Illuminating function of the understudied druggable kinome.

The human kinome, with more than 500 proteins, is crucial for cell signaling and disease. Yet, about one-third of kinases lack in-depth study. The Data and Resource Generating Center for Understudied Kinases has developed multiple resources to address this challenge including creation of a heavy amino acid peptide library for parallel reaction monitoring and quantitation of protein kinase expression, use of understudied kinases tagged with a miniTurbo-biotin ligase to determine interaction networks by proximity-dependent protein biotinylation, NanoBRET probe development for screening chemical tool target specificity in live cells, characterization of small molecule chemical tools inhibiting understudied kinases, and computational tools for defining kinome architecture. These resources are available through the Dark Kinase Knowledgebase, supporting further research into these understudied protein kinases.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.

Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy.

Non-small-cell lung cancer (NSCLC) and glioblastoma (GB) have poor prognoses. Discovery of new molecular targets is needed to improve therapy. Tax interacting protein 1 (TIP1), which plays a role in cancer progression, is overexpressed and radiation-inducible in NSCLC and GB. We evaluated the effect of an anti-TIP1 antibody alone and in combination with ionizing radiation (XRT) on NSCLC and GB in vitro and in vivo. NSCLC and GB cells were treated with anti-TIP1 antibodies and evaluated for proliferation, colony formation, endocytosis, and cell death. The efficacy of anti-TIP1 antibodies in combination with XRT on tumor growth was measured in mouse models of NSCLC and GB. mRNA sequencing was performed to understand the molecular mechanisms involved in the action of anti-TIP1 antibodies. We found that targeting the functional domain of TIP1 leads to endocytosis of the anti-TIP1 antibody followed by reduced proliferation and increased apoptosis-mediated cell death. Anti-TIP1 antibodies bound specifically (with high affinity) to cancer cells and synergized with XRT to significantly increase cytotoxicity in vitro and reduce tumor growth in mouse models of NSCLC and GB. Importantly, downregulation of cancer survival signaling pathways was found in vitro and in vivo following treatment with anti-TIP1 antibodies. TIP1 is a new therapeutic target for cancer treatment. Antibodies targeting the functional domain of TIP1 exhibited antitumor activity and enhanced the efficacy of radiation both in vitro and in vivo. Anti-TIP1 antibodies interrupt TIP1 function and are effective cancer therapy alone or in combination with XRT in mouse models of human cancer.

Sleep deprivation exacerbates microglial reactivity and Aß deposition in a TREM2-dependent manner in mice.

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer's disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-ß (Aß) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression. Sleep deprivation not only enhanced TREM2-dependent Aß plaque deposition compared with 5xFAD mice with normal sleeping patterns but also induced microglial reactivity that was independent of the presence of parenchymal Aß plaques. We investigated lysosomal morphology using transmission electron microscopy and found abnormalities particularly in mice without Aß plaques and also observed lysosomal maturation impairments in a TREM2-dependent manner in both microglia and neurons, suggesting that changes in sleep modified neuro-immune cross-talk. Unbiased transcriptome and proteome profiling provided mechanistic insights into functional pathways triggered by sleep deprivation that were unique to TREM2 and Aß pathology and that converged on metabolic dyshomeostasis. Our findings highlight that sleep deprivation directly affects microglial reactivity, for which TREM2 is required, by altering the metabolic ability to cope with the energy demands of prolonged wakefulness, leading to further Aß deposition, and underlines the importance of sleep modulation as a promising future therapeutic approach.

Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification.

Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.

Functional characterization of the dural sinuses as a neuroimmune interface.

Despite the established dogma of central nervous system (CNS) immune privilege, neuroimmune interactions play an active role in diverse neurological disorders. However, the precise mechanisms underlying CNS immune surveillance remain elusive; particularly, the anatomical sites where peripheral adaptive immunity can sample CNS-derived antigens and the cellular and molecular mediators orchestrating this surveillance. Here, we demonstrate that CNS-derived antigens in the cerebrospinal fluid (CSF) accumulate around the dural sinuses, are captured by local antigen-presenting cells, and are presented to patrolling T cells. This surveillance is enabled by endothelial and mural cells forming the sinus stromal niche. T cell recognition of CSF-derived antigens at this site promoted tissue resident phenotypes and effector functions within the dural meninges. These findings highlight the critical role of dural sinuses as a neuroimmune interface, where brain antigens are surveyed under steady-state conditions, and shed light on age-related dysfunction and neuroinflammatory attack in animal models of multiple sclerosis.

Paenibacillus infection with frequent viral coinfection contributes to postinfectious hydrocephalus in Ugandan infants.

Postinfectious hydrocephalus (PIH), which often follows neonatal sepsis, is the most common cause of pediatric hydrocephalus worldwide, yet the microbial pathogens underlying this disease remain to be elucidated. Characterization of the microbial agents causing PIH would enable a shift from surgical palliation of cerebrospinal fluid (CSF) accumulation to prevention of the disease. Here, we examined blood and CSF samples collected from 100 consecutive infant cases of PIH and control cases comprising infants with non-postinfectious hydrocephalus in Uganda. Genomic sequencing of samples was undertaken to test for bacterial, fungal, and parasitic DNA; DNA and RNA sequencing was used to identify viruses; and bacterial culture recovery was used to identify potential causative organisms. We found that infection with the bacterium Paenibacillus, together with frequent cytomegalovirus (CMV) coinfection, was associated with PIH in our infant cohort. Assembly of the genome of a facultative anaerobic bacterial isolate recovered from cultures of CSF samples from PIH cases identified a strain of Paenibacillus thiaminolyticus This strain, designated Mbale, was lethal when injected into mice in contrast to the benign reference Paenibacillus strain. These findings show that an unbiased pan-microbial approach enabled characterization of Paenibacillus in CSF samples from PIH cases, and point toward a pathway of more optimal treatment and prevention for PIH and other proximate neonatal infections.

Diurnal Rhythms Spatially and Temporally Organize Autophagy.

Circadian rhythms are a hallmark of physiology, but how such daily rhythms organize cellular catabolism is poorly understood. Here, we used proteomics to map daily oscillations in autophagic flux in mouse liver and related these rhythms to proteasome activity. We also explored how systemic inflammation affects the temporal structure of autophagy. Our data identified a globally harmonized rhythm for basal macroautophagy, chaperone-mediated autophagy, and proteasomal activity, which concentrates liver proteolysis during the daytime. Basal autophagy rhythms could be resolved into two antiphase clusters that were distinguished by the subcellular location of targeted proteins. Inflammation induced by lipopolysaccharide reprogrammed autophagic flux away from a temporal pattern that favors cytosolic targets and toward the turnover of mitochondrial targets. Our data detail how daily biological rhythms connect the temporal, spatial, and metabolic aspects of protein catabolism.

Integrative omics analyses broaden treatment targets in human cancer.

BACKGROUND: Although large-scale, next-generation sequencing (NGS) studies of cancers hold promise for enabling precision oncology, challenges remain in integrating NGS with clinically validated biomarkers. METHODS: To overcome such challenges, we utilized the Database of Evidence for Precision Oncology (DEPO) to link druggability to genomic, transcriptomic, and proteomic biomarkers. Using a pan-cancer cohort of 6570 tumors, we identified tumors with potentially druggable biomarkers consisting of drug-associated mutations, mRNA expression outliers, and protein/phosphoprotein expression outliers identified by DEPO. RESULTS: Within the pan-cancer cohort of 6570 tumors, we found that 3% are druggable based on FDA-approved drug-mutation interactions in specific cancer types. However, mRNA/phosphoprotein/protein expression outliers and drug repurposing across cancer types suggest potential druggability in up to 16% of tumors. The percentage of potential drug-associated tumors can increase to 48% if we consider preclinical evidence. Further, our analyses showed co-occurring potentially druggable multi-omics alterations in 32% of tumors, indicating a role for individualized combinational therapy, with evidence supporting mTOR/PI3K/ESR1 co-inhibition and BRAF/AKT co-inhibition in 1.6 and 0.8% of tumors, respectively. We experimentally validated a subset of putative druggable mutations in BRAF identified by a protein structure-based computational tool. Finally, analysis of a large-scale drug screening dataset lent further evidence supporting repurposing of drugs across cancer types and the use of expression outliers for inferring druggability. CONCLUSIONS: Our results suggest that an integrated analysis platform can nominate multi-omics alterations as biomarkers of druggability and aid ongoing efforts to bring precision oncology to patients.

Interleukin-17 limits hypoxia-inducible factor 1α and development of hypoxic granulomas during tuberculosis.

Mycobacterium tuberculosis (Mtb) is a global health threat, compounded by the emergence of drug-resistant strains. A hallmark of pulmonary tuberculosis (TB) is the formation of hypoxic necrotic granulomas, which upon disintegration, release infectious Mtb. Furthermore, hypoxic necrotic granulomas are associated with increased disease severity and provide a niche for drug-resistant Mtb. However, the host immune responses that promote the development of hypoxic TB granulomas are not well described. Using a necrotic Mtb mouse model, we show that loss of Mtb virulence factors, such as phenolic glycolipids, decreases the production of the proinflammatory cytokine IL-17 (also referred to as IL-17A). IL-17 production negatively regulates the development of hypoxic TB granulomas by limiting the expression of the transcription factor hypoxia-inducible factor 1α (HIF1α). In human TB patients, HIF1α mRNA expression is increased. Through genotyping and association analyses in human samples, we identified a link between the single nucleotide polymorphism rs2275913 in the IL-17 promoter (-197G/G), which is associated with decreased IL-17 production upon stimulation with Mtb cell wall. Together, our data highlight a potentially novel role for IL-17 in limiting the development of hypoxic necrotic granulomas and reducing disease severity in TB.

5MeCDDO Blocks Metabolic Activation but not Progression of Breast, Intestine, and Tongue Cancers. Is Antioxidant Response Element a Prevention Target?

The preventive efficacy of the triterpenoid 5MeCDDO was tested in two models of mammary cancer, the Min model of intestinal cancer, and a chemically induced model of head and neck cancer. In one model of mammary cancer, female Sprague-Dawley rats were administered MNU at 50 days of age, and 5MeCDDO (27 ppm) was administered in the diet beginning 5 days later for the duration of the study; 5MeCDDO was ineffective. In contrast, in a model examining initiation of mammary cancers by the procarcinogen dimethyl-benzanthracene, 5, 6-benzoflavone (500 ppm, an Ah receptor agonist) or 5MeCDDO (27 or 2.7 ppm) decreased tumor multiplicity by 90%, 80%, and 50%, respectively. This anti-initiating effect which is presumably mediated by altered metabolic activation parallels our observation that 5MeCDDO induced proteins of various antioxidant response element (ARE)-related phase II drug-metabolizing enzymes [e.g., GST Pi, AKR 7A3 (aflatoxicol), epoxide hydrolase, and quinone reductase] in the liver. 5MeCDDO tested in the 4-nitroquinoline-l-oxide (4-NQO) head and neck cancer model failed to decrease tumor incidence or invasiveness. In the Min mouse model of intestinal cancer, a high dose of 5MeCDDO (80 ppm) was weakly effective in reducing adenoma multiplicity [∼30% (P < 0.05)]; however, a lower dose was totally ineffective. These findings question whether measuring increased levels of certain ARE-related genes (e.g., quinone reductase, GST Pi), indicating decreased carcinogen activation are sufficient to imply general chemopreventive efficacy of a given agent or mixture. Cancer Prev Res; 9(7); 616-23. ©2016 AACR.

Proteomic Profiling of Early Chronic Pulmonary Hypertension: Evidence for Both Adaptive and Maladaptive Pathology.

BACKGROUND: The molecular mechanisms governing right atrial (RA) and ventricular (RV) hypertrophy and failure in chronic pulmonary hypertension (CPH) remain unclear. The purpose of this investigation was to characterize RA and RV protein changes in CPH and determine their adaptive versus maladaptive role on hypertrophic development. METHODS: Nine dogs underwent sternotomy and RA injection with 3 mg/kg dehydromonocrotaline (DMCT) to induce CPH (n=5) or sternotomy without DMCT (n=4). At 8-10 weeks, RA and RV proteomic analyses were completed after trypsinization of cut 2-D gel electrophoresis spots and peptide sequencing using mass spectrometry. RESULTS: In the RV, 13 protein spots were significantly altered with DMCT compared to Sham. Downregulated RV proteins included contractile elements: troponin T and C (-1.6 fold change), myosin regulatory light chain 2 (-1.9), cellular energetics modifier: fatty-acid binding protein (-1.5), and (3) ROS scavenger: superoxide dismutase 1 (-1.7). Conversely, beta-myosin heavy chain was upregulated (+1.7). In the RA, 22 proteins spots were altered including the following downregulated proteins contractile elements: tropomyosin 1 alpha chain (-1.9), cellular energetic proteins: ATP synthase (-1.5), fatty-acid binding protein (-2.5), and (3) polyubiquitin (-3.5). Crystallin alpha B (hypertrophy inhibitor) was upregulated in both the RV (+2.2) and RA (+2.6). CONCLUSIONS: In early stage hypertrophy there is adaptive upregulation of major RA and RV contractile substituents and attenuation of the hypertrophic response. However, there are multiple indices of maladaptive pathology including considerable cellular stress associated with aberrancy of actin machinery activity, decreased efficiency of energy utilization, and potentially decreased protein quality control.

Functional and phylogenetic characterization of proteins detected in various nematode intestinal compartments.

The parasitic nematode intestine is responsible for nutrient digestion and absorption, and many other processes essential for reproduction and survival, making it a valuable target for anthelmintic drug treatment. However, nematodes display extreme biological diversity (including occupying distinct trophic habitats), resulting in limited knowledge of intestinal cell/protein functions of fundamental or adaptive significance. We developed a perfusion model for isolating intestinal proteins in Ascaris suum (a parasite of humans and swine), allowing for the identification of over 1000 intestinal A. suum proteins (using mass spectrometry), which were assigned to several different intestinal cell compartments (intestinal tissue, the integral and peripheral intestinal membranes, and the intestinal lumen). A multi-omics analysis approach identified a large diversity of biological functions across intestinal compartments, based on both functional enrichment analysis (identifying terms related to detoxification, proteolysis, and host-parasite interactions) and regulatory binding sequence analysis to identify putatively active compartment-specific transcription factors (identifying many related to intestinal sex differentiation or lifespan regulation). Orthologs of A. suum proteins in 15 other nematodes species, five host species, and two outgroups were identified and analyzed. Different cellular compartments demonstrated markedly different levels of protein conservation; e.g. integral intestinal membrane proteins were the most conserved among nematodes (up to 96% conservation), whereas intestinal lumen proteins were the most diverse (only 6% conservation across all nematodes, and 71% with no host orthologs). Finally, this integrated multi-omics analysis identified conserved nematode-specific intestinal proteins likely performing essential functions (including V-type ATPases and ABC transporters), which may serve as promising anthelmintic drug or vaccine targets in future research. Collectively, the findings provide valuable new insights on conserved and adaptive features of nematode intestinal cells, membranes and the intestinal lumen, and potential targets for parasite treatment and control.

Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.

Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis.

Multiplex proteomics analysis of gender-associated proteins in Brugia malayi.

Gender-associated (GA) genes are important for the development and reproduction of filarial nematodes. Identification and characterization of GA genes may provide insight into major pathways and processes involved in development and reproduction. The recent completion of the Brugia malayi genome has provided a good foundation for proteomics studies. Multiplex protein labelling and two-dimensional difference in-gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF tandem MS were used to identify GA proteins. Thirty male and 32 female associated proteins were identified in this study. Many of these GA genes have homologues in Caenorhabditis elegans (83% male and 69% female), and most of the homologues have severe RNA interference (RNAi) phenotypes (72% male and 55% female) in C. elegans. Functional analysis showed that the male-associated genes are enriched for energy production, metabolic processes and cytoskeleton, while the female-associated genes are enriched for RNA modification and transcription. GA genes encode many excreted/secreted proteins. In situ localization studies showed that GA genes are mainly expressed in reproductive organs, and this is further evidence for their involvement in reproduction. Improved understanding of the basic biology of filarial nematodes may lead to improved tools for prevention and treatment of filarial infections. This study combined proteomics, in situ hybridization (ISH) and bioinformatics in a systems biology approach to improve understanding of gender differences and key proteins involved in reproduction in filarial worms. Advanced proteomics methods and bioinformatics led to the identification of 62 GA proteins for B. malayi. ISH revealed that most of those GA genes are expressed during embryogenesis or spermatogenesis. ISH results were consistent with RNAi data for C. elegans that linked the homologues of the B. malayi proteins to gamete production and embryogenesis.

Efficacy of the EGFr inhibitor Iressa on development of chemically-induced urinary bladder cancers: dose dependency and modulation of biomarkers.

The effects of the EGFr inhibitor Iressa on development of urinary bladder cancers induced by hydroxybutyl(butyl)nitrosamine (OH-BBN) in rats were examined. Iressa treatment (4.5 or 1.5 mg/kg BW/day) beginning one week after the last dose of OH-BBN decreased the occurrence of large (>200 mg) bladder cancers at termination of the study by 75 and 52%, respectively. Treatment with Iressa (10 mg/kg BW/day) beginning one week or three months (delayed initiation) after the last dose of OH-BBN also significantly increased tumor latency and decreased the incidence of palpable bladder cancers. In the delayed initiation study, microscopic cancers already existed when treatment was initiated; implying that the effects of Iressa occur late in tumor progression. Potential pharmacodynamics and/or efficacy biomarkers modulated by short-term exposure (5 day) to Iressa (10 mg/kg BW/day) were determined in palpable bladder lesions by using three different approaches: i) direct immunohistochemical examination of EGFr related proteins; which showed that phosphorylated EGFr, AKT and ERK were significantly decreased; ii) measurement of protein expression by two dimensional gel electrophoresis and tandem mass spectrometry. This showed that the Annexin A2, MAP kinase kinase and nucleolin (all proteins associated with the VEGF pathway) were decreased in treated tumors; and iii) measurement of gene expression determined in gene microarrays demonstrated that numerous pathways were markedly altered by Iressa treatment. In particular, cell cycle genes related to the anaphase protein complex (APC) pathway, including CDC 20, cyclin B1, BUB1 and both of the Aurora kinases, were significantly decreased.

A bioinformatics approach for biomarker identification in radiation-induced lung inflammation from limited proteomics data.

Many efforts have been made to discover novel bio-markers for early disease detection in oncology. However, the lack of efficient computational strategies impedes the discovery of disease-specific biomarkers for better understanding and management of treatment outcomes. In this study, we propose a novel graph-based scoring function to rank and identify the most robust biomarkers from limited proteomics data. The proposed method measures the proximity between candidate proteins identified by mass spectrometry (MS) analysis utilizing prior reported knowledge in the literature. Recent advances in mass spectrometry provide new opportunities to identify unique biomarkers from peripheral blood samples in complex treatment modalities such as radiation therapy (radiotherapy), which enables early disease detection, disease progression monitoring, and targeted intervention. Specifically, the dose-limiting role of radiation-induced lung injury known as radiation pneumonitis (RP) in lung cancer patients receiving radiotherapy motivates the search for robust predictive biomarkers. In this case study, plasma from 26 locally advanced non-small cell lung cancer (NSCLC) patients treated with radiotherapy in a longitudinal 3 × 3 matched-control cohort was fractionated using in-line, sequential multiaffinity chromatography. The complex peptide mixtures from endoprotease digestions were analyzed using comparative, high-resolution liquid chromatography (LC)-MS to identify and quantify differential peptide signals. Through analysis of survey mass spectra and annotations of peptides from the tandem spectra, we found candidate proteins that appear to be associated with RP. On the basis of the proposed methodology, α-2-macroglobulin (α2M) was unambiguously ranked as the top candidate protein. As independent validation of this candidate protein, enzyme-linked immunosorbent assay (ELISA) experiments were performed on independent cohort of 20 patients' samples resulting in early significant discrimination between RP and non-RP patients (p = 0.002). These results suggest that the proposed methodology based on longitudinal proteomics analysis and a novel bioinformatics ranking algorithm is a potentially promising approach for the challenging problem of identifying relevant biomarkers in sample-limited clinical applications.

Cross-species comparison of orthologous gene expression in human bladder cancer and carcinogen-induced rodent models.

Genes differentially expressed by tumor cells represent promising drug targets for anti-cancer therapy. Such candidate genes need to be validated in appropriate animal models. This study examined the suitability of rodent models of bladder cancer in B6D2F1 mice and Fischer-344 rats to model clinical bladder cancer specimens in humans. Using a global gene expression approach cross-species analysis showed that 13-34% of total genes in the genome were differentially expressed between tumor and normal tissues in each of five datasets from humans, rats, and mice. About 20% of these differentially expressed genes overlapped among species, corresponding to 2.6 to 4.8% of total genes in the genome. Several genes were consistently dysregulated in bladder tumors in both humans and rodents. Notably, CNN1, MYL9, PDLIM3, ITIH5, MYH11, PCP4 and FM05 were found to commonly down-regulated; while T0P2A, CCNB2, KIF20A and RRM2 were up-regulated. These genes are likely to have conserved functions contributing to bladder carcinogenesis. Gene set enrichment analysis detected a number of molecular pathways commonly activated in both humans and rodent bladder cancer. These pathways affect the cell cycle, HIF-1 and MYC expression, and regulation of apoptosis. We also compared expression changes at mRNA and protein levels in the rat model and identified several genes/proteins exhibiting concordant changes in bladder tumors, including ANXA1, ANXA2, CA2, KRT14, LDHA, LGALS4, SERPINA1, KRT18 and LDHB. In general, rodent models of bladder cancer represent the clinical disease to an extent that will allow successful mining of target genes and permit studies on the molecular mechanisms of bladder carcinogenesis.

Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells.

The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.