Adjacent Normal Bladder Tissues Research Articles

Expression of Beclin 1 in Bladder Cancer and Its Clinical Significance

The aim of this study is to explore the expression of beclin 1, an autophagy gene, in bladder cancer and to evaluate its clinical and prognostic significance in patients with bladder cancer. Beclin 1 expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry in bladder cancer tissues and adjacent normal bladder tissues. The relationship between the expression of beclin 1 and clinicopathological characteristics and prognosis was statistically analyzed. mRNA level, protein expression and immunoreactivity of beclin 1 were decreased in bladder cancer tissues compared with adjacent normal tissues. Downregulation of beclin 1 was more frequent in tumors with higher histological grades (the expression of beclin 1 was reduced by 49.0% in G1 and G2, and by 71.8% in G3, p=0.010), and was also reduced by 69.5% in the muscle invasive type and by 51.1% in the non-muscle invasive type (p=0.04). Reduced beclin 1 expression was positively associated with higher histological grade and more advanced clinical stage (p<0.05). Kaplan-Meier survival analysis revealed that patients exhibiting lower beclin 1 expression experienced a shorter survival than those with higher expression (p=0.006). Cox proportional hazards regression analysis showed that beclin 1 protein is an independent predictor of survival (p=0.005). Beclin 1 has an influence on the progression of bladder cancer and might serve as a potential prognostic factor for patients with bladder cancer.

Tissue fatty acid composition in human urothelial carcinoma

Bladder cancer cells appear to have an altered lipid metabolism as evidenced by modulated lipogenic enzymes. The aim of this study is to investigate differences in tissue fat composition between malignant and adjacent normal urinary bladder tissue. Normal-appearing and malignant bladder tissues were collected from 31 patients with highgrade (Ta) urothelial carcinoma during transurethral resection (TUR). The fatty acid composition in the tissue was determined by gas liquid chromatography. In the bladder cancer tissue, levels of stearic acid (18:0; P=0.01) and oleic acid (18:1n-9; P=0.03) were higher, and the level of arachidonic aid (20:4n-6; P<0.001) was lower than that in the normal-appearing bladder. Overall, bladder cancer tissue showed a significant reduction in total n-6 polyunsaturated fatty acid (–15.1%; P<0.001). The change in the fatty acid composition may be regarded as an indicator of altered lipid metabolism occurring in vivo during human bladder tumourigenesis.

CIP2A is a predictor of survival and a novel therapeutic target in bladder urothelial cell carcinoma

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified human oncoprotein that stabilizes the c-MYC protein. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in urothelial cell carcinoma (UCC) of the bladder. CIP2A expression was examined in 20 fresh bladder UCC tissues and paired adjacent normal bladder tissues by RT-PCR and Western blot. Immunohistochemistry for CIP2A was performed on additional 117 bladder UCC tissues. The clinical significance of CIP2A expression was analyzed. CIP2A downregulation was performed in bladder UCC cell line T24 with high abundance of CIP2A, and the effects of CIP2A silencing on cell proliferation, migration, invasion in vitro, and tumor growth in vivo were evaluated. We found that CIP2A expression was upregulated in bladder UCC tissues relative to adjacent normal bladder tissues. Clinicopathological analysis showed that CIP2A expression was significantly associated with tumor stage (P = 0.004), histological grade (P = 0.007), and lymph node status (P = 0.001). The Kaplan-Meier survival curves revealed that CIP2A expression was associated with poor prognosis in bladder UCC patients (log-rank value = 14.704, P < 0.001). CIP2A expression was an independent prognostic marker of overall patient survival in a multivariate analysis (P = 0.015). Knockdown of the CIP2A expression reduced cell proliferation, anchorage-independent growth, migration, invasion, and tumor growth in xenograft model mice. Our findings suggest that CIP2A is an independent predictor of poor prognosis of bladder UCC patients, and inhibition of its expression might be of therapeutic significance.

Non-muscle myosin II is an independent predictor of overall survival for cystectomy candidates with early-stage bladder cancer

Non-muscle myosin heavy chain IIA (NMHC IIA) plays a significant role in tumor progression and metastasis. The aim of this study was to explore the relationship between the expression levels of NMHC IIA and the characteristics, prognosis of patients who were cystectomy candidates with early-stage bladder cancer. Real-time PCR was used to examine the expression of NMHC IIA mRNA in 16 paired bladder cancer and the adjacent normal tissues. The expression of NMHC IIA protein in 167 specimens of bladder cancer was determined by immunohistochemistry assay. Statistical analyses were performed to evaluate the association between the expression of NMHC IIA, and clinicopathological features and prognosis. Compared with adjacent normal bladder tissues, upregulated expression of NMHC IIA mRNA was observed in 81.3% of bladder cancer tissues (P=0.011). Moreover, the higher levels of NMHC IIA expression were positively correlated with the histopathological classification (P=0.021), lymph node metastasis (P=0.047) and cancer-related mortality (P=0.030). The 5-year survival rate of patients with higher NMHC IIA expression was significantly lower than that of patients with lower NMHC IIA expression (P=0.004). Furthermore, in multivariate analysis by Cox regression model, high NMHC IIA expression was confirmed to be an independent molecular marker (P=0.047), while grade (P=0.020) and clinical T stage (P=0.049) were also significant prognostic factors. Expression of NMHC IIA mRNA was higher in bladder cancer compared to the adjacent normal tissues. The detection of NMHC IIA protein expression is potentially useful in prognostic evaluation of cystectomy candidates with early-stage bladder cancer.

Integrin-linked Kinase Functions as a Tumor Promoter in Bladder Transitional Cell Carcinoma

The aim of this study was to elucidate the role of the integrin-linked kinase (ILK) gene in development of human bladder transitional cell carcinoma (BTCC). Expression of ILK protein and ILK mRNA in 56 cases of human BTCC tissue and in 30 cases of adjacent normal bladder tissue was detected by immunohistochemistry S-P and reverse transcription polymerase chain reaction (RT-PCR), respectively. Four specific miRNA RNAi vectors targeting human ILK were synthesized and transfected into BIU-87 cells by liposome to obtain stable expression cell strains. The influence of ILK on proliferation of BTCC was detected by MTT, FCM on athymic mouse tumorigenesis. The positive rate of ILK protein in BTCC tissue (53.6%) was much higher than adjacent normal bladder tissue (10.0%) (p<0.05). Similarly, expression of ILK mRNA in BTCC tissue (0.540 ± 0.083) was significantly higher than in adjacent normal bladder tissue (0.492 ± 0.070) (p<0.05). MTT showed that the proliferation ability of miRNA-ILK transfected group was clearly decreased (p<0.05), the cell cycle being arrested in G0/G1-S, an tumorigenesis in vivo was also significantly reduced (p<0.05). ILK gene transcription and protein expression may be involved in the development of BTCC, so that ILK might be the new marker for early diagnosis and the new target for gene treatment.

Feature Extraction Approach for Mass Spectrometry Imaging Data Using Non-negative Matrix Factorization

Mass spectrometry imaging (MSI) provides molecules composition information and corresponding spatial information on complex biological surfaces in a single experiment without label. It is a hotspot for getting significant amount of attention in the mass spectrometric community currently. However, the MSI data are large and complexity, which makes the reduction and feature extraction difficult. Some multivariate statistical analysis methods, for example, the famous principal component analysis (PCA), were developed to address this issue. But the results with negative value are hard to be interpreted as features about molecules. In this study, a feature extraction approach for MSI data by applying non-negative matrix factorization was developed. It could extract single molecules composition feature and corresponding distribution (basic images) feature, and further integrated the basic images to create a profile showing the whole sample by RGB (red, green and blue) color overlaid model clearly. The MSI data of a mouse brain section was used to test the efficiency of this approach. The white matter regions, the grey matter regions and the background regions were clearly observed and the corresponding molecules mass spectrums were extracted, which indicated that the approach was easier than PCA approach in results interpreting. Moreover, the MSI data of a human cancerous and adjacent normal bladder tissue sections on the same sample target were analyzed by the approach, and the cancerous regions and the normal regions were clearly differentiated. The software developed in this paper could be downloaded from the website http://www.msimaging.net.

Reduced expression of MTUS1 mRNA is correlated with poor prognosis in bladder cancer

Mitochondrial tumor suppressor 1 (MTUS1) is a newly identified candidate tumor suppressor gene. Previous studies have demonstrated that the expression status of MTUS1 is altered in several types of tumors. However, its clinical significance for bladder cancer patients remains undetermined. In this study, we detected the expression of MTUS1 mRNA in bladder tumors and paired normal samples obtained from 5 patients using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). A significant downregulation of MTUS1 mRNA expression was observed in the tumor tissues compared with the corresponding normal bladder tissue (P<0.001). We further tested the expression of MTUS1 mRNA in 55 bladder cancer tissues and 10 adjacent normal bladder tissues by quantitative real-time RT-PCR. Correlations between MTUS1 and clinicopathological features and prognosis were investigated by statistical analyses. The results showed that MTUS1 expression was correlated with tumor grade, stage, size and number (P<0.001, P<0.001, P=0.034 and P=0.029, respectively). Patients with low levels of MTUS1 mRNA expression had a poor prognosis compared with those with a high expression (P<0.001). Univariate and multivariate logistic regression prognostic analyses revealed that MTUS1 mRNA was an independent prognostic factor for disease-free survival in bladder cancer (P<0.05). In conclusion, these data suggest that MTUS1 is significant in the progression of bladder cancer and that the status of MTUS1 mRNA expression is a novel prognostic marker for predicting bladder tumor disease-free survival.

Study of the expression and function of PIWIL2 in the bladder urothelial carcinoma

Objective To investigate the gene expression of PIWIL2 in the bladder urothelial carcinoma (BTCC) and siRNA interact on PIWIL2 gene expression in human bladder cancer cell line BIU-87.Methods Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to detect the PIWIL2 expressions in tissues of BTCC (46 cases),cystitis glandularis(21 cases),adjacent non-cancerous tissues (17 cases) and normal bladder tissues (7 cases). 3 specific siRNA targeted PIWIL2 gene were synthesized after designed and transferred. After siRNA was transferred into BIU-87 cells, MTI and TUNEL methods were applied to detect the proliferation inhibitory rate (IR) and apoptosis index (AI) in BIU-87 cells,qRT-PCR and Western blot were used to examine effects of siRNA on the expressions of the PIWIL2 gene and protein,respectively.Results The expression rate of PIWIL2 mRNA in BTCC tissues was 76.08 ％(35/46) and significantly higher than those in the cystitis glandularis tissues (42.86 ％,9/21),adjacent non-cancerous tissues (41.17 ％,7/17) and normal tissues (7.14 ％,1/14) (P =0.008,P =0.010,P =0.000).The IR [(37.52±8.84) ％,(64.36±9.64)％] and (62.94±8.43) ％] and AI [(26.18±5.42) ％,(38.75±6.19) ％ and (40.02±5.64) ％] of BIU-87 cells in the siRNA 1～3 groups were respectively significantly higher than those [(1.97±0.02) ％ and (3.35±0.47) ％] in the control group(P=0.000),and expressions of PIWIL2 mRNA and protein in the siRNA groups were both lower than those in the control group. Moreover, the effects of siRNA 2 group and siRNA 3 group on inhibiting PIWIL2 expression, IR and AI of BIU-87 cells were stronger than siRNA 1 group. Conclusion The over-expression of PIWIL2 suggested that it played an important role in the mechanism of development and malignant progression of BTCC. The siRNA of transcription can significantly inhibit its expression, induce cell apoptosis and inhibit the growth of BIU-87 cells which might provide the experimental evidence for the gene targeting therapy of bladder tumor. Key words: PIWIL2; siRNA; Bladder neoplasms

Overexpression of RIN1 associates with tumor grade and progression in patients of bladder urothelial carcinoma

Ras and Rab interactor 1 (RIN1) is an effector of H-Ras, which plays an important role in the development and progression of carcinomas, but it has not been reported in bladder cancer. Hence, the association of RIN1 expression with prognosis of bladder urothelial carcinoma (UC) was examined. RIN1 mRNA and protein expression in 20 paired UCs and the adjacent normal tissues was detected by quantitative reverse transcription polymerase chain reaction and Western blot. The expression of RIN1 protein in 96 specimens of UCs and 22 specimens of adjacent normal bladder tissues were analyzed by immunohistochemistry. The overall survival (OS) was assessed by univariate and multivariate analysis. Moreover, the progression-free survival (PFS) and recurrence-free survival (RFS), classified by the clinicopathologic features with RIN1 expression, were assessed by multivariate analysis. RIN1 mRNA and protein level was higher in UCs than in the adjacent normal tissues (P < 0.01). Enhanced RIN1 immunoexpression was associated with high histologic grades (P = 0.046), cancer progression (P = 0.047) as well as Ki-67 expression (P = 0.023). Furthermore, the 5-year survival rate was 29% in the subgroup with high level of RIN1 expression, while it was 43% in the subgroup with normal level of RIN1 expression (P < 0.05). Importantly, RIN1 level was revealed as the significant independent prognostic factor for death (P = 0.023) and progression (P = 0.003), but a weak contribution for recurrence (P = 0.063). Collectively, RIN1 expression could be a potential prognostic predictor for UC patients.

Artificial Neural Networks for Classification and Identification of Data of Biological Tissue Obtained by Mass-Spectrometry Imaging

Mass-spectrometry imaging (MSI), the combination of molecular mass analysis and spatial information, providing visualization of molecules on complex biological surfaces, is currently receiving a significant amount of attention among the mass-spectrometry community. One important problem in this research field concerns the development of an effective method for the classification and identification of MSI data, especially for both differentiating a cancerous tissue from adjacent normal tissues and classifying the different functional regions in a complex biological tissue. For this purpose, we developed a new method, which involved image reconstruction from raw mass-spectral data; preprocessing of MSI data; classification of tissue regions with reference to the background regions using self-organizing feature maps; and identification of regions of special interest in whole-tissue samples by learning-vector quantization. The MSI data of samples of six pairs (12 tissue samples) of human cancerous and adjacent normal bladder tissues were used to test the efficacy of this method. The results showed an error rate of less than 23.38% for identification of cancerous regions and an error rate of less than 9.08% for identification of the adjacent normal regions. The method was also tested with reference to classification of the regions of the white matter and gray matter of three adjacent slices of mouse brain tissue. The slice in the middle was used to establish an identification model, and the other two slices were used to test the model. The inconsistency rate of the results obtained by identification using a self-organizing feature map was less than 4% compared with the results using learning-vector quantization. This indicated that the method could be carried out simply and efficiently to extend the capability of MSI and underlined its potential to be a regular tool in the studies related to clinical applications.

The expression of type III transforming growth factor-β receptor in urothelial carcinoma of the bladder and its clinical significance

Objective To investigate the expression of type Ⅲ transforming growth factor-β receptor (TGFBR3) mRNA in urothelial carcinoma of the bladder (BUC) and its clinical significance.Methods Fresh tissues of BUC and adjacent normal bladder tissues were obtained from 32 patients with BUC.TGFBR3 mRNA expression in these specimens was measured by Real-time polymerase chain reaction (PCR).Results Lower expression of TGFBR3 transcripts was detected in BUC (2.25 ± 0.65 ) than in adjacent normal tissues (6.42 ± 1.53,P ＜ 0.05 ).The expression level of TGFBR3 mRNA in stage Tis-T1,T2-T4 was 1.94 ± 0.76,2.46 ± 0.97; in patients with and without lymph nodes metastasis was 6.05 ± 2.55,1.37 ±0.42;in grade Ⅰ,Ⅱ and Ⅲ was 4.15 ± 1.69,1.96 ±0.68 and 0.90 ±0.65;in male and female group was 2.25 ± 0.72,2.26 ± 1.42.TGFBR3 mRNA level did not correlate with grade,stage and gender ( P ＞ 0.05 ).Higher level of expression was associated with lymph nodes metastasis ( P ＜ 0.05 ).Conclusion Abnormal expression of TGFBR3 plays an important role in BUC.TGFBR3 may participate in the pathogenesis and development of BUC. Key words: Urothelial carcinoma of the bladder; TGFBR3

Expression of Hedgehog Pathway Components is Associated with Bladder Cancer Progression and Clinical Outcome

Hedgehog (Hh) pathway has been implicated in the tumorigenesis of a large number of human tumors. But its effects on the progression and prognosis of bladder cancer remain poorly understood. The aim of this study was to investigate expression patterns of Hh pathway components in bladder cancer and to elucidate their prognostic values in this tumor. The expression of sonic hedgehog (Shh), its receptor Patched (Ptch1), and downstream transcription factor Gli1 in 118 specimens of bladder cancer and 30 specimens of adjacent normal bladder tissue was determined by immunohistochemistry. Statistical analyses were applied to test the relationship between the expression of these three proteins and clinicopathologic features and prognosis. Immunohistochemical staining results showed the localizations of Shh and Ptch1 proteins to be mainly located in the cytoplasm of bladder cancer cells, whereas Gli1 was mainly localized in the nuclear of tumor cells. Additionally, positive expression of Shh, Ptch1 and Gli1 proteins was correlated with pathological stage (P = 0.006, 0.006 and 0.008, respectively), venous invasion (P = 0.01, 0.01 and 0.012, respectively) and lymph node metastasis (P = 0.009, 0.01 and 0.013, respectively), but not with other factors including age, gender, tumor grade and recurrence of superficial cancer. Moreover, patients with positive expression of Shh, Ptch1 and Gli1 proteins respectively showed poorer disease-free (P = 0.002, 0.002 and 0.001, respectively) and overall survival (all P < 0.001) than those with negative expression of these three proteins. Univariate and multivariate analysis of prognostic factors in bladder cancer patients indicated that the expression patterns of Shh, Ptch1 and Gli1 proteins were independent unfavorable prognostic factors (all P < 0.001). This is the first report describing about the correlation between Hh pathway and the prognosis of bladder cancer. Expression of Shh, Ptch1 and Gli1 proteins was greater in bladder cancers than in the adjacent normal tissues. The examination of their expression is potentially valuable in prognostic evaluation of bladder cancer.

Abstract 4974: Allele-specific mRNA and protein expression on genetic variants of CCNE1 associated with risk of bladder cancer

Abstract A single nucleotide polymorphism (SNP), rs8102137, located 6 Kb upstream from the cyclin E1 gene (CCNE1) on chromosome 19q12, has recently been identified as a risk factor for bladder cancer (OR, p=1.7×10-11, Rothman et al, Nat Gen, 2010). We found a higher expression of CCNE1 mRNA in cDNA samples from bladder tumors (n=42) compared to adjacent normal bladder tissue (n=41, 3.7 fold, p=2.7×10-12). However, the expression did not correlate with genotypes of rs8102137. We also performed allelic expression imbalance (AEI) using a coding synonymous variation in the last exon (rs7257694, Ser390Ser) that is in high LD with the GWAS rs8102137 (normal bladder tissue samples, n = 41, D’=1.0, r2=0.815 and in HapMap CEU samples, n=60, D’=0.95, r2=0.68). In normal and tumor tissue samples heterozygous for both SNPs, the risk variant of rs8102137 was associated with lower expression of allele T of rs7257694 (p=2.2×10-4 in normal samples and 1.11×10-10 in tumor samples). Western blot protein analysis with bladder tissue and prostate cell lines lysates showed that this allele-specific difference in mRNA expression is likely to be related to two protein isoforms that showed a differential pattern of expression dependent on rs8102137 and rs7257694 genotypes. CCNE1 regulates cell cycle G1/S transition, and alternative protein forms may have differential functions in cell cycle regulation, thereby affecting the risk of cancer. Alternative splicing forms of CCNE1 have been cloned and their functional analysis is ongoing. In conclusion, our results suggest that bladder cancer associated genetic variants within the CCNE1 gene may affect mRNA splicing and protein structure of the gene, contributing to altered regulation of cell cycle and risk of bladder and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4974. doi:10.1158/1538-7445.AM2011-4974

Multivariate statistical identification of human bladder carcinomas using ambient ionization imaging mass spectrometry.

Diagnosis of human bladder cancer in untreated tissue sections is achieved by using imaging data from desorption electrospray ionization mass spectrometry (DESI-MS) combined with multivariate statistical analysis. We use the distinctive DESI-MS glycerophospholipid (GP) mass spectral profiles to visually characterize and formally classify twenty pairs (40 tissue samples) of human cancerous and adjacent normal bladder tissue samples. The individual ion images derived from the acquired profiles correlate with standard histological hematoxylin and eosin (H&E)-stained serial sections. The profiles allow us to classify the disease status of the tissue samples with high accuracy as judged by reference histological data. To achieve this, the data from the twenty pairs were divided into a training set and a validation set. Spectra from the tumor and normal regions of each of the tissue sections in the training set were used for orthogonal projection to latent structures (O-PLS) treated partial least-square discriminate analysis (PLS-DA). This predictive model was then validated by using the validation set and showed a 5% error rate for classification and a misclassification rate of 12%. It was also used to create synthetic images of the tissue sections showing pixel-by-pixel disease classification of the tissue and these data agreed well with the independent classification that uses histological data by a certified pathologist. This represents the first application of multivariate statistical methods for classification by ambient ionization although these methods have been applied previously to other MS imaging methods. The results are encouraging in terms of the development of a method that could be utilized in a clinical setting through visualization and diagnosis of intact tissue.

Overexpression of HMGA2 in bladder cancer and its association with clinicopathologic features and prognosis: HMGA2 as a prognostic marker of bladder cancer

PurposeTo examine HMGA2 expression and investigate its clinical and prognostic significance in human urothelial bladder cancer (BUC). MethodsWe detected HMGA2 mRNA and protein expression by quantitative reverse-transcription polymerase chain reaction and western blotting, respectively in 44 frozen bladder cancer tissues and 18 adjacent normal bladder tissues. HMGA2 protein expression was assessed by immunohistochemical analysis of 148 paraffin-embedded specimens of human BUC and 30 specimens of adjacent normal bladder tissue. Correlations between HMGA2 and clinicopathologic features and prognosis were tested by statistical analyses. ResultsHMGA2 mRNA and protein levels in bladder cancer samples were significantly increased compared with adjacent normal bladder tissues (P < 0.001). mRNA overexpression correlated with high stage and grade of the bladder cancer (P < 0.001 and P = 0.002 respectively). HMGA2 protein expression was negative in all normal urothelial tissue samples, but positive in 52% (77/148) of bladder cancers (P < 0.001). HMGA2 expression correlated with tumor grade and stage (P < 0.001 and P = 0.003 respectively), Overexpression of HMGA2 protein in non-muscle-invasive bladder cancer was significantly associated with shorter recurrence-free survival (P < 0.001), and progression-free survival (P = 0.0004). Multivariate analysis showed that HMGA2 expression was an independent prognostic factor for both tumor recurrence (P < 0.001) and tumor progression (P = 0.006). ConclusionsHMGA2 is up-regulated in bladder cancer at both the transcriptional and translational levels compared with normal bladder tissue, HMGA2 protein is thus a potential prognostic marker for predicting tumor recurrence and progression.

Tumor-specific upregulation of human leukocyte antigen–G expression in bladder transitional cell carcinoma

Human leukocyte antigen–G (HLA-G) is a potent immunosuppressive molecule that induces functional silencing of both innate and adaptive immune responses. The relevance of the aberrant HLA-G expression in malignant contexts has been intensively investigated. However, its expression status and clinical significance in bladder cancer remain to be elucidated. In the current study, HLA-G expression in 75 primary bladder transitional cell carcinoma (TCC) lesions was analyzed with immunohistochemistry, and relationship between HLA-G expression and clinical parameters, including disease stage was evaluated. Plasma soluble HLA-G levels were analyzed in 15 TCC patients and 109 normal controls. Data revealed that HLA-G was expressed in 68.0% (51/75) primary TCC lesions, whereas it was undetectable in adjacent normal bladder tissues. The proportion of HLA-G expression in TCC samples varied from negative to 100%, and no significant association was observed for the HLA-G expression status with the patient age, gender, and disease stage. Furthermore, no significance for sHLA-G levels was observed between the TCC patients and normal controls (median 10.75 vs 8.69 U/ml, p = 0.578). Given its immunotolerant properties, our finding suggested that lesion HLA-G expression upregulated in bladder TCC lesions might be an additional mechanism for tumor cells evading from host immunosurveillance; however, its clinical relevance needs further investigation.

Expression of Bmi-1 is a prognostic marker in bladder cancer

BackgroundThe molecular mechanisms of the development and progression of bladder cancer are poorly understood. The objective of this study was to analyze the expression of Bmi-1 protein and its clinical significance in human bladder cancer.MethodsWe examined the expression of Bmi-1 mRNA and Bmi-1 protein by RT-PCR and Western blot, respectively in 14 paired bladder cancers and the adjacent normal tissues. The expression of Bmi-1 protein in 137 specimens of bladder cancer and 30 specimens of adjacent normal bladder tissue was determined by immunohistochemistry. Statistical analyses were applied to test the relationship between expression of Bmi-1, and clinicopathologic features and prognosis.ResultsExpression of Bmi-1 mRNA and protein was higher in bladder cancers than in the adjacent normal tissues in 14 paired samples (P < 0.01). By immunohistochemical examination, five of 30 adjacent normal bladder specimens (16.7%) versus 75 of 137 bladder cancers (54.3%) showed Bmi-1 protein expression (P < 0.05). Bmi-1 protein expression was intense in 20.6%, 54.3%, and 78.8% of tumors of histopathological stages G1, G2, and G3, respectively (P < 0.05). Expression of Bmi-1 protein was greater in invasive bladder cancers than in superficial bladder cancers (81.5% versus 32.5%, P < 0.05). In invasive bladder cancers, the expression of Bmi-1 protein in progression-free cancers was similar to that of cancers that have progressed (80.0% versus 82.4%, P > 0.5). In superficial bladder cancers, the expression of Bmi-1 protein in recurrent cases was higher than in recurrence-free cases (62.5% versus 13.7%, P < 0.05). Bmi-1 expression was positively correlated with tumor classification and TNM stage (P < 0.05), but not with tumor number (P > 0.05). Five-year survival in the group with higher Bmi-1 expression was 50.8%, while it was 78.5% in the group with lower Bmi-1 expression (P < 0.05). Patients with higher Bmi-1 expression had shorter survival time, whereas patients with lower Bmi-1 expression had longer survival time (P < 0.05).ConclusionExpression of Bmi-1 was greater in bladder cancers than in the adjacent normal tissues. The examination of Bmi-1 protein expression is potentially valuable in prognostic evaluation of bladder cancer.

Overexpression of EIF-5A2 Is an Independent Predictor of Outcome in Patients of Urothelial Carcinoma of the Bladder Treated with Radical Cystectomy

Our previous study has suggested an oncogenic role of eIF-5A2 in ovarian tumorigenesis. Abnormalities of eIF-5A2 and its clinical/prognostic significance, however, in urothelial carcinoma of the bladder (UC) are unclear. In this study, the methods of reverse transcription-PCR, immunohistochemistry, and fluorescence in situ hybridization were used to examine mRNA/protein expression and amplification of eIF-5A2 in a large cohort of UCs treated with radical cystectomy. Up-regulated expression of eIF-5A2 mRNA was observed in 50% (8 of 16) of UCs, when compared with adjacent normal bladder tissues. Overexpression of EIF-5A2 protein and amplification of eIF-5A2 was examined informatively in 45.3% (39 of 86) and 10.6% (5 of 47) of UCs, respectively. In univariate survival analysis of the UC cohorts, a significant association of overexpression of EIF-5A2 with shortened patient survival (mean, 38.2 months versus 52.9 months, P = 0.001, log-rank test) was shown. In different subsets of UC patients, overexpression of EIF-5A2 was also a prognostic indicator in grade 1/2 (P = 0.0009) and grade 3 (P = 0.016) tumor patients, and in pT1 (P = 0.0089), pT2 (P = 0.0354), pT3/4 (P = 0.0058), pN0 (P = 0.0039), and pN1-2 (P = 0.0093) tumor patients. Importantly, EIF-5A2 expression (P = 0.0007) together with pT stage (P = 0.0001) provided significant independent prognostic variables in multivariate analysis. These findings indicate that overexpression of EIF-5A2 in UCs is coincident with acquisition of a poor prognostic phenotype, suggesting that the expression of EIF-5A2, as detected by immunohistochemistry, is an independent molecular marker for shortened survival time of UC patients treated with radical cystectomy.

Proteomic‐based approach for biomarkers discovery in early detection of invasive urothelial carcinoma

Identification of reliable non-invasive markers for the detection of invasive phenotype of urothelial carcinoma is needed. This study characterizes and compares protein expression profiles of adjacent non-neoplastic urothelium and invasive urothelial carcinoma to identify biomarkers for early detection of de novo bladder cancer. Differences in protein expression between adjacent non-neoplastic and high-grade, stage T4, grade 3 invasive urothelial carcinoma tissues were investigated using 2-DE, MALDI-TOF-MS, and data processing. Ingenuity Pathway Analysis (IPA) was applied to examine the biological mechanisms represented by the altered proteins. The 2-DE of the adjacent non-neoplastic urothelium and invasive urothelial carcinoma showed reproducibly similar proteomic mapping for each group distinguishing adjacent non-neoplastic urothelium from invasive urothelial carcinoma. Twenty-one proteins were altered in expression and one of these proteins, Choroideremia-like protein (CHML) was significantly overexpressed (p<0.005) and therefore was analyzed further using IHC and Western blot. Urothelial carcinoma presented an elevated expression of CHML but not adjacent non-neoplastic or normal bladder tissues. IPA revealed the involvement of CHML in cell morphology, cellular assembly, and organization. Further investigation is warranted to elucidate the biological significance of CHML and to validate its role as a biomarker for early detection of invasive urothelial carcinoma de novo.

Aurora-A/STK15/BTAK enhances chromosomal instability in bladder cancer cells

Chromosomal aneuploidy is associated with invasive bladder cancer and one of the genes implicated in these changes is Aurora-A/STK15/BTAK, that is localized on chromosome 20q13 and encodes a centrosome-associated serine/threonine kinase. To better understand the association between Aurora-A/STK15 expression, tumor aneuploidy and clinical prognosis, we sought to determine whether overexpression of Aurora-A/STK15 in cultured urothelial cells facilitated chromosomal instability. Using immunofluorescence staining, Northern and Western blot analyses, we verified that overexpression of Aurora-A/STK15 in bladder tumor cell lines enhanced chromosomal instability. Additionally, we observed that some bladder tumor cell lines expressed more Aurora-A/STK15 than cultured normal urothelial cells and that Aurora-A/STK15 expression was higher in an immortalized E7 urothelial cell line having 20q amplification than in an E6 line lacking 20q amplification. These results were consistent with our observations of higher mRNA levels in some T3 invasive bladder tumors than in T1 superficial tumors and adjacent normal bladder tissue. Overall our results suggest that overexpression of Aurora-A/STK15 in bladder tumor cells contributes to tumor progression by promoting chromosomal instability leading to aneuploidy.