Graduation Date

Fall 12-18-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Pankaj Singh

Abstract

Hepatocellular carcinomas exhibit metabolic alterations to support their proliferative and biosynthetic needs. We identified that elevated expression of the mitochondrial oxidative carboxylase, malic enzyme 2 (ME2), correlates with poorer hepatocellular carcinoma patient survival. Hepatocellular carcinoma patient tumors with high ME2 expression exhibit transcriptomic alterations indicative of PI3K/AKT/mTOR and c-Myc signaling as well as elevated central carbon, fatty acid, and redox metabolism pathways. Depletion of ME2 in the hepatocellular carcinoma cell line PLC or in the livers of mice treated with diethylnitrosamine to chemically induce hepatocellular carcinomas, results in impaired proliferation and reduced tumor formation. Additionally, the loss of ME2 results in reduced protein levels of c-Myc in both models and diminished activation of the PI3K/AKT/mTOR signaling pathway in PLC cells. Increased c-Myc levels in ME2 expressing cells may be due to the overexpression of CIP2A, which stabilizes c-Myc by preventing its proteolytic degradation. Inhibition of mTOR with Everolimus was equally effective at impairing PLC cell viability, regardless of ME2 expression status. Liquid chromatography-tandem mass spectrometric analysis revealed that the loss of ME2 not only reduces glycolysis and TCA Cycle intermediates but also diminishes metabolites involved in the glutathione antioxidant pathway and fatty acid metabolism. Overall, these data suggest that ME2 supports hepatocellular carcinoma cell proliferation, and that targeting the enriched c-Myc and PI3K/AKT/mTOR pathways may be an effective therapeutic, particularly in combination therapy, even though no selectivity was observed in the cell line we tested.

Cancer patients commonly experience a complex metabolic syndrome called cachexia, which notably results in body weight loss and muscle wasting. Significant muscle wasting not only impairs the quality of life but also impacts the treatment efficacy for patients. Skeletal muscle protein turnover is imparted by increased expression of ubiquitin-proteasome pathway components. Mitogen-activated protein kinases p38 and ERK have been shown to augment E3 ubiquitin ligase expression. Utilizing reverse-phase protein arrays, we identified pancreatic cancer cell-conditioned media-induced activation of JNK signaling in myotubes differentiated from C2C12 myoblasts. Inhibition of JNK signaling with SP600125 reduced cancer cell-conditioned media-induced myotube atrophy, myosin heavy chain protein turnover, and mRNA expression of cachexia-specific ubiquitin ligases Trim63 and Fbxo32. Furthermore, utilizing an orthotopic pancreatic cancer cachexia mouse model, we demonstrated that treatment of tumor-bearing mice with SP600125 improved longitudinal measurements of forelimb grip strength. Post-necropsy measurements demonstrated that SP600125 treatment rescued body weight, carcass weight, and gastrocnemius muscle weight loss without impacting tumor growth. JNK inhibitor treatment also rescued myofiber degeneration and reduced the muscle expression of Trim63 and Fbxo32. These data demonstrate that JNK signaling contributes to muscle wasting in cancer cachexia, and its inhibition has the potential to be utilized as an anti-cachectic therapy.

Available for download on Sunday, December 11, 2022

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