CONSEQUENCES OF NUCLEAR CONFINEMENT IN CANCER METASTASIS

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