The focus of this dissertation is to identify alterations in melanomas that influence the tumor microenvironment to promote melanoma progression. Under normal conditions, type I collagen, which constitutes ∼90% of the dermal extracellular matrix maintains tissue integrity and homeostasis.
1. Loss of Krüppel-like factor 6 uncouples signaling from the collagen I rich extracellular environment of the skin to promote early melanoma development. Early stage melanoma cells need to overcome inhibitory signals present in skin in order to invade into the deeper dermal layers, ultimately culminating in metastasis. To identify genes regulating this process, fragments of 10p15, a chromosomal region frequently lost in early melanoma, were transferred into melanoma cells. Significantly smaller tumors formed by these cells having fewer proliferating cells and alterations in extracellular matrix organization resulting in a phenotype resembling more benign-like tumors with looping collagen I surrounding nests of tumor cells. To identify the gene on 10p15, an in vitro collagen I gel model that mimicked the skin microenvironment was established by plating cells onto type I collagen in culture dishes. As observed in animals, type I collagen inhibited proliferation of melanoma cells containing the 10p15 region but not those lacking it. In contrast, growth on plastic or anchorage independent growth did not differ. KLF6 (Krüppel-like factor 6), which is functionally inactivated in several human cancers was present on the transferred 10p15 fragment suggesting it may be the putative candidate suppressor gene.
2. Macrophage inhibitory cytokine-1 regulated the tumorigenic and metastatic potential of melanoma. Melanoma cells secrete various growth factors and cytokines that can deregulate surrounding cells, which are important for promoting proliferation, invasion and angiogenesis. Macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor-β superfamily cytokine, is elevated in blood serum as well as in cells of cancers patients, but its function in melanoma development remains unknown. This study unravels the role played by MIC-1 in melanoma development. Melanoma patient tumors and serum were examined for MIC-1 levels by western blot analysis and ELISA. MIC-1 protein expression was significantly elevated in 67% of melanoma tumors compared to normal melanocytes. Furthermore, melanoma patients had high MIC-1 concentrations in blood serum ranging from 500 pg/mL to 3000 pg/mL whereas control blood donors had low MIC-1 concentrations ranging from 100–400 pg/mL. MIC-1 was found to be regulated by the most mutated gene in melanomas called V600EB-Raf.
3. Reducing Interleukin-8 secretion from melanoma cells disrupts interaction with neutrophils thereby decreasing metastasis development. Circulating tumor cells are frequently trapped in lung capillaries but only a minority form metastases. To unravel involvement of immune cells in lung metastasis development, human melanoma cells were intravenously injected into the lateral tail vein of nude mice leading to entrapment of many cancer cells in the lungs. However, 24 hours later very few remained in the lungs. In contrast, injection of human neutrophils an hour after tumor cell injection, increased cancer cell retention by ∼2-fold. Entrapped metastatic melanoma cells were found to produce and secrete high levels of a cytokine called IL-8, attracting neutrophils to transiently held melanoma cells and increasing β 2 integrin expression on neutrophils by 75–100%. ICAM-1 on melanoma cells and β2 integrin on neutrophils interacted to promote tethering of melanoma cells to the vascular endothelium via the neutrophil. Decreasing IL-8 secretion from melanoma cells lowered extracellular levels by 20–50%, decreased β2 integrin on neutrophils by ∼50% and reduced neutrophil-mediated extravasation by 25–60%, resulting in ∼50% fewer melanoma cells being tethered to endothelium and retained in the lung. As a result, transendothelial migration and lung metastasis development decreased by ∼50%. Thus, therapeutically targeting IL-8 in melanoma cells has the potential to decrease metastasis development by disrupting interaction with neutrophils. (Abstract shortened by UMI.)