges and opportunities. Pigment Cell Melanoma Res 29:404-16 |
| Cataisson, Christophe; Michalowski, Aleksandra M; Shibuya, Kelly et al. (2016) MET signaling in keratinocytes activates EGFR and initiates squamous carcinogenesis. Sci Signal 9:ra62 |
| Day, Chi-Ping; Merlino, Glenn; Van Dyke, Terry (2015) Preclinical mouse cancer models: a maze of opportunities and challenges. Cell 163:39-53 |
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