Publication Date

2016

Document Type

Thesis

Committee Members

Madhavi Kadakia (Advisor), Weiwen Long (Committee Member), Michael Raymer (Committee Member)

Degree Name

Master of Science (MS)

Abstract

ΔNp63α, the most physiologically relevant isoform of p63, a p53 family member protein, maintains the proliferative capacity of the basal layer of epithelial tissues and is highly expressed in non-melanoma skin cancer. It has been implicated in a variety of different cancers, including breast, lung, and prostate cancers, however the complex role it plays in disease remains poorly understood. Elucidating ΔNp63α’s regulatory network may provide insight into its role in epithelial tissues and in cancer progression. The objective of this thesis is to investigate ΔNp63α’s upstream regulation in the context of non-melanoma skin cancer as well as expand on ΔNp63α’s downstream function through its modulation of target microRNAs (miRNAs). Non-melanoma skin cancer is primarily a result of excessive exposure to ultraviolet (UV) radiation. ΔNp63α appears to be regulated by post-translational modifications such as phosphorylation, which can lead to its degradation in a ubiquitin-mediated, proteasome-dependent manner. cJun N-terminal kinase (JNK), a stress activated protein kinase (SAPK), is potently activated by UV stress and has been shown to phosphorylate p53 and p73. I hypothesized that UV-induced JNK activation can similarly regulate ΔNp63α via phosphorylation. Anisomycin and UV induced JNK activation were shown to produce an electrophoretic mobility shift in ΔNp63α, suggesting it was phosphorylated. Co-expression of JNK and ΔNp63α also exhibited an enhanced electrophoretic mobility shift in ΔNp63α following UV and anisomycin treatment. Phosphorylation was confirmed using an in vitro kinase assay with recombinant JNK and p63 proteins. Mass spectrometry analysis of immunoprecipitated ΔNp63α protein from UV treated H1299 cells transfected with ΔNp63α and in vitro kinase assay samples identified several phosphorylated residues, some of which are highly conserved in p73. Mutation of these potential JNK targeted residues alone did not prevent an electrophoretic mobility shift, however, a combination of mutated residues resulted in a stabilization of ΔNp63α levels. These results suggest that JNK plays a role in the phosphorylation of ΔNp63α, implicating it as a potential mediator of ΔNp63α degradation and apoptosis. ΔNp63α has been shown to regulate a number of miRNAs that affect a wide range of biological processes. Advances in high-throughput sequencing technologies have made it possible to profile differentially expressed miRNAs to gain insight into the role they play in the pathogenesis of cancer. Interestingly, although next-generation sequencing (NGS) platforms have become popular for studying miRNA expression profiles through small RNA-Seq, a consensus on the data processing methodology has not yet been reached. In this dissertation, I sought to both optimize and validate a pipeline for analyzing small RNA-Seq data and, with the resulting dataset, gain insight into the regulation of miRNA expression by ΔNp63α. A test NGS dataset was generated by sequencing small RNA from non-silencing control (NSC) and p63-silenced (sip63) HaCaTs cells using the Ion Proton System. I optimized data collection and analysis parameters using this dataset and established a functional pipeline workflow involving alignment to the miRBase reference database, normalization using the Trimmed Mean of M-values (TMM) normalization method, and differential expression using a lognormal with shrinkage response distribution model. This pipeline configuration resulted in a shortlist of differentially expressed miRNAs which was enriched for known p63-regulated miRNAs, thus empirically confirming pipeline function. Finally, I validated the differential expression of several novel p63-regulated mi...

Page Count

92

Department or Program

Biochemistry and Molecular Biology

Year Degree Awarded

2016

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