Date of Completion

12-22-2016

Embargo Period

6-22-2017

Keywords

Colon cancer, DNA methylation, PRC2, AP-1, folate, Dclk1, metabolomics, bile acid, fatty acid oxidation

Major Advisor

Daniel W. Rosenberg

Associate Advisor

Charles Giardina

Associate Advisor

Blanka Rogina

Associate Advisor

Marc Lalande

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Colorectal cancer (CRC) is the 3rd most common form of cancer in the United States, and the 2nd most common cause of cancer-related death. CRC incidence has declined over the past several decades, however, there is a continued need for improved methods of CRC detection and prevention. Methyl group homeostasis, which is dysregulated in CRC and impacts several fundamental biological processes, represents a promising target for novel CRC detection and prevention strategies. DNA methylation is influenced by methyl group homeostasis and is known to be frequently dysregulated in CRC. However, less is known about the extent of aberrant methylation in early colonic neoplasia. We utilized reduced representation bisulfite sequencing (RRBS) to identify cancer-associated methylation changes in 10 KRAS-mutant human aberrant crypt foci (ACF), the earliest precursor to CRC, and 10 primary CRCs. Using this approach, extensive methylation changes were identified in both ACF and CRCs, including hypermethylation of homeobox genes and Polycomb repressive complex 2 (PRC2) gene targets. Furthermore, we show that intergenic binding sites for the transcription factor AP-1 undergo significant hypomethylation. Importantly, 75% of the methylation changes identified in ACF were also present in CRC samples. These data identify epigenomic features of early colonic neoplasia that may provide new targets for CRC detection and prevention.

Folate one-carbon metabolism (FOCM), which regulates methyl group homeostasis, has been proposed as a target for CRC prevention strategies. The present work shows that dietary restriction of the methyl donors folate, choline, methionine and vitamin B12 provides long-lasting tumor protection to the intestine. Tumor protection was associated with persistent remodeling of intestinal morphology, inhibition of crypt fission, reduced crypt cell mitosis, and increased apoptosis in both normal crypts and tumors. MDD also reduced numbers of crypt cells expressing Dclk1, which have tumor-initiating potential. In a follow-up study, we used untargeted metabolomic profiling to identify MDD-associated changes to several metabolic pathways, including the methionine cycle, the transsulfuration pathway, secondary bile acid synthesis, and fatty acid β-oxidation (FAO). Together, these results indicate that temporary MDD can provide long-lasting tumor protection and identify several pathways that may serve as novel targets for CRC chemoprevention.

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