Liver Expression of a MiniATP7B Gene Results in Long-Term Restoration of Copper Homeostasis in a Wilson Disease Model in Mice.

Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.

Long-term metabolic correction of Wilson's disease in a murine model by gene therapy.

BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. METHODS: We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. RESULTS: We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. CONCLUSIONS: Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Using microRNA profiling in urine samples to develop a non-invasive test for bladder cancer.

Current standard methods used to detect and monitor bladder urothelial cell carcinoma (UCC) are invasive or have low sensitivity. The incorporation into clinical practice of a non-invasive tool for UCC assessment would enormously improve patients' quality of life and outcome. This study aimed to examine the microRNA (miRNA) expression profiles in urines of UCC patients in order to develop a non-invasive accurate and reliable tool to diagnose and provide information on the aggressiveness of the tumor. We performed a global miRNA expression profiling analysis of the urinary cells from 40 UCC patients and controls using TaqMan Human MicroRNA Array followed by validation of 22 selected potentially diagnostic and prognostic miRNAs in a separate cohort of 277 samples using a miRCURY LNA qPCR system. miRNA-based signatures were developed by multivariate logistic regression analysis and internally cross-validated. In the initial cohort of patients, we identified 40 and 30 aberrantly expressed miRNA in UCC compared with control urines and in high compared with low grade tumors, respectively. Quantification of 22 key miRNAs in an independent cohort resulted in the identification of a six miRNA diagnostic signature with a sensitivity of 84.8% and specificity of 86.5% (AUC = 0.92) and a two miRNA prognostic model with a sensitivity of 84.95% and a specificity of 74.14% (AUC = 0.83). Internal cross-validation analysis confirmed the accuracy rates of both models, reinforcing the strength of our findings. Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable tool for the non-invasive assessment of UCC.

Biomarkers vs conventional histological analysis to detect lymph node micrometastases in bladder cancer: a real improvement?

UNLABELLED: Study Type - Therapy (case series) Level of Evidence 4. What's known on the subject? and What does the study add? Muscle invasive bladder cancer has a mortality rate at 5 years of 50%, despite radical therapy, as a result of tumour progression and dissemination. This suggests that half of patients have disseminated disease at the time of diagnosis, which is not detected by the staging techniques currently used. The prognostic factors (histological grade and tumour stage) and current staging techniques do not discriminate between those patients who will be cured with surgical treatment and those who will die from metastatic spread. New diagnostic and prognostic tools that complement the existing methods and provide a proper assessment of carcinoma invading bladder muscle are therefore essential. Molecular staging techniques using specific biomarkers have been applied in various solid tumours to determine the presence of missed tumour cells in lymph nodes (LNs) during routine pathological examination. These techniques could identify patients with LN micrometastases who may potentially benefit from early treatment with chemotherapy. This study compares the performance of conventional histological analysis and molecular biomarkers in detecting bladder cancer LN micrometastases and predicting patient's clinical outcome. The study found that, even though a clear trend to a worse outcome was shown in those patients who became node-positive after molecular analysis, no statistical differences were found in cancer-specific and recurrence-free survival analysis between those patients who were negative by histology but positive by molecular analysis and those who were negative by both techniques. We concluded that molecular analysis of LN spreading in bladder cancer has a better detection rate than conventional histological examination. OBJECTIVE: • To improve the sensitivity of histological examination in detecting occult lymph node (LN) dissemination of bladder cancer using gene expression analysis. PATIENTS AND METHODS: • We carried out a retrospective study that included 504 formalin-fixed paraffin-embedded LNs from 90 patients with muscle invasive bladder cancer and 35 controls. • Gene expression values of two molecular biomarkers (FXYD3 and KRT20) were analysed using reverse transcription real-time quantitative PCR (RT-qPCR). • Molecular results were compared with histological status and patients' clinical outcomes. RESULTS: • Of the 90 patients analysed, 16 were positive and 74 were negative by histological analysis. Of these 74, 19 were classified as positive using RT-qPCR. • Significant differences in cancer-specific (P= 0.011) and recurrence-free (P= 0.009) survival were found between the three patient groups (patients positive by both techniques, patients negative by both techniques, and patients negative by histological but positive by molecular analysis). • A significant difference was not found between histologically negative but molecularly positive patients and patients who were negative by both techniques, but a clear trend to a worse outcome was found in those patients who became node-positive after molecular analysis. CONCLUSIONS: • The analysis of FXYD3 and KRT20 could improve current pathological examination for the detection of micrometastases in LNs. • Further and more extensive studies will determine the real prognostic value of such LN micrometastases.

High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease.

Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.

Molecular characterization of upper urinary tract tumours.

OBJECTIVES: To assess gene-expression patterns of BIRC5, FGFR3, IGF2, KRT20, UPK2, EBF1, CDH1, FXYD3, HTERT, TP53, AGR2, HER2 and VEGF, widely known markers of bladder urothelial carcinoma (UC) in upper tract UC, and to determine their value as prognostic factors of tumour progression and cancer-specific survival. PATIENTS AND METHODS: The study included 83 formalin-fixed paraffin-embedded tissue specimens (68 and 15 from patients with UTUC and controls, respectively) collected between 1990 and 2004. Thirteen bladder cancer-related genes were selected from previous reports and analysed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in all samples. RESULTS: Six genes were over-expressed (BIRC5, FGFR3, KRT20, UPK2, FXYD3 and hTERT) and three under-expressed (AGR2, TP53 and VEGF) in the tumour group (P < 0.05). For four genes (IGF2, EBF1, CDH1 and HER2) there was no statistically significant difference between the tumour and control groups. Overall, 21 patients developed tumour progression and 13 died from UTUC after a mean follow-up of 35.24 months. The 5-year disease-free progression and cancer-specific survival rates were 65.8% and 72.9%, respectively. In a multivariate regression analysis, the independent predictive variable for tumour progression and cancer-specific survival was pathological stage (hazard ratio 3.60, P < 0.001; and 3.73, P < 0.005, respectively), but none of the studied genes were identified as prognostic factors of tumour progression or cancer-specific survival. CONCLUSIONS: Our data suggest that bladder cancer and UTUC share some characteristics, but have differences in gene expression. None of BIRC5, FGFR3, IGF2, KRT20, UPK2, EBF1, CDH1, FXYD3, HTERT, TP53, AGR2, HER2 and VEGF were correlated either tumour progression or survival.

Maturation of NAA20 Aminoterminal End Is Essential to Assemble NatB N-Terminal Acetyltransferase Complex.

Protein lifespan is regulated by co-translational modification by several enzymes, including methionine aminopeptidases and N-alpha-aminoterminal acetyltransferases. The NatB enzymatic complex is an N-terminal acetyltransferase constituted by two subunits, NAA20 and NAA25, whose interaction is necessary to avoid NAA20 catalytic subunit degradation. We found that deletion of the first five amino acids of hNAA20 or fusion of a peptide to its amino terminal end abolishes its interaction with hNAA25. Substitution of the second residue of hNAA20 with amino acids with small, uncharged side-chains allows NatB enzymatic complex formation. However, replacement by residues with large or charged side-chains interferes with its hNAA25 interaction, limiting functional NatB complex formation. Comparison of NAA20 eukaryotic sequences showed that the residue following the initial methionine, an amino acid with a small uncharged side-chain, has been evolutionarily conserved. We have confirmed the relevance of second amino acid characteristics of NAA20 in NatB enzymatic complex formation in Drosophila melanogaster. Moreover, we have evidenced the significance of NAA20 second residue in Saccharomyces cerevisiae using different NAA20 versions to reconstitute NatB formation in a yNAA20-KO yeast strain. The requirement in humans and in fruit flies of an amino acid with a small uncharged side-chain following the initial methionine of NAA20 suggests that methionine aminopeptidase action may be necessary for the NAA20 and NAA25 interaction. We showed that inhibition of MetAP2 expression blocked hNatB enzymatic complex formation by retaining the initial methionine of NAA20. Therefore, NatB-mediated protein N-terminal acetylation is dependent on methionine aminopeptidase, providing a regulatory mechanism for protein N-terminal maturation.

NatB-mediated protein N-α-terminal acetylation is a potential therapeutic target in hepatocellular carcinoma.

The identification of new targets for systemic therapy of hepatocellular carcinoma (HCC) is an urgent medical need. Recently, we showed that hNatB catalyzes the N-α-terminal acetylation of 15% of the human proteome and that this action is necessary for proper actin cytoskeleton structure and function. In tumors, cytoskeletal changes influence motility, invasion, survival, cell growth and tumor progression, making the cytoskeleton a very attractive antitumor target. Here, we show that hNatB subunits are upregulated in in over 59% HCC tumors compared to non-tumor tissue and that this upregulation is associated with microscopic vascular invasion. We found that hNatB silencing blocks proliferation and tumor formation in HCC cell lines in association with hampered DNA synthesis and impaired progression through the S and the G2/M phases. Growth inhibition is mediated by the degradation of two hNatB substrates, tropomyosin and CDK2, which occurs when these proteins lack N-α-terminal acetylation. In addition, hNatB inhibition disrupts the actin cytoskeleton, focal adhesions and tight/adherens junctions, abrogating two proliferative signaling pathways, Hippo/YAP and ERK1/2. Therefore, inhibition of NatB activity represents an interesting new approach to treating HCC by blocking cell proliferation and disrupting actin cytoskeleton function.

Utility of urothelial mRNA markers in blood for staging and monitoring bladder cancer.

OBJECTIVE: To test the efficiency of 6 mRNA bladder markers in staging urothelial cell carcinoma (UCC) and monitoring UCC dissemination from blood samples. METHODS: From 2002 to 2009, 347 blood samples were collected from 150 patients with UCC and 29 healthy controls. Sequential blood sampling was performed in patients undergoing cystectomy at surgery and 6, 12, 18, and 24 months postoperatively. The median follow-up was 33 months. The presence of KRT20, FXYD3, C10orf116, UPK2, AGR2, and KRT19 markers in blood was evaluated in all patients and controls by measuring the gene expression using preamplified cDNA and reverse transcriptase quantitative polymerase chain reaction. Gene expression data were correlated with the tumor risk, follow-up, and outcomes data. RESULTS: Expression of C10orf116 and KRT19 genes differed between patients and controls (P<.001). KRT20, C10orf116, and AGR2 differentiated between low- and high-risk nonmuscle-invasive bladder cancer (P=.001, P=.011, and P=.001, respectively). FXYD3 differentiated between patients with high-risk nonmuscle-invasive bladder cancer and those with muscle-invasive bladder cancer (P=.009). In contrast, the 6 markers showed no differences in gene expression between metastatic and patients without metastases who had not undergone cystectomy (P=NS). None of the markers were significantly increased in the metastatic patients at 6, 12, 18, or 24 months after surgery. CONCLUSION: The gene expression of bladder-specific mRNA markers in blood was different among the various tumor risk groups of patients with UCC. However, this gene expression analysis is not suitable for predicting metastases or monitoring UCC hematogenous dissemination in patients who have undergone cystectomy.

Characterization of the human Nalpha-terminal acetyltransferase B enzymatic complex.

BACKGROUND: Human Nalpha-acetyltransferase complex B (hNatB) is integrated by hNaa20p (hNAT5/hNAT3) and hNaa25p (hMDM20) proteins. Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells. METHODS: We have silenced hNAA20 expression in hepatic cell lines using recombinant adenoviruses that express specific siRNAs against this gene and analyzed cell cycle progression and apoptosis induction after this treatment. Immunopurified hNatB enzymatic complexes from human cell lines were used for analyzing hNatB in vitro enzymatic activity using as substrate peptides predicted to be acetylated by NatB. RESULTS: hNAA20 silencing in hepatic cell lines reduces cell proliferation in a p53 dependent and independent manner. At the same time this treatment sensitizes the cells to a proapototic stimulus. We have observed that the hNatB complex isolated from human cell lines can acetylate in vitro peptides that present an aspartic or glutamic acid in their second position as has been described in yeast. CONCLUSION: hNatB enzymatic complex is implicated in cell cycle progression but it exerts its effects through different mechanisms depending on the cellular characteristics. This is achievable because it can acetylate a great number of peptides composed by an aspartic or glutamic acid at their second residue and therefore it can regulate the activity of a great number of proteins.

Protamine 2 precursors (Pre-P2), protamine 1 to protamine 2 ratio (P1/P2), and assisted reproduction outcome.

OBJECTIVE: To determine whether the presence of protamine 2 precursors (pre-P2/P2 ratio) and the protamine 1 to protamine 2 ratio (P1/P2) are related to the assisted reproduction outcome. DESIGN: Prospective study. SETTING: Assisted Reproduction Unit and University laboratory. PATIENT(S): One hundred two infertile patients undergoing treatment at the Assisted Reproduction Unit of the Hospital Clinic of Barcelona. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) and/or IVF treatment of the infertile patients, sperm protamine analysis through electrophoresis and densitometry, and pre-P2 analysis through Western blot. MAIN OUTCOME MEASURE(S): The presence of protamine 2 precursors (pre-P2/P2 ratio), sperm P1/P2 ratio, fertilization rates by IVF and/or ICSI, and pregnancy outcome. RESULT(S): Pre-P2/P2 and P1/P2 ratios are positively associated with the pregnancy rate. In addition, the P1/P2 ratio is positively associated with the proportion of embryos obtained by IVF, but not by ICSI. The pre-P2/P2 ratio was not related to the fertilization rate. CONCLUSION(S): Decreased pre-P2/P2 and P1/P2 ratios are related to a poor pregnancy outcome, but not with the proportion of embryos obtained after ISCI.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

Proteomics of human spermatozoa, protamine content and assisted reproduction outcome.

It is well known that alterations in the expression of the major sperm nuclear proteins (protamines) are related to infertility in man. In addition, other minor proteins extracted from human spermatozoa are being analysed by 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and identified by MALDI-TOF MS analysis. The function of the identified proteins turns out to be energy production, transcription, protein synthesis, transport, folding and turnover, cell cycle, apoptosis and oxidative stress, signal transduction, cytoskeleton, flagella and cell movement, cell recognition, metabolism and unknown function. Many of the proteins identified using MALDI-TOF had not been yet been described as being expressed in human spermatozoa. Substantial differences have been detected in the levels of some of the newly identified human sperm proteins in the different groups of infertile patients. Present research efforts are targeting the potential correlations among changes in the proteomic composition, protamine content, DNA integrity and assisted reproduction outcome.

High frequency of gr/gr chromosome Y deletions in consecutive oligospermic ICSI candidates.

BACKGROUND: The Y chromosome gr/gr microdeletion eliminates two copies of the DAZ gene and several additional transcriptional units and has been associated as a risk factor for infertility. Our objective was to study the presence of the gr/gr deletion in ICSI candidates in our population and to determine whether the laboratory, clinical and ICSI outcome were different in the gr/gr deleted patients. METHODS: Two hundred and eighty-three ICSI candidates were studied. Semen analysis, serum FSH, LH, testosterone, inhibin B, karyotype and detection of sequence tagged sites in the Y chromosome were performed. RESULTS: gr/gr deletions were detected in 11 (5.07%) of 217 oligospermic and in one (1.52%) of 66 azoospermic consecutive ICSI candidates, but in none of 232 controls (P=0.002). The fertility rate was not different in the four patients of the gr/gr deleted group treated by ICSI (64.38%; 47/73) as compared to average results at our center (65.49%; 2393/3654). CONCLUSIONS: gr/gr deletions are a risk factor for spermatogenic failure at our population, but the prognosis of the four patients of the gr/gr deleted group treated by ICSI is not different from that of other ICSI patients.