Novel ZEB1 expression in bladder tumorigenesis.

UNLABELLED: What's known on the subject? and What does the study add? Epithelial-mesenchymal transition (EMT) is involved in tumor progression where the underlying cellular changes associated with EMT have been identified in in vitro models and confirmed in a limited number of in vivo studies. ZEB1, which targets E-cadherin repression, is a transcriptional regulator that has been implicated in EMT, and is associated with uterine and colorectal cancers. Regulation of ZEB1 expression has been shown to involve different microRNAs (miRNAs), identifying a potential role for miRNA in EMT. In the present study we have identified novel expression of ZEB1 in bladder tumours and shown a role for ZEB1 in enhanced migration and invasion potential in in vitro assays. Confirmation of ZEB1 expression in bladder tumours was shown in tissue microarrays (TMAs). OBJECTIVE: To evaluate ZEB1 expression in bladder tumorigenesis and define a possible role for this transcription factor in urothelial carcinomas of the bladder (UCBs). MATERIALS AND METHODS: Five hundred and fifty-eight samples were assembled in 10 tissue microarrays (TMAs; 263 non-muscle-invasive Ta/T1/Tis, 295 muscle-invasive T2-T4). All tumours were transitional cell carcinomas (TCCs) and processed for immunohistochemistry to assess nuclear ZEB1 expression. Expression levels of ZEB1 were modulated in bladder carcinoma cell lines CUBIII or UM-UC-3 after forced expression or shRNA knockdown, respectively. Protein expression levels were determined using western blot analysis and transfectants were assessed for migration and invasion potential in standard in vitro assays. RESULTS: Nuclear ZEB1 expression was recorded in 22.8% of non-muscle-invasive UCBs and 21.7% of muscle-invasive UCBs, including 24.1% grade I/II and 21.1% grade III tumours, and absent in normal bladder mucosa. No significant correlation was observed for tumour stage and grade, nodal involvement, vascular invasion, metastasis and overall or cancer-specific survival. The introduction or knockdown of ZEB1 expression in bladder carcinoma cell lines showed enhanced or reduced migration and invasive potential, respectively. Changes in ZEB1 expression were accompanied by altered microRNA (miRNA) expression underlying events linked to epithelial-mesenchymal transition (EMT). CONCLUSION: The results in the present study showed novel expression of ZEB1 in bladder cancer in the absence of a link to clinical variables of change, including metastasis and survival. However, in vitro assays showed enhanced or reduced migration and invasion after the introduction or reduction of ZEB1, respectively, in transfected bladder cell lines. Modulation in expression of ZEB1 was closely linked to changes in the miR-200 family along with alternative known prognostic indicators of bladder tumour progression.

A MicroRNA expression profile defining the invasive bladder tumor phenotype.

OBJECTIVE: The purpose of this study was to identify microRNA (miRNA) involved in the transition between the noninvasive and invasive urothelial carcinoma of the bladder (UCB) phenotype. METHODS: Differential expression of miRNA was identified in a microarray format between noninvasive and invasive UCB cell lines and confirmed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) within this cell panel. Normalization of qRT-PCR with miR-222 was established from the microarray data and validated within a panel of 57 UCB tumors (26 noninvasive lesions (Ta/G1) and 31 invasive lesions (T2-T4). Pre-miR constructs were transfected into appropriate UCB cell lines to establish a change in invasive potential. RESULTS: Differential expression of miRNAs was identified from microarray analysis and included reduced expression associated with miR-30b, miR-31, miR-141, miR-200a, miR-200b, miR-200c, miR-205, miR-21 in invasive lesions and elevated miR-99a in noninvasive UCB lesions. Reduced invasion potential was recorded in UM-UC-3, following pre-miR transfection, in all UCB cell lines with the exception of UM-UC-3/miR-30b transfectants. Our results identify a panel of miRNA modulated and expressed in invasive UCB tumors and demonstrates a role for them in the invasive phenotype. CONCLUSIONS: The diagnostic test, based on the three most discriminatory miRNAs in our panel (miR-200c, miR-141, and miR-30b), showed a sensitivity of 100% and a specificity of 96.2%. Such a panel of miRNAs has the potential to identify invasive bladder tumors misclassified in pathologic assessment of bladder biopsy specimens.

Robotic enterocystoplasty: technique and early outcomes.

OBJECTIVE: Enterocystoplasty is an established treatment for patients with refractory neurogenic bladder symptoms. We assessed the feasibility, safety, and efficacy of a robot-assisted enterocystoplasty in this population. MATERIALS AND METHODS: Five neurogenic bladder patients, median age of 43.8 years, underwent the procedure. Using a five-port technique, intraperitoneal robotic enterocystoplasty was performed through the following steps: (1) creation of a U-shaped full-thickness detrusor cystotomy, (2) intracorporeal harvesting of 30 cm of ileum, (3) intracorporeal construction of a detubularized ileal patch, and (4) anastomosis of the ileal patch to the cystotomy. An extracorporeal side-to-side bowel anastomosis re-established bowel continuity. After surgery, urinary continence, bladder capacity, upper tract protection, and complications were assessed. RESULTS: Mean operative time was 6.4 hours, estimated blood loss was 180 mL, and length of stay was 7 days. Postoperatively, all patients had a functioning enterocystoplasty, urethral continence, and normal upper tract imaging. One patient was rehospitalized for an ileus/urinoma, which resolved with conservative treatment. CONCLUSIONS: Robot-assisted enterocystoplasty can be effectively and safely performed with minimal morbidity.

Identification of tumor and invasion suppressor gene modulators in bladder cancer by different classes of histone deacetylase inhibitors using reverse phase protein arrays.

PURPOSE: We assessed the ability of different classes of histone deacetylase inhibitors to target tumor and invasive suppressor genes in a panel of bladder carcinoma cell lines using reverse phase protein arrays. MATERIALS AND METHODS: Three poorly, moderately and highly invasive cell lines were exposed to histone deacetylase inhibitors, trichostatin A, apicidin, valproic acid (Sigma) and MS-275 (AXXORA) for 0 to 36 hours. Lysates were harvested and arrayed in a 10-fold dilution series in duplicate. Data points were collected and analyzed using a concentration interpolation methodology after normalization. RESULTS: Protein expression profiles revealed up-regulation of gamma-catenin in highly invasive lines, and alpha-catenin in moderately and highly invasive lines after exposure to all histone deacetylase inhibitors, apicidin and MS-275, respectively. Gelsolin was up-regulated in poorly and moderately invasive lines after exposure to all histone deacetylase inhibitors. Desmoglein was down-regulated in poorly and moderately invasive cell lines by all 4 histone deacetylase inhibitors, in addition to decreased FAK (Transduction Laboratories) expression in moderately and highly invasive lines exposed to valproic acid and MS-275. CONCLUSIONS: Different histone deacetylase inhibitor classes have the potential to modulate tumor and invasive suppressor gene expression, identifying histone deacetylase inhibitors as potential therapeutic agents for bladder cancer. Reverse phase protein arrays enable high throughput screening of multiple compounds to assess the expression profile of specific protein groups targeted for therapy.

Face, content, and construct validity of dV-trainer, a novel virtual reality simulator for robotic surgery.

OBJECTIVES: To assess the face, content, and construct validity of the dV-Trainer. The dV-Trainer is a virtual reality simulator for the da Vinci Surgical System that is in beta development. METHODS: Medical students, residents, and attending surgeons were enrolled in a prospective, institutional review board-approved study. The subjects were prospectively categorized as novice or experienced. Each subject completed 2 EndoWrist modules and 2 needle-driving modules. The performance was recorded using a built-in scoring algorithm. Each subject completed a questionnaire after finishing the modules. RESULTS: The novice group (n = 19) consisted of 3 students (16%), 11 residents (58%), and 5 attending surgeons (26%). The novices had operated an average of 1.3 +/- 2.2 hours at the da Vinci console before using the simulator. The experienced subjects (n = 7) had performed an average of 140 robotic cases (range 30-320). Experienced robotic surgeons outperformed novices in nearly all variables, including total score, total task time, total instrument motion, and number of instrument collisions (P < .01). All experienced surgeons ranked the simulator as useful for training and agreed with incorporating the simulator into a residency curriculum. The virtual reality and instrumentation achieved acceptability. The needle-driving modules did not exceed the acceptability threshold. CONCLUSIONS: The results of the present study have shown that the dV-Trainer has face, content, and construct validity as a virtual reality simulator for the da Vinci Surgical System. The needle-driving modules need to be refined. Studies are underway to assess the concurrent and predictive criterion validity. The dV-Trainer could become a beneficial training simulator for robotic surgery.