Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Kevin R. Jones

Second Advisor

Rui Yi

Third Advisor

Joaquin M. Espinosa

Fourth Advisor

Robert L. Garcea

Fifth Advisor

Dylan J. Taatjes

Abstract

miRNAs are small non-coding RNAs, approximately 22 nucleotide in length, that mediate post-transcriptional repression of target mRNAs. Since their discovery in mammals in the early 2000s, miRNAs have been intensely studied and determined to be an important mechanism to regulate gene expression in diverse biological processes. In human cancers, miRNAs are known to act as tumor suppressors or oncogenes and are being actively explored as a possible mechanism for therapeutic intervention. In the mouse, multistage skin carcinogenesis is a well-established model for studying tumor development however the functions of miRNAs in this model are poorly understood.

The Ras oncogene was the first discovered oncogene in human cancers, and in mouse models of skin cancer it is required for tumor development. In order to elucidate the mechanisms by which oncogenic Ras initiates tumorigenesis in the skin, I conducted genome-wide profiling experiments to define the mRNA and miRNA landscape regulated by oncogenic Ras. From these datasets I identified a microRNA, miR-203, that is strongly suppressed by oncogenic Ras. Furthermore, I demonstrate that miR-203 is commonly silenced in mouse models of Squamous Cell Carcinoma (SCC), and in human skin SCC samples, strongly suggesting that miR-203 may act to suppress SCC tumor development.

miR-203 is a broadly conserved miRNA that accounts for ~25% of the miRNA found in the epidermis of the skin. This miRNA was previously shown to promote keratinocyte differentiation by antagonizing epidermal cell proliferation, yet it's requirement for epidermal development and in skin cancer prior to this work were unknown. To address these important questions, a conditional miR-203 knockout mouse model was generated. I showed that loss of miR-203 perturbs epidermal embryonic development and enhances keratinocyte self-renewal. I next conducted mouse skin chemical carcinogenesis studies to analyze the functions of miR-203 in skin cancer and demonstrated that loss of miR-203 sensitizes skin to chemical carcinogenesis, resulting in enhanced tumor burden. Lastly, to identify the molecular mechanisms by which miR-203 restricts tumorigenesis, I utilized genome-wide techniques to detect miR-203 targets in keratinocytes. Ago2-HITS-CLIP, which identifies direct miRNA/mRNA interactions, was combined with genome-wide expression profiling to identify high confidence miR-203 targets. From these studies novel miR-203 targets that are involved in the regulation of Ras/MAPK signaling and cell division were identified. Collectively, these studies provide insight into the mechanisms and functions of miR-203 in the skin.

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