Graduation Date

5-2018

Document Type

Master's Thesis

Degree Name

Master of Science

Department or Program

Biological Sciences

Department or Program Chair

Meredith Protas, PhD

First Reader

Christopher Benz, MD

Second Reader

Ekaterina Kalashnikova, PhD

Abstract

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane protein linked to electron transport and capable of generating mitochondrial glutamate and intracellular ATP, especially under cellular stress conditions. Among a panel of 51 human breast cancer cell lines, PRODH and glutaminase (GLS1) expression levels were found to be inversely correlated (1) implicating two independent and alternative mitochondrial pathways supplying anaplerotic glutamate for cancer cell energy production and macromolecular synthesis. Proposing PRODH to be a promising cancer therapeutic target, we compared the in vitro cellular effects of PRODH knockdown by siRNA as well as competitive (L-tetrahydrofuroic acid, THFA; or 5-oxo-2-tetrahydrofurancarboxylic acid, 5-oxo) and irreversible/suicide (PPG) inhibitors of PRODH using cultured human breast cancer cells. PRODH knockdown or enzymatic inhibition each inhibited cell growth and induced variable degrees of apoptosis against malignant breast epithelial cell lines (ZR-75-1, DU4475, MCF-7) without affecting immortalized and non-malignant breast epithelial cells (MCF-10A). Loss of PRODH function produced additive in vitro anticancer effects when combined with either a p53 upregulator (MI-63) or a glutaminase inhibitor (CB-839) (2). Unlike the competitive inhibitors, PPG not only demonstrated irreversible inhibition of PRODH enzymatic activity on isolated breast cancer cell mitochondria (ZR-75-1) but it also induced loss of mitochondrial PRODH expression prior to its induction of cancer cell apoptosis. Computer modeling of PRODH’s enzymatic pocket occupied by either 5-oxo or PPG revealed that PPG likely induces molecular distortion, suggesting its unique ability to activate apoptosis by first triggering mitochondrial stress; however, it remains unclear whether the cancer cell’s ultimate response to this initial mitochondrial stress is mediated by mitophagy or by the mitochondrial unfolded protein response (UPRmt). The two objectives of this current project address both the mechanistic questions about mitochondrial stress induced by the suicide PRODH inhibitor, PPG, as well as a more translational question about the in vivo feasibility of systemically administering PPG as a potential therapeutic. To address the first objective, we employed confocal intracellular imaging to investigate the mitochondrial stress induced by PPG. To address the second objective, PPG was administered in vivo to flies and mice (implanted with xenografted human breast tumors) to observe both whole organism and tissue-specific effects of this mitochondrial stress inducing suicide inhibitor of PRODH. These studies form the basis for more extended in vivo preclinical testing of PRODH suicide inhibitors as a potential new breast cancer treatment strategy.

Available for download on Tuesday, May 12, 2020

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