Publication Date

2024

Document Type

Dissertation/Thesis

First Advisor

Bode, Barrie P.

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Biological Sciences

Abstract

Hepatocellular Carcinoma (HCC) is a liver cancer that accounts for a significant proportion of cancer-related deaths globally; likewise, as the primary metabolic regulatory center in the body, the liver undergoes profound metabolic shifts in carcinogenesis, with net glucose and glutamine consumption. Much attention has been focused on the myriad roles of glutamine in supporting cellular growth over the past several decades, but more recently, the relationship between glutamine and the other amide-bearing amino acid, asparagine, has garnered attention. Asparagine is synthesized from glutamine, aspartic acid, and ATP via the enzyme Asparagine Synthetase (ASNS). Extracellular asparagine reliance has been targeted in L-asparaginase therapy for Acute Lymphoblastic Leukemia with low ASNS expression. Here, we investigated the metabolic interaction between these amino acids in epithelial (Type I) and mesenchymal (Type II) human HCC cell lines to identify potential therapeutic targets for drug development. We further examined the response of glutamine metabolic enzymes, including ASNS, under conditions of glutamine deprivation and created ASNS null cell lines through the CRISPR/Cas9 gene editing system to assess the role of glutamine-dependent asparagine production in cancer cell proliferation. Epithelial (Type I) and mesenchymal (Type II) human HCC cells exhibited a differential response to asparagine substitution for glutamine. Epithelial HCC survived in the absence of extracellular glutamine if asparagine was provided, while mesenchymal HCC failed to survive without glutamine even in the presence of asparagine. In Huh7 (epithelial) and SK-HEP-1 (mesenchymal), glutamine withdrawal induces expression of the biosynthetic enzymes glutamine synthetase (GLUL) and Asparagine synthetase (ASNS); however, induction of linked anaplerotic enzymes like glutamic oxaloacetic transaminase (GOT), glutamine dehydrogenase (GDH) was observed only in SK-HEP-1. Elimination of ASNS expression in the cell lines significantly impacts growth, increases their sensitivity to asparaginase treatment, and created tools to test the role of asparagine signaling in cancer. A model has been proposed where ASNS-generated asparagine is a signal and proxy for glutamine sufficiency in cancer. In conclusion, these results provide insights into the relationship between the two amide amino acids, glutamine and asparagine, and will hopefully contribute to the development of differentially targeted metabolic therapies for primary and metastatic cancer.

Extent

103 pages

Language

en

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

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