Date of Award

2020

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Dr. Emine Koc, Committee Co-Chairperson

Second Advisor

Dr. Hasan Koc, Committee Co-Chairperson

Third Advisor

Dr. Richard Egleton

Fourth Advisor

Dr. Jung Han Kim

Fifth Advisor

Dr. Nalini Santanam

Abstract

Mitochondria are essential organelles that play crucial roles in many aspects of cellular homeostasis. More importantly, the mitochondria are home to the majority of the metabolic pathways within the cell and are responsible for producing most of the cell’s useable energy in the form of adenine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). In mammals, the majority of OXPHOS complex subunits are encoded by nuclear deoxyribonucleic acid (DNA); however, 13 core subunits essential for the function of OXPHOS complexes I, III, IV, and V are encoded in the mitochondrial (mt) DNA (mtDNA) and are synthesized within the mitochondria by its own transcription and translation machinery. Changes in the expression and post-translational modifications (PTMs) of OXPHOS subunits and mitochondrial proteins can be detrimental to mitochondrial energy production. In fact, alterations in mitochondrial functions impact cellular energy metabolism as well as influence whole-body metabolism and have been identified as underlying causes for several diseases including neurological disorders, insulin resistance, type 2 diabetes (T2D), and numerous cancer types. This has led to an extensive search for a better understanding of key players involved in mitochondrial function in disease states, in addition to new preventative and therapeutic strategies that are aimed at exploiting key components of mitochondrial biogenesis and energy metabolism. Our laboratory has shown that differences in mitochondrial biogenesis can be caused by changes in the sequence and/or PTMs of mitochondrial proteins, resulting in altered mitochondrial function and energy metabolism. In the present studies, we investigated changes in mitochondrial energy metabolism in two major metabolic diseases, T2D and liver cancer, and evaluated potential targets and therapies. We first investigated mitochondrial biogenesis and energy metabolism in T2D by studying the differences in the expression and activity of OXPHOS complexes in the liver and kidney of the polygenic T2D model, TALLYHO/Jng (TH), and normal, C57BL/6J (B6), mice. A significant decrease was observed in the expression of both nuclear- and mitochondrial-encoded subunits of complexes I and IV, respectively, in TH mice compared to B6, which coincided with significant reductions in their enzymatic activities. Furthermore, we identified sequence variants in several mitochondrial proteins including OXPHOS complex subunits, a mitochondrial transfer ribonucleic acid (tRNA) synthetase, and mitochondrial ribosomal proteins (MRPs). The sequence variants identified in mitochondrial proteins might contribute to impaired mitochondrial biogenesis and energy metabolism by diminishing OXPHOS expression and activity in TH mice. In addition to T2D, we investigated liver cancer and demonstrated impaired mitochondrial OXPHOS complex expression and activity in cancerous liver biopsies compared to non-cancerous biopsies. The expression of the Src family kinases (SFKs), c-Src and Fyn, were increased in liver cancer cell lines and tissues. In fact, aberrant expression of c-Src kinase was observed in metastatic liver cancer tissues and the hepatic cell line Hep3B, which was correlated to a significant reduction in OXPHOS complex expression and activity. Additionally, increased c-Src expression was associated with decreased OXPHOS expression and activity in mouse embryonic fibroblast (MEF) cell lines, demonstrating the role of c-Src on mitochondrial OXPHOS in both health and disease. An inhibition of c-Src with the SFK inhibitor, PP2, and cSrc-specific small interfering ribonucleic acid (siRNA) alleviated the negative impact of c-Src on OXPHOS expression and improved mitochondrial energy metabolism while significantly impairing cell proliferation in normal and cancerous cells. In contrast, increased expression of Fyn kinase was associated with an elevated OXPHOS subunit expression in the liver cancer cell line, HepG2. Continuous OXPHOS activity can lead to high production of reactive oxygen species (ROS), which can cause mitochondrial damage; therefore, we explored the effects of Fyn kinase, along with the effects of its inhibition on OXPHOS expression and cell proliferation, by treating cells with the SFK inhibitor SU6656 and natural antioxidants kaempferol and resveratrol. An increase in Fyn expression was associated with high OXPHOS expression, which was reduced in the presence of SU6656 and kaempferol. Furthermore, the treatment of the HepG2 cell line with SU6656, kaempferol, and resveratrol significantly inhibited cell proliferation. These treatments reduced OXPHOS activity and slowed cell growth by impairing cell proliferation in HepG2 cells, possibly by inhibiting Fyn activity. Evidence provided in these studies indicate that increased expression of c-Src and Fyn are found in liver cancer and more importantly regulate mitochondrial energy metabolism by altering the expression of OXPHOS complexes, which can contribute to the development of cancer and other metabolic diseases. Our data suggests that the suppression of SFKs can reduce cell proliferation and improve mitochondrial function and energy metabolism and should be further evaluated as targets for the treatment of liver cancer. Together, our studies demonstrated that changes in the expression of OXPHOS complexes due to sequence variants or PTMs, can significantly impair mitochondrial energy metabolism and may be underlying factors in both T2D and liver cancer. The identification of factors contributing to mitochondrial dysfunction will allow us to improve disease prognosis and treatments in various disease states.

Subject(s)

Mitochondria -- Research.

Mitochondrial pathology.

Liver -- Cancer -- Research.

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