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Genotyping NAT2 with only two SNPs (rs1041983 and rs1801280) outperforms the tagging SNP rs1495741 and is equivalent to the conventional 7-SNP NAT2 genotype.

Genotyping N-acetyltransferase 2 (NAT2) is of high relevance for individualized dosing of antituberculosis drugs and bladder cancer epidemiology. In this study we compared a recently published tagging single nucleotide polymorphism (SNP) (rs1495741) to the conventional 7-SNP genotype (G191A, C282T, T341C, C481T, G590A, A803G and G857A haplotype pairs) and systematically analysed if novel SNP combinations outperform the latter. For this purpose, we studied 3177 individuals by PCR and phenotyped 344 individuals by the caffeine test. Although the tagSNP and the 7-SNP genotype showed a high degree of correlation (R=0.933, P<0.0001) the 7-SNP genotype nevertheless outperformed the tagging SNP with respect to specificity (1.0 vs. 0.9444, P=0.0065). Considering all possible SNP combinations in a receiver operating characteristic analysis we identified a 2-SNP genotype (C282T, T341C) that outperformed the tagging SNP and was equivalent to the 7-SNP genotype. The 2-SNP genotype predicted the correct phenotype with a sensitivity of 0.8643 and a specificity of 1.0. In addition, it predicted the 7-SNP genotype with sensitivity and specificity of 0.9993 and 0.9880, respectively. The prediction of the NAT2 genotype by the 2-SNP genotype performed similar in populations of Caucasian, Venezuelan and Pakistani background. A 2-SNP genotype predicts NAT2 phenotypes with similar sensitivity and specificity as the conventional 7-SNP genotype. This procedure represents a facilitation in individualized dosing of NAT2 substrates without losing sensitivity or specificity.

The long noncoding RNA HOTAIR has tissue and cell type-dependent effects on HOX gene expression and phenotype of urothelial cancer cells

BackgroundUrothelial carcinoma (UC) is the fifth most common cancer in the developed world. Delineation of differentiation subtypes in UC highlighted the importance of aberrant differentiation. Understanding underlying mechanisms may facilitate diagnosis and development of efficient therapy strategies. It is well accepted that epigenetic mechanisms are involved. Long noncoding RNAs (lncRNAs), a new class of epigenetic factors, are thought to mediate molecular differences between cell types to control cellular identity. The present study focuses on the lncRNA HOTAIR, originating from the HOXC locus. Its overexpression induces an aggressive phenotype in many cancers and aberrant expression of homeotic HOX transcription factors, especially HOXD10, that regulate differentiation and tissue homeostasis. The aim of the present study was to determine the functional role of HOTAIR in UC with regard to aggressive phenotype, regulation of aberrant differentiation and altered HOX gene expression.MethodsWe determined RNA expression levels of HOTAIR and HOX genes in UC tissues and cell lines. Knockdown of HOTAIR and ectopic overexpression was performed to determine the effect on reported target genes in UC. Cell lines were stably transfected with HOTAIR to investigate changes in phenotype and HOX gene expression.ResultsHOTAIR was overexpressed in approximately half of UC tissues and cell lines. Effects of HOTAIR overexpression differed between cell lines. Whereas VM-CUB1 cells acquired the expected phenotype with increased proliferation, clonogenicity, anchorage independent growth, migratory activity and epithelial-to-mesenchymal transition, 5637 cells grew more slowly displaying induction of senescence and related immune response genes. Other UC lines showed intermediate effects. Expression profiling revealed divergent effects on HOX genes, cell cycle regulators and differentiation according with the phenotypic differences between HOTAIR-overexpressing VM-CUB1 and 5637 cells.ConclusionsOur data indicate that HOTAIR overexpression may affect differentiation state and aggressiveness of UC cells, but in a cell-type dependent manner. Our functional studies and the comparison of our expression data sets with those from other cancer cell types, which revealed minimal overlaps, indicate that effects of HOTAIR are strongly tissue-dependent and can even differ within one cancer type. Thus, HOTAIR functions and target genes cannot simply be transferred from one cancer type to the other.

Changes in histone deacetylase (HDAC) expression patterns and activity of HDAC inhibitors in urothelial cancers

OBJECTIVE To determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat. MATERIALS AND METHODS Expression of HDAC mRNAs was determined by quantitative RT-PCR in 18 urothelial cancer cell lines (UCC), normal uroepithelial controls (NUC), 24 urothelial cancer tissues, and 12 benign controls. Results were compared with published microarray data. Effects of pan-HDAC inhibitor vorinostat and on UCCs were determined by viability and apoptosis assays, cell cycle analysis, and measurements of p21(CIP1), thymidylate synthase (TS), and EZH2. In addition, protein expression levels of HDACs were investigated in UCCs. RESULTS Prominent changes in UCCs were HDAC2 and/or HDAC8 up-regulation in 11 of 18 cell lines and decreased expression of HDAC4, HDAC5, and/or HDAC7 mRNA in 15 of 18 cell lines. In cancer tissues, HDAC8 was likewise significantly up-regulated (P = 0.002), whereas HDAC2 up-regulation was detected only in a subset of tumors (9/24, P = 0.085). Overexpression of HDAC2 and HDAC8 mRNA did not correspond with the protein level. Vorinostat induced G2/M arrest, an increase in the sub-G1 fraction, up-regulation of p21, and down-regulation of TS in all UCC. Effects on EZH2 and PARP cleavage as well as activation of caspase 3/7 differed between cell lines. Associations between the overall sensitivity to the pan-HDACi vorinostat and overexpression of HDAC2 and HDAC8 mRNA were not observed. CONCLUSIONS In urothelial cancer, up-regulation of HDAC2 and HDAC8 and down-regulation of HDAC4, HDAC5, and HDAC7 mRNA are common findings. The treatment effect of the pan-HDAC inhibitor vorinostat was variable in UCCs and up-regulation of HDAC2 and HDAC8 was not predictive for treatment response. Whether selective targeting of HDAC2, HDAC8, or other HDACs deregulated in urothelial cancer (e.g., HDAC4, HDAC5, and HDAC7) result in a more consistent treatment response needs further investigation.

Neoadjuvant Chemotherapy in Patients with Muscle-invasive Bladder Cancer: Which Patients Benefit?

Level I evidence supports neoadjuvant chemotherapy in the treatment of advanced bladder cancer. For the most benefit, it is suggested that neoadjuvant chemotherapy be restricted to patients with clinical T3 disease and/or clinical N+ disease.

Prognostic Factors in Second-Line Treatment of Urothelial Cancers With Gemcitabine and Paclitaxel (German Association of Urological Oncology Trial AB20/99)

BACKGROUND In the treatment of urothelial cancer, identification of patients who are likely to benefit from further therapy after cisplatin failure is crucial for reasonable treatment decisions. OBJECTIVE Validate the prognostic factor model (PFM) for survival developed by Bellmunt et al. in a different patient cohort with a different chemotherapy regimen. DESIGN, SETTING, AND PARTICIPANTS Baseline parameters of 102 patients treated within a randomized phase 3 trial of second-line gemcitabine and paclitaxel (GP) comparing short-term to prolonged chemotherapy (German Association of Urological Oncology trial AB20/99) were analyzed. Patients were stratified according to the PFM based on a score including performance status, presence of hepatic metastases, and hemoglobin levels. MEASUREMENTS The baseline parameters of the GP cohort were compared with those of patients treated in the phase 3 trial of vinflunine versus best supportive care. Univariate and multivariate analyses of baseline parameters with respect to overall survival (OS) and treatment response were performed. OS of patients stratified according to the PFM was compared by log-rank test. RESULTS AND LIMITATIONS The vinflunine and the GP cohorts differed, as patients after perioperative (neoadjuvant or adjuvant) treatment were included in the latter cohort. According to the PFM, prognostic subgroups with significant difference in OS (11.8 mo [95% confidence interval (CI), 6.3-17.3], 8.1 mo [95% CI, 4.8-11.4], 3.2 mo [95% CI, 0.0-7.9]; p=0.007) were identified. The PFM identified risk groups in patients with failed treatment of metastatic disease (14.1 mo [95% CI, 8.9-19.3], 7.3 mo [95% CI, 0.0-17.8], 3.8 mo [95% CI, 0.0-9.0]; p=0.006) but not in patients treated (neo)adjuvantly. Lymph node-only disease was a strong predictor of treatment response that overruled every other single predictive parameter (0.284, p=0.0266). CONCLUSIONS The PFM was successfully validated in the GP and should be used to tailor second-line treatment strategy. Patients with lymph node-only disease may benefit from second-line treatment even if anemia or impaired performance status is present. TRIAL REGISTRATION German Cancer Society 01-09 (www.krebsgesellschaft.de).

HERV-K and LINE-1 DNA Methylation and Reexpression in Urothelial Carcinoma

Changes in DNA methylation frequently accompany cancer development. One prominent change is an apparently genome-wide decrease in methylcytosine that is often ascribed to DNA hypomethylation at retroelements comprising nearly half the genome. DNA hypomethylation may allow reactivation of retroelements, enabling retrotransposition, and causing gene expression disturbances favoring tumor development. However, neither the extent of hypomethylation nor of retroelement reactivation are precisely known. We therefore assessed DNA methylation and expression of three major classes of retroelements (LINE-1, HERV-K, and AluY) in human urinary bladder cancer tissues and cell lines by pyrosequencing and quantitative reverse transcription–polymerase chain reaction, respectively. We found substantial global LINE-1 DNA hypomethylation in bladder cancer going along with a shift toward full-length LINE-1 expression. Thus, pronounced differences in LINE-1 expression were observed, which may be promoted, among others, by LINE-1 hypomethylation. Significant DNA hypomethylation was found at the HERV-K_22q11.23 proviral long terminal repeat (LTR) in bladder cancer tissues but without reactivation of its expression. DNA methylation of HERVK17, essentially absent from normal urothelial cells, was elevated in cell lines from invasive bladder cancers. Accordingly, the faint expression of HERVK17 in normal urothelial cells disappeared in such cancer cell lines. Of 16 additional HERV-Ks, expression of 7 could be detected in the bladder, albeit generally at low levels. Unlike in prostate cancers, none of these showed significant expression changes in bladder cancer. In contrast, expression of the AluYb8 but not of the AluYa5 family was significantly increased in bladder cancer tissues. Collectively, our findings demonstrate a remarkable specificity of changes in expression and DNA methylation of retroelements in bladder cancer with a significantly different pattern from that in prostate cancer.

Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment

BackgroundPrevious studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.MethodsWe conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.ResultsEfficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC50 9 – 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.ConclusionsDeregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.

A nomogram including baseline prognostic factors to estimate the activity of second-line therapy for advanced urothelial carcinoma

To study the impact of the prognostic factors liver metastasis (LM), anaemia (haemoglobin [Hb] <10 g/dL), Eastern Cooperative Oncology Group performance status (ECOG‐PS) ≥1 and time from previous chemotherapy (TFPC) on the activity of second‐line therapy for advanced urothelial carcinoma (UC).

Multiple Mechanisms Mediate Resistance to Sorafenib in Urothelial Cancer

Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor α/-receptor β, PDGFR-α/-β; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.

Concomitant downregulation of the imprinted genes DLK1 and MEG3 at 14q32.2 by epigenetic mechanisms in urothelial carcinoma

BackgroundThe two oppositely imprinted and expressed genes, DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are located in this region, but these have not been identified.ResultsWe observed that both DLK1 and MEG3 are frequently silenced in urothelial cancer tissues and cell lines. The concomitant downregulation of the two genes is difficult to explain by known mechanisms for inactivating imprinted genes, namely deletion of active alleles or epitype switching. Indeed, quantitative PCR revealed more frequent copy number gains than losses in the gene cluster that were, moreover, consistent within each sample, excluding gene losses as the cause of downregulation. Instead, we observed distinctive epigenetic alterations at the three regions controlling DLK1 and MEG3 expression, namely the DLK1 promoter; the intergenic (IG) and MEG3 differentially methylated regions (DMRs). Bisulfite sequencing and pyrosequencing revealed novel patterns of DNA methylation in tumor cells, which were distinct from that of either paternal allele. Furthermore, chromatin immunoprecipitation demonstrated loss of active and gain of repressive histone modifications at all regulatory sequences.ConclusionsOur data support the idea that the main cause of the prevalent downregulation of DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.