Date of Award

Winter 12-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Altered cellular metabolism in cancer has been shown to support the energetic and biosynthetic requirements for malignancy. Specifically, alterations in cellular redox homeostasis are hypothesized to play an essential role in driving biochemical processes such as proliferation. Reactions involving reactive oxygen species (ROS) are diffusion-limited; therefore, the compartmentalized production and detoxification of these species are thought to have regulatory implications. How compartmentalized redox balance affects the proliferative metabolism of cancer cells, however, requires further investigation. In my thesis, I investigated the role of three metabolic processes related to compartmentalized redox balance in cancer: (i) lactate production during fermentation, (ii) pyruvate carboxylase (PC) activity in mitochondria, and (iii) altered isocitrate dehydrogenase (IDH) activity due to commonly occurring mutations. In the first project, I found that lactate, often regarded as a fermentation waste product secreted to the extracellular matrix, can serve as an important source of carbon for metabolic reactions in mitochondria of cancer cells. Silencing lactate dehydrogenase (LDH) led to a decreased redox ratio (NAD+/NADH), which resulted in elevated PC activity in cancer cells. PC activity is required to replenish carbon in the TCA cycle and is known to be regulated by acetyl-CoA and aspartate. In the second project of my thesis, I demonstrated that PC activity is closely correlated with cellular redox ratio (NAD+/NADH). In addition, I found that alterations in cellular redox affect the amount of glutamine that is oxidatively and reductively metabolized in proliferating cancer cells. Lastly, in the third project of my thesis, I evaluated how changes in redox metabolism are affected by gain-of-function mutations in IDH1 and IDH2 that lead to production of 2-hydroxyglutarate (2HG). To understand redox homeostasis in each compartment, I established an approach to trace deuterium from glucose to intermediates of proline biosynthesis that localize to either the cytosol or mitochondria. By using this method to resolve cytosolic and mitochondrial NADPH fluxes, I demonstrated that IDH1 mutations altered NADPH flux distributions in the cytosol, and IDH2 mutations altered NADPH flux distributions in mitochondria as expected. Interestingly, however, IDH1 mutations increased the flux of the pentose phosphate pathway (PPP) in the cytosol but did not influence NADPH fluxes in mitochondria. In contrast, IDH2 mutations increased the flux of one-carbon metabolism in mitochondria but did not influence NADPH fluxes in the cytosol. This work highlights that NADPH homeostasis in the cytosolic and mitochondrial locations of a cell are independently regulated, with no evidence for NADPH shuttle activity.

Language

English (en)

Chair and Committee

Gary J Patti

Committee Members

Meredith Jackrel

Comments

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Available for download on Tuesday, August 18, 2122

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