Melanoma incidence in the United States has grown continuously at a rate of 1.5% each year for the last decade. Disease detected early can be cured by surgery, but metastatic disease is lethal. Recent discoveries have led to promising-targeted MAPK-pathway inhibitors (MAPKih) and immunotherapies. However, low response rates and acquired drug resistance remain as significant challenges to improved outcomes. The mechanisms that drive resistance to MAPKih are elusive. On the other hand, our data suggests that drug-induced alterations in oxidative metabolism and cellular antioxidant systems (e.g., glutathione; GSH and superoxide dismutase; SOD) in melanoma cells play prominent roles in acquisition of resistance to melanoma drugs. Our studies further indicate a correlative relationship between changes in cellular/mitochondrial reactive oxygen species (superoxide, hydrogen peroxide) levels and adaptation of melanoma cells to MAPK pathway inhibition (MAPKi). Interestingly, our findings suggest that drug-induced alterations in the oxidized and reduced GSH balance facilitate acquisition of drug resistance by activating restorative pathways (e.g., the unfolded protein response; UPR and autophagy). Our data further show that inhibiting GSH synthesis using FDA-approved drug buthionine sulfoximine (BSO) in the presence of MAPKih prevents the acquisition of resistance to MAPKih in vitro. Further, simultaneously attenuating ER-stress responses using sodium 4-phenylbutyrate (PBA) and inhibiting autophagy using hydroxychloroquine (HCQ) in combination with MAPKih prevented adaptation to MAPKi in vitro and significantly improved tumor response and overall survival of mice bearing metastatic melanoma xenograft tumors (BRAFi-resistant and sensitive) in vivo. This thesis research focuses on exploring the role of MAPKi-mediated metabolic reprogramming and changes in the oxidative state in melanoma cells and tumors with the acquisition of resistance to MAPKih in metastatic melanoma.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is an experimental chemotherapeutic agent, believed to form free radicals in vivo, and cancer cell resistance to 17AAG is believed to be a thiol-dependent process. Inhibitors of thiol-dependent hydroperoxide metabolism [L-buthionine-S,R-sulfoximine (BSO) and auranofin (AUR)] were combined with the inhibitor of glucose metabolism [2-deoxy-D-glucose (2DG)] to determine if 17AAG-mediated cancer cell killing could be enhanced. When 2DG (20 mM, 24 h), BSO (1 mM, 24 h), and auranofin (500 nM, 3 h) were combined with 17AAG, they significantly increased cell killing in three human cancer cell lines (PC-3, SUM159, MDA-MB-231), relative to 17AAG alone. Increases in toxicity seen with this drug combination also correlated with increased glutathione (GSH) and thioredoxin (Trx) oxidation and depletion. Furthermore, treatment with the thiol antioxidant, N-acetyl cysetine (NAC, 15 mM, 24 h), was able to significantly protect from drug-induced toxicity and ameliorate GSH oxidation, Trx oxidation, and Trx depletion. These data strongly support the hypothesis that simultaneous inhibition of GSH and Trx dependent metabolism is necessary to sensitize human cancer cells to 2DG + 17AAG-mediated cancer cell killing by enhancing thiol-dependent oxidative stress and suggest that simultaneous targeting of both GSH and Trx metabolism could represent an effective strategy for chemo-sensitization in human cancer cells.

This thesis describes studies that are designed to investigates the hypothesis that mitochondrial production of reactive oxygen species (O2*- and H2O2) cause oxidative stress during PCB exposure and this increased production of ROS contributes to the biological effects of PCBs on cell proliferation in human breast and prostate epithelial cells. Exponentially growing non-malignant human breast epithelial cells (MCF-10A) and non-malignant human prostate epithelial cells (RWPE-1) were treated with selected PCBs and their metabolites (PCB3, 77, 153, Aroclor and 4ClBQ). Results showed that PCBs and their metabolites could significantly inhibit MCF-10A and RWPE-1 cell growth as well as inducing clonogenic cell killing. These PCBs were also found to increase steady-state levels of mitochondrial O2*- and H2O2. Furthermore, the same PCBs were also found to induce alterations in SOD activities in MCF-10A and RWPE-1 cells. Finally, treatment with either N-acetyl-cysteine (NAC), or the combination of polyethylene glycol (PEG) conjugated CuZnSOD and PEG-catalase added 1 hour after PCBs, significantly protected MCF-10A and RWPE-1 cells from PCB-induced toxicity even when added following PCB exposure. Similar experiments were also accomplished using airborne PCBs treated RWPE-1 cells. 4-OH-PCB11, a metabolite of airborne PCB 11 is shown to lead to steady-state increases in superoxide and hydroperoxides in exponentially growing RWPE-1 human nonmalignant prostate epithelial cells. This increased level of ROS was accompanied by the inhibition of cell growth and clonogenic cell killing. Furthermore treatment of cells with antioxidants one hour following exposure to 4-OH-PCB11 was able to significantly diminish the toxicity in human prostate epithelial cells. These results strongly supported the hypothesis that exposure to PCBs or their metabolites can induce the cytotoxicity and alterations in cellular proliferation as well as causing oxidative stress in exponentially growing human breast and prostate epithelial cells. More importantly, the data also provide clear evidence that antioxidant manipulations after PCB exposure are capable of protecting human cells against PCB-induced cytotoxicity. Based on these observations, the long term goal of this work is to develop a mechanism based biochemical rationale for the development of pharmaceutical manipulations to protect humans from PCB intoxication.

Melanoma incidence is increasing faster than any other cancer worldwide.1 Early detection is often curative, but metastatic melanoma is lethal (5-year survival <20%) due to the development of resistance to all approved drugs.1 However, emerging evidence suggests that differences in melanoma metabolism relative to non-malignant cells may provide a target to improve treatment.2-14 Specifically, melanoma cells have increased mitochondrial electron transport chain (ETC) activity, elevated levels of reactive oxygen species, and a simultaneous hyperpolarized mitochondrial membrane potential relative to non-malignant cells.4, 8, 11, 15-17 Furthermore, melanoma cells have upregulated glucose consumption and concurrent increased levels of glucose transporters (GLUTs) relative to non-malignant cells; the products of glycolysis (pyruvate and NADPH) aid in the detoxification reactive oxygen species (ROS), while the intermediates are utilized in energy production via increased oxidative metabolism.15, 18 Collectively, melanoma cells exhibit alterations in metabolic, mitochondrial, and cell-surface targets that can be potentially exploited for therapeutic strategies for selective cancer cell killing relative to non-malignant cells. The research presented here demonstrates the therapeutic potential for a new class of mitochondrial-targeted fluorescent lipophilic-cations: pyridinium derivatives (UIRF 17023.186PV1 U.S. Provisional Patent Application No. 62/268,980 Patent Pending). Importantly, the pyridinium derivatives presented in this study have not been previously investigated as a mitochondrial-targeted therapy.19-21 Furthermore, the research presented outlines the feasibility of improving melanoma cellular accumulation of these pyridinium derivatives by including a GLUT targeting moiety in the form of a hexosamine. The addition of a hexosamine molecule to pyridinium derivatives has the potential to increase melanoma cell accumulation by targeting upregulation of GLUT expression in melanoma cells relative to normal cells. Thus, the results of this study identified: (1) a triphenylvinylpyridine (TPVP) lipophilic cation derivative that increased melanoma oxidative metabolism and decreased melanoma cell viability; and (2) the targeting potential for GLUT-mediated melanoma cell specific delivery of glucosamine-modified TPVP derivatives. These findings support the hypothesis that TPVP-based therapies can be developed to exploit fundamental differences in glucose and mitochondrial metabolism to selectively kill melanoma cells relative to non-malignant cells.

Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified by a class of enzymes called glucosyltransferases. This class of enzymes is of interest to this lab due to their unique structural and functional properties. Glucosides of flavonoids produced by glucosyltransferases are a critical part of plant metabolism and survival; many have health benefits when consumed. One such glucosyltransferase, found in Duncan grapefruit (Citrus paradisi), was identified, recombinantly expressed, and shown through biochemical characterization to exclusively glucosylate the flavonol class of flavonoids at the 3-OH position. The structural basis that accounts for a glucosyltransferase’s selectivity is not currently known, however great advances have been realized through the protein crystallization of 6 different secondary product glucosyltransferases. None of these show the same specificity exhibited by this flavonol-specific glucosyltransferase, CP3GT. The WT enzyme and two mutants have undergone site-directed mutagenesis to insert thrombin cleavage sites for removal of recombinant protein tags. The plasmid was transformed into yeast and protein was expressed through methanol induction. Cobalt column affinity chromatography was used to purify the protein. An aliquot of protein was treated with thrombin to remove tags and both tagged and native protein were assayed for activity with the flavonol quercetin. The data show that the reaction is linear for at least 15 minutes when 2ug of enzyme is used. Thus, kinetics assays will be conducted for 10 minutes. The presence of tags on the enzyme does not appear to impact activity with respect to the time course, however, more assays must be conducted to reliable confirm this with kinetic assays under different conditions. It is hypothesized that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, it is hypothesized that a thrombin cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.

Eukaryotic gene expression is a complex process that can be controlled at the level of transcription, post-transcription, translation, or post-translation. In recent years there has been growing interest in understanding the role of the 3'-untranslated region (UTR) in post-transcriptional regulation. The 3'-UTR contains many regulatory sequences, including microRNA (miR) target sites and AU-rich elements (AREs). Although a relatively recent discovery, miRs have been shown to downregulate target gene expression and have important roles in regulating many cellular processes, including cellular growth. Cellular growth consists of two distinct states, proliferation and quiescence. The proliferative state consists of G1, S, G2, and M phases while quiescence is the G0 phase. In response to mitogenic stimuli, quiescent cells enter the proliferative cycle and may transit back to the quiescent state. Reentry into quiescence is essential to prevent aberrant proliferation as well as to protect the cellular life span. Cells that remain in quiescence for an extended period of time lose their ability to proliferate. It has been shown that the redox status of the cells may regulate quiescence and proliferative capacity since overexpression of SOD2 protects the proliferative capacity of quiescent cells. We hypothesized that the redox environment regulates proliferative capacity through miR expression and regulation of miR targets. Early results showed treatment with hydroxytyrosol (HT), an olive-derived catechol, was able to protect the proliferative capacity of quiescent normal human fibroblasts. HT was shown to use hydrogen peroxide and produce superoxide in a catechol-semiquinone-quinone redox cycle. Interestingly, HT also induced SOD2 expression. Further results from microRNA PCR arrays and Taqman PCR assays showed a significant decrease (4-fold) in miR-302 levels during quiescence compared to proliferating normal human fibroblasts, suggesting that miR-302 could regulate cellular growth states. Results from a Q-RT-PCR and dual luciferase-3'-UTR reporter assays identified ARID4a (AT-Rich Interacting Domain 4a, also known as RBP1) and CCL5 (C-C motif Ligand 1) as targets for miR-302. Ionizing radiation, that is well known to induce oxidative stress and delay cell cycle progression, decreased miR-302 levels, which was associated with an increase in its target mRNA levels, ARID4a and CCL5. Such an inverse correlation was also observed in cells treated with hydrogen peroxide, SOD2 overexpressing cells, and HT treated cells. Overexpression of miR-302 suppresses ARID4a and CCL5 mRNA levels, and increased the percentage of S-phase cells. These results identified miR-302 as a redox-sensitive regulator of ARID4a and CCL5 mRNAs as well as demonstrate a regulatory role of miR-302 during quiescence and proliferation.

Breast cancer is a heterogeneous disease with multiple phenotypes that specify both treatment options and prognosis. The Luminal A-type breast cancers, characterized by high levels of estrogen receptor (ER) expression and transcriptional activity, have a stable of hormone-related treatment options and a favorable prognosis. Recent efforts to identify mechanisms governing the Luminal A phenotype have identified transcription factor activator protein 2C (TFAP2C) as a critical regulator of both ER and ER-associated gene expression, making it a prime target for manipulation in breast cancer therapy. To that end, we sought to establish specific contributions of TFAP2C in both normal development and cancer progression, with the overarching hypothesis that TFAP2C is an integral transcription factor in maintaining a luminal differentiation and expression pattern in human breast cancer. To address this, we utilized several parallel approaches to identify potential TFAP2C-related contributions to carcinogenesis. In the first approach, we identified that tissue-specific abrogation of the mouse homolog TCFAP2C in mammary epithelium produced a reduction in luminal populations and a concomitant increase in basal populations, producing mild phenotypic alterations in gland structure. In the second, we stably manipulated TFAP2C expression in the established Luminal A model cell line MCF-7, establishing that TFAP2C loss promotes a loss of luminal differentiation characteristics and a gain of basal / mesenchymal traits, mirroring the results found in the mouse model. As a final approach, we examined the role of a TFAP2C target gene, manganese superoxide dismutase (MnSOD), in modulating redox biological and epigenetic parameters that may contribute to carcinogenesis. We found that loss of MnSOD in the murine liver, chosen because of its utility as a model system, promoted subtle changes in the redox buffering capacity and identified preliminary changes in the epigenome, suggesting that MnSOD modulation by TFAP2C could play a role in cancer development. Overall, these results further establish a role of TFAP2C in the genesis of Luminal A breast cancer, and serve as a foundation for more comprehensive future work evaluating specific contributions in carcinogenesis.

Combination radiation and chemotherapy are commonly used to treat locoregionally advanced head and neck squamous cell carcinoma. Aggressive dosing of these therapies is significantly hampered by side effects due to normal tissue toxicity. Selenium represents an adjuvant that selectively sensitizes cancer cells to therapy, potentially by inducing lipid peroxidation (LPO). This study investigated whether one such selenium compound, methylseleninic acid (MSA), induces LPO and radiation sensitivity in HNSCC cells. Results from BODIPY C11 oxidation and ferric thiocyanate assay revealed that MSA induced LPO in HNSCC cells rapidly and persistently. Cell counts and propidium iodide viability assays showed that MSA was more toxic to HNSCC cells than other related selenium compounds, and this toxicity was abrogated by treatment with α-tocopherol, an LPO inhibitor. MSA was also found to sensitize HNSCC cells to radiation by clonogenic assay. The addition of MSA to a cell-free solution of glutathione (GSH) resulted in an increase in oxygen consumption as measured by Clark electrode, suggesting the formation of reactive oxygen species. Intracellular GSH in HNSCC was depleted following MSA treatment. Supplementation of the intracellular GSH pool with N-acetylcysteine (NAC) rendered the cells more sensitive to MSA. Results from this study identify MSA as an inducer of LPO, and reveal its capability to sensitize HNSCC to radiation. MSA may represent a potent adjuvant to combination therapy in HNSCC.

The SOD2 gene encodes the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), which converts superoxide (O2●-) to hydrogen peroxide (H2O2). Down-regulation of SOD2 has been reported in many cancer cells of diverse tissue origins, and forced over expression of this enzyme in carcinoma cells decreases their tumorigenicity. These findings suggest that SOD2 functions as an intiation or promotion tumor suppressor; however, it remains to be determined whether loss of SOD2 expression is sufficient for tumor formation since homozygous SOD2 knock-out mice die within weeks after birth. Additionally, due to the shortened lifespan of the constitutive SOD2 knock-out mouse limited studies have been performed in assessing the role of SOD2 in tissue-specific development in vivo. Using Cre-loxP genetics, we now have the capability to generate mice in which SOD2 may be knocked out in a cell type-specific manner and these mice can be used to query the effect of loss of SOD2 expression in tissue development, function, and oncogenesis. We primarily focused our studies on the hematopoietic system, but expanded our research to solid organs such as the liver and mammary gland as well. Our findings demonstrate that SOD2 does not act as a tumor suppressing enzyme in any of these unchallenged systems, and in fact the loss of SOD2 may act to delay tumor formation. T-cell specific SOD2 knock-out demonstrated significant immunodeficiency as illustrated by a decreased immune response to influenza challenge secondary to decreased T-cell populations. Furthermore, hematopoietic stem cell specific SOD2 knock-out showed a severe anemia in the red blood cell population due to increased mitochondrial superoxide. Additional analysis revealed the anemia to be a result of a porphyria due to the inactivation of the heme synthesis enzyme ferrochelatase. The observed phenotypes in all conditional hematological SOD2 knock-out models were rescued by the addition of mitochondrially targeted anti-oxidants. In conclusion, the tissue specific loss of SOD2 in various hematological organs appeared to demonstrate significant developmental and functional aberrations, but the role in tumor initiation appears to be limited if any. The data presented here also suggest the potential for novel anti-oxidant therapy in a variety of hematological diseases.

Apoptosis is a cell death pathway that regulates tissue homeostasis, and is often altered in oncogenesis. Autophagy is a lysosome degradation pathway that mediates cellular adaptation in response to stresses. Altered autophagy pathways are proposed to be associated with pathogenesis of neurodegenerative diseases and oncogenesis. The goal of this work is to study the complex link between apoptosis and autophagy pathways, and their possible roles in the development of cancer. Using transgenic mice models, we found that impaired apoptosis by overexpression of a dominant negative form of Caspase-9 (Casp9DN) failed to accelerate T-cell lymphoma either by itself or in tumor-prone Bax overexpressing transgenic mice. Additionally, heterozygous disruption of Beclin 1, a central upstream autophagy regulator, failed to promote T-cell lymphoma in either Casp9DN or tumor-prone Bax overexpressing transgenic mice. However, caspase inhibition enhanced a unique form of selective mitochondrial autophagy, referred to as mitochondrial outer membrane permeabilization (MOMP)-induced mitophagy. Parkin, a protein mutated in early-onset Parkinson's disease, mediates mitophagy following protonophore (CCCP) treatment, suggesting that Parkin may also play a role in MOMP-induced mitophagy. Thus, two different types of mitochondrial stresses, MOMP and CCCP, cause mitochondrial depolarization and induce mitophagy. We therefore examined if there is a mechanistic link between two mitophagy pathways. Focusing on the roles of autophagy and apoptosis regulators using isogenic hematopoietic cell lines, our studies demonstrate that MOMP-induced mitophagy is dependent upon Bcl-2 family members, but independent of Parkin or ULK1 (an autophagy regulator). In contrast, CCCP-induced mitophagy is dependent upon Parkin and ULK1, but independent of Bcl-2 family members. However, we found that both pathways ultimately result in the following properties: reduced mitochondrial respiration rate, altered cellular metabolism, and high sensitivity to 2-DG (an inhibitor of glycolysis). Interestingly, 2-DG induced cell death in cells following Parkin-dependent mitophagy is independent of Bcl-2 and Bax/Bak. Overall, the work in this dissertation demonstrates that the two different mitochondrial stresses, MOMP and protonophore (CCCP) treatment, lead to two mechanistically distinct mitophagy pathways, but both alter mitochondrial respiration and cellular metabolism.