Thesis Examination: MSc Student Dean Reddick

Wed. Dec. 4 09:00 AM - Wed. Dec. 4 11:00 AM
Contact: Dylan Armitage
Location: Room 1RC028

Dean Reddick - MSc Student in Bioscience, Technology, Public Policy

The Role of N-myristoyltransferase in the Progression of Estrogen Receptor Positive Breast Cancer

N-myristoyltransferase (NMT) catalyzes the attachment of a 14-carbon fatty acid onto the N-terminal glycine residue of a target protein. In higher eukaryotes the enzyme has been found to exist in two different non redundant isoforms, NMT1 and NMT2. The myristoylation reaction has been shown to be essential for the regulation of cellular activities such as signal transduction, proliferation, migration, differentiation and transformation. Abnormal expression of NMT has been implicated in the development of several types of cancer including colorectal and brain cancers. So far, little is known about the regulation of NMT or the mechanisms linking cancer cell development with increased NMT expression. Various oncoproteins such as c-Src and Ras are activated upon myristoylation, thus increased expression of NMT is consistent with increased demand of myristoylation for oncoproteins in cancer cells. Recent findings from our laboratory indicate that altered localization and expression of NMT is linked to estrogen receptor breast cancer prognosis. It was found that nuclear NMT1 in primary ER breast cancer tissues predicts better treatment outcome when patients underwent endocrine therapy. These observations have prompted further studies focused on understanding the mechanism of NMT translocation into the nucleus and its role in cancer onset and progression. It is conceivable that recruitment of NMT to the nucleus may allow it to interact with transcription factor complexes, which regulate expression of oncogenes. It is likely that regulation of NMT is at least in part dependent on the subcellular localization patterns of the enzyme. Currently, the recruitment system used to locate NMT to a specific area of the cell, such as the nucleus, is unknown. This study focused on investigating the mechanisms by which NMT2 may translocate into the nucleus of ER positive breast cancer cells (MCF7 breast cancer cells). More specifically, the possibility that NMT2 localization to the nucleus may be regulated by its phosphorylation status at specific positions. Utilizing bioinformatics tools, these positions were identified as serine (S)38 and S68 residues that flank an NMT2 putative nuclear localization sequence. MCF7 cells were transfected with pcDNA3-NMT2-GFP clover plasmids to produce NMT2-GFP fusion proteins. The wild type NMT2 gene sequence and altered forms of NMT2 gene sequence with S38 ->A (S38A), S38E, S68A and S68E mutations were engineered into the plasmid. The mutation of serine to alanine (S->A) mimicked phospho-dead, whereas, mutation of serine to glutamic acid (S->E) mimicked constitutively phosphorylated serine. Wild type and mutated NMT2-GFP fusion protein expression patterns were visualized by fluorescence microscopy. MCF7-GFP-NMT2-S38E cells displayed an increased nuclear localisation of NMT2-GFP fusion protein, whereas, MCF7-GFP-NMT2-S38A failed to demonstrate nuclear localization of NMT2. This is suggestive of the fact that phosphorylation of NMT2 at S38 is an important regulator in the nuclear localisation of NMT2. Further investigation was conducted to determine the possible roles played by nuclear NMT2 in cell proliferation and regulation of growth related IGF1R gene. Results indicate that compared to parental MCF7 cells, MCF7-GFP-NMT2-S38E cells demonstrated increased cell proliferation, whereas, MCF7-GFP-NMT2-S38A cells demonstrated reduced proliferation. IGF1R gene expression was determined by RT-qPCR in MCF7, MCF7-GFP-NMT2, MCF7-GFP-NMT2-S38A and MCF7-GFP-NMT2-S38E cell lines. The results show an upregulation of the IGF1R gene in the S38E cell line and downregulation in the S38A line. Western blot analysis displayed a very similar trend for the expression of IGF1R protein. IGF1R is a transmembrane receptor responsible for activation of the insulin like growth factor pathway, which is a cellular growth and proliferation promotion pathway. The mutation of S38 to A or E also impacted the cell adhesion properties of MCF7 cells. The S38A cells displayed a distinctive clumped tightly associated adherence pattern while the S38E cells exhibited loose connections between cells. Therefore, the expression of tight junction protein, claudin 1, was determined in these cell lines. Western blot analysis revealed claudin 1 was upregulated in the S38A cells relative to MCF7, MCF7-GFP-NMT2 and S38E cells. This overexpression of claudin 1 is likely causing the distinctive non-metastatic growth patterns observed in the S38A cell line. All of this data combined demonstrates the importance of NMT2 regulation via phosphorylation in the development and proliferation of MCF7 ER+ breast cancer cells