Prostaglandin E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation.We generated HEK 293 EBNA cell lines expressing the EP3-Ia, EP3-II, or EP3-III isoforms. After confirming the functional expression of each of these isoforms, we examined their activation of cellular signal transduction pathways.We found that each of these isoforms utilize distinct mechanisms to regulate ERK 1/2 phosphorylation and that these differences lead to unique regulation of the downstream effectors ELK-1 and AP-1. We also found MAPK dependent differences in regulation of cell proliferation. The EP3-III isoform increases cell proliferation in a MAPK dependent manner while the EP3-Ia dose dependently regulates cell proliferation via Gαi and not ERK 1/2. Activation of the EP3-II receptor had no effects on cell proliferation.To study differential gene regulation by these three EP3 receptor isoforms, we conducted microarray studies. Over 300 genes were differentially regulated by these isoforms. Quantitative real-time PCR analysis was used to validate 15 candidate genes. Five genes were chosen for further analysis of protein expression using immunoblotting, but only one of these, WT-1, was significantly increased following treatment with PGE2. WT-1, a transcription factor important for kidney and heart development, was strongly upregulated by PGE2 stimulation of the EP3-II receptor, but only weakly by the other isoforms.In conclusion, these studies show that the human EP3 prostanoid receptor isoforms are capable of distinct regulation of both signal transduction pathways and gene transcription. Elucidating the differential functions of EP3 receptor isoforms may allow for greater understanding of the diverse functions attributed to this receptor and their physiological functions.

Activation of pathways resulting in an overexpression of oncoproteins, reliant on cap-dependent translation, or mutations of key proteins in a pathway can be advantageous to cancer cells but creates heightened protein quality control pressure. Because of this, there has been an interest in targeting enzymes involved in protein synthesis and protein quality control: such as the eukaryotic initiation factor, eIF4A, a DEAD-box RNA helicase involved in translation initiation, and p97, an AAA+ chaperone involved in protein quality control. Despite some successes in discovering both eIF4A and p97 inhibitors, many of these compounds have pharmacological setbacks. The work in this dissertation defines new inhibitors of eIF4A and p97 with unique mechanisms of action. As described in chapter 2, we demonstrated that a marine-derived sesquiterpene, elatol, can modulate the ATPase activity of eIF4A. We provide further evidence that this molecule inhibits cap-dependent translation. Because there is no clear consensus on the mechanism of action for elatol, we hypothesized that the mechanism of toxicity attributed to elatol is likely through inhibition of cap-dependent translation initiation by targeting eIF4A. In chapter 3, we adapted a colorimetric assay to identify natural products that modulate the ATPase activity of p97 from which withaferin A (WFA) was identified. Because proteostasis modulation can connect each of the reported modes of action of WFA, we hypothesized that the primary mode of cytotoxic action of WFA is through inhibition of protein quality control machinery. Through medicinal chemistry efforts, we were able to improve WFA's biochemical and cellular activities as well as shifting the activity toward p97 and away from the proteasome. The work described in chapter 4 reports that dehydrocurvularin (DHC) and its chlorinated analogs are covalent modifiers of p97 and that the selectivity toward p97 can be attributed, in part, to the electronic effects of the chlorines. Taken together, this work highlights the significance of targeting protein translation and quality control, by modulation of eIF4A and p97 activity respectively, as promising anticancer therapeutics.

Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates a battery of downstream genes that contain the antioxidant response element (ARE) in their promoter regions, including intracellular redox-balancing proteins, phase II detoxifying enzymes, and transporters. These Nrf2-dependent proteins work in collaboration to protect against many diseases where oxidative stress plays an essential role in disease onset and progression. Consequently, it is imperative to understand the basic molecular mechanisms of how Nrf2 is regulated so that this pathway can be targeted for disease prevention and treatment.Nrf2 is mainly regulated at the protein level by the ubiquitin proteasome system. Under basal conditions Nrf2 is constantly ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and subsequently degraded by the 26S proteasome. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, other mechanism responsible for modulating Nrf2-ARE signal remains to be explored. This dissertation identifies three molecular mechanisms that are important in understanding how the Nrf2-Keap1 pathway is regulated: (i) In Chapter 2, KPNA6 was identified and characterized as a negative regulatory mechanism of the Nrf2 pathway, which mediates Keap1 nuclear import and represses the Nrf2-dependent antioxidant response at post-induction phase. (ii) In Chapter 3, I identified PARP-1 as a new transcription co-activator of Nrf2, which augments ARE-specific DNA binding of Nrf2 and enhances the transcription of Nrf2 target genes. This indicates a novel function of PARP-1 and reveals another layer of regulation of Nrf2. (iii) In Chapter 4, I demonstrated that XBP1 and SYVN1 are involved in regulating the Nrf2 pathway in a Keap1-independent mechanism. During ER stress, XBP1s upregulates transcription of SYVN1, which is an ubiquitin E3 ligase. SYVN1 accelerates the clearance of Nrf2 protein through promoting ubiquitination of Nrf2, and subsequent proteasomal degradation. Moreover, we observed an inverse correlation between XBP1s/SYVN1 and Nrf2 expression in the end stage alcoholic cirrhosis liver samples, implying a pathological role of ER stress-oxidative stress crosstalk. Taken together, these findings further our understanding of how the Nrf2-Keap1 pathway is regulated, providing novel targets of chemoprevention or chemotherapy.

Cumulative evidence suggests that constitutively elevated levels of proteotoxic stress represent a specific vulnerability of malignant cells that can be targeted by pharmacological modulation of the intracellular proteotoxic stress response. According to this emerging mechanism, small molecule stress modulators may induce deviations from protein homeostasis causing cytotoxicity confined to malignant cells already at a high set point of constitutive proteotoxic stress leading to functional impairment and even cell death. In contrast, normal cells with sufficient protein degradation capacity can tolerate the extra dysfunctional protein overload. My graduate research has focused on testing the feasibility of repurposing clinically used non-oncological drugs for experimental chemotherapy targeting metastatic melanoma cells. The following specific aims were pursued: (1) To identify clinically used non-oncological drugs that preferentially induce cytotoxicity in melanoma cells but not primary melanocytes through upregulation of proteotoxic and/or oxidative stress; (2) To explore the specific molecular mechanisms underlying induction of melanoma cell apoptosis by lead compounds focusing on oxidative and proteotoxic stress modulation; (3) To explore efficacy of selected lead compounds for antimelanoma intervention in a murine xenograft model. First, we demonstrate feasibility of using the FDA-approved redox-active D-cysteine-derivative D- penicillamine for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo through induction of the unfolded protein response (UPR). Second, we demonstrate that the antimicrobial oligopeptide thiostrepton displays dual activity as a selective prooxidant and proteasome inhibitor causing proteotoxic stress that preferentially targets malignant melanoma and multiple myeloma cells. Third, we demonstrate for the first time that the clinically used 4-aminoquinoline antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced melanoma cell death. Taken together, our data indicate the chemotherapeutic potential of small molecule proteotoxic stress inducers and strongly suggest feasibility of repurposing specific non-oncological drugs for proteotoxic stress-directed antimelanoma intervention.

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In the studies presented herein, MTK1 is shown to be required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced extracellular acidification and cell migration in MCF-7 breast cancer cells. Furthermore, it was shown that HRG stimulation leads to association of MTK1 with tyrosine phosphorylated HER3 in MCF-7 and T-47D breast cancer cells. The MTK1/HER3 association was dependent on HER2 activation and was decreased by pre-treatment with the HER2 inhibitor, lapatinib. Furthermore, HER2 does not directly associate with MTK1, but phosphorylates HER3 transiently. MTK1 also has a role in the ERK1/2 MAPK signaling pathway in response to heregulin (HRG) stimulation in T-47D and MCF-7 breast cancer cells. In addition to MTK1, Shc, Grb2 and GIT1 proteins are all involved in the ERK1/2 MAPK pathway in response to growth factor stimulation. MTK1 was also shown to associate with activated ERK1/2, GIT1, Shc, Grb2 and p85 of PI3K in response to heregulin stimulation. ERK1/2 kinase activity is involved in aberrant signaling that leads breast cancer progression. GIT1 is a scaffolding protein that is linked to growth factor mediated ERK1/2 signaling in cell migration. Moreover, we also identify the actin interacting region (AIR) on MTK1 and disruption of actin cytoskeletal polymerization with cytochalasin D inhibited the interaction between HER3 and MTK1, indicating that f-actin (which is needed for cell migration) is required for the MTK1/HER3 association. Additionally, HRG stimulation leads to extracellar acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.

Although curable in the majority of cases, Diffuse Large B-cell Lymphoma (DLBCL), the most prevalent Non-Hodgkin Lymphoma (NHL) throughout the world, is still fatal for 30-40% patients. This patient population could benefit from the addition of new drugs to the current DLBCL chemotherapy regimen. Histone deacetylase inhibitors (HDIs) are a promising group of drugs for the treatment of hematological malignancies. In the current study we tested the HDI PXD101 in a panel of the two most common DLBCL subtypes, GCB (germinal center) and ABC (activated B-cell like), ABC being the least curable subtype. Cell viability assays showed that PXD101 induces antiproliferative effects at submicromolar concentrations in DLBCL cell lines regardless of DLBCL subtype. Flow cytometry demonstrated that upon PXD101 treatment two GCB cell lines (DB and OCILY19) undergo G2M cell cycle arrest followed by apoptosis, while two GCB (SUDHL4 and SUDHL8) and one ABC (U2932) cell line undergo G1 arrest with little apoptosis. Further experiments demonstrated that upon PXD101 removal G1-arresting cells recover their normal proliferative state, while in G2M-arresting cells only 8h exposure to PXD101 is sufficient to induce considerable apoptosis. We classified as PXD101-resistant cell lines that re-enter the cell cycle after drug removal, and PXD101-sensitive cell lines that commit to apoptosis after short periods of drug exposure. Kinase assays established that upon PXD101 treatment G1 phase cyclin dependent kinase 2 (CDK2)-cyclin E complex activity significantly decreases in resistant but not in sensitive cells lines. Furthermore, pull-down assays revealed that CDK inhibitors (CDKIs) p21 and/or p27 in resistant, but not sensitive cell lines persistently bind the CDK2-cyclin E complex throughout PXD101 treatment, thereby explaining why resistant lines stop at the G1 phase. CDKIs induction by PXD101 was p53-independent. This is the first time that an in vitro model of sensitivity and resistance to HDIs in DLBCL is established. We have also performed preliminary genomic and proteomic analysis in DLBCL cell lines treated with PXD101. We anticipate that further analysis of the genomic response and the functional impact of protein acetylation induced by HDIs will offer additional insight into mechanisms of sensitivity and resistance to HDIs in DLBCL.

Arsenic is common metalloid that is found globally. Its ubiquitous nature means that large portions of the global population are exposed through a variety of pathways. Arsenic is a known human carcinogen and its role in the development of cardiovascular and metabolic disease has become more completely characterized in the past decades. However, the examination of arsenic exposure during embryonic development at relatively low level exposures is an emergent area where lots of questions remain unanswered. As arsenic is difficult and costly to remove from water, the investigation of exposures in vulnerable populations at relevant concentrations is necessary to justify remediation efforts. This dissertation work examines fetal arsenic metabolism contributing to the understanding of tissue specific arsenic effects during embryonic development. Following that, the focus shifts to fetal and early life exposure to arsenite at 100 parts per billion in drinking water (10 times higher than the EPA mandated limit in municipal water but common in well water globally) and how it contributes to the severity and incidence of diet-induced non-alcoholic fatty liver disease (NAFLD). NAFLD is the most prevalent chronic liver disease in the United States and it contributes to increased cardiovascular morbidity and mortality. NAFLD also results in alterations in hepatic drug metabolism and disposition which contributes to adverse drug reactions. We demonstrate similar effects in arsenic potentiated NAFLD in addition to changes in transporter expression with arsenic exposure alone independent of triglyceride accumulation associated with NAFLD. Taken together, this work highlights deleterious health effects of low level arsenic exposure during development and demonstrates the need for further investigation of developmental arsenic effects.

The risk of breast cancer transiently increases immediately following pregnancy. Hispanic women have one of the highest rates of postpartum breast cancers of all racial/ethnic minority groups in the US. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. In this study, we measured the miRNA expression of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n=12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n=44). We identified 15 miRNAs that are differentially expressed between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression; miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 were overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were under expressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Taken together, the results of this study show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.

Adverse drug reactions (ADRs) are a pervasive complication in the realm of pharmacotherapy. At the root of ADRs lies interindividual variability in drug response, which can range from allergic reactions, to genetic variability, to any factors that influence the pharmacokinetics of a drug. Nonalcoholic steatohepatitis (NASH) is the late-stage of non-alcoholic fatty liver disease (NAFLD), characterized by fat deposition, oxidative stress, inflammation, and fibrosis. Over the last several years, alterations in drug metabolizing enzymes and transporters have been broadly characterized through NAFLD progression. Multidrug resistance-associated protein 2 (MRP2) is a canalicular efflux transporter that directs the biliary elimination of a wide variety of xenobiotics and metabolites. In NASH, MRP2 is mislocalized away from the canalicular membrane in a post-translational event. The mechanism and extent of this mislocalization has yet to be elucidated. While transporter misregulation has been shown to influence the disposition of a variety of substrates, the direct impact of MRP2 mislocalization on its overall transport capacity, and pharmacologic consequence of this change, is unknown. The purpose of this study was to elucidate the mechanism behind, and functional consequence of, MRP2/Mrp2 mislocalization in NASH, predominantly using the rodent methionine-and-choline-deficient (MCD) dietary model.To identify the mechanism of MRP2/Mrp2 mislocalization, a comparison of the activation status of various mediators of MRP2/Mrp2 retrieval was conducted between healthy and NASH livers. Results in rat samples and human NASH samples indicate that activation changes of these mediators, including radixin, PKCα, PKCδ, and PKA, are consistent with a shift toward active retrieval of MRP2/Mrp2 from the membrane, and some evidence of impaired membrane insertion is also present. Measurement of Mrp2 transport capacity was completed using pemetrexed, a novel Mrp2 probe substrate. Comparison of biliary excretion of pemetrexed between wild-type and Mrp2^(-/-) rats shows a 100% decrease, confirming that it relies upon Mrp2 for biliary excretion. NASH rats exhibited a 60% decrease in pemetrexed levels in the bile compared to their control counterparts, indicating that Mrp2 transport capacity is severely impaired in NASH rats. Finally, to ascertain the pharmacologic consequence of impaired Mrp2 transport, a study was conducted measuring the effects of the active morphine glucuronide on control and NASH rats. NASH rats exhibited a decreased biliary excretion, and increased systemic retention, of M3G. While they did also exhibit increased antinociception of M6G, the definitive impact of altered disposition on pharmacologic response was masked due to the interference of an MCD dietary effect on antinociception. Overall, the data reported herein identify active membrane retrieval as a mechanism of MRP2/Mrp2 mislocalization in NASH, and that mislocalization results in a 60% decrease in overall Mrp2 transport capacity. This decrease significantly hinders biliary excretion of Mrp2 substrates, and may result in ADRs by contributing to interindividual variability in drug response.

Uranium has been mined for many years and used for fuel for nuclear reactors and materials for atomic weapons, ammunition, and armor. While the radioactivity associated with uranium mining has been linked to the development of lung and kidney cancers, and leukemia, little is known about the direct chemical genotoxicity of uranium. The overall hypothesis of the current research is that uranium can produce DNA damage by chemical genotoxicity mechanisms. Three specific aims were tested. In Aim 1, specific DNA lesions caused by direct interaction of uranium and DNA were investigated. Chinese Hamster Ovary cells (CHO) with mutations in various DNA repair pathways were exposed to 0 – 300 μM of soluble depleted uranium (DU) as uranyl acetate (UA) for 0 – 48 hr. Results indicate that UA readily enters CHO cells, with the highest concentration localizing in the nucleus. Clonogenics assay shows that UA is cytotoxic in each cell line with the greatest cytotoxicity in the base excision repair deficient EM9 cells and the nuclear excision repair deficient UV5 cells compared to the non-homologous end joining deficient V3.3 cells and the parental AA8 cells after 48 hr. This indicates that UA is forming DNA adducts that may be producing single strand breaks through hydrolysis rather than double strand breaks in CHO cells. Fast Micromethod® results indicate an increased amount of single strand breaks in the EM9 cells after 48 hr UA exposure compared to the V3.3 and AA8 cells. In Aim 2, the role of oxidative stress in producing DNA lesions was determined. Cellular oxidative stress has been implicated in the genotoxicity of many heavy metals as a mechanism of induced DNA damage. To investigate this possible mechanism, human bronchial epithelial cells (16HBE14o⁻) were exposed to 30 ppb (0.13 μM U) UA for 2 – 24 hr. UA did not significantly induce oxidative stress compared to untreated cells at 3 – 4 hr time points. These results suggest that cellular oxidative stress is not a major pathway of DU genotoxicity at low concentrations. In Aim 3, DNA damage response to uranium-induced DNA damage was investigated. It has been widely reported that metals can be genotoxic by inhibiting DNA repair. Cultured cells were co-exposed to 0.13 μM UA in the presence of 0 – 25 μM of etoposide for 0 – 48 hr. Results indicate that UA inhibited double strand break repair. Coexposures of etoposide and UA synergistically induced cytotoxicity compared to individual treatments and untreated cells. Co-exposed UA and etoposide treated 16HBE14o⁻ cells exhibited a decrease in phosphorylation of DNA repair proteins compared to etoposide treatments. Untreated and UA-treated 16HBE14o⁻ cells did not induce phosphorylation of DNA repair proteins. These results suggest that DU inhibits double strand break DNA repair at low concentrations in the presence of a known DNA double-strand damaging agent, etoposide. The inhibition of DNA repair by DU at environmentally relevant concentrations suggests a novel means by which uranium may exert its genotoxic effects. Results found at low dose exposures are not consistent with alterations seen with radioactivity, suggesting that the effects of uranium at low doses are due to its chemical genotoxic effects. Understanding how uranium reacts with DNA is important to better understand how this suspected carcinogen induces cancer and to help to elucidate mechanisms that produce cancers in people exposed to uranium.