Role of human arylamine n-acetyltransferase 1 in tumorigenesis and cancer biology.

Stepp, Marcus W.

doi:10.18297/etd/2862

Cited by 3 publications

(4 citation statements)

References 97 publications

(132 reference statements)

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“…Based on results of this study, our laboratory and others are utilizing CRISPR/Cas9 to make permanent deletions at the NAT1 gene open reading frame causing 100% knockout of NAT1. In preliminary studies, our laboratory has reported that NAT1 knockout in MDA‐MB‐231 breast cancer cell lines with two different guide RNAs replicates the results reported here with shRNA partial NAT1 knockdown . In summary, inhibition of NAT1 by either partial knockdown (shRNA) or small molecule inhibition reduced anchorage‐independent growth in the MDA‐MB‐231 human breast cancer cell line.…”

Section: Discussionsupporting

confidence: 79%

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Genetic and small molecule inhibition of arylamine N‐acetyltransferase 1 reduces anchorage‐independent growth in human breast cancer cell line MDA‐MB‐231

Stepp

Doll

Carlisle

et al. 2018

Molecular Carcinogenesis

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Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer.

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Section: Discussionsupporting

confidence: 79%

“…Portions of this work constituted partial fulfillment of a PhD in Pharmacology and Toxicology at the University of Louisville for Marcus W. Stepp . This work was supported in part by the National Institute of Environmental Sciences [Grant USPHS T32‐ES011564].…”

Section: Acknowledgmentsmentioning

confidence: 99%

Genetic and small molecule inhibition of arylamine N‐acetyltransferase 1 reduces anchorage‐independent growth in human breast cancer cell line MDA‐MB‐231

Stepp

Doll

Carlisle

et al. 2018

Molecular Carcinogenesis

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“…Individual knockout cell lines representing the knockout of NAT1 activity for gRNA #2 or #5 were chosen based on NAT1 enzymatic activity and genomic sequence. Further details on NAT1 knockout cell line construction and characterization are described elsewhere [25].…”

Section: Methodsmentioning

confidence: 99%

Knockout of human arylamine N‐acetyltransferase 1 (NAT1) in MDA‐MB‐231 breast cancer cells leads to increased reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity

Carlisle

Trainor

Doll³

et al. 2018

Molecular Carcinogenesis

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Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can also hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. Expression of NAT1 varies between individuals and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. To date, however, the exact mechanism by which NAT1 expression affects mitochondrial bioenergetics in breast cancer cells has not been described. To further evaluate the role of NAT1 in energy metabolism MDA-MB-231 breast cancer cells with parental, increased, and knockout levels of NAT1 activity were compared for bioenergetics profile. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured followed by programmed sequential injection of Oligomycin (ATP synthase inhibitor), FCCP (ETC uncoupler), Antimycin A (Complex III inhibitor), and Rotenone (Complex I inhibitor) to evaluate mitochondrial bioenergetics. Compared to the cell lines with parental NAT1 activity, NAT1 knockout MDA-MB-231 cell lines exhibited significant differences in bioenergetics profile, while those with increased NAT1 did not. Significant increases in reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity were observed in NAT1 knockout MDA-MB-231 cell lines compared to those with parental and increased NAT1 activity. These data indicate that NAT1 knockout in MDA-MB-231 breast cancer cells may enhance adaptation to stress by increasing plasticity in response to energy demand.

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“…The construction and characterization of the siRNA generated cell lines has been described extensively elsewhere 82,84,115,116 . Briefly, three cell lines were constructed, Scrambled,…”

Section: Construction Of Sirna Generated Cell Linesmentioning

confidence: 99%

Deciphering the role of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer cell metabolism using a systems biology approach.

Carlisle¹

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who constructed the cell lines used as samples in the studies presented in this dissertation, Dr. Carolyn Klinge who I worked with on the Bioenergetics experiments presented in Chapter 3 of this dissertation, Dr. Wolfgang Zacharias, Sabine Waigel, and Vennila Arumugam of the University of Louisville Genomics Core who graciously allowed me to be a part of the library preparation of the RNA-seq (transcriptomics) samples, presented in Chapter 5, Dr. Eric Rouchka and Dr. Xiaohong Li of the KBRIN Bioinformatics Core who did the preprocessing of the transcriptomics data, and Dr. Patrick Trainor who was never too busy to serve as my own personal StackOverflow database. I want to thank all of my collaborators for their kindness, help, and willingness to let me be involved. Furthermore, I would like to thank the Bioinformatics Journal Club Group and Dr. Julia Chariker for providing such a welcoming and supportive environment in which to broaden my bioinformatics knowledge. Thank you to my fellow Hein Lab members, Dr. Raul Salazar-González, Dr. Mariam Habil, and Angeliki Likoudi, for providing a listening ear when times were tough and their friendship. Lastly, I would like to acknowledge and thank all of my friends and family who have provided unwavering encouragement and support in all of my endeavors. I am tremendously grateful for all of you. Several chapters included in this dissertation have been published; Chapter 2 has been published in the International Journal of Oncology and Chapter 3 has been published in Molecular Carcinogenesis; additionally, we intend to publish Chapters 4-7 soon.

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Role of human arylamine n-acetyltransferase 1 in tumorigenesis and cancer biology.

Cited by 3 publications

References 97 publications

Genetic and small molecule inhibition of arylamine N‐acetyltransferase 1 reduces anchorage‐independent growth in human breast cancer cell line MDA‐MB‐231

Genetic and small molecule inhibition of arylamine N‐acetyltransferase 1 reduces anchorage‐independent growth in human breast cancer cell line MDA‐MB‐231

Knockout of human arylamine N‐acetyltransferase 1 (NAT1) in MDA‐MB‐231 breast cancer cells leads to increased reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity

Deciphering the role of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer cell metabolism using a systems biology approach.

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