UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
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Item CONSEQUENCES OF NUCLEAR CONFINEMENT IN CANCER METASTASIS(2021) Baird, Michelle; Waterman, Clare M; Stroka, Kimberly M; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Malignant melanoma is characterized by its mutational heterogeneity and aggressive metastatic spread. During metastasis, melanoma cells migrate through diverse microenvironments, including regions of dense tissue confinement to reach the vasculature. Microenvironmental confinement of tumor cells causes nuclear deformation, which can lead to loss of nuclear envelope (NE) integrity and DNA damage, improper repair of which leads to genomic aberrations and heterogeneity. We hypothesize that during metastatic progression, expression levels of NE genes are altered, facilitating nuclear deformability and NE fragility, mediating an increase in genetic heterogeneity within the population. In this dissertation, we show a novel bioinformatic analysis of orthogonal RNA-seq data sets from patient samples of metastatic melanoma and benign nevi, revealing several NE proteins upregulated in metastatic disease. Performing a targeted siRNA-based screen using a PDMS confinement device to assay for nuclear fragility, we found reduction of lamin B receptor (LBR) dramatically reduced NE fragility in melanoma cells, and ectopic overexpression of LBR was sufficient to increase NE fragility in benign melanocytes. Utilizing functional protein domain truncations and point mutations in LBR, we found the cholesterol synthase activity of LBR was specifically required for increased NE fragility, independent of LBRs additional roles tethering heterochromatin and lamin B to the NE. Additionally, we found that reduction of LBR in melanoma cells results in a reorganization of cholesterol in the NE. Thus, LBR generated cholesterol in the NE promotes NE fragility. To determine if LBR-mediated NE fragility was correlated with increased nuclear deformability, we assayed NE mechanics with atomic force microscopy. In melanoma cells, we find reduction of LBR results in an increase in nuclear stiffness and a decrease in deformability, while LBR overexpression in benign melanocytes results in an increase in nuclear deformability. These results show for the first time that upregulation of LBR in metastatic melanoma plays dual roles in reducing nuclear deformability and increasing NE rupture, specifically through alterations in cholesterol organization in the NE and open an exciting new direction to the role of cholesterol in NE integrity and mechanics.Item INITIATION AND PROGRESSION OF BRAF/NRAS WILDTYPE MELANOMA IN UV-INDUCED MOUSE MODELS OF CUTANEOUS MELANOMA(2018) Michael, Helen Thompson; Samal, Siba; Merlino, Glenn; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Melanoma is the deadliest skin cancer and is responsible for nearly 60,000 deaths worldwide each year. At least some melanomas are believed to arise from stepwise progression from normal melanocytes through a benign nevus stage to malignant melanoma and finally metastatic disease. Approximately 20-50% of melanomas have evidence of a pre-existing nevus, indicating that progression is an important route of melanomagenesis. Ultraviolet radiation exposure is believed to play an important role in nevus and melanoma formation, although the mechanisms of this remain unclear. Childhood sunburn and intermittent sun exposure are epidemiologically linked to increased melanoma risk. While most melanomas have activation of the mitogen activated protein kinase pathway, often due to mutations in BRAF or RAS genes, nearly 15% of cutaneous melanomas do not have an identified strong driver. Despite targeted therapies and immunotherapy, the death rate from melanoma has remained nearly static for several decades, so there is a need to identify additional genes and pathways to provide novel therapeutic targets. We hypothesized that progression of melanocytic lesions from benign to malignant is associated with the acquisition of additional genomic mutations. Unlike wildtype mice, hepatocyte growth factor (HGF) transgenic mice have “humanized” distribution of melanocytes along the dermal-epidermal junction. Following a single dose of UV at 3 days of age, HGF mice develop melanocytic nevi and melanomas. In this project, two HGF models were used to generate melanocytic lesions. The first model, on an albino FVB background had a tumor incidence of only 10% and used melanocyte-specific green fluorescent protein expression to identify early nevi and melanomas. The second model, on a C57BL/6 had a high tumor incidence (80%), and 60% of tumor-bearing mice have metastatic lesions. Sequencing of melanocytic lesions at different stages revealed a variety of driver mutations, including Nf1, Gnaq, and Gna11, as well as genes and pathways with less established roles in melanoma development. Our data provide a broad overview of genes and pathways involved in progression of non-BRAF, non-NRAS melanoma. Additionally, we present the first potential germline variants that may increase metastatic susceptibility for melanoma patients. These genes suggest potential biomarkers for progression of melanocytic lesions.