Normal Urothelium Research Articles

Intravesical bacillus Calmette-Guérin Efficiently Reduces p70S6K1 but Not 4E-BP1 Phosphorylation in Nonmuscle Invasive Bladder Cancer

We characterized the functional consequences of intravesical bacillus Calmette-Guérin on the molecular mechanism of the AKT/mTOR signaling pathway in nonmuscle invasive bladder cancer. To our knowledge this has not been reported previously. At age 7 weeks female Fischer 344 rats received 1.5 mg/kg MNU intravesically every other week for 6 weeks. They were randomized at 10 per group to MNU (0.2 ml vehicle), bacillus Calmette-Guérin (10(6) cfu Connaught strain), rapamycin (15 μg/ml) and bacillus Calmette-Guérin plus simultaneous rapamycin, each intravesically for 6 weeks. At week 15 the bladders were collected for histopathology, immunohistochemistry and immunoblot to determine p-AKT, Rictor, Raptor, p-4E-BP1, p-p70S6K1, p-AMPK-α, p-mTOR and p-p53. Papillary carcinoma (pTa) and high grade intraepithelial neoplasia (pTis) predominated in the MNU group while normal urothelium, papillary and flat hyperplasia were more common in treated groups. Nonmuscle invasive bladder cancer treated with bacillus Calmette-Guérin showed suppression of p70S6K1 but not 4E-BP1 phosphorylation. This suggests that 4E-BP1 is regulated differently than p70S6K1, escaping the bacillus Calmette-Guérin action that occurs in a mTOR independent manner. The association of bacillus Calmette-Guérin with rapamycin but not rapamycin monotherapy affected p70S6K1 and 4E-BP1 phosphorylation with no features of in situ carcinoma (pTis). The activation status of p70S6K1 and 4E-BP1 might be used to stratify patients who could benefit from targeting such molecular elements with multitarget/multidrug intravesical therapy. In the future 4E-BP1 might be a worthwhile new target for bacillus Calmette-Guérin refractory nonmuscle invasive bladder cancer.

Bladder cancer: ESMO Practice Guidelines for diagnosis, treatment and follow-up

Bladder cancer: ESMO Practice Guidelines for diagnosis, treatment and follow-up

Canonical Notch signalling is inactive in urothelial carcinoma

BackgroundNotch signalling regulates cell fate in most tissues, promoting precursor cell proliferation in some, but differentiation in others. Accordingly, downregulation or overactivity variously contributes to cancer development. So far, little is known about Notch pathway activity and function in the normal urothelium and in urothelial carcinoma (UC). We have therefore investigated expression of Notch pathway components in UC tissues and cell lines and studied the function of one receptor, NOTCH1, in detail.MethodsExpression of canonical Notch pathway components were studied in UC and normal bladder tissues by immunohistochemistry and quantitative RT-PCR and in UC cell lines and normal cultured urothelial cells by qRT-PCR, immunocytochemistry and Western blotting. Pathway activity was measured by reporter gene assays. Its influence on cell proliferation was investigated by γ-secretase inhibition. Effects of NOTCH1 restoration were followed by measuring cell cycle distribution, proliferation, clonogenicity and nuclear morphology.ResultsNOTCH1 and its ligand, DLL1, were expressed at plasma membranes and in the cytoplasm of cells in the upper normal urothelium layer, but became downregulated in UC tissues, especially in high-stage tumours. In addition, the proteins were often delocalized intracellularly. According differences were observed in UC cell lines compared to normal urothelial cells. Canonical Notch pathway activity in reporter assays was repressed in UC cell lines compared to normal cells and a mammary carcinoma cell line, but was induced by transfected NOTCH1. Inhibitors of Notch signalling acting at the γ-secretase step did not affect UC cell proliferation at concentrations efficacious against a cell line with known Notch activity. Surprisingly, overexpression of NOTCH1 into UC cell lines did not significantly affect short-term cell proliferation, but induced nuclear abnormalities and diminished clonogenicity.ConclusionOur data indicate that canonical Notch signalling is suppressed in urothelial carcinoma mainly through downregulation of NOTCH1. These findings can be explained by proposing that canonical Notch signalling may promote differentiation in the urothelium, like in many squamous epithelia, and its suppression may therefore be advantageous for tumour progression. As an important corollary, inhibition of canonical Notch signalling is unlikely to be efficacious and might be counter-productive in the treatment of urothelial carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-628) contains supplementary material, which is available to authorized users.

The differential expression of EphB2 and EphB4 receptor kinases in normal bladder and in transitional cell carcinoma of the bladder.

Effective treatment of transitional cell carcinoma (TCC) of the bladder requires early diagnosis. Identifying novel molecular markers in TCC would guide the development of diagnostic and therapeutic targets. Ephrins mediate signals via tyrosine kinase activity that modulates diverse physiologic and developmental processes, and ephrins are increasingly implicated in carcinogenesis. The aim of our study was to examine the differential regulation of EphB4 and EphB2 in normal bladder and in TCC of the bladder in 40 patients undergoing radical cystectomy for curative intent. Immunostaining and Western blotting revealed that normal urothelium expresses EphB2 (20 of 24 cases, 83% of the time) not EphB4 (0 of 24 cases, 0%). In sharp contrast, TCC specimens show loss of EphB2 expression (0 of 34 cases, 0%) and gain of EphB4 expression (32 of 34, 94%). Furthermore, EphB4 signal strength statistically correlated with higher tumor stage, and trended toward the presence of carcinoma in situ (CIS). These results are confirmed by analysis of normal urothelial and tumor cell lines. EphB2 is not a survival factor in normal urothelium, while EphB4 is a survival factor in TCC. Treatment of bladder tumor xenograft with an EphB4 inhibitor sEphB4-HSA leads to 62% tumor regression and complete remission when combined with Bevacizumab. Furthermore, tissue analysis revealed that sEphB4-HSA led to increased apoptosis, decreased proliferation, and reduced vessel density, implicating direct tumor cell targeting as well as anti-angiogenesis effect. In summary loss of EphB2 and gain of EphB4 expression represents an inflection point in the development, growth and possibly progression of TCC. Therapeutic compounds targeting EphB4 have potential for diagnosing and treating TCC.

Molecular ultrastructure of the urothelial surface: insights from a combination of various microscopic techniques.

The urothelium forms the blood-urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno-TEM), and freeze-fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno-FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues.

The role of PTEN tumor suppressor pathway staining in carcinoma in situ of the bladder

ObjectivesThe PI3k/Akt pathway has been associated with the development and progression of bladder tumors, with most studies focused on papillary or muscle-invasive tumors. We sought to characterize the expression patterns of the PI3K/Akt pathway in a large cohort of high-risk preinvasive carcinoma in situ (CIS) tumors of the bladder. Our goal was to understand whether PI3K/Akt pathway alterations associated with CIS resemble early- or late-stage bladder cancers. Material and methodsWe evaluated tissue specimens from 97 patients with CIS of the bladder, of which 14 had a concomitant papillary tumor. All patients were treated with intravesical bacillus Calmette-Guerin. All specimens were evaluated for PTEN, p-AKT, and p-S6 immunoreactivity. Markers were evaluated for percentage and intensity of staining and were scored using a 0 to 3+grading system. ResultsPTEN staining was noted as least intense in 67% of tumor specimens and 22% of normal urothelium. P-Akt and p-S6 had intense staining in 77% and 90% of tumor specimens vs. 44% and 68% in normal tissue, respectively. Low-intensity staining for PTEN at 12 months correlated with higher recurrence risk (P = 0.026). ConclusionWe describe a large cohort of CIS bladder tumors with decreased staining intensity of PTEN and increased staining intensity of p-AKT and p-S6, similar to high-grade and high-stage papillary tumors. Low-intensity staining of PTEN at 12 months was associated with an increased risk of recurrence.

The clinical significance of intra-operative ureteral frozen section analysis at radical cystectomy for urothelial carcinoma of the bladder.

To evaluate the clinical significance of intra-operative ureteral frozen section analysis (FSA) at the time of radical cystectomy. A total of 402 patients underwent radical cystectomy for urothelial carcinoma of the bladder at our institution from January 1991 to December 2011. Except for 35 ureters who underwent nephroureterectomy, 769 ureters were finally identified. Among these, FSA was performed at 645 ureters (83.8 %). If the first FSA result was positive for malignancy (carcinoma in situ or carcinoma) or demonstrated atypia or dysplasia, sequential ureteral resection was undertaken until normal urothelium was identified, when possible. A total of 54 ureters (8.4 %) of 46 patients (11.2 %) had ureteral involvement (positive) on the first FSA. On permanent section analysis (PSA), ureteral involvement was noted in 40 ureters (6.2 %) of 35 patients (8.7 %). The sensitivity, specificity, and accuracy of FSA were approximately 75, 96, and 95 %, respectively. Intramural tumor invasion was correlated with ureteral involvement on both FSA and PSA (p < 0.05). There was no correlation between upper urinary tract (UUT) recurrence and ureteral involvement on FSA and PSA. Positive FSA and PSA were not risk factors associated with overall and cancer-specific survival (CSS) on multivariate analysis. Intra-operative FSA can reasonably detect ureteral involvement. However, a relatively high false-positive rate (44 %) may be problematic and has the likelihood to overestimate disease at the ureteral margin. Overall, routine FSA of the ureters shows no correlation with UUT recurrence as well as overall and CSS.

NOV is upregulated and promotes migration and invasion in bladder cancer

NOV is pro-tumourigenic via epithelial-mesenchymal transition (EMT) in several malignancies but is not studied in bladder cancer (BCa). Whether NOV is responsible for bladder carcinogenesis and the underlying mechanism is unclear. Using immunohistochemical staining, we quantified expressions of NOV, pS6, Vimentin and E-cadherin in 66 bladder cancer and 10 normal bladder urothelium samples. EMT was profiled by EMT index (EMTi) calculated as the ratio of Vimentin to E-cadherin. In vitro and in vivo studies were carried out to profile the role of NOV in the tumourigenesis of BCa. NOV was upregulated in bladder cancer compared to normal tissue, and its expression was correlated to pS6 and EMTi. Expression of NOV was higher in recurrent and multiple tumours and was increased with progression of tumour grade. NOV expression was also higher in BCa cell lines. Silence of NOV attenuated EMT, decreased invasion and migration of BCa cells. Silence of NOV also inhibited xenograft tumour growth and decreased tumour EMT. NOV is pro-tumourigenic in bladder cancer especially in nonmuscle-invasive entities (NMIBC). NOV may promote carcinogenesis via promotion of EMT and association with increased mTOR activity.

MP39-01 CALCIUM MEDIATED AUTOPHAGY INDUCTION PRESERVE CELL SURVIVAL DURING ENDOPLASMIC RETICULUM STRESS IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2014MP39-01 CALCIUM MEDIATED AUTOPHAGY INDUCTION PRESERVE CELL SURVIVAL DURING ENDOPLASMIC RETICULUM STRESS IN BLADDER CANCER Rani Ojha, Shrawan Singh, Arup Mandal, Shalmoli Bhattacharyya, and Vivekanand Jha Rani OjhaRani Ojha More articles by this author , Shrawan SinghShrawan Singh More articles by this author , Arup MandalArup Mandal More articles by this author , Shalmoli BhattacharyyaShalmoli Bhattacharyya More articles by this author , and Vivekanand JhaVivekanand Jha More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1316AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Anticancer agents have been shown to induce endoplasmic reticulum (ER) stress and autophagy in various cancer cells. Autophagy acts as an adaptive mechanism for cancer cell survival. However, the mechanism and the effect of ER stress induced autophagy in urothelial cancer cell survival is not well understood. METHODS Materials consisted of high and low grade urothelial cancer (UC) tissue and normal urothelium. ER stress was induced by tunicamycin and thapsigargin and measured by expression of various ER stress marker i.e. Grp-78, Ero-1Lα, PDI, IRE1α and PERK. ER stress induction was also validated by the measurement of Ca+2, IP3 and DAG assay. Autophagy was studied by lysotracker and LC3 immuno-staining. Expression of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) was also studied for determination of autophagy. ER stress induced autophagy on cell survival was studied in UC derived primary culture cells and T24 bladder cancer cell line, by the measurement of cell viability, mitochondrial membrane potential, and caspases activation. RESULTS Treatment of tunicamycin and thapsigargin induced the expression of ER stress marker at six hours and increased up-to 12 hours in UC cells as well as in normal urothelium (Fig.1A). There was increased Ca+2 flux, IP3 and DAG activity. Autophagosomes also increased in number, more so in high grade UC (Fig.1B). The expression of phophorylated-AMPK increased and the expression of phosphorylated-mTOR decreased. Addition of autophagy inhibitor, wortmannin (Wm) along with tunicamycin or thapsigargin induced cell death in both grade of UC (Fig.2A). Treatment of calcium chelater (EGTA) or pan caspase inhibitor, z-VAD-fmk blocked the cell death induced by Wm (Fig.1C). There was increased caspase-12, -9 and -3 expression (Fig.1D). CONCLUSIONS Ca+2 serves as a potent inducer of autophagy during ER stress in UC as well as in normal urothelium. Ca+2 mediated autophagy is regulated by IP3-AMPK-mTOR pathways and inhibition of Ca+2 mediated autophagy leads to caspase mediated intrinsic apoptotic cell death, suggesting that autophagy plays important roles in cell survival. Further elucidation of the molecular mechanisms linking ER stress to autophagy is needed for therapeutic intervention in urothelial carcinoma. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e426 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Rani Ojha More articles by this author Shrawan Singh More articles by this author Arup Mandal More articles by this author Shalmoli Bhattacharyya More articles by this author Vivekanand Jha More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Redistribution of calcium‐independent phospholipase A2 isoforms in IC/PBS urothelial cells (488.2)

Interstitial cystitis/painful bladder syndrome (IC/PBS) is associated with impairment of urothelial barrier function, activated mast cells and neurogenic inflammation. We have demonstrated that urothelial cell calcium‐independent PLA2 (iPLA2) is activated by tryptase, resulting in platelet‐activating factor (PAF) production. We used immortalized urothelial cells isolated from normal and IC/PBS patients to determine whether there are differences in PAF production. We identified the two predominant mammalian iPLA2 isoforms, iPLA2β and iPLA2γ, in urothelial cells. IC/PBS cells demonstrated a decrease in iPLA2γ and an increase in iPLA2β immunoprotein when compared to normal cells. Selective inhibition of iPLA2 isoforms demonstrated that the majority of iPLA2 activity in normal urothelium represents iPLA2γ whereas that in IC/PBS urothelium is iPLA2β. Greater PAF production and PMN adherence was observed in tryptase‐stimulated IC/PBS when compared to normal urothelial cells. both normal and IC/PBS urothelial cells. Increased PAF production and PMN adherence was completely blocked when tryptase‐stimulated cells were pretreated with (S)‐BEL to inhibit iPLA2β. PMN adherence was blocked when PMN were pretreated with ginkgolide B to block the PAF receptor. A redistribution of iPLA2 isoforms in IC/PBS may represent a novel therapeutic target to manage IC/PBS.Grant Funding Source: Supported by DK 66119

OP1-03 INTRAVESICAL BACILLUS CALMETTE-GUERIN (BCG) EFFICIENTLY TACKLES S6K1 BUT NOT 4E-BP1 PHOSPHORYLATION IN NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyOutstanding Posters: Research1 Apr 2014OP1-03 INTRAVESICAL BACILLUS CALMETTE-GUERIN (BCG) EFFICIENTLY TACKLES S6K1 BUT NOT 4E-BP1 PHOSPHORYLATION IN NON-MUSCLE INVASIVE BLADDER CANCER Karen Ferrari, Juliana A. de Camargo, Guilherme Z. Rocha, José B.C. Carvalheira, Athanase Billis, Mário J.A. Saad, and Leonardo O. Reis Karen FerrariKaren Ferrari More articles by this author , Juliana A. de CamargoJuliana A. de Camargo More articles by this author , Guilherme Z. RochaGuilherme Z. Rocha More articles by this author , José B.C. CarvalheiraJosé B.C. Carvalheira More articles by this author , Athanase BillisAthanase Billis More articles by this author , Mário J.A. SaadMário J.A. Saad More articles by this author , and Leonardo O. ReisLeonardo O. Reis More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.611AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bacillus Calmette-Guérin (BCG) is the gold standard treatment of non-muscle invasive bladder cancer (NMIBC). We aimed to characterize the functional consequences of intravesical BCG on the molecular mechanism of the PI3K/AKT/mTOR signaling pathway in NMIBC, never reported to date. METHODS Female (Fischer 344) rats, 6 weeks old, received 1.5 mg/kg MNU (N-methyl-n-nitrosourea) intravesically every other week for 6 weeks and were randomly divided into 2 groups: - MNU tumor group (n=10) received 0.3 ml saline and - BCG treatment group (n=10) received 106 cfu of BCG intravesically for 6 weeks. At week 15 all bladders were collected for histopathology, immunohistochemistry, proliferation index (Ki-67), and immunoblotting (pAKT, pAMPK, pp53, pp70S6K, RICTOR, RAPTOR, mTOR, p4EBP1). Student’s t-test was used to compare groups. Two tailed p<0.05 was considered statistically significant. RESULTS Papillary carcinoma (pTa) and high-grade intraepithelial neoplasia (carcinoma in situ) predominated in the MNU group while normal urothelium, papillary and flat hyperplasia were more common in BCG treatment. NMIBC treated with BCG exhibited suppression of S6K1 (Figure 1 B) but not 4E-BP1 phosphorylation (Figure 1 A), suggesting that 4E-BP1 is regulated differently that S6K1, escaping the BCG action. The cellular proliferation index decreased in the BCG treatment compared to MNU (Figure 1 C). Table 1 shows the mammalian target of rapamycin pathway phosphorylation status of BCG treatment compared to MNU. CONCLUSIONS 4E-BP1 might be a worthwhile new target for BCG refractory NMIBC. In the future, activation status of S6K1 and 4E-BP1 might stratify patients that could benefit from targeting such molecular elements. This is the first study to explore the mammalian target of rapamycin pathway in the intravesical BCG treatment of NMIBC. Table 1. Mammalian target of rapamycin pathway phosphorylation status of BCG treatment compared to MNU Immunoblotting BCG/MNU (%) p values mTOR 66.84 0.5299 pAKT 56.74 0.3278 pAMPK 121.33 0.6399 pp53 73.40 0.5166 RICTOR 80.28 0.6448 RAPTOR 88.62 0.8867 p4EBP1 140.16 0.5069 pp70S6K 17.08 0.0034 © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e162-e163 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Karen Ferrari More articles by this author Juliana A. de Camargo More articles by this author Guilherme Z. Rocha More articles by this author José B.C. Carvalheira More articles by this author Athanase Billis More articles by this author Mário J.A. Saad More articles by this author Leonardo O. Reis More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP77-11 ENDOTHELIAL EXPRESSION OF ENDOGLIN IN UPPER URINARY TRACT UROTHELIAL CARCINOMA IS ASSOCIATED WITH INTRAVESICAL RECURRENCE AFTER RADICAL NEPHROURETERECTOMY

You have accessJournal of UrologyBladder Cancer: Upper Tract Tumors II1 Apr 2014MP77-11 ENDOTHELIAL EXPRESSION OF ENDOGLIN IN UPPER URINARY TRACT UROTHELIAL CARCINOMA IS ASSOCIATED WITH INTRAVESICAL RECURRENCE AFTER RADICAL NEPHROURETERECTOMY Kazutoshi Fujita, Motohide Uemura, Akira Nagahara, Yasutomo Nakai, Go Tanigawa, Masahiro Nakagawa, Ryoichi Imamura, Wataru Nakata, Mototaka Sato, Koki Shimazu, Hiroaki Fushimi, Seiji Yamaguchi, and Norio Nonomura Kazutoshi FujitaKazutoshi Fujita More articles by this author , Motohide UemuraMotohide Uemura More articles by this author , Akira NagaharaAkira Nagahara More articles by this author , Yasutomo NakaiYasutomo Nakai More articles by this author , Go TanigawaGo Tanigawa More articles by this author , Masahiro NakagawaMasahiro Nakagawa More articles by this author , Ryoichi ImamuraRyoichi Imamura More articles by this author , Wataru NakataWataru Nakata More articles by this author , Mototaka SatoMototaka Sato More articles by this author , Koki ShimazuKoki Shimazu More articles by this author , Hiroaki FushimiHiroaki Fushimi More articles by this author , Seiji YamaguchiSeiji Yamaguchi More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2474AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Endoglin (CD105) is a type I homodimeric integral trasnmenbrane glycoprotein and accessory TGF-beta receptor. Endoglin is highly expressed on endothelial cells during tumor angiogenesis and inflammation. Endoglin levels in serum or urine have been reported as biomarkers in several cancers. Anti-endoglin therapy was also developed and now under the clinical trial. In the current study, we evaluated the expression status and prognostic significance of endoglin in upper urinary tract urothelial carcinoma (UTUC). METHODS Archival fomain-fixed and paraffin-embedded tissues from 99 primary UTUCs were retrieved. Tissued microarrays were constructed with triplicate tumor samples and paired nonneoplastic urothelium. Tissue microarrays were analyzed using immunohistochemistry for endoglin; association with clinicopathological parameters and outcome was studied. RESULTS We found that endoglin was expressed in the endothelium and significantly highly in UTUC compared with paired benign urothelium (P<0.05). Endoglin expression was not associated with pathological T stage or tumor grade. Whereas endoglin expression was not associated with increased hazard ratios for cancer-specific mortality or tumr recurrence in lymph node or distant metastasis, endolign expression was associated with intravesical recurrence, when adjusting for relevant clinicopathologic variables (P <0.05). CONCLUSIONS Upregulation of endolglin was observed in the endothelium of UTUC compared with normal urothelium, and associated with intravesical recurrence. The endoglin could be a biomarker to predict the intravesical recurrence as well as the therapeutic target. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e915-e916 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kazutoshi Fujita More articles by this author Motohide Uemura More articles by this author Akira Nagahara More articles by this author Yasutomo Nakai More articles by this author Go Tanigawa More articles by this author Masahiro Nakagawa More articles by this author Ryoichi Imamura More articles by this author Wataru Nakata More articles by this author Mototaka Sato More articles by this author Koki Shimazu More articles by this author Hiroaki Fushimi More articles by this author Seiji Yamaguchi More articles by this author Norio Nonomura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Altered Expression of Transmembrane Mucins, MUC1 and MUC4, in Bladder Cancer: Pathological Implications in Diagnosis

PurposeRadical changes in both expression and glycosylation pattern of transmembrane mucins have been observed in various malignancies. We and others have shown that MUC1 and MUC4, two transmembrane mucins, play a sentinel role in cell signaling events that drive several epithelial malignancies. In the present study, we investigated the expression profile of MUC1 and MUC4 in the non-neoplastic bladder urothelium, in various malignant neoplasms of bladder and in bladder carcinoma cell lines.Material and MethodsImmunohistochemistry was performed on tissue sections from the urinary bladder biopsies, resection samples and tissue microarrays (TMAs) with monoclonal antibodies specific for MUC1 and MUC4. We also investigated their expression in bladder carcinoma cell lines by RT-PCR and immunoblotting.ResultsMUC1 is expressed on the apical surface or in umbrella cells of the normal non-neoplastic bladder urothelium. Strong expression of MUC1 was also observed in urothelial carcinoma (UC). MUC1 staining increased from normal urothelium (n = 27, 0.35±0.12) to urothelial carcinoma (UC, n = 323, H-score, 2.4±0.22, p≤0.0001). In contrast to MUC1, MUC4 was expressed in all the layers of non-neoplastic bladder urothelium (n = 14, 2.5±0.28), both in the cell membrane and cytoplasm. In comparison to non-neoplastic urothelium, the loss of MUC4 expression was observed during urothelial carcinoma (n = 211, 0.56±0.06). However, re-expression of MUC4 was observed in a subset of metastatic cases of urothelial carcinoma (mean H-score 0.734±0.9).ConclusionThe expression of MUC1 is increased while that of MUC4 decreased in UC compared to the normal non-neoplastic urothelium. Expression of both MUC1 and MUC4, however, are significantly higher in urothelial carcinoma metastatic cases compared to localized UC. These results suggest differential expression of MUC1 and MUC4 during development and progression of bladder carcinoma.

Reduced membranous MET expression is linked to bladder cancer progression

The MET protein is involved in the malignant progression of different tumors. This study aimed to analyze the relationship of MET expression with tumor phenotype and clinical outcome in bladder cancer and the role of gene amplification for MET overexpression. A bladder cancer tissue microarray containing 686 bladder cancers was analyzed by immunohistochemistry and by fluorescence in situ hybridization. MET immunostaining was seen in normal urothelium and was recorded in 459 of 560 analyzable urothelial carcinomas (82.0%). Low MET staining was associated with a more unfavorable tumor phenotype. MET staining was seen in 89.8% of 266 pTa, 81.1% of 132 pT1, and 69.4% of 160 pT2-4 cancers (P < 0.0001). MET staining was detectable in 92.4% of 66 grade 1, 85.6% of 257 grade 2, and 75.1% of 237 grade 3 cancers (P = 0.001). MET expression status was not associated with overall or tumor-specific survival in muscle-invasive cancers (pT2-4), tumor progression in pT1 cancers, or recurrences in pTa tumors. Only four of the analyzed tumors (0.8%) showed amplification of the MET gene. We conclude that MET is not overexpressed in urothelial cancer but rather downregulated in a fraction of cancers. Accordingly, rare amplification of the genomic area including the MET gene was not associated with MET protein overexpression.

A Cell of Origin Gene Signature Indicates Human Bladder Cancer Has Distinct Cellular Progenitors

There are two distinct forms of urothelial (bladder) cancer: muscle-invasive (MI) and nonmuscle invasive (NMI) disease. Since it is currently believed that bladder cancer arises by transformation of urothelial cells of the basal layer, bladder cancer stem cells (CSCs) have been isolated based on expression markers found in such cells. However, these CSCs have only been identified in MI tumors raising the intriguing hypothesis that NMI tumor progenitors do not arise from the basal compartment. To test this hypothesis, we carried out genome-wide expression profiling of laser capture microdissected basal and umbrella cells, the two most histologically distinct cell types in normal urothelium and developed a cell of origin (COO) gene signature that distinguishes these. The COO signature was a better predictor of stage and survival than other bladder, generic, or breast CSC signatures and bladder cell differentiation markers in multiple patient cohorts. To assess whether NMI and MI tumors arise from a distinct progenitor cell (DPC) or common progenitor cell, we developed a novel statistical framework that predicts COO score as a function of known genetic alterations (TP53, HRAS, KDM6A, and FGFR3) that drive either MI or NMI bladder cancer and compared this to the observed COO score of the tumor. Analysis of 874 patients in five cohorts established the DPC model as the best fit to the available data. This observation supports distinct progenitor cells in NMI and MI tumors and provides a paradigm shift in our understanding of bladder cancer biology that has significant diagnostic and therapeutic implications.

Expression and prognostic significance of cancer stem cell markers CD24 and CD44 in urothelial bladder cancer xenografts and patients undergoing radical cystectomy

ObjectivesTo evaluate CD24/CD44/CD47 cancer stem cell marker expressions in bladder cancer (BCa) and provide data on their prognostic significance for clinical outcome in patients undergoing radical cystectomy (RC). Material and methodsPrimary BCa tissue was used for xenograft studies. A tissue microarray was prepared using specimens from a cohort of 132 patients. All patients underwent RC for urothelial BCa between 2001 and 2010. Expression of CD24, CD44, and CD47 was examined in primary samples and xenografts by fluorescence-activated cell sorting. Populations of CD24low- and CD24high- expressing cells were sorted and evaluated for tumorigenicity in vivo. Tissue microarray was analyzed for CD24/CD44 staining intensity and tumor-specific vs. stromal cell staining. Associations with BCa survival, BCa stage, and lymph node status were evaluated by univariate and multivariate analyses. ResultsCD24 and CD44/CD47 expressions mark distinct cell populations within the normal urothelium as well as in BCa. CD24high/low expression was not sufficient to characterize CD24 as a BCa-initiating marker in in vivo primary xenotransplants. CD24 and CD44 expressions correlated with lower cancer-specific survival in patients. However, multivariate analyses of CD24 or CD44 did not demonstrate significantly increased hazards for cancer-specific death if analyzed together with stage, grade, and nodal status of patients. ConclusionsCancer stem cell markers CD24/CD44/CD47 are differentially expressed in cells of urothelial BCa in patients undergoing RC and influence cancer-specific survival of patients. Further evaluation of CD24/CD44/CD47 protein expression could be of high therapeutic value in BCa. However, both CD24 and CD44 expressions cannot be regarded as independent prognostic parameters for patients undergoing RC.

Short-Term Resveratrol Exposure Causes In Vitro and In Vivo Growth Inhibition and Apoptosis of Bladder Cancer Cells

Conventional adjuvant chemotherapies for bladder transitional cell carcinomas (TCCs) may cause strong systemic toxicity and local irritation. Non-toxic resveratrol inhibits TCC cell growth but its feasibility in clinical management of TCCs remains obscure. This study aimed to evaluate the safety and anti-TCC efficacy of resveratrol, using the experimental models closer to the clinical treatment condition. Human TCC EJ cells were exposed to 100 µM, 150 µM and 200 µM resveratrol respectively for 1 hour and 2 hours to mimic intravesical drug instillation and the cell responses were analyzed by multiple experimental approaches. An orthotopic TCC nude mouse model was established by injecting EJ cells into the sub-urothelial layer and used for short-term intravesical resveratrol instillation. The safety of resveratrol instillation was evaluated and compared with that of MCC. The results revealed that 2 h 150 µM or 200 µM resveratrol treatment leaded to remarkable S phase arrest and apoptosis at 72 h time-point, accompanied with attenuated phosphorylation, nuclear translocation and transcription of STAT3, down-regulation of STAT3 downstream genes (survivin, cyclinD1, c-Myc and VEGF) and nuclear translocations of Sirt1 and p53. The importance of STAT3 signaling in cell growth was confirmed by treating EJ cells with JAK2 inhibitor tyrphostin AG490. The efficacy and safety of resveratrol instillation were proved by the findings from nude mouse orthotopic xenograft models, because this treatment caused growth suppression, distinctive apoptosis and STAT3 inactivation of the transplanted tumors without affecting normal urothelium. Our results thus suggest for the first time the practical values of resveratrol as a safe and effective agent in the post-operative treatment of TCCs.

Alterations of Global Histone H3K9 and H3K27 Methylation Levels in Bladder Cancer

Background: Epigenetic alterations, including histone modifications, play an important role during carcinogenesis. This study was designed to systematically investigate histone H3K9 and H3K27 methylation levels in bladder cancer (BCa) tissue. Methods: A tissue microarray with urothelial BCa (150 non-muscle-invasive BCa, NMIBC; 121 muscle-invasive BCa, MIBC; 31 metastatic BCa, MET) and normal urothelium (29, CTRL) specimen was used to determine the global levels of H3K9 and H3K27 mono-, di- and tri-methylation. Results: Global levels of H3K9 and H3K27 methylation were significantly higher in CTRL than in BCa, and levels in NMIBC were higher compared to MIBC. Histone methylation levels of MET resembled MIBC. We observed furthermore a correlation of histone methylation levels with pT stage (H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me3) and grade (H3K9me2, H3K9me3, H3K27me1) in NMIBC. H3K9me1, H3K9me3, H3K27me1 and H3K27me3 levels were also correlated with pT stage in MIBC. Histone modifications were not associated with recurrence-free or cancer-specific survival. Conclusions: Global histone H3K9 and H3K27 methylation levels are altered in BCa.

The significance of the expression of interferon regulatory factor 3 and its alternative splicing isoforms in bladder cancer

Objective To evaluate the role of interferon regulatory factor 3 (IRF-3) and its alternative splicing isoforms in bladder cancer.Methods RNA was isolated from 3 cell lines (normal human urothelium cells,769-P,EJ) and 48 cases of bladder cancer tissues and adjacent normal tissues from Peking university first hospital between Jun.2012 to Jan.2013 by Trlzol method.RT-PCR was used to examine IRF-3 and its four alternative splicing isoforms (IRF-3a,-3c,-3d,-3e).Sequencing technology was used to find the point mutations in open reading frame of IRF-3 cDNA.Results The expression of IRF-3d is negative in normal human urothelium cells and the level of IRF-3d is lower than that in 769-P and EJ cell lines.In the 48 cases of pathological specimens,IRF-3 and IRF-3a expressed widely and no significant difference was observed between tumor and normal tissues.IRF-3d and-3e showed tumor-specific expression (P＜0.05).Although IRF-3c expressed more widely in tumor than in normal tissues,no statistical significance was observed.There was a correlation between IRF-3a and bladder tumor staging.Conclusions IRF-3 ubiquitously expressed in tumor and normal tissue (P =0.01).Its alternative splicing isoforms IRF-3d and-3e had significant correlations with bladder tumor occurrence.IRF-3a expression was also associated with bladder tumor staging. Key words: Interferon regulatory factor 3; Alternative splicing isoforms; Point mutation; Bladder tumor

A global profile of gene promoter methylation in treatment-naïve urothelial cancer

The epigenetic alteration of aberrant hypermethylation in the promoter CpG island of a gene is associated with repression of transcription. In neoplastic cells, aberrant hypermethylation is well described as a mechanism of allele inactivation of particular genes with a tumor suppressor function. To investigate the role of aberrant hypermethylation in the biology and progression of urothelial cancer, we examined 101 urothelial (transitional cell) carcinomas (UC), broadly representative of the disease at presentation, with no prior immunotherapy, chemotherapy or radiotherapy, by Infinium HM27 containing 14,495 genes. The genome-wide signature of aberrant promoter hypermethylation in UC consisted of 729 genes significant by a Wilcoxon test, hypermethylated in a CpG island within 1 kb of the transcriptional start site and unmethylated in normal urothelium from aged individuals. We examined differences in gene methylation between the two main groups of UC: the 75% that are superficial, which often recur but rarely progress, and the 25% with muscle invasion and poor prognosis. We further examined pairwise comparisons of the pathologic subgroups of high or low grade, invasive or non-invasive (pTa), and high grade superficial or low grade superficial UC. Pathways analysis indicated over-representation of genes involved in cell adhesion or metabolism in muscle-invasive UC. Notably, the TET2 epigenetic regulator was one of only two genes more frequently methylated in superficial tumors and the sole gene in low grade UC. Other chromatin remodeling genes, MLL3 and ACTL6B, also showed aberrant hypermethylation. The Infinium methylation value for representative genes was verified by pyrosequencing. An available mRNA expression data set indicated many of the hypermethylated genes of interest to be downregulated in UC. Unsupervised clustering of the most differentially methylated genes distinguished muscle invasive from superficial UC. After filtering, cluster analysis showed a CpG Island Methylator Phenotype (CIMP)-like pattern of widespread methylation in 11 (11%) tumors. Nine of these 11 tumors had hypermethylation of TET2. Our analysis provides a basis for further studies of hypermethylation in the development and progression of UC.