NAT1 and Cancer Biology: The Effects of NAT1 Silencing

Jacky Tiang (2010). NAT1 and Cancer Biology: The Effects of NAT1 Silencing PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Jacky Tiang
Thesis Title NAT1 and Cancer Biology: The Effects of NAT1 Silencing
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2010-07
Thesis type PhD Thesis
Supervisor Professor Rodney Minchin
Dr Neville Butcher
Total pages 143
Total colour pages 32
Total black and white pages 111
Subjects 11 Medical and Health Sciences
Abstract/Summary Arylamine N-acetyltransferase I (NAT1; EC is a phase II xenobiotic metabolising enzyme that catalyses the biotransformation of a wide range of arylamine and hydrazine substrates. The protein is widely expressed in the body and has received considerable attention because of its potential role in the metabolic activation and detoxification of arylamine carcinogens. However, unlike the closely related isozyme NAT2, the link between NAT1 and cancer susceptibility is relatively weak. Recent studies of NAT1 expression in various cancers of the breast and prostate suggest that this protein may be involved in other aspects of cancer biology. However, a direct role for NAT1 in cancer has not been established. Initially, we knocked down NAT1 in the colon adenocarcinoma cell line HT-29 using RNAi technology and found a marked change in cell morphology that was accompanied by an increase in cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar, which correlated with the up-regulation of E-cadherin. This change in E-cadherin expression was not attributed to RNAi off-target effects and was also observed in the prostate cell line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology and suggests a role for NAT1 in the regulation of epithelial-mesenchymal plasticity. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics by attenuating cancer growth. Anchorage-independent growth of cancer cells has been shown as a useful predictor of cell invasion potential. Loss of E-cadherin promotes cancer invasion and metastasis. Attenuation of anchorage-independent growth and re-expression of E-cadherin following NAT1 knock-down suggest that NAT1 may have a role in cancer invasion and metastasis. To address this, we used a lentivirus based shRNA system to knock-down NAT1 in MDA-MB-231 cells. The Lentilox 3.7 lentivirus system is specifically engineered for stable RNAi expression in cells. It allows the expression of shRNA in cells tagged with enhanced green fluorescence protein for identification. MDA-MB-231 is a breast adenocarcinoma cell line that is invasive and exhibit high metastatic potential. Knocking-down NAT1 inhibited invasion and metastasis of the cells, both in vitro and in vivo. The reduced invasiveness and metastatic ability was independent of cell migration and mesenchymal-epithelial transition. Since MDA-MB-231 cells require filopodia-like membrane protrusion to invade, we examined the effects of NAT1 knock-down on the cytoskeleton structure of MDA-MB-231 cells. Loss of NAT1 reduced number of filopodia in MDA-MB-231 cells and it can be rescued by re-introducing NAT1. NAT1 subcellular localisation study indicated that the protein distributes to the cortical cytoskeleton and extends into filopodia, suggesting that NAT1 may act locally to maintain filopodia structure of the cell. The present study also characterised a small molecule inhibitor of NAT1, Rhod-o-hp. Inhibition of NAT1 by Rhod-o-hp significantly reduced the invasiveness of MDA-MB-231 cells. Collectively, the study suggests that NAT1 is a novel target to prevent or attenuate metastatic cancers.
Keyword NAT1
contact inhibition
small molecule inhibitor
Additional Notes Colour pages: 18, 24, 44, 50, 51, 55, 61, 64, 73, 74, 76, 81, 83, 84, 86, 90, 92, 94, 95, 98, 104, 106, 107, 108, 110, 112, 114, 115, 117, 119, 124, 133 Landscape pages: 50, 51, 55, 61, 64, 73, 76, 77, 78, 79, 81, 83, 86, 98, 110, 112, 115, 117, 119, 133

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Created: Mon, 25 Oct 2010, 08:51:16 EST by Mr Jacky Tiang on behalf of Library - Information Access Service