PhD, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Clear cell renal cell carcinoma (ccRCC) is the most common cancer of the kidneys. Understanding the mechanisms that govern tumor suppression and tumor progression is critical to facilitate the discovery of novel therapeutic avenues for treatment of ccRCC and to develop biomarkers for predicting relapse and response to treatment. Presented here are three studies investigating several of these mechanisms that we have recently identified. The first part elucidates the contribution of copper in driving tumor progression through metabolic rewiring and the pathways involved in maintaining cellular homeostasis in this condition. Namely, copper accumulation and allocation to cytochrome c oxidase is associated with higher stage tumors. Furthermore, copper augments tumor growth in xenograft models. Copper drives oxidative phosphorylation (oxphos) supported by mitochondrial remodeling which contributes to energy and biosynthesis. This induction of ETC/oxphos is coordinated with synthesis of a pool of glutathione derived from glucose through the activity of glutamate pyruvate transaminase 2 (GPT2) which couples the two pathways. Synthesis of glutathione is required to maintain redox homeostasis in these cells and represents an area of possible therapeutic vulnerability. The second part focuses first on the regulation of the non-canonical tumor-suppressing autophagic program requiring Microtubule Associated Protein 1 Light Chain 3 Gamma (MAP1LC3C/LC3C). LC3C is regulated by non-canonical upstream regulatory complex components whose assembly is coordinated by the unique C-terminal peptide that is present on the LC3C. The post-division midbody, associated with cancer cell stemness, is a LC3C specific target. Finally, we show that high lysosomal exocytosis is induced by loss of LC3C and leads to a decrease in intracellular zinc and transcriptomic reprogramming. This drives aggressive ccRCC tumor formation in mice. Together these studies deepen our understanding of the pathogenesis (open full item for complete abstract)

Committee: Maria Czyzyk-Krzeska M.D. Ph.D. (Committee Chair); David Plas Ph.D. (Committee Member); Atsuo Sasaki Ph.D. (Committee Member); Krushna Patra Ph.D. (Committee Member); Nicolas Nassar Ph.D M.A B.A. (Committee Member); John Cunningham Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy (PhD), University of Toledo, 2023, Experimental Therapeutics

Triple-negative breast cancer (TNBC), the most lethal and aggressive subtype of breast cancer, lacks estrogen receptors, progesterone receptors, and human epidermal receptors, rendering it unsuitable with targeted-based treatment. TNBC has higher relapse rate, worst prognosis and higher metastasis rate compared to non-TNBC because of their tendency to resist to apoptosis, a form of programmed cell death, induced by chemotherapy. Hence, non-apoptotic cell death inducers could be a potential alternative to circumvent the apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induce non-apoptotic cell death in TNBC cells. These lead compounds were 15 to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death characterized by no cellular shrinkage, absence of nuclear fragmentation and f apoptotic blebs. Although TPH104c and TPH104m produced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase total cytochrome c and intracellular ROS, lacked caspases activation, and was not rescued by pan-caspase inhibitor, zVAD-FMK. Moreover, TPH104c and TPH104m significantly downregulated mitochondrial fission protein, Drp1 and its levels determined their cytotoxic efficacy. Studies have shown that protein, Bcl-2 interacting protein 3 (BNIP3), mediates a non-apoptotic, necrosis-like cell death similar to that produced by TPH104c and TPH104m that lacked activation of caspases and reduced mitochondrial transmembrane potential. Therefore, we determined the effect of TPH104c and TPH104m on various mitochondrial functions, in triple negative breast cancer (TNBC) cells, BT-20 and MDA-MB-231. TPH104c and TPH104m (2 and 5 μM), compared to vehicle, significantly increased the levels of reactive oxygen species (ROS) BNIP3 and c-Jun (open full item for complete abstract)

Committee: Amit K. Tiwari (Committee Chair); Aniruddha Ray (Committee Member); Ana Maria Oyarce (Committee Member); Frederick E. Williams (Committee Member) Subjects: Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2022, Pathology

Mitochondrial dynamics, the fission and fusion of mitochondria, are vital to cellular health and homeostasis. Altered dynamics are associated with a number of diseases, one of which is the most aggressive and prevalent brain cancer, glioblastoma (GBM). Once diagnosed, patients live a median of less than 15 months with nearly all experiencing tumor recurrence, highlighting the need for therapeutic advancement. Also, increased mitochondrial fragmentation is often observed within the GBM stem cells (GSCs), known to be resistant to conventional therapies and believed to contribute to tumor recurrence. The mitochondrial fission machinery, dynamin-related protein-1 (Drp1) and mitochondrial fission factor (Mff), have both been implicated in contributing to the GBM disease state. To understand the altered regulation of these proteins in GBM, we investigate the post-translation modifications, O-GlcNAcylation and phosphorylation on them, respectively. We found that Drp1 O-GlcNAcylation and Mff phosphorylation were both increased in patient GSCs. Specifically, elevated O-GlcNAcylation of Drp1 correlated with increased mitochondrial fragmentation and mass, as well as altered mitochondrial ETC complex function. We also found that adenosine monophosphate (AMP)-activated protein kinase (AMPK) was the primary kinase phosphorylating Mff in one, but not all, of the GSC patient samples examined, demonstrating the importance of individual patient tumor profiling. Altogether, these studies demonstrate the importance of proper PTM regulation of Drp1and Mff in GBM, which may serve as future therapeutic points of intervention.

Committee: Jason Mears (Advisor); Brian Cobb (Committee Chair); Clive Hamlin (Committee Member); Justin Lathia (Committee Member); Ruth Keri (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Molecular Biology; Morphology; Science Education

PhD, University of Cincinnati, 2019, Medicine: Pathobiology and Molecular Medicine

Although great advances have been made in the development of therapies for Acute Lymphoid Leukemia (ALL) and Acute Myeloid Leukemia (AML), patients with reciprocal translocations of the 11q23 locus develop acute leukemias (MLL-r leukemias) resistant to conventional chemotherapies. The translocations generates an oncogenic fusion protein comprised of an amino terminus derived from MLL (now called KMT2A) gene fused to a carboxyl terminus derived from one of several different genes. Unfortunately, this disease is highly prevalent in infants accounting for 80% of ALL and 35-50% of AMLs. Novel therapies like small molecules inhibitors and immunotherapies have focused on inhibiting the functions of the MLL-fusion protein (MLL-FP) complex and associated proteins or target proteins that are expressed in a multitude of healthy cells leading to on-target off tumor effects and high toxicities. Therefore, further research is required to understand better the molecular pathobiology of the disease and develop more targeted therapies. Transcriptome studies are one approach to understand the pathobiology and potential vulnerabilities of diseases. MEIS1 and LAMP5 are two genes that have been identified in independent studies as being highly expressed in MLL-r leukemias regardless of age, lineage or fusion partner. MEIS1 have been shown to be required for normal maintenance of Hematopoietic Stem Cells (HSC) by limiting oxidative metabolism and ROS levels in these cells. In MLL-r leukemia, MEIS1 is essential for the maintenance of MLL-FP induced transformation. Furthermore, MEIS1 is required for the propagation of MLL-r leukemias in vivo. Similar to healthy HSC, MEIS1 regulate the hypoxic state through blocking of the induction of oxidative phosphorylation and generation of ROS, a mechanism required to retain stem cell properties in MLL-r leukemias. MEIS1 control this effects partly by regulating the expression of the Hepatic Leukemia Factor (HLF). MLL-r leukemias are greatly affecte (open full item for complete abstract)

Committee: Ashish Kumar M.D. Ph.D. (Committee Chair); Jose Cancelas-Perez M.D. (Committee Member); Gang Huang Ph.D. (Committee Member); James Mulloy Ph.D. (Committee Member); Daniel Starczynowski Ph.D. (Committee Member) Subjects: Molecular Biology

Master of Science (MS), Wright State University, 2015, Physiology and Neuroscience

Malignant transformation is the process by which cells develop cancer properties. While many causes for malignant transformation are known (i.e. common genetic mutations and/or exposure to toxins or viruses), the basic requirements that allow a cell to stay alive with altered nutrient and energy requirements are just now being studied. In some tumor types malignant cells undergo changes that result in metabolic differences compared to normal cells. These can include defects in mitophagy resulting in an accumulation of dysfunctional mitochondria and/or a metabolic switch resulting in increased glycolysis, termed the Warburg effect. Increased tumor growth and metastasis have also been associated with mitochondrial DNA mutations in some tumor types. In this study, we characterized the mitochondrial function of malignant peripheral nerve sheath tumor (MPNST) cell lines commonly used to study malignant transformation in Neurofibromatosis Type I. We identified metabolic differences between NF1-wildtype (STS26T) and NF1-deficient (ST88-14, 90-8, and S462) MPNST cell lines by measuring extracellular acidification and oxygen consumption, mitochondrial respiration protein expression, and ROS levels. Similar to findings from other malignant tumors, all MPNST cell lines were more glycolytic compared to non-tumorigenic normal human Schwann cells and surprisingly NF1-deficiency correlated with lower glycolytic and mitochondrial respiratory rate compared to wildtype MPNST. Mitochondrial respiratory rates and respiratory protein expression were significantly lower in the NF1-deficient MPNST cell lines when compared to NF1-wildtype MPNST cells. These findings demonstrate that neurofibromin affects glycolysis and mitochondrial respiration in malignant cells.

Committee: Debra Mayes Ph.D. (Advisor); Robert Putnam Ph.D. (Committee Member); Christopher Wyatt Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Neurosciences

Master of Science, The Ohio State University, 2009, Evolution, Ecology, and Organismal Biology

Differences in organismal life-history characteristics are often related to variation in an organisms' energy expenditure. For example, tropical bird species show a significantly slower “pace of life” in terms of increased longevity, reduced fecundity and faster maturation when compared with temperate species and this slower pace is correlated with a reduced basal metabolic rate (BMR). Differences in BMR could have a genetic basis yet the mechanisms governing energy expenditure remain unclear. Genes encoding proteins involved in crucial energetic pathways present logical candidates to explore the role of selection as a cause of energetic differences between species. The distribution of certain bird families in both tropical and temperate environments offers a unique opportunity to study the molecular evolution of these physiological adaptations through paired comparisons of mitochondrial DNA evolution among confamilial species. Here, I explored the role of evolution of mitochondrial proteins as a genetic cause for variation in mass-organismal energy expenditure by examining the role of selection on the 13 mitochondrial genes encoding proteins involved in the oxidative phosphorylation system (OXPHOS). I sequenced the OXPHOS mitogenome of 14 species of birds from the Order Passeriformes in paired confamilial groupings from both temperate and tropical environments and analyzed the molecular evolution of these genes as well as how the amino acid differences I observed may influence life-history traits between the regional groupings. Overall, I found an overwhelming signature of purifying selection operating on these genes across each complex of the OXPHOS system. However, variation in the level of functional conservation does vary between the ND and CO OXPHOS complexes. In addition I found 26 sites with elevated dN/dS ratios that occur in functionally important regions but do not show patterns of substitutions consistent with a role in metabolic governance. Therefore, t (open full item for complete abstract)

Committee: H. Lisle Gibbs PhD (Advisor); Joseph B. Williams PhD (Committee Member); Laura S. Kubatko PhD (Committee Member) Subjects: Biology; Genetics; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2008, Physiology and Biophysics

Lipid accumulation in non-adipose tissue may play a role in the pathophysiology of heart failure. Accumulation of myocardial lipids and ceramide is associated with decreased contractile function, mitochondrial oxidative phosphorylation, and electron transport chain (ETC) complex activities. We hypothesized that the progression of heart failure would be exacerbated by elevated myocardial lipids and a ceramide-induced inhibition of oxidative phosphorylation and ETC activities. Rats were fed a normal (14% kcal fat) or high fat diet (60% kcal fat) for two weeks. Heart failure was induced by coronary artery ligation. High fat feeding prior to ligation surgery increased surgical mortality, consequently the study was modified so that all rats remained on the normal diet prior to ligation surgery. Following ligation surgery, rats were fed a normal (HF) or high fat diet (HF+FAT) for eight weeks. Sham-operated animals were fed a normal diet. State 3 respiration and ETC complex activities were increased in subsarcolemmal mitochondria (SSM) of HF+FAT, despite elevated myocardial ceramide. No further progression of left ventricular dysfunction was evident in HF+FAT.We then investigated possible mechanisms by which high fat improved mitochondrial function in heart failure. We hypothesized that a high fat diet during heart failure would increase mitochondrial fatty acid oxidation and state 3 respiration by activating genes involved in fatty acid uptake and utilization. Rats underwent ligation or sham surgery and were fed a normal (SHAM, HF) or high fat diet (SHAM+FAT, HF+FAT) for eight weeks. State 3 respiration using lipid substrates was elevated in SSM of HF+FAT and correlated to increased activities of short-, medium- and long-chain acyl-CoA dehydrogenase. This was associated with improved myocardial contractility as assessed by LV +dP/dt max. Despite decreased medium-chain acyl-CoA dehydrogenase mRNA in HF and HF+FAT, protein content was unchanged. Though high fat improved myo (open full item for complete abstract)

Committee: Margaret Chandler PhD (Advisor); Nosek Thomas PhD (Committee Chair); Hoppel Charles MD (Committee Member); Fisher Steven MD (Committee Member); Romani Andrea PhD (Committee Member); Nagy Laura PhD (Committee Member); Kirwan John PhD (Committee Member) Subjects: Anatomy and Physiology

Doctor of Philosophy, Case Western Reserve University, 2005, Pathology

Protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) are prominent features of oxidative damage in neuronal cell bodies in Alzheimer disease (AD). Such adducts are also seen in axons of normal as well as diseased individuals. These HNE adducts are constant throughout the axons and the aging process of humans, mice, and rats, indicating that HNE adduction is physiological and regulated from birth to senility. In axons, HNE is primarily adducted to the neurofilament heavy subunit (NFH) and the neurofilament medium subunit (NFM), and limited to lysine residues. Interestingly, we found that phosphorylation is essential since formation and removal of HNE adducts are controlled by the NFH/M phosphorylation state. Studies using immunochemistry, synthetic peptides, mass spectrometry and chemical stabilization of HNE adducts, demonstrated that XKSPX, the most repeated sequences in NFH/M, are the major component in neurons highly susceptible to phosphorylation regulated aldehyde adduction. To our knowledge, this is the first study to directly show phosphorylation can regulate NFH-HNE levels in axons and the multiple KSP repeats are the critical motifs which preserve the phosphorylation-dependent regulation. This study provides the new evidence that indicates signal transduction could modulate oxidative modification in brain through activation of kinases and phosphatases.

Committee: George Perry (Advisor) Subjects: