Mechanisms of coordinating hyaluronan and glycosaminoglycan production by nucleotide sugars.

Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it.

Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

Glucuronidation controls androgen levels in the prostate and the dysregulation of enzymes in this pathway is associated with castration resistant prostate cancer. UDP-glucose dehydrogenase (UGDH) produces UDP-glucuronate, the essential precursor for glucuronidation, and its expression is elevated in prostate cancer. We compared protein and metabolite levels relevant to the glucuronidation pathway in five prostate cancer patient-derived xenograft models paired with their isogenic counterparts that were selected for castration resistant (CR) recurrence.

Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase.

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels.

Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy.

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids.

Hyaluronan Pericellular Matrix: Particle Exclusion Assay.

Particle exclusion assays are used to visualize pericellular envelopes with a high content of hyaluronan. Pericellular hyaluronan is basally abundant in certain cell types while in others it is deposited in a highly dynamic manner in response to specific conditions and its presence may indicate cellular status. This assay, described here, is a quick semiquantitative approach to detecting pericellular hyaluronan using the hyaluronan-binding proteoglycan, aggrecan, to stabilize and amplify the surface matrix.

The NCI Transcriptional Pharmacodynamics Workbench: A Tool to Examine Dynamic Expression Profiling of Therapeutic Response in the NCI-60 Cell Line Panel.

: The intracellular effects and overall efficacies of anticancer therapies can vary significantly by tumor type. To identify patterns of drug-induced gene modulation that occur in different cancer cell types, we measured gene-expression changes across the NCI-60 cell line panel after exposure to 15 anticancer agents. The results were integrated into a combined database and set of interactive analysis tools, designated the NCI Transcriptional Pharmacodynamics Workbench (NCI TPW), that allows exploration of gene-expression modulation by molecular pathway, drug target, and association with drug sensitivity.

Prostate tumor cell exosomes containing hyaluronidase Hyal1 stimulate prostate stromal cell motility by engagement of FAK-mediated integrin signaling.

The hyaluronidase Hyal1 is clinically and functionally implicated in prostate cancer progression and metastasis. Elevated Hyal1 accelerates vesicular trafficking in prostate tumor cells, thereby enhancing their metastatic potential in an autocrine manner through increased motility and proliferation. In this report, we found Hyal1 protein is a component of exosomes produced by prostate tumor cell lines overexpressing Hyal1.

Molecular Pharmacodynamics-Guided Scheduling of Biologically Effective Doses: A Drug Development Paradigm Applied to MET Tyrosine Kinase Inhibitors.

The development of molecularly targeted agents has benefited from use of pharmacodynamic markers to identify "biologically effective doses" (BED) below MTDs, yet this knowledge remains underutilized in selecting dosage regimens and in comparing the effectiveness of targeted agents within a class. We sought to establish preclinical proof-of-concept for such pharmacodynamics-based BED regimens and effectiveness comparisons using MET kinase small-molecule inhibitors. Utilizing pharmacodynamic biomarker measurements of MET signaling (tumor pYMET/total MET ratio) in a phase 0-like preclinical setting, we developed optimal dosage regimens for several MET kinase inhibitors and compared their antitumor efficacy in a -amplified gastric cancer xenograft model (SNU-5).

Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD(+)-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate and fuels UGT-catalyzed glucuronidation.

Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form.

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking.

Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin.

Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis.

DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood.

Emerging roles for hyaluronidase in cancer metastasis and therapy.

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past 5 years have clarified many apparent contradictions: (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high-molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature supporting these conclusions and discusses novel mechanisms by which hyaluronidases impact invasive tumor cell processes.

Emerging regulatory paradigms in glutathione metabolism.

One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms.

Pharmacokinetic and Biodistribution Assessment of a Near Infrared-Labeled PSMA-Specific Small Molecule in Tumor-Bearing Mice.

Prostate cancer is the most frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27.

UDP-glucose dehydrogenase activity and optimal downstream cellular function require dynamic reorganization at the dimer-dimer subunit interfaces.

UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme.

Essential role for Dnmt1 in the prevention and maintenance of MYC-induced T-cell lymphomas.

DNA cytosine methylation is an epigenetic modification involved in the transcriptional repression of genes controlling a variety of physiological processes, including hematopoiesis. DNA methyltransferase 1 (Dnmt1) is a key enzyme involved in the somatic inheritance of DNA methylation and thus plays a critical role in epigenomic stability. Aberrant methylation contributes to the pathogenesis of human cancer and of hematologic malignancies in particular.

Characterization of IRDye 800CW chlorotoxin as a targeting agent for brain tumors.

Primary brain tumors present significant challenges for surgical resection because of their location and the frequent occurrence of malignant projections extending beyond the primary tumor. Visualization of the tumor margins during surgery is critical for a favorable outcome. We report the use of IRDye 800CW chlorotoxin (CLTX) as a targeted imaging agent for brain tumors in a spontaneous mouse model of medulloblastoma, ND2:SmoA1.

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves.

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr).

UDP-glucose dehydrogenase polymorphisms from patients with congenital heart valve defects disrupt enzyme stability and quaternary assembly.

Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects.

Systemic blockade of the hyaluronan receptor for endocytosis prevents lymph node metastasis of prostate cancer.

Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA receptor for endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis.

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis.

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis.