Student Theses and Dissertations

Nora Pencheva

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Tavazoie Laboratory

Abstract

Metastatic melanoma, a prevalent and deadly form of skin cancer, remains an aggressive clinical outcome that lacks effective preventative and/or curative therapeutics. Patients diagnosed with advanced metastatic melanoma face a dreaded 10-year survival rate of less than 10%. By performing in vivo selection for highly metastatic melanoma cells, we systematically identified three microRNAs (miRNAs) that promoted metastasis across multiple mutationally diverse human melanoma subtypes. These miRNAs were found to promote metastasis by enhancing the invasive capacity of melanoma cells and also by allowing melanoma cells to recruit endothelial cells into the metastatic niche, leading to enhanced metastatic angiogenesis. Importantly, the expression levels of these miRNAs served as prognostic biomarkers in stratifying melanoma patients at high risk from those at low risk for metastatic relapse. Notably, therapeutic delivery of specific locked nucleic acids (LNAs) antisense to the three miRNAs synergistically suppressed metastasis in pre-clinical models, attesting to the therapeutic potential of combinatorial miRNA silencing for the prevention of melanoma metastasis. Through systematic transcriptomic analyses and mutagenesis studies, I found that the three miRNAs silenced a common set of target genes: the heat-shock factor DNAJA4 and the metabolic protein apolipoprotein E (ApoE). Interestingly, this work also revealed that DNAJA4 positively regulated ApoE expression, establishing ApoE as the central molecule in this convergent network. Consistent with these findings, treatment of multiple melanoma lines with ApoE blocked their metastatic capacity, while ApoE expression in patients’ tumors was significantly prognostic of melanoma progression outcomes. Systematic loss-of-function experiments revealed that melanoma-derived extracellular ApoE exerted dual cell-intrinsic/ cell-extrinsic effects on metastasis: ApoE acting on melanoma cell LRP1 receptors inhibited melanoma invasiveness, while ApoE acting on endothelial cell LRP8 receptors suppressed metastatic endothelial recruitment (MER). In light of ApoE’s key metastasis-suppressive role, we next investigated therapeutic approaches to chronically activate ApoE in metastatic melanoma. We identified the nuclear hormone liver-X receptor (LXRβ), a known transcriptional trans-activator of ApoE, as an ideal target given its ubiquitous expression across all normal tissues as well as melanoma subtypes. Oral delivery of LXR agonists strongly suppressed melanoma tumor progression and metastasis across multiple mutationally diverse and genetically initiated pre-clinical melanoma models, consistent with broad-spectrum in vivo efficacy of LXRβ activation therapy in melanoma. Importantly, combining LXR agonist treatment with approved melanoma drugs elicited additive melanoma-suppressive responses, revealing therapeutic cooperativty between LXR activation and modern frontline agents. Complementary molecular and genetic approaches revealed that LXRβ agonism mediated melanoma suppression through the coordinate transcriptional induction of ApoE in both melanoma cells as well as stromal tissues. Collectively, this thesis provides molecular, genetic, and pharmacologic lines of evidence that establish ApoE as a robust metastasis suppressor in melanoma.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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