Functional Relevance of N-myristoyltransferase as a Biomarker for Colorectal Cancer

Abstract

Colorectal cancer (CRC) is the second leading cause of cancer deaths in Canada. The high mortality rate for CRC patients demonstrate the urgent need for a convenient, accurate and cost-effective screening test, which could triage patients for more intensive procedures such as colonoscopy. Current CRC screening tests are limited by their efficacy, invasiveness, and poor acceptability by the patients. N-myristoylation refers to the covalent attachment of the 14-carbon fatty acid myristoyl group to the N-terminal glycine residue of a target protein, which ensures its proper functioning and intracellular trafficking. This reaction is common amongst signalling proteins and is often integral to their activity. Myristoylation is catalyzed by N-myristoyltransferase which transfers the myristoyl moiety from myristoyl-coenzyme A to the N-terminal glycine residue. The foremost aim of my study was to optimize and quantify NMT1 and METAP2 gene expression by using quantitative Polymerase Chain Reaction (qPCR). Due to its capacity to generate both qualitative and quantitative results, qPCR is considered a quick and accurate technique to begin with. NMT1 and METAP2 oncogenes overexpressed in colorectal cancer cells are directly associated with aggressive clinical behaviour. The NMT1 and METAP2 oncogenes and their respective protein products (NMT1 and METAP2) have been utilized for disease diagnosis and as predictive markers for treatment. To support accurate quantification and analysis in this study, I have optimized and standardized qPCR analysis to analyze colorectal cancer samples, obtaining significant and clinically useful results. We recently demonstrated that NMT1 is expressed in the peripheral blood mononuclear cells (PBMC) and T cells of colorectal cancer patients. The aims of this study are to quantify NMT1 and METAP2 expression in individuals undergoing colonoscopy and determine whether NMT1 and METAP2 gene expression could serve as a screening marker for CRC. Chapter two: From chapter 1, it was determined that NMT1 and METAP2 expressions in peripheral blood and PBMC are potential biomarkers for CRC screening. These biomarkers are overexpressed in individuals with polyps and CRC. To understand the mechanism and the functional relevance of NMT1 and METAP2 overexpression in PBMC, Nmt1 was knocked out in mouse embryonic stem cells and its effect was investigated on metabolic pathway. The generation of homozygous (Nmt1-/-) deficient embryonic stem cells was confirmed by qPCR and Western analysis. The depletion of NMT1 leads to enhanced phosphorylation of key proteins within the PI3K/Akt signalling pathway that are normally stimulated by insulin and inhibited by rapamycin. The Nmt1- deficiency resulted in the over activation of the PI3K/Akt signalling pathway and Igf1r gene expression, thus, NMT1 likely has a vital role in regulating metabolic disorders.