The G-protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4) plays an important role in prostate cancer metastasis. CXCR4 is regarded as a plasma membrane receptor, that it transmits signals that support transformation, progression and metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutic approaches such as antagonists and monoclonal antibodies have focused on receptors the located at the plasma membrane. An emerging concept for GPCRs is that they can localize to the nucleus where they may retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 is highly expressed in high grade metastatic prostate cancer tissues. Increased expression of CXCR4 is also detected in several prostate cancer cell lines as compared to normal prostate epithelial cells. Our studies identify a nuclear pool of CXCR4 and also define a mechanism for nuclear targeting of CXCR4. A classical nuclear localization sequence (cNLS), "RPRK", in CXCR4 can contribute to nuclear localization. In addition, CXCR4 interacts with the nuclear transport receptor, Transportin βi, to promote nuclear accumulation of CXCR4. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicate that nuclear CXCR4 is functional and can participate in G-protein signaling revealing that the nuclear pool of CXCR4 can retain function. Localization of functional CXCR4 to the nucleus may be a mechanism by which prostate cancer cells evade treatment, thus contributing to increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4. This study addresses the mechanism of nuclear targeting for CXCR4 and demonstrates that CXCR4 can retain function within the nucleus and provides important new information to illuminate what have previously been primarily clinical observations of nuclear CXCR4.

Melanoma is the deadliest form of skin cancer due to its high propensity to metastasize and resistance to current therapies. We have created a spontaneous mouse model of metastatic melanoma (Dct-Grm1/K5-Edn3) where metastasis to the lungs is 80% penetrant. The primary tumors of these mice present cellular heterogeneity with cells at varying levels of differentiation. The main goal of this study was to determine the metastatic potential of the primary tumor resident Tyrosinase positive cells and evaluate the dynamic phenotypic changes as those cells move from the primary tumors to the sites of metastasis. To accomplish this aim I crossed the Dct-Grm1/K5-Edn3 mice to CreERT2/mT/mG mice to indelibly label Tyrosinase cell populations within the primary tumor with Green Fluorescent Protein (GFP) by topical application of 4-hydroxytamoxifen (4HT) at the tumor site. In vivo lineage tracing and characterization of GFP+ cells were performed in the metastatic lesions. In the 4HT treated Dct-Grm1/ K5-Edn3/Tyr-CreERT2/mT/mG mice, primary tumor derived Tyrosinase positive cells or their progeny (GFP+) established successful metastases in the distant organs indicating the tumorigenic capacity of the differentiated cell populations. Numerous metastatic melanoma cells were identified in the vasculature of the metastatic organs and established close association with the vascular endothelium. The intravascular cells lost pigmentation and did not express melanocytic markers; however, they mimicked endothelial cell properties and gained the expression of CD31 (also known as platelet endothelial cell adhesion molecule PECAM-1) and vascular endothelial (VE)-Cadherin. In the lung metastatic foci, GFP+ cells resumed pigmentation production and lost the expression of endothelial cell markers. Evidence from other metastatic organs in the mice further supported the phenotypic plasticity of metastatic melanoma cells. The in vivo lineage tracing system established in the melanoma mouse model revealed tumor phenotypic plasticity and will be a powerful model to evaluate and help us understand the etiology and pathogenesis of melanoma metastasis. Further characterization of those more aggressive cells in melanoma will allow for the development of new prognostic tests and novel therapeutic strategies to eliminate metastasis.

This report describes the case of a 56-year-old man who presented with blurry vision, increased intraocular pressure, and conjunctival injection after posterior chamber intraocular lens implantation. Initially post-operative endophalmitis and foreign body inflammation were considered as differential diagnoses, but after further examination pan-uveitis was diagnosed. Uveitis is an ocular finding that may indicate several diseases, one of which is Behçet’s Disease. During the interview, the patient mentioned a history of apthous ulcers and genital ulcers which then lead to the clinical diagnosis of Behçet’s Disease. This report emphasizes that Behçet’s Disease is rare in Caucasians. Therefore, is frequently misdiagnosed in North America due to variable presentations and by not exploring the option when analyzing differential diagnoses. Early diagnosis and intervention will prevent the development of blindness and fatality due to complications of the disease.

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.

The overall objective of this thesis work was to develop new insights into the etiology of scleroderma, a human systemic autoimmune disease, by analyzing the autoantibodies to centrosome antigens that develop during the disease. Centrosomes are perinuclear organelles that form microtubule arrays, including mitotic spindles that ensure the faithful segregation of chromosomes during mitosis. These studies used a novel methodology to determine the prevalence of anti-centrosome autoantibodies in patients with scleroderma. Recombinant centrosome antigens were used to determine the antigenic specificity of anti-centrosome antibody subsets by immunoblotting. Centrosome marker antibodies were used in indirect immunofluorescence assays to distinguish centrosomes within the polymorphic staining pattern frequently given by scleroderma sera. We found that 43% of patients are autoreactive to centrosomes, a prevalence higher than has been reported for any other scleroderma autoantigen. Half of the centrosome-positive patients also had autoantibodies against other antigens used in scleroderma diagnosis. However, in the remaining half of these patients, anti-centrosome antibodies represented the sole class of autoantibodies that was detectable. Anti-centrosome antibodies were detected in only a small percentage of normal individuals and patients with other connective tissue diseases. These data suggest that anti-centrosome autoantibodies may represent a new diagnostic tool in scleroderma. Upon examination of anti-centrosome autoantibody development in an animal model, it appeared that this autoantibody specificity may develop in mice as a consequence of an infection. An infectious agent was isolated by plaque-formation from carrier mice. Further characterization of the infectious agent was undertaken to obtain information on its physical, morphological and cytopathological properties. The infectious agent was identified by sequence and unique antigenic properties to be homologous to the pig pathogen Mycoplasma hyorhinis. When reintroduced into naive mice, the murine mycoplasma triggered anti-centrosome autoantibody development. While anti-centrosome autoantibodies of IgM isotype are part of the repertoire of naive unimmunized mice, mycoplasma infection specifically triggered the development of anti-centrosome IgG. Moreover, centrosome autoreactivity was prevented by antibiotic treatment. The autoantibody response evolved to recruit additional specificities, having IgM isotypes, reactive to endoplasmic reticulum-associated autoantigens.

Cancer cells are most clearly characterized by their abnormal and uncontrolled cell growth. One of the most notable theories that explains the vast proliferative capacity of tumorigenic cells is the Warburg effect, a significant shift in metabolism wherein cancer cells preferentially fuel cell division using aerobic glycolysis instead of aerobic respiration. This upregulation of glycolytic fermentation in aerobic environments is highly unusual - glycolysis is typically utilized in anaerobic conditions, but nonetheless dominates cancer metabolic activity in spite of the presence of oxygen. Since the discovery the Warburg effect in the 1920s, researchers have struggled to identify whether aerobic glycolysis is a cause or consequence of carcinogenesis. Interestingly, a new theory recently emerged that challenges this widely-accepted metabolic paradigm for cancer. Known as the reverse Warburg effect, this new mechanism shows that in carcinomas such as breast cancer, the Warburg effect occurs not in cancer cells, but rather in tumor-adjacent stromal fibroblasts. These cancer-associated fibroblasts (CAFs) in the greater tumor microenvironment produce lactate - a high-energy metabolite formed as a byproduct of aerobic glycolysis - to fuel aerobic respiration and rapid tumorigenesis in neighboring cancer cells. This emerging theory emphasizes the pivotal role of the tumor microenvironment in determining whether cancer cells undergo aerobic glycolysis or aerobic respiration. Central to this lactate-linked metabolic intersection are two critical enzymes that regulate a cell's metabolic commitment - lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHc). In order to clarify the mechanisms through which CAFs induce tumorigenesis in breast cancer, we plan to carry out two specific aims: (1) evaluate the enzymatic activity of LDH and PDHc, and (2) compare LDH and PDHc enzyme content. Using co-culture techniques to study the breast cancer tumor microenvironment in vitro, we will compare the enzymatic activity and enzyme content of both MCF7 breast cancer cells and CAFs to identify whether the reverse Warburg effect occurs due to post-translational enzyme activation or increased enzyme synthesis.

Vitiligo is an autoimmune disease in which CD8+ T cells selectively destroy melanocytes, leading to a patchy, disfiguring depigmentation of the skin. Our group and others have highlighted the central role of IFN-γ-dependent chemokines in the progression of disease; however, IFN-γ is also reported to have pleiotropic effects on melanocyte biology. We examined whether IFN-γ has a direct role in melanocyte killing. We tested the T-cell effector functions IFN-γ, Fas ligand and perforin by deleting them from autoreactive T cells used to induce vitiligo in mice. We found that disease incidence, disease severity and T cell accumulation in the skin was reduced in mice receiving adoptive transfer of either IFN-γ deficient or Fas ligand deficient gp100-specific T cells; however, perforin was dispensable and led to increased disease scores and T cell accumulation. To determine how melanocytes are affected by IFN-γ signaling during vitiligo, we performed single-cell RNA-sequencing on suction blister biopsies obtained from vitiligo and healthy subjects. We discovered that integrin expression and TGFb2 signaling was decreased only in lesional melanocyte transcriptomes. Moreover, melanocytes appear to participate in their own demise by increasing HLA expression and recruiting effector cells through the chemotactic ligand CCL18. The loss of melanocyte retention factors may explain their clean disappearance from the skin during keratinocyte turnover. Taken together, we believe IFN-γ production by autoreactive T cells in the skin leads to clean loss of melanocytes by downregulation of melanocyte retention factors and by increasing their potential to be detected by effector cells during vitiligo.

Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, I hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, I developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. I characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.