Interactions of melanomas and the tumor microenvironment.
Open Access
- Author:
- Huh, Sung Jin
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 10, 2009
- Committee Members:
- Gavin Peter Robertson, Dissertation Advisor/Co-Advisor
Gavin Peter Robertson, Committee Chair/Co-Chair
Henry Joseph Donahue, Committee Member
Craig Matthew Meyers, Committee Member
"Thomas P Loughran, Jr", Committee Member - Keywords:
- KLF6
tumor microenvironment
melanoma
IL-8
MIC-1 - Abstract:
- Malignant melanoma is an aggressive tumor of melanocytes in the skin with rapidly increasing incidence. Accumulations of gene mutations in melanocytes initiate cells to gain the ability to grow abnormally to develop into malignant melanomas. However, these genetic changes in the cell do not only alter the properties of tumor cells itself but also interact with extracellular surrounding of the tumor cell-called the tumor microenvironment. The microenvironment includes extracellular matrix, stromal fibroblasts, infiltrating immune cells, the blood and lymphatic networks. Dynamic bidirectional interaction between the cancer cell and tumor microenvironment can produce a variety of growth factors, cytokines and matrix-degrading enzymes that enhance melanoma development. Therefore, research understanding the complex networks between melanoma cells and the surrounding microenvironment will lead to discovery of novel therapeutics for cancer prevention and treatment. The focus of this dissertation is to identify alterations in melanomas that influence the tumor microenvironment to promote melanoma progression. Under normal conditions, type Ι collagen, which constitutes ~90% of the dermal extracellular matrix maintains tissue integrity and homeostasis. However, for tumorigenesis to occur, cells need to uncouple this regulation in order to proliferate and survive under condition in which cells would not normally thrive. In the first part of the dissertation, I have studied how the loss of KLF6 gene located, on chromosome 10, uncouples the cancer cell’s communication with collagen I thereby enabling proliferation. The angiogenesis process is another key process that involves cross-talks between melanoma cells and the surrounding microenvironment. Angiogenesis creates new blood vessels from existing vasculature to provide tumor oxygen and nutrient supply and removal of waste products to grow beyond a certain size. In the second part of this dissertation, I have investigated the role of MIC-1, which is secreted by melanomas, in angiogenesis its involvement in promoting tumor growth and metastasis. In the third part, I have identified that IL-8 secretion by melanoma cells functions to increase neutrophils-melanoma interaction, which aids melanoma cell adherence to the endothelium thereby promoting metastasis. The major findings are summarized in the following: 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. Introduction of ectopically expressed KLF6 into melanoma cells reduced growth on type I collagen mediated by decreasing levels of pErk and cyclin D1. Furthermore, siRNA-mediated knockdown of KLF6 in cells containing the transferred 10p15 enhanced proliferation on collagen I. Finally, high KLF6 staining by immunohistochemistry was observed in nevi surrounded by the type I collagen-rich dermis. In more advanced melanocytic lesions, KLF6 expression was only present at the periphery of tumor nodules but not in the center and was largely absent in metastatic melanomas. In protein lysates, KLF6 protein expression was found to be lacking in 100% of advanced stage patient tumors further supporting a role in melanoma development. These data suggest that KLF6 communicates directly with type I collagen of the dermis to inhibit cellular proliferation mediated by decreased MAP kinase-signaling. Advanced melanomas lacked KLF6 expression, functioning to uncouple the cancer cell’s communication with the collagen I rich environment of the skin thereby enabling proliferation by promoting increased MAP kinase signaling. Restoration of functional KLF6 in early melanocytic may be a useful strategy to prevent or treat melanoma. 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. Knockdown of mutant V600EB-Raf using siRNA decreased expression and secretion of MIC-1 from melanoma cells by 3-4 fold. However, decreased MIC-1 protein expression did not significantly alter melanoma growth in vitro, but dramatically inhibited tumor development by ~3-fold. To identify the mechanism action of MIC-1 for promoting tumor development, rates of tumor angiogenesis, cell proliferation and apoptosis were analyzed in time and size matched melanoma tumors following siRNA-mediated inhibition of MIC-1. Increased tumor angiogenesis was identified as the key event leading to tumor development by MIC-1. Furthermore, role of MIC-1 on tumor angiogenesis was validated by treating chick chorioallantoic membrane of developing embryos and Matrigel plug in vivo assay with recombinant MIC-1. MIC-1 significantly induced angiogenesis by ~2-4-fold. In conclusion, mutant V600EB-Raf signaling increases MIC-1 expression and secretion to enhance angiogenesis and thereby promote melanoma development. Thus, MIC-1 may be a useful prognostic marker as well as an important therapeutic target for melanoma treatment. 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 β2 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.