Date of Award

Spring 1-1-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Natalie G. Ahn

Second Advisor

William M. Old

Abstract

Genome sequencing studies have identified oncogenic B-Raf mutations in over 70% of all sporadic melanomas. The B-RafV600E mutation leads to elevated kinase activity and constitutive activation of the mitogen-activated protein (MAP) kinase signaling pathway, which supports a variety of oncogenic functions critical to melanoma formation and progression. Metastatic melanomas are highly resistant to conventional chemotherapeutic treatments, and pharmacological inhibitors of B-RafV600E have been incompletely effective in pre-clinical studies. Understanding the global molecular responses to constitutive MAP kinase signaling in melanomas would allow the development of more effective therapeutics.

Manipulation of microRNA (miRNA) expression is emerging as an important molecular mechanism by which oncogenes can broadly support tumorigenesis. In this study, strategic microarray profiling of melanoma cells identified a network of over 25 miRNAs that are controlled by B-RafV600E. Functional screening of all regulated microRNAs revealed that many were capable of altering cell growth and/or invasion, and I was able to identify important melanoma genes that were targeted by one or more B-RafV600E miRNA(s). Interestingly, several genes suppressed by multiple miRNAs were direct targets for both induced and repressed miRNAs, suggesting a highly complex interaction of co-regulated miRNAs may contribute to overall gene regulation. The strong overlap in targets and functions suggested that BRafV600E miRNAs might work cooperatively, and I showed that miRNAs work combinatorially to further augment cellular invasion responses.

Previously, a similar DNA microarray profiling analysis discovered a set of axon guidance genes repressed by activated MAP kinase signaling in melanoma cells. I validated one of these genes, semaphorin 3C (sema3C), as suppressed by B-RafV600E through a transcriptional mechanism involving the co-regulation of TFAP2A, ATF3, and JUN transcription factors. Functional analyses in both primary and metastatic melanoma cell lines using strategies for sema3C inhibition or over-expression identified a novel role for this semaphorin in melanoma cell migration and invasion, as well as tumor growth and metastasis. By using microarray profiling studies to identify global molecular responses to activated MAP kinase signaling in melanoma cells, I established multiple microRNAs and an axon guidance gene, sema3C, as novel molecular targets controlled by B-RafV600E and functionally relevant in melanoma progression.

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