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SummaryMuscle-invasive bladder cancers (MIBCs) are biologically heterogeneous and have widely variable clinical outcomes and responses to conventional chemotherapy. We discovered three molecular subtypes of MIBC that resembled established molecular subtypes of breast cancer. Basal MIBCs shared biomarkers with basal breast cancers and were characterized by p63 activation, squamous differentiation, and more aggressive disease at presentation. Luminal MIBCs contained features of active PPARγ and estrogen receptor transcription and were enriched with activating FGFR3 mutations and potential FGFR inhibitor sensitivity. p53-like MIBCs were consistently resistant to neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin chemotherapy, and all chemoresistant tumors adopted a p53-like phenotype after therapy. Our observations have important implications for prognostication, the future clinical development of targeted agents, and disease management with conventional chemotherapy.

Antimitotics such as taxanes are being considered as alternatives to conventional cisplatin-based chemotherapy in patients with bladder cancer, but the molecular determinants of sensitivity or resistance to these agents in bladder cancer cells have not been defined. Here we examined the cytotoxic effects of a novel antimitotic, the Eg5 inhibitor AZD4877, in a molecularly diverse panel of human bladder cancer cell lines. The cells displayed heterogeneous responses to the drug that correlated closely with sensitivity to docetaxel but not with sensitivity to cisplatin. Global gene expression profiling identified p63 as the top gene that was differentially expressed between sensitive and resistant cell lines. Stable knockdown of p63 inhibited cell death induced by either AZD4877 or docetaxel and was associated with decreased proliferation and decreased expression of c-myc. Furthermore, c-myc knockdown also rendered cells resistant to AZD4877 or docetaxel. Together, our results implicate p63 and its downstream target c-myc as determinants of sensitivity to anti-mitotics in bladder cancer cells. Our data also suggest that anti-mitotics and cisplatin target different subsets of bladder cancer cells, a conclusion that may have important implications for the therapy of muscle-invasive bladder cancers.

Purpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo. Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-UC-9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with RAD001 or placebo. Tumors were harvested for immunohistochemical analysis. Results: In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001, whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and 4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However, inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells. Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin Cancer Res; 17(9); 2863–73. ©2011 AACR.

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed ∼1,900 unique genes differentially expressed (≥3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers. Cancer Prev Res; 3(6); 776–86. ©2010 AACR.

Purpose: In smooth muscle (SM), myosin heavy chain (MHC) is expressed predominantly as two isoforms, SM1 and SM2, which are encoded by a single gene and expressed by alternative splicing mechanisms. Although functional differences of these isoforms are unknown, changes in SM1/SM2 ratio have been reported in various pathophysiologic conditions. We analyzed MHC composition of bladder detrusor SM from rabbits of different ages to determine whether SM1 and SM2 isoform expressions are developmentally regulated.Materials and Methods: Rabbit bladders on the −11, −4, 1, 7, 14, 21, and 90th days of life were analyzed for SM MHC isoform expression at protein and mRNA levels. Porous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), S1 protection assay, and histological analysis were employed.Results: The predominant MHC isoform in fetal and neonatal bladders was SM1. In the third postnatal week, the SM1/SM2 ratio decreased from 2.3 to 1.0. A stable SM1/SM2 ratio of 0.6 was observed in the adult a...

KU7 is a popular urothelial carcinoma cell line that was isolated from the bladder of a patient at Keio University in 1980. It has subsequently been widely used in laboratories around the world. We describe how routine cell line authentication revealed that KU7 was cross contaminated almost 30 years ago with HeLa, a cervical carcinoma cell line.Presumed KU7 clones dating from 1984 to 1999 were provided by M.D. Anderson Cancer Center, Vancouver Prostate Centre, Kyoto University, Tokyo Medical University and Keio University. HeLa was obtained from ATCC. Genomic DNA was isolated and short tandem repeat analysis was performed at the M.D. Anderson Cancer Center Characterized Cell Line Core Facility, Johns Hopkins University Fragment Analysis Facility and RIKEN BioResource Center, Ibaraki, Japan. Comparative genomic hybridization was performed on a platform (Agilent Technologies, Santa Clara, California) at Vancouver Prostate Centre.The short tandem repeat profile of all KU7 clones was an exact match with that of HeLa. Comparative genomic hybridization of all samples revealed an abundance of shared chromosomal aberrations. Slight differences in some genomic areas were explained by genomic drift in different KU7 clones separated by many years.Our analysis identified that cross contamination of KU7 with HeLa occurred before 1984 at the source institution. All KU7 clones in the urological literature should be considered HeLa and experimental results should be viewed in this light. Our results emphasize the need to authenticate cell lines in oncological research.

Background p63 is a member of the p53 family that has been implicated in maintenance of epithelial stem cell compartments. Previous studies demonstrated that p63 is downregulated in muscle-invasive bladder cancers, but the relationship between p63 expression and survival is not clear. Methodology/Principal Findings We used real-time PCR to characterize p63 expression and several genes implicated in epithelial-to-mesenchymal transition (EMT) in human bladder cancer cell lines (n = 15) and primary tumors (n = 101). We correlated tumor marker expression with stage, disease-specific (DSS), and overall survival (OS). Expression of E-cadherin and p63 correlated directly with one another and inversely with expression of the mesenchymal markers Zeb-1, Zeb-2, and vimentin. Non-muscle-invasive (Ta and T1) bladder cancers uniformly expressed high levels of E-cadherin and p63 and low levels of the mesenchymal markers. Interestingly, a subset of muscle-invasive (T2–T4) tumors maintained high levels of E-cadherin and p63 expression. As expected, there was a strongly significant correlation between EMT marker expression and muscle invasion (p

Objectives Cyclooxygenase 2 (COX-2) is aberrantly expressed in multiple tumor types including bladder cancer and is associated with enhanced growth, resistance to apoptosis, invasion, and angiogenesis. To evaluate the mechanisms through which COX-2 expression alters normal urothelium, we transfected the SV-40 immortalized human urothelial cell line SV-HUC with COX-2. Methods SV-HUC cells were stably transfected with a plasmid containing COX-2 under a CMV promoter. Following isolation of monoclonal transfectants, COX-2 expression was determined by Western and Northern analyses. Prostaglandin E2 (PGE2) in the culture supernatant was measured by ELISA. Cell growth was measured by crystal violet assay. Cellular invasion through Matrigel and anchorage-independent growth in 0.4% agarose were assessed. Tumorigenicity was evaluated by subcutaneous injection of cells in nude mice with and without Matrigel. Results Four of 12 clones stably overexpressing COX-2 at high levels relative to vector-transfected control cells were chosen for further study. Cell growth rates of these 4 clones were higher than vector control cells. PGE2 production was elevated in 3 of these 4 clones, and PGE2 levels correlated significantly with invasion through Matrigel. COX-2-transfected cells did not form colonies in soft agarose or tumors in nude mice. Conclusions Forced COX-2 expression in SV-HUC immortalized urothelial cells contributes to increased PGE2 production and increased invasion through Matrigel. However, it is insufficient to induce malignant transformation.

Areca nut (AN) is a popular carcinogen used by about 0.6-1.2 billion people worldwide. Although AN contains apoptosis-inducing ingredients, we previously demonstrated that both AN extract (ANE) and its 30-100 kDa fraction (ANE 30-100K) predominantly induce autophagic cell death in both normal and malignant cells. In this study, we further explored the action mechanism of ANE 30-100K-induced autophagy (AIA) in Jurkat T lymphocytes and carcinoma cell lines including OECM-1 (mouth), CE81T/VGH (esophagus), SCC25 (tongue), and SCC-15 (tongue). The results showed that chemical- and small hairpin RNA (shRNA)-mediated inhibition of AMP-activated protein kinase (AMPK) resulted in the attenuation of AIA in Jurkat T but not in OECM-1 cells. Knockdown of Atg5 and Beclin 1 expressions ameliorated AIA in OECM-1/CE81T/VGH/Jurkat T and OECM-1/SCC25/SCC-15, respectively. Furthermore, ANE 30-100K could activate caspase-3 after inhibition of Beclin 1 expression in OECM-1/SCC25/SCC15 cells. Meanwhile, AMPK was demonstrated to be the upstream activator of the extracellular-regulated kinase (ERK) in Jurkat T cells, and inhibition of MEK attenuated AIA in Jurkat T/OECM-1/CE81T/VGH cells. Finally, we also found that multiple myeloma RPMI8226, lymphoma U937, and SCC15 cells survived from long-term non-cytotoxic ANE 30-100K treatment exhibited stronger resistance against serum deprivation through upregulated autophagy. Collectively, our studies indicate that Beclin-1 and Atg5 but not AMPK are commonly required for AIA, and MEK/ERK pathway is involved in AIA. Meanwhile, it is also suggested that long-term AN usage might increase the resistance of survived tumor cells against serum-limited conditions.

Selective COX-2 inhibitors such as celecoxib and NS-398 are being evaluated as chemopreventive and therapeutic agents for bladder and other cancers. We investigated the effects of these nonsteroidal anti-inflammatory agents on a panel of bladder cancer cell lines, and assessed their effects on anchorage-dependent and -independent growth, cell cycle, apoptosis and morphology. The human bladder cancer cell lines UM-UC-1, -3, and -6 were assayed for COX-2 expression by Western analysis using a monoclonal antibody to COX-2. UM-UC-1, -3, and -6 cells were grown in the presence of increasing concentrations of NS-398 and celecoxib, and cell growth was quantitated over 7 days by crystal violet elution. The cell lines were treated with NS-398 and celecoxib for 48 h and analyzed by flow cytometry with propidium iodide staining and Br-dUTP staining for apoptosis. Anchorage-independent growth was assessed using an agarose growth assay. Western analysis demonstrated that COX-2 expression in UM-UC-1, -6, and -3 was high, low, and undetectable, respectively. NS-398 and celecoxib produced dose-dependent growth inhibition of UM-UC-1 and -6. Both NS-398 and celecoxib also inhibited anchorage-dependent and -independent growth of UM-UC-3 in a dose-dependent fashion, despite the low basal expression of COX-2 in this cell line. Cell cycle analyses of UM-UC-1 and -6 revealed a 50% reduction in S-phase in the presence of 100 microM NS-398 whereas a smaller reduction in S-phase was noted in UM-UC-3 cells. Furthermore, treatment with 100 microM celecoxib resulted in significant apoptosis in all three cell lines, which was associated with downregulation of Bcl-2. COX-2 selective inhibitors NS-398 and celecoxib produced dose-dependent growth inhibition of bladder cancer cells associated with a significant reduction in S-phase. Induction of apoptosis in all three cell lines by celecoxib was associated with downregulation of Bcl-2. These changes occur independently of COX-2 expression levels suggesting the presence of a COX-2 independent pathway.

Cell lines have become an essential component for the investigation of cancer. We have developed a panel of cell lines derived from human urothelial cancers and we describe some of their important characteristics.Ten human urothelial cancer cell lines were characterized by their growth in athymic nude mice, CAR expression and their susceptibility to adenoviral mediated transfer of the green fluorescence protein gene. TP53 mutation status and immunochemical analysis of p53, pRB and p16 were also examined.Five cell lines rapidly produced tumors in athymic nude mice. Two cell lines produced tumors in 1 month, 1 produced them in 3 months and 2 were nontumorigenic. The cell lines varied in CAR expression and in their susceptibility to adenoviral mediated gene transduction. There was no direct correlation between CAR expression and susceptibility to adenoviral mediated gene transduction. Seven cell lines had TP53 mutations, of which 2 had large deletions and did not express p53 protein by immunostaining. All cell lines expressed abnormal pRB by immunochemical analysis (3 had no staining and 7 had homogenously strong staining) and 8 did not express p16 (7 showed homogeneously strong pRB staining).Our panel of 10 human urothelial cell lines differed in genetic alterations, growth in nude mice, susceptibility to adenoviral mediated gene transduction, and expression of p53, p16 and pRB. The availability of various urothelial cancer cell lines with differing genotypic and phenotypic features will facilitate further research into bladder cancer.

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.

Urothelial cell carcinoma of the bladder (UCC) ranks 5th in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. A few urine-based tests for screening and surveillance are commercially available for bladder cancer, but there are no reliable non-invasive methods for detecting premalignant UCC. In order to determine molecular mechanisms involved in early UCC development and to identify new biomarkers for detection, diagnosis and prognosis of UCC, a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) was analyzed by DNA microarray technology. Genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild type (WT) littermates at 3, 6, 20 and 30 weeks of age were identified. The ages selected correspond to premalignant, carcinoma in-situ, and early and later stage papillary UCC, respectively. Approximately 1,900 unique genes were differentially expressed (≥ 3-fold difference at one or more time points) between WT and UPII-SV40Tag urothelium during the time course of tumor development. A high proportion of cell cycle regulatory genes and proliferation signaling genes were more strongly expressed in the UPII-SV40Tag bladder urothelium. Several of the genes upregulated and downregulated in UPII-SV40Tag urothelium were tested as biomarkers for premalignancy, including autocrine motility factor (AMFR), hyaluronan-mediated motility receptor (Hmmr), proliferating cell nuclear antigen (PCNA), Rac GTPase activating protein 1 (RacGAP1), superoxide dismutase 3 (SOD3), collagen 1a2 (Col1a2), and others. These tests were performed by analyzing urine samples from a recently completed Phase II, randomized, placebo controlled chemoprevention trial (N01 CN85186, PI: A. Sabichi) that was designed to test whether celecoxib can prevent recurrence in patients successfully treated by transurethral resection (TUR) for non-muscle invasive bladder cancer. Positive markers, Hmmr and RacGAP1, and a negative marker, Col1a2, have been identified in patient urine samples that show promise for detecting UCC recurrence. We are currently attempting to determine the biological function of Hmmr, and RacGAP1 in bladder premalignancy. 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 4861. doi:10.1158/1538-7445.AM2011-4861

Transitional cell carcinoma of the bladder (TCC) ranks 4th in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are a few commercially available urine-based tests for screening and surveillance for bladder cancer, but none of these can detect premalignancy. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology, in order to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild type (WT) littermates at 3, 6, 20 and 30 weeks of age. These are ages which correspond to premalignant, carcinoma in-situ, and early and later stage papillary TCC, respectively. There were approximately 1,900 unique genes differentially expressed (>= 3-fold difference at one or more time points) between WT and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We have tested several of the genes upregulated in UPII-SV40Tag urothelium, including autocrine motility factor (AMFR), hyaluronan-mediated motility receptor (Hmmr), proliferating cell nuclear antigen (PCNA), Rac GTPase activating protein 1 (RacGAP 1) and others as biomarkers for premalignancy, in urine samples from a recently completed Phase II, randomized, placebo controlled chemoprevention trial (N01 CN85186, PI: A. Sabichi) that was designed to test whether celecoxib can prevent recurrence in patients successfully treated by TUR for non-muscle invasive bladder cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A16.

B52 Transitional cell carcinoma of the bladder (TCC) ranks 4th in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40T mice) with DNA microarray technology, in order to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40T mice and their age-matched wild type (WT) littermates at 3, 6, 20 and 30 weeks of age. These are times which correspond to premalignant, carcinoma in-situ, and early and later stage papillary TCC, respectively. There were approximately 1,900 unique genes differentially expressed (≥ 3-fold difference at one or more time points) between WT and UPII-SV40T urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and cytokinesis genes that are more strongly expressed in the UPII-SV40T bladder urothelium. We have tested several of the genes upregulated in UPII-SV40T urothelium, including autocrine motility factor (AMFR), Caspase 3, Cox-2, PCNA and others as biomarkers for premalignancy in urine samples from a completed chemoprevention trial. These results will be discussed in the context of the UPII-SV40T model. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B52.