膀胱尿路上皮癌组织中Snail蛋白表达与E-cadherin蛋白、T细胞亚群的相关性分析

目的 研究膀胱尿路上皮癌组织中Snail蛋白表达与E-cadherin蛋白、T细胞亚群的相关性。 方法 采用免疫组化SP法检测156例膀胱尿路上皮癌组织和80例癌旁组织中Snail蛋白、E-cadherin蛋白的表达情况,比较二者在不同病理组织中的阳性表达率,并分析二者表达的相关性;分析膀胱尿路上皮癌组织中Snail蛋白阳性表达和CD4+、CD8+细胞数量及CD4+/CD8+的相关性。结果膀胱尿路上皮癌组织Snail蛋白阳性表达率65.4％(102/156)显著高于癌旁组织的48.8％(39/80),差异有统计学意义(P＜0.05);其表达与膀胱尿路上皮癌的临床分期、病理分级、肿瘤数量、远处转移及复发有关（P值均＜0.05）。膀胱尿路上皮癌中Snail蛋白与E-cadherin蛋白表达呈负相关（r= -0.186,P＜0.05）;Snail蛋白阳性表达与CD4+细胞数量以及CD4+/CDt+值呈负相关(r=-0.313,P＜0.05;r=-0.305,P＜0.05),而与CDs+细胞数量无关（r= -0.250,P＞0.05）。 结论 Snail蛋白可能通过抑制E-cadherin蛋白表达及诱导膀胱肿瘤局部免疫抑制,促进膀胱尿路上皮癌的浸润、转移。     

High expression of polo-like kinase 1 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

ObjectivesPolo-like kinase 1 (Plk1) has been widely pursued as an oncology target because it is overexpressed in several human tumor types. To investigate whether Plk1 plays a general role in bladder urothelial carcinoma, we examined the expression of Plk1 protein in bladder urothelial carcinoma and cell lines, and analyzed the relationship among Plk1 protein expression, metastasis, and recurrence of urinary bladder urothelial carcinoma.MethodsImmunohistochemistry was used to detect the expression of Plk1 in 120 bladder urothelial carcinoma. Moreover, the expression of Plk1 was analyzed by Western blot in 60 bladder urothelial carcinoma and 21 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of scytonemin (the inhibitor of Plk1). Statistical analysis was used to discuss the association between Plk1 expression and clinicopathologic parameters, tumor metastasis and recurrence, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells.ResultsThere was a significantly higher Plk1expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Plk1 expression was positively correlated with histologic grade, pT stage, recurrence, and metastasis. With the increasing concentration of scytonemin, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G2/M stage.ConclusionPlk1 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Plk1 played an important function on the bladder cancer cells' proliferation by regulating the cancer cell cycle from G1/S to G2/M and probably promoted the invasion and metastasis of bladder cancer.     

A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT.Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay.ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration.ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.     

Expression of pigment epithelium-derived factor and tumor necrosis factor-α is correlated in bladder tumor and is related to tumor angiogenesis

ObjectiveAngiogenesis is a pivotal process on which solid tumor growth is substantially dependent. Pigment epithelium-derived factor (PEDF) is the most potent natural anti-angiogenic factor, which has seldom been studied in bladder tumor, and whose functioning pathway remains unclear. We have thus investigated PEDF expression in relation to tumor necrosis factor-α (TNF-α) and microvessel density (MVD) with immunohistochemistry.MethodsAntibodies of PEDF and TNF-α were examined by Western blotting before immunohistochemistry. Sixty-four urothelial tumor sections and 23 normal controls were stained and expression of PEDF, TNF-α, and MVD were studied.ResultsDecreased PEDF expression and increased TNF-α expression was noticed in tumorous tissue compared with healthy urothelium. Lower PEDF expression was related to higher tumor grade but stage. Increased TNF-α expression was noticed in recurrent, larger tumors as well as in tumors with progression in grade and stage. Expression of PEDF and TNF-α was correlated in bladder tumor. PEDF or TNF-α was correlated with MVD negatively or positively, respectively, in cancerous tissue and tumorous grouping without correlation in papillary urothelial neoplasm of low malignant potential.ConclusionExpressional change of PEDF and TNF-α is in relation to angiogenesis of bladder tumor, especially in bladder cancer development.     

Fascin is a predictor for invasiveness and recurrence of urothelial carcinoma of bladder

ObjectivesTo evaluate the expression of fascin in bladder urothelial carcinoma, and to analyze its association with clinicopathologic features and prognosis of urinary bladder urothelial carcinoma.Materials and methodsImmunohistochemistry was used to detect the expression of fascin, Ki-67, p53, CK20, and multidrug resistance gene (MDR) in 111 bladder urothelial carcinoma and 42 normal epithelial tissues. The association between fascin expression and clinicopathologic parameters and prognostic factors on tumor recurrence was analyzed by Kaplan-Meier method, log-rank test, and Cox proportional hazards model.ResultsNinety-four of 111 cases of bladder urothelial carcinoma showed positive fascin expression, while no fascin expression was detected in normal transitional epithelium. There was a significant difference in the expression of fascin in normal epithelium and bladder urothelial carcinoma (P = 0.000). Fascin expression was positively correlated with pT stage (P = 0.001) and tumor size (P = 0.011), while it had no association with age, gender, and tumor grade (P > 0.05). pT stage and the expression of fascin, Ki-67, p53, and CK20 were significantly correlated with urothelial carcinoma recurrence, and fascin expression was an independent factor predicting tumor recurrence.ConclusionsFascin expression was up-regulated in bladder urothelial carcinoma. Over-expression of fascin might play an important role in invasiveness and recurrence of bladder urothelial carcinoma. Fascin may be used as a prognostic marker and a new target for the treatment of bladder urothelial carcinoma.     

Phospho-Akt pathway activation and inhibition depends on N-cadherin or phospho-EGFR expression in invasive human bladder cancer cell lines

ObjectivesA particular interest in epithelial-mesenchymal transition (EMT), which takes place during embryonic development, provided potential mechanisms involved in the progression of many epithelial tumors, including bladder cancer (BC). The phospho-Akt signaling pathway is supposed to be involved in invasion and progression of human tumors, including BC. Moreover, it has been demonstrated in bladder cancer cell lines that N-cadherin or phospho-epithelial growth factor receptor (EGFR) expression are correlated to tumor progression. Our objectives were to evaluate the potential phospho-Akt pathway involvement in N-cadherin and/or phospho-EGFR positive BC cell lines and to evaluate the prognostic value of E- and N-cadherin expression in patients undergoing cystectomy for invasive BC.Materials and methodsWe screened a panel of invasive and noninvasive BC cell lines for E- and N-cadherin, phospho-EGFR, and phospho-Akt expression using the Western blot technique (WB). The potential role of N-cadherin in invasion was assessed by Matrigel assays with and without the N-cadherin blocking monoclonal antibody GC-4. Then we used the Affymetrix microarray technique to evaluate the prognostic value of E- and N-cadherin expression in 30 patients undergoing a cystectomy for invasive BC.ResultsN-cadherin and phospho-EGFR expression are associated with Akt activation and with invasive behavior modulation. Even if Akt activation is sufficient in promoting invasion, its inactivation by LY294002 (PI-3 kinase inhibitor) is less efficient on invasion than inhibition of N-cadherin and phospho-EGFR by GC-4 (monoclonal antibody) and gefitinib (anti-tyrosine kinase), respectively. N-cadherin and phospho-EGFR inhibition decreased phospho-Akt activation but also caused restoration and reinforcing of E-cadherin expression, respectively, while phospho-Akt inhibition did not have any impact on E-cadherin expression. In a group of high-risk bladder tumors (T₁G₃), N- and E-cadherin expression could be considered as a prognostic marker. In a group of patients with invasive BC (pT₂-T₄) undergoing cystectomy, we showed a shorter overall survival when BC expressed N-cadherin (P = 0.0064) and when E-cadherin expression was down-regulated (P = 0.00165). The N _positive /E _negative profile has the worst prognosis (P = 0.00153).ConclusionsWe confirmed the partial responsibility of p-Akt activation in invasion of some BC cell lines expressing N-cadherin or p-EGFR and also the potential role of N-cadherin and p-EGFR as target in cancer therapy. N/E- cadherin expression profile has a significant prognostic value in invasive BC.     

MK2206 potentiates cisplatin-induced cytotoxicity and apoptosis through an interaction of inactivated Akt signaling pathway

ObjectivesTo improve conventional chemotherapeutic efficacy, it is important to detect new molecular markers for chemosensitivity and possible accelerating cell-killing mechanisms. In this study, we investigated how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in urothelial cancer cells.Materials and methodsWe examined bladder cancer cell lines for the expression of phospho(p)-Akt and its downstream targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was examined by transwell invasion assay, and the activities of the Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot.ResultsThe expression of p-Akt and its downstream targets was increased in invasive bladder cancer cell lines vs. in noninvasive bladder cancer cell lines. MK2206 (500 nM) inhibited cell invasion in UMUC3 cell line and significantly increased the susceptibility of bladder cancer cell lines to CDDP. When used in combination with CDDP, MK2206 (500 nM) enhanced CDDP-induced cytotoxicity and apoptosis, with suppressed expression of p-Akt and its downstream targets. In vivo MK2206 combined with CDDP effectively suppressed tumor growth in subcutaneous xenograft models.ConclusionsThese results suggest that concomitant use of MK2206 could promote the CDDP-induced cytotoxicity and apoptosis in urothelial cancer cell lines through the inhibited expression of the Akt pathway. This combined treatment may provide a new therapeutic option to enhance chemosensitivity in bladder cancer.     

Loss of APKC expression independently predicts tumor recurrence in superficial bladder cancers

ObjectivesEpithelial-mesenchymal transition (EMT) is known to play an important role in the development of tumor invasion and progression in tumors of epithelial origin. Our aim was to investigate the role of tight junction proteins, Par3/Par6/atypical protein kinase C (APKC), Discs large (Dlg), and Scribble in human bladder pathogenesis.MethodsWe evaluated levels of APKC, Dlg, and Scribble in 92 superficial bladder tumors using tissue microarrays and immunohistochemistry, and correlated expression with pathologic variables and clinical outcomes.ResultsThere was a slight apparent enrichment in strong vs. weak staining for APKC (54.9% vs. 45.1%), Dlg (65.7% vs. 34.3%), and a marked enrichment for Scribble (75% vs. 25%) in the superficial bladder tumors. Univariate analysis determined that both tumor focality and APKC expression were significantly associated with tumor recurrence (P < 0.05). Multivariate analysis using the Cox's proportional hazards model revealed that only APKC (P = 0.025) as well as tumor focality (P = 0.018) were independent and significant prognostic factors for tumor recurrence in all patients. We found that no immunohistochemical staining of any of the cell polarity proteins significantly predicted for tumor progression on either univariate or multivariate analysis.ConclusionsLoss of APKC expression in superficial bladder tumors is a strong predictor of tumor recurrence.     

An experimental model of the epithelial to mesenchymal transition and pro-fibrogenesis in urothelial cells related to bladder pain syndrome/interstitial cystitis

BackgroundSuitable in vitro models are needed to investigate urothelial epithelial to mesenchymal transition (EMT) and pro-fibrogenesis phenotype in bladder pain syndrome/interstitial cystitis (BPS/IC). This study is to establish a novel experimental BPS/IC cell model and explore how different concentrations of tumor necrosis factor (TNF)-α influence the EMT and pro-fibrogenesis phenotype of urothelial cells.MethodsSV-HUC-1 urothelial cells were cultured with 2, 10, or 50 ng/mL TNF-α to mimic chronic inflammatory stimulation. The EMT and pro-fibrogenesis phenotype, including production of collagen I and pro-fibrosis cytokines, were estimated after 72 h of culture.ResultsThe bladder urothelial cells of BPS/IC exhibited upregulated vimentin, TNF-α and TNF receptor, downregulated E-cadherin, and increased collagen I. Higher concentrations of TNF-α (10 and 50 ng/mL) produced an obvious mesenchymal morphology, enhanced invasion and migratory capacity, increased expression of vimentin, and decreased expression of E-cadherin. Collagen I was increased in cells treated with 2 and 10 ng/mL TNF-α after 72 h. Secretion of interleukin (IL)-6 and IL-8 was promoted with 10 and 50 ng/mL TNF-α, while that of IL-1β or transforming growth factor-β was unaffected. Slug and Smad2 were upregulated by TNF-α after 72 h. The Smad pathway was activated most strongly with 10 ng/mL TNF-α and Slug pathway activation was positively correlated with the concentration of TNF-α.ConclusionsSustained 10 ng/mL TNF-α stimulation induced the EMT and pro-fibrogenesis phenotype resembling BPS/IC in SV-HUC-1 cells. Minor inflammatory stimulation induced the pro-fibrogenesis phenotype while severe inflammatory stimulation was more likely to produce significant EMT changes. Different degrees of activation of the Slug and Smad pathways may underlie this phenomenon.     

Tumor Location Based Segmentation in Upper-Tract Urothelial Carcinoma Impacts on the Urothelial Recurrence-Free Survival: A Multi-Institutional Database Study

Introduction and ObjectivesThe predictive impact of primary tumor location for patients with upper-tract urothelial carcinoma (UTUC) in the presence of concomitant urothelial bladder cancer, along with urothelial recurrence after the curative treatment is still contentious. We evaluated the association between precise tumor location and concomitant presence of urothelial bladder cancer and urothelial recurrence-free survival in patients with UTUC treated by radical nephroureterectomy with a bladder cuff.MethodsA total of 1,349 patients with localized UTUC (Ta-4N0M0) from a retrospective multi-institutional cohort were studied. We queried four UTUC databases. This retrospective clinical series was of patients with localized UTUC managed by nephroureter-ectomy with a bladder cuff, for whom data were from the Nishinihon Uro-Oncology Collaborative Group registries. Patients with a history of chemotherapy or radiotherapy were excluded from the study. Associations between the location of the tumor and subsequent outcome following nephroureterectomy were assessed using COX multivariate analysis. The location of the tumor was verified by pathological samples. Urothelial recurrence was defined as tumor relapse in any local urothelium, and coded apart from distant metastasis. The median follow-up was 34 months.ResultsA total of 887 patients had an evaluation of the tumor location in which 475 patients had pelvic tumors (53.6%), 96 had ureteral tumors in the U1 segment (10.8%), 87 in the U2 segment (9.8%), and 176 in the U3 segment (19.8%). There were 52 patients who had multifocal tumors (5.9%) as follows: 8 (0.9%) in the pelvis and ureter, 11 (1.2%) in U1 + U2, 1 (0.1%) in U1 + U3, 27 (3.0 %) in U2 + U3, and 6 (0.7%) in U1 + U2 + U3. In all, 145 (16.3%) had concomitant bladder tumors. Logistic regression analysis of gender, age, hydronephrosis, cytology, performance status, grade, lymphovascular invasion, pT, pN, and tumor focality showed that tumor location was associated with the presence of concomitant bladder cancer (p = 0.004, HR = 1.265). When the tumor location was stratified into 8 segments, including multifocal tumors, only the U3 segment remained as a predictor for the presence of concomitant bladder cancer (p = 0.002, HR = 2.872). Kaplan-Meier analysis for unifocal disease showed that lower ureter tumors (a combination of U2 and U3) had a worse prognosis for urothelial recurrence than pelvic tumors or upper ureteral tumors (U1) (p < 0.001 for lower ureteral tumors versus pelvic tumors, p = 0.322 for upper ureteral tumor versus pelvic tumor by log rank). Multivariate analysis showed that lower ureter remained as a prognostic factor for urothelial recurrence after adjusting for gender, age, hydronephrosis, urine cytology, lymphovascular invasion, pT, and pN (p < 0.001, HR = 1.469), and a similar tendency was found when the analysis was run for patients without concomitant bladder tumors (p = 0.003, HR = 1.446). Patients with lower ureteral tumors had a higher prevalence of deaths (HR = 2.227) compared to patients with upper ureter tumors.ConclusionsThis multi-institutional study showed that the primary tumor locations were independently associated with the presence of concomitant bladder tumors and subsequent urothelial recurrence.Key Words: Upper-tract urothelial carcinoma, Prognosis, Tumor location     

Immunohistochemical negative expression of ezrin predicts poor prognosis in clear cell renal cell carcinoma

PurposeTo analyze the immunohistochemical expression of ezrin and moesin in clear cell renal cell carcinoma (ccRCC). These proteins, as part of the ezrin-radixin-moesin complex link the cell membrane to the actin cytoskeleton, affecting such processes as cell adhesion, cell survival, cell motility, and signal transduction. Our aim was to examine the impact of their expression on clinical outcomes and survival rates.Patients and methodsFive hundred seventy-five consecutive patients who had been treated surgically for ccRCC in a single center between 1985 and 2016 were selected. A single pathologist reviewed all cases to perform a uniform reclassification and determined the most representative tumor areas for construction of a tissue microarray.ResultsOf all ccRCC specimens, 106 (18.3%) were negative for ezrin, and 469 (81.7%) had positive ezrin expression; 16 (2.8%) were negative and 559 (97.2%) were positive for moesin, respectively. Ezrin expression was associated with pT stage (P < 0.001), clinical stage (P = 0.012), synchronic metastasis (P < 0.001), incidental tumors (P = 0.007), and International Society of Urological Pathology histological grade (P = 0.025). There was a correlation between moesin expression and clinical stage (P = 0.027), pT stage (P = 0.025), and pN stage (P = 0.007). Ezrin expression significantly influenced tumor-related deaths. By multivariate analysis, negative ezrin expression was an independent risk factor for disease-specific survival (HR 1.89; 95% CI 1.11–3.20).ConclusionsNegativity for ezrin in ccRCC patients significantly impacts survival rates. We encourage further prospective studies to analyze ezrin analysis to evaluate its significance in the prognosis of ccRCC.     

E-钙粘素与壶腹部癌的分化、浸润及转移的关系

目的：探讨E－钙粘素（E－cadherin,E-cd)在壶腹部癌中的表达,及其与壶腹部癌的分化、浸润及转移的关系。方法：采用免疫组化S－P法检测39例壶腹部癌组织和11例炎性壶腹部粘膜组织E－cadherin的表达。结果：（1）11例炎性壶腹部粘膜中5例可见表达,2例粘膜不典型增生处呈杂合性表达;（2）66．7％（26／39）壶腹癌E－cd异常表达;（3）83．3％的中、低分化肿瘤（10／12）E－cd表达异常;侵犯全层及邻近组织的肿瘤78．6％E－cd表达异常。结论：E－cd表达异常是壶腹部部的恶性表型之一,且其异常表达率较高;E－cd表达异常与壶腹部癌的分化、浸润有关,提示E－cd表达异常率越高,肿瘤细胞分化程度越低,浸润深度越深。     

Urokinase-type plasminogen activator receptor (uPAR) expression is associated with T-stage and survival in urothelial carcinoma of the bladder

ObjectivesTo evaluate the expression-and localization pattern of the urokinase-type plasminogen activator receptor (uPAR), focusing on its clinical implications in patients with urothelial neoplasia of the bladder treated with radical cystectomy. uPAR is a central molecule in tissue remodeling during cancer invasion and metastasis and is an established prognostic marker in cancer. The expression and localization of uPAR and its prognostic significance is only limitedly investigated in urothelial bladder neoplasia.Materials and methodsThe expression-and localization pattern of uPAR was investigated in formalin-fixed paraffin-embedded tumor tissue from 149 patients treated with radical cystectomy between 1988 and 2005. uPAR expression was determined by immunohistochemistry and scored as either negative or positive. Separate values were obtained for cancer cells, macrophages, and myofibroblasts at the invasive front and tumor core, respectively. Statistical analyses were performed to evaluate the association of uPAR localization and score with clinicopathologic covariates and survival.ResultsuPAR positivity was seen in 122/137 (89%) and 118/149 (74%) of the neoplasias at the invasive front and tumor core, respectively. uPAR was primarily expressed by myofibroblasts and macrophages in the surrounding stroma as well as some cancer cells. A significant association between uPAR positivity and T-stage as well as grade was found for all 3 cell types in tumor core (P≤0.04 for all comparisons). In univariate analysis, the uPAR positive group had a shorter survival than the uPAR negative group (hazard ratio = 2.39; 95% CI: 1.15–5.01; P = 0.020).ConclusionsThe expression of uPAR is a possible prognostic marker that could be useful in identification of patients with aggressive, highly invasive tumors that could benefit from additional chemotherapy or more intensive follow-up after cystectomy.     

Chromosomal imbalances in successive moments of human bladder urothelial carcinoma

ObjectiveTo understand developmental characteristics of urinary bladder carcinomas (UBC) by evaluating genomic alterations and p53 protein expression in primary tumors, their recurrences, and in the morphologically normal urothelium of UBC patients.MethodsTumors and their respective recurrences, six low-grade and five high-grade cases, provided 19 samples that were submitted to laser microdissection capture followed by high resolution comparative genomic hybridization (HR-CGH). HR-CGH profiles went through two different analyses—all tumors combined or classified according to their respective histologic grades. In a supplementary analysis, 124 primary urothelial tumors, their recurrences, and normal urothelium biopsied during the period between tumor surgical resection and recurrence, were submitted to immunohistochemical analyses of the p53 protein. During the follow-up of at least 21 patients, urinary bladder washes citologically negative for neoplastic cells were submitted to fluorescence in situ hybridization (FISH) to detect copy number alterations in centromeres 7, 17, and 9p21 region.Results and ConclusionsHR-CGH indicated high frequencies (80%) of gains in 11p12 and losses in 16p12, in line with suggestions that these chromosome regions contain genes critical for urinary bladder carcinogenesis. Within a same patient, tumors and their respective recurrences showed common genomic losses and gains, which implies that the genomic profile acquired by primary tumors was relatively stable. There were exclusive genomic alterations in low and in high grade tumors. Genes mapped in these regions should be investigated on their involvement in the urinary bladder carcinogenesis. Successive tumors from same patient did not present similar levels of protein p53 expression; however, when cases were grouped according to tumor histologic grades, p53 expression was directly proportional to tumor grades. Biopsies taken during the follow-up of patients with history of previously resected UBC revealed that 5/15 patients with no histologic alterations had more than 25% of urothelial cells expressing the p53 protein, suggesting that the apparently normal urothelium was genomically unstable. No numerical alterations of the chromosomes 7, 17, and 9p21 region were found by FISH during the periods “free-of-neoplasia.” Our data are informative for further studies to better understand urinary bladder urothelial carcinogenesis.     

Rictor-dependent AKT activation and inhibition of urothelial carcinoma by rapamycin

ObjectiveWe previously reported a very high cumulative incidence of urothelial carcinoma in Taiwanese kidney transplant recipients. Rapamycin, the inhibitor of mTOR Complex 1, provides alternative immunosuppressive therapy after kidney transplantation with less neoplastic potential. We examined the in vivo and in vitro effects of rapamycin on urothelial carcinoma.Materials and methodsThe rat model of urothelial carcinoma was induced by 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in Fischer F344 rats. The anti-tumor effect of rapamycin was assessed grossly, microscopically, and by Western blot analysis. The mechanism of rapamycin's attenuation of urothelial carcinoma was also evaluated by T24 cells.ResultsRapamycin significantly reduced urinary bladder tumor growth in the rat model of 0.05% BBN-induced urothelial carcinoma (P < 0.001). The blood trough levels of rapamycin were correlated with the occurrence of urothelial carcinoma. In vitro, rapamycin also inhibited the cell proliferation, migration, and invasion, as well as the protein expression of vascular endothelial growth factor-A of T24 urothelial carcinoma cells, whereas rapamycin did not induce significant apoptosis in T24 cells. Rapamycin decreased the expression of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A. Rapamycin also activated AKT in T24 cells in the rat model of urothelial carcinoma. The rapamycin-associated activation of AKT was inhibited by rictor siRNA, but not raptor siRNA.ConclusionsThis study provides in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings also suggest rictor-dependent AKT activation as a consequence of mTORC1 inhibition.     

Estrogen receptor β (ERβ) is a novel prognostic marker of recurrence survival in non-muscle-invasive bladder cancer potentially by inhibiting cadherin switch

Objective

The function and significance of estrogen receptor β (ERβ) in bladder cancer remains a field of hot debate. In this study, we aimed to (a) evaluate ERβ as a novel prognostic marker of recurrence free survival; and (b) digest the underlying mechanism by elucidating the relationship between ERβ expression and cadherin switch.

Methods

We examined the expression levels of ERβ, E-cadherin and N-cadherin in 42 initial non-muscle-invasive urothelial bladder carcinomas via immunohistochemistry. Correlation analysis was performed among ERβ expression, cadherin switch and recurrence free survival. Moreover, in vitro studies were performed to validate the identified correlation using two bladder cancer cell lines RT4 and 253J. Upon stimulation with an ERβ selective agonist diarylpropionitrile, E-cadherin, N-cadherin expressions; cell migration and invasion capacity were assessed.

Results

Expression of ERβ protein was seen in 34 bladder cancer cases (80.9 %), and 21 (50 %) specimens showed non-cadherin switch (positive E-cadherin and negative N-cadherin). ERβ expression and the non-cadherin switch are both accompanied with better recurrence free survival. Also, the least ERβ expression was observed in specimens that undergo cadherin switch. Moreover, these results were consistent with our observations in bladder cancer RT4 and 253J cell lines studies. Diarylpropionitrile stimulation resulted in an increase in E-cadherin, a decrease in N-cadherin expression and abolished cell migration and invasion.

Conclusion

ERβ is a prognostic marker of recurrence free rate in non-muscle-invasive bladder cancer, potentially through suppressing cadherin switch, and may act as a potential target for bladder cancer therapy.