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The goal of this proposal is to investigate the role of exposure to environmental contaminants and UV in altering enzymes involved in matrix remodeling, specifically the matrix metalloproteinases (MMPs). Metastatic melanoma relies on the expression of these MMPs to destroy the extracellular matrix and basement membrane to allow cell migration. Expression of these enzymes correlates with the invasive potential of melanoma cell lines, and are therefore markers of an unfavorable clinical prognosis. <P>We have chosen to use 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a representative environmental contaminant. TCDD is one of the strongest tumor promoters tested in rodent model systems. TCDD appears non-mutagenic, and it is believed to act by promoting neoplastic transformation in cells that have been initiated. TCDD is an unintentional by-product of industrial combustion, aluminum and coal production, and also of municipal/ medical waste incineration. <P>It is of particular interest due to its resistance to chemical and biological metabolism, and its persistence in biological tissues. TCDD is an agricultural contaminant and accumulates in the adipose tissue of farm animals and aquatic species. Exposure occurs mainly through oral ingestion of exposed animal tissue, and, as it is associated with fat, it can also be passed on through dairy products, such as milk and cheese, from exposed animals.<P> It is concentrated upward through food chain and a human's body burden of TCDD will increase as they age. Therefore, our goal is to investigate a causal link between UV and TCDD exposure on invasiveness of normal human melanocytes and keratinocytes, and to identify the cellular signaling pathways that are involved in mediating melanoma invasion and metastasis. <P>The importance of keratinocyte-melanocyte interactions in metastatic progression will be investigated. Further, these experiments aim to identify novel pathways involved in melanoma progression as potential targets for prevention and chemotherapy. <P>Specifically, we will: <OL> <LI>Demonstrate the importance of the AhR on melanoma invasion and metastasis. <LI>Investigate the interaction between the UVB and AhR signaling pathway on gene expression and melanoma invasion.<LI> Determine the importance of melanocyte-keratinocyte interactions on the invasive characteristics of melanocytes and keratinocytes.
Non-Technical Summary: Melanoma is a significant health concern for New Jersey. A report published by the Center for Disease Control (CDC: MMWR, 1995) documents New Jersey as having one of the highest mortality rates from metastatic melanoma, in addition to being in the top 5 for total melanoma related deaths. Newark Bay has been declared an environmental area of concern due to the high concentrations of environmental chemicals found in the water, sediment and wildlife. Exposure to the PAH/HAH and UVB is a concern for agriculture workers as well, as they spend a considerable amount of time exposed to these environmental stressors. The health effects of the interaction between UVB and xenobiotic compounds are unclear. These studies will provide important mechanistic information on the molecular interaction between the UVB and environmental toxin signaling pathways. In addition, it will further our understanding of the role of environmental exposure in metastatic melanoma. <P> Approach: 1. Demonstrate the importance of the AhR signaling pathway on melanoma invasion and metastasis. We hypothesize that the expression level of AhR relates to MMP expression, as the cells that express the highest levels of AhR demonstrate increased MMP expression upon TCDD exposure. Therefore, we will generate two cell lines that will determine the importance of the AhR in melanoma invasion. First, we will generated a cell line containing a constitutively active form of the AhR (CA-AhR). In addition, we will knock down expression of AhR in the high-AhR A2058 melanoma cell line, to determine whether increased AhR expression is related to these cells invasive potential. Two strategies will be employed to accomplish this. One, we will use short inhibitory RNA (siRNA) to transiently knockdown AhR expression. In addition, we will transfect the cells with a dominant negative AhR (dnAhR) to knockdown its function. Following generation of the cell lines, we will examine the expression and activity of MMPs, as well as on the expression of proteins involved in cell adhesion. We will use quantitative RT-PCR and fluorescent activity assays (BioTrak, Calbiochem) to demonstrate changes in MMP expression and activity. Expression of cell surface markers, including E- and N-cadherin, that are important for cell-cell interaction; the integrins, that are important for cell-matrix interactions; and EMMPRIN, that is a cell-surface protein associated with MMP expression, will be examined. Finally, we will examine changes in invasiveness, using the Fluoroblok Invasion Assays (BD-Falcon). 2. Investigate the interaction between the UV and AhR signaling pathway on gene expression and melanoma invasion. Recent data show that the UV-photoproduct FITZ binds to the AhR with similar affinity as TCDD. These data suggest that UV activation of the AhR pathway may contribute to carcinogenic progression. Therefore the goals of the experiments in this aim is to investigate the effect of UV and TCDD on expression of genes/proteins involved in melanoma progression, as well as their impact of melanoma invasiveness. The cell models generated in specific aim 1 will be used to examine the effect of UV and AhR signaling in melanoma invasion. In addition to endpoints described above, we will also determine whether UV exposure results in changes in cellular proliferation and apoptosis using flow cytometery. 3. Determine the importance of melanocyte-keratinocyte interactions on invasive characteristics. Data from other laboratories demonstrates that cell-cell interaction is important to tumor invasiveness. To investigate the role of melanocyte-keratinocyte interactions on the response to UV irradiation and TCDD, we will co-culture these cell types. Melanocytes and melanoma cells will be mixed with keratinocytes at a 1:10 ratio and seeded onto tissue culture plates in half minimal melanocyte media and half keratinocyte media. Co-cultures will be exposed to UV irradiation, TCDD (10-8M) or both as described in Specific Aim I. Experiments examining MMP expression, cell adhesion markers, proliferation and invasion will be performed as described above.