Date on Master's Thesis/Doctoral Dissertation

12-2013

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemistry

Committee Chair

Fan, Teresa

Author's Keywords

Cancer; Metabolism; Pyruvate carboxylase

Subject

Pyruvate carboxylase; Lungs--Cancer

Abstract

Non-small cell lung cancer (NSCLC) is the number one cause of cancer-related death and, like all cancers, requires metabolic reprogramming to support the energetic and biosynthetic demands of the tumor cells to proliferate and survive the tumor microenvironment. This dissertation sought to uncover novel metabolic dysfunctions in NSCLC that may lead to new, targeted therapeutic strategies. To this end, a stable isotope-resolved metabolomics approach was used to study NSCLC in human patients in vivo, in cell culture, and in mouse xenograft tumors. This approach involves treating biological samples with tracers containing stable isotopes, such as 13C6-glucose, and monitoring the incorporation of the isotope into metabolites by mass spectrometry and nuclear magnetic resonance spectroscopy. In Chapter 2, NSCLC-associated metabolic dysfunctions were elucidated by infusing human NSCLC patients with 13C6-glucose prior to tumor resection. Metabolite concentrations and labeling patterns in tumor tissues were compared to surrounding benign lung tissue from the same patient. The major findings were that tumor tissues had enhanced flux through glycolysis, the pentose phosphate pathway, glycogen synthesis and utilization, pyruvate dehydrogenase, malic enzyme, nucleotide biosynthesis, glycine biosynthesis, and UDP-GlcNAc biosynthesis. Particularly interesting was the increased rate of glucose oxidation through the TCA cycle, accompanied by increased utilization of TCA cycle intermediates for biosynthesis. To support this increase in TCA cycle flux, the expression and activity of the anapleurotic enzyme pyruvate carboxylase (PC) was enhanced in human NSCLC tumors. In Chapter 3, PC expression was suppressed in NSCLC cell lines by shRNA. The phenotypic and metabolic consequences were monitored in vitro and in mouse xenografts. Silencing PC reduced NSCLC cell growth, tumorigenicity, and survival. It was demonstrated that PC serves several important functions in NSCLC, including supporting mitochondrial activity, macromolecule biosynthesis especially lipids, and maintaining the redox status of the cell. In Chapter 4, small molecules were employed to inhibit PC activity in vitro, including a known anticancer agent methylseleninic acid (MSA). This is the first report demonstrating that MSA inhibits PC activity in vitro and in cell culture. Several other molecules with anti-PC activity were elucidated by in silico screening. Like PC knockdown, these inhibitors reduced NSCLC cell growth, tumorigenicity, and macromolecule biosynthesis. Together, these data indicate that PC is upregulated in NSCLC and functions to support anabolic processes critical to NSCLC proliferation and survival.

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