Urothelial Cell Lines Research Articles

Expression of PAK1 in bladder cancer and its influence on invasion of bladder cancer cells

Objective: To investigate the expression of p21-activated kinase 1 (PAK1) in bladder cancer and its biological influence on invasion ability of bladder cancer cells. Methods: A total of 54 paraffin-embedded bladder cancer tissue samples and 12 normal bladder tissue specimens were retrieved in Jinshan Hospital Affiliated to Fudan University between January 2009 and December 2012. The expression of PAK1 in these tissues was detected by immunohistochemical staining. The PAK1 mRNA and protein levels were measured in bladder cancer cell lines and human normal bladder epithelial cell line using real-time, fluorescence-based quantitative polymerase chain reaction (RT-PCR) and Western blot, respectively. A stable PAK1 gene silencing bladder cancer cell strain 5637 were successfully constructed. After treatment with PAK1 RNA interference(RNAi), the ability of migration and invasion of the 5637 cell was evaluated using a Transwell system. Results: The expression of PAK1 proteins was significantly higher in bladder cancer tissues than in normal bladder tissues (28/54 vs 1/12, P<0.05). The overexpression of PAK1 was positively correlated with high histological grade, lymph node metastasis, and tumor size (all P<0.05). The mRNA level and protein level of PAK1 was much higher in bladder cancer cell lines T24, 5637 than human normal urothelial cell line SV-HUC-1.PCR and Western blot showed satisfactory inhibitory effect of PAK1 short hairpin RNA (shRNA) on PAK1 expression in 5637 bladder cancer cells. The number of PAK1 RNAi-treated 5637 cells traversed the membrane was decreased compared with the control group in migration and invasion assays. Conclusions: Overexpression of PAK1 in bladder cancer tissues may be an important feature of bladder cancer and related with the metastasis and invasion of bladder cancer. The molecular mechanisms involved in regulation of PAK1 expression needs further research.

Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP.

To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal‐regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two‐photon excitation microscope and a vacuum‐stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch‐induced ERK activation was reproduced in an hTERT‐immortalized human urothelial cell line (TRT‐HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch‐induced ERK activation in TRT‐HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP‐induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor‐expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks.

Erratum to: The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies.

Following the publication of our recent article in BMC Genomics [1] a number of aspects were called to our attention. We have carefully reviewed the experiments reported in this manuscript, as well as additional data from our laboratories, and would like to make the following points.

Dasatinib enhances tumor growth in gemcitabine-resistant orthotopic bladder cancer xenografts.

BackgroundSystemic chemotherapy with gemcitabine and cisplatin is standard of care for patients with metastatic urothelial bladder cancer. However, resistance formation is common after initial response. The protein Src is known as a proto-oncogene, which is overexpressed in various human cancers. Since there are controversial reports about the role of Src in bladder cancer, we evaluated the efficacy of the Src kinase inhibitor dasatinib in the urothelial bladder cancer cell line RT112 and its gemcitabine-resistant sub-line RT112rGEMCI20 in vitro and in vivo.MethodsRT112 urothelial cancer cells were adapted to growth in the presence of 20 ng/ml gemcitabine (RT112rGEMCI20) by continuous cultivation at increasing drug concentrations. Cell viability was determined by MTT assay, cell growth kinetics were determined by cell count, protein levels were measured by western blot, and cell migration was evaluated by scratch assays. In vivo tumor growth was tested in a murine orthotopic xenograft model using bioluminescent imaging.ResultsDasatinib exerted similar effects on Src signaling in RT112 and RT112rGEMCI20 cells but RT112rGEMCI20 cells were less sensitive to dasatinib-induced anti-cancer effects (half maximal inhibitory concentration (IC50) of dasatinib in RT112 cells: 349.2 ± 67.2 nM; IC50 of dasatinib in RT112rGEMCI20 cells: 1081.1 ± 239.2 nM). Dasatinib inhibited migration of chemo-naive and gemcitabine-resistant cells. Most strikingly, dasatinib treatment reduced RT112 tumor growth and muscle invasion in orthotopic xenografts, while it was associated with increased size and muscle-invasive growth in RT112rGEMCI20 tumors.ConclusionDasatinib should be considered with care for the treatment of urothelial cancer, in particular for therapy-refractory cases.

Immunomodulatory proteins FIP-gts and chloroquine induce caspase-independent cell death via autophagy for resensitizing cisplatin-resistant urothelial cancer cells

BackgroundChloroquine, a lysosomal inhibitor, is used for malaria, rheumatoid arthritis, and lupus erythematosus therapy. In our previous study, FIP-gts, an immunomodulatory protein from Ganoderma tsugae, inhibited cell viability in lung cancer cells and urothelial cancer cells. Urothelial carcinoma is the most common type of bladder cancer. Cisplatin resistance is an important issue in urothelial carcinoma therapy. PurposeThe aim of this study is to investigate the effect of combination treatment with FIP-gts and chloroquine on cytotoxicity to resensitize the cisplatin-resistant cells. MethodsFIP-gts and chloroquine cytotoxicity were determined by evaluating CCK-8 assay. Cell death pathways, ROS and cell cycle arrested were analysed through flow cytometry and Western blot. ShRNA targeting to autophagy-related genes were tested to evaluate their autophagic cell death for resistant urothelial cells. ResultsUsing CCK-8 assay, chloroquine increased FIP-gts-induced cytotoxicity in parental and cisplatin-resistant urothelial cancer cell lines. On flow cytometry, chloroquine enhanced FIP-gts-mediated sub-G1 accumulation, annexin V positive signal and mitochondrial membrane potential loss. Caspase-3/PARP cascade and z-VAD-fmk were performed to prove that FIP-gts and chloroquine induced caspase-independent cell death. Using H2DCFDA staining and flow cytometry, FIP-gts and chloroquine did not induce ROS production. N-acetyl cysteine, a ROS scavenger, inhibited the cytotoxicity and LC3-II accumulation in FIP-gts and chloroquine-treated N/P cells. To elucidate the role of autophagy in caspase-independent cell death by FIP-gts and chloroquine, LC3 shRNA were used to inhibit autophagy in N/P cells. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells. This is the first study to reveal the novel function of FIP-gts in triggering caspase-independent cell death in cisplatin-resistant urothelial cancer cells. ConclusionChloroquine enhanced FIP-gts-induced autophagy dependent caspase-independent cell death via abundant autophagosome accumulation. Combination treatment with FIP-gts and chloroquine may provide a new strategy for urothelial cancer therapy.

Elevated connexin 43 expression in arsenite-and cadmium-transformed human bladder cancer cells, tumor transplants and selected high grade human bladder cancers

Connexin 43 has been shown to play a role in cell migration and invasion; however, its role in bladder cancer is not well defined. Previous studies from our laboratory have shown that the environmental pollutants arsenite and cadmium can cause malignant transformation of the immortalized urothelial cell line UROtsa. These transformed cells can form tumors in immune-compromised mice. The goal of the present study was to determine if connexin 43 is expressed in the normal human bladder, the arsenite and cadmiun-transformed UROtsa cells as well as human urothelial cancer. The results obtained showed that connexin 43 is not expressed in the epithelial cells of the human bladder but is expressed in immortalized cultures of human urothelial cells and the expression is variable in the arsenite and cadmium- transformed urothelial cell lines derived from these immortalized cells. Tumor heterotransplants generated from the transformed cells expressed connexin 43 and the expression was localized to areas of squamous differentiation. Immuno-histochemical analysis of human bladder cancers also showed that the expression of connexin 43 was localized to areas of the tumor that showed early features of squamous differentiation. Treatment of UROtsa cells with various concentrations of arsenite or cadmium did not significantly alter the expression level of connexin 43. In conclusion, our results show that the expression of connexin 43 is localized to the areas of the tumor that show squamous differentiation, which may be an indicator of poor prognosis. This suggests that connexin 43 has the potential to be developed as a biomarker for bladder cancer that may have the ability to invade and metastasize.

Urothelium muscarinic activation phosphorylates CBS(Ser227) via cGMP/PKG pathway causing human bladder relaxation through H2S production.

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser227 following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders.

Epigenetic modification suppresses proliferation, migration and invasion of urothelial cancer cell lines.

Epigenetic approaches offer additional therapeutic options, including apoptosis induction, modification of cell cycle regulating proteins and the re-expression of pharmaceutical targets, such as hormone receptors. The present study analyzed the effect of the epigenetic modifiers 5-aza-2′-deoxycytidine and Trichostatin A on the proliferative, migratory and invasive behavior of four urinary bladder cancer cell lines (RT-4, RT-112, VMCUB-1 and T-24), and the expression of various matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). Cell proliferation, migration and invasion assays revealed that treatment with the two epigenetic modifiers resulted in proliferation inhibition in all cell lines, and migration and invasion inhibition in RT-4, RT-112 and T-24 cell lines. Quantitative polymerase chain reaction demonstrated that the mRNA expression of a broad selection of MMPs and their TIMPs was induced in all cell lines, and MMP-14 mRNA expression was suppressed in all cell lines, with the exception of RT-4. In conclusion, epigenetic modifications suppressed the motility and invasiveness of three out of four urothelial cancer cell lines. The inhibitory effect on cell motility appears to be crucial for reduced invasive properties. However, even a broad spectrum of mRNA analysis does not sufficiently explain the loss of invasiveness, as it does not allow for functional conclusions. Further complex urothelial tumour models should be applied to investigate whether epigenetic therapeutic approaches may be an option in urothelial cancer.

Abstract 4506: Identification of druggable transcriptional outliers in urothelial carcinoma

Abstract Background: Urothelial carcinoma (UC) is a major public health problem affecting new 75,000 patients and resulting in 15,000 deaths annually. Despite a variety of genetic and epigenetic alterations described in UC, few therapeutically exploitable drivers have been identified and validated. We hypothesized that transcriptional outliers from primary tumor biopsies could reveal such therapeutically exploitable weaknesses. Identification of these druggable outliers would be critical for individualized therapy of patients with UC. Methods: In order to identify transcriptional outliers we calculated Z-scores for a set of 72 druggable cancer-related genes from RNA sequencing data from our Institute of Precision Medicine (IPM) cohort of advanced platinum-resistant UC. We performed similar outlier analyses on The Cancer Genome Atlas (TCGA) cohort of UC and the Cancer Cell Line Encyclopedia (CCLE) database of urothelial cell lines. Common outliers between IPM and CCLE datasets or between TCGA and CCLE datasets were nominated for further in-vitro testing. Sensitivity scores of cell lines for 130 drugs were extracted from the publicly available cell line browser database and the variance these sensitivity scores across (640) cell lines was calculated. The drugs targeting the outlier genes (real sensitivity z-score) were compared to the mean z-scores of randomly chosen drugs associated with those genes (control sensitive z-score). To investigate the mechanisms underlying comparison of outliers methylation patterns of the promoter regions of outlier genes were examined. Results: 6 druggable outlier genes were common between IPM and CCLE cohorts, and 15 outlier genes between CCLE and TCGA cohorts. Common outliers included FGFR3, KIT, BCL2, ABL1, and SMO. The presence of outlier genes in cell lines correlated with higher sensitivity to drugs predicted to target these genes in comparison to randomly selected drugs (p&amp;lt;0.05). In vitro cell viability assays confirmed sensitivity of RT112 and RT4 cell lines to Dovitinib, an FGFR3 inhibitor as predicted by our algorithm. Outlier genes were frequently involved in gene fusions, amplification or DNA hypomethylation (p&amp;lt;0.02). Conclusions: Identification of druggable transcriptional outliers can elucidate novel oncogenic dependencies and aid in selecting precision therapies for UC patients. Citation Format: Bishoy S. M. Faltas, Rohan Bareja, Ethan Shelkey, Rebecca Mayer, Andrea Sboner, Mark A. Rubin, Olivier Elemento. Identification of druggable transcriptional outliers in urothelial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4506.

Abstract 1512: Functional validation of microRNA activity inferred by ActMiR in bladder cancer

Abstract Background: MicroRNAs (miRs) play a key role in cancer, both in tumorigenesis and tumor progression. In the past years, miR expression signatures have been reported as prognostic biomarkers in different tumor types including bladder cancer (BC). However, miR's expression does not always correlate with activity. We recently developed a novel computational method, named ActMiR, for explicitly inferring the activity of miRs based on the changes in expression levels of target genes. The main objective of this study was to validate the inferred miRs’ activity using BC as a model. Design: We applied ActMiR in 405 BC cases from The Cancer Genome Atlas (TCGA) database for which information from both mRNA and miR expression was available. Different BC cell lines (5637 and HT1376 for basal BC, SW780 and HT1197 for p53-like BC, and RT4 and RT112 for luminal BC) as well as the immortalized urothelial cell line [Human Urothelium cell (HUC)] were used to perform the in vitro functional validation. RNA was extracted from these cells basally and after inhibition of miR106-5p with specific anti-miR inhibitor (Taqman, Life Technologies); miR and target genes’ expression was assessed by qRT-PCR. Results: ActMir analyses revealed that a subset of 306 out of 1044 miRNAs were differentially expressed between tumor and normal samples at p-value&amp;lt;10-4. More importantly, at p-value&amp;lt;10-4, 155 out of 556 miRNAs were functionally active. From these, only four (miR106b, miR532, mir556, and mir134) were significantly differentially expressed, functionally active and showed prognostic significance. We chose to further analyze mir106b because it showed the biggest and most significant difference in activity between normal and cancer cells (p-value&amp;lt;2*10-11). As inferred by ActMir, mir106-5p was significantly overexpressed in all cancer cells when compared to HUC. Basal cells showed the highest fold increase (mean of 39; range: 31-47), followed by luminal (mean of 19; range: 5-32) and p53-like cells (mean of 8; range: 3-13). Inhibition of mir106b-5p was performed to assess whether the predicted target genes were consequently upregulated. Conclusion: Our results underscore the value of ActMir for inferring miR activity in BC from a systems biology perspective. It endorses ActMir as a promising tool for studying casual effects of miR activity on target genes, and ultimately for determining survival outcomes of BC patients. Citation Format: Mireia Castillo-Martin, Ana Collazo Lorduy, EunJee Li, Jun Zhu. Functional validation of microRNA activity inferred by ActMiR in bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1512.

615 - Induction of EMT is WNT-independent in cisplatin resistant urothelial carcinoma cell lines

615 - Induction of EMT is WNT-independent in cisplatin resistant urothelial carcinoma cell lines

Molecular analysis of urothelial cancer cell lines for modeling tumor biology and drug response

The utility of tumor-derived cell lines is dependent on their ability to recapitulate underlying genomic aberrations and primary tumor biology. Here, we sequenced the exomes of 25 bladder cancer (BCa) cell lines and compared mutations, copy number alterations (CNAs), gene expression and drug response to BCa patient profiles in The Cancer Genome Atlas (TCGA). We observed a mutation pattern associated with altered CpGs and APOBEC-family cytosine deaminases similar to mutation signatures derived from somatic alterations in muscle-invasive (MI) primary tumors, highlighting a major mechanism(s) contributing to cancer-associated alterations in the BCa cell line exomes. Non-silent sequence alterations were confirmed in 76 cancer-associated genes, including mutations that likely activate oncogenes TERT and PIK3CA, and alter chromatin-associated proteins (MLL3, ARID1A, CHD6 and KDM6A) and established BCa genes (TP53, RB1, CDKN2A and TSC1). We identified alterations in signaling pathways and proteins with related functions, including the PI3K/mTOR pathway, altered in 60% of lines; BRCA DNA repair, 44%; and SYNE1–SYNE2, 60%. Homozygous deletions of chromosome 9p21 are known to target the cell cycle regulators CDKN2A and CDKN2B. This loci was commonly lost in BCa cell lines and we show the deletions extended to the polyamine enzyme methylthioadenosine (MTA) phosphorylase (MTAP) in 36% of lines, transcription factor DMRTA1 (27%) and antiviral interferon epsilon (IFNE, 19%). Overall, the BCa cell line genomic aberrations were concordant with those found in BCa patient tumors. We used gene expression and copy number data to infer pathway activities for cell lines, then used the inferred pathway activities to build a predictive model of cisplatin response. When applied to platinum-treated patients gathered from TCGA, the model predicted treatment-specific response. Together, these data and analysis represent a valuable community resource to model basic tumor biology and to study the pharmacogenomics of BCa.

Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines.

BackgroundTargeting of class I histone deacetylases (HDACs) exerts antineoplastic actions in various cancer types by modulation of transcription, upregulation of tumor suppressors, induction of cell cycle arrest, replication stress and promotion of apoptosis. Class I HDACs are often deregulated in urothelial cancer. 4SC-202, a novel oral benzamide type HDAC inhibitor (HDACi) specific for class I HDACs HDAC1, HDAC2 and HDAC3 and the histone demethylase LSD1, shows substantial anti-tumor activity in a broad range of cancer cell lines and xenograft tumor models.AimThe aim of this study was to investigate the therapeutic potential of 4SC-202 in urothelial carcinoma (UC) cell lines.MethodsWe determined dose response curves of 4SC-202 by MTT assay in seven UC cell lines with distinct HDAC1, HDAC2 and HDAC3 expression profiles. Cellular effects were further analyzed in VM-CUB1 and UM-UC-3 cells by colony forming assay, caspase-3/7 assay, flow cytometry, senescence assay, LDH release assay, and immunofluorescence staining. Response markers were followed by quantitative real-time PCR and western blotting. Treatment with the class I HDAC specific inhibitor SAHA (vorinostat) served as a general control.Results4SC-202 significantly reduced proliferation of all epithelial and mesenchymal UC cell lines (IC50 0.15–0.51 μM), inhibited clonogenic growth and induced caspase activity. Flow cytometry revealed increased G2/M and subG1 fractions in VM-CUB1 and UM-UC-3 cells. Both effects were stronger than with SAHA treatment.ConclusionSpecific pharmacological inhibition of class I HDACs by 4SC-202 impairs UC cell viability, inducing cell cycle disturbances and cell death. Combined inhibition of HDAC1, HDAC2 and HDAC3 seems to be a promising treatment strategy for UC.Electronic supplementary materialThe online version of this article (doi:10.1007/s11523-016-0444-7) contains supplementary material, which is available to authorized users.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines.

BackgroundEpithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown.MethodsReal-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice.ResultsThis study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin.ConclusionsThe finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells “seeding” a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth.

Distinct mechanisms contribute to acquired cisplatin resistance of urothelial carcinoma cells.

Cisplatin (CisPt) is frequently used in the therapy of urothelial carcinoma (UC). Its therapeutic efficacy is limited by inherent or acquired drug resistance. Here, we comparatively investigated the CisPt-induced response of two different parental urothelial carcinoma cell lines (RT-112, J-82) with that of respective drug resistant variants (RT-112R, J-82R) obtained upon month-long CisPt selection. Parental RT-112 cells were ~2.5 fold more resistant to CisPt than J-82 cells and showed a different expression pattern of CisPt-related resistance factors. CisPt resistant RT-112R and J-82R variants revealed a 2–3-fold increased CisPt resistance as compared to their corresponding parental counterparts. Acquired CisPt resistance was accompanied by morphological alterations resembling epithelial mesenchymal transition (EMT). RT-112R cells revealed lower apoptotic frequency and more pronounced G2/M arrest following CisPt exposure than RT-112 cells, whereas no differences in death induction were observed between J-82 and J-82R cells. CisPt resistant J-82R cells however were characterized by a reduced formation of CisPt-induced DNA damage and related DNA damage response (DDR) as compared to J-82 cells. Such difference was not observed between RT-112R and RT-112 cells. J-82R cells showed an enhanced sensitivity to pharmacological inhibition of checkpoint kinase 1 (Chk1) and, moreover, could be re-sensitized to CisPt upon Chk1 inhibition. Based on the data we suggest that mechanisms of acquired CisPt resistance of individual UC cells are substantially different, with apoptosis- and DDR-related mechanisms being of particular relevance. Moreover, the findings indicate that targeting of Chk1 might be useful to overcome acquired CisPt resistance of certain subtypes of UC.

AB264. MiR-186 inhibits the malignant behaviours of urothelial bladder cancer cell lines by targeting HMGN5

BackgroundTo investigate the role of miR-186 in the carcinogenesis and metastasis of human urothelial bladder cancer and its potential target protein.MethodsQuantitative real-time PCR (qRT-PCR) was performed to detect miR-186 expression in human urothelial bladder cancer tissues and cell lines. Then, Bioinformatics analysis, combined with luciferase reporter assay demonstrated the miR-186’s target gene and protein. Finally, the roles of miR-186 in regulation of tumor proliferation and invasion were further investigated by MTT assay and transwell assay.ResultsMiR-186 was down-regulated in human urothelial bladder cancer tissues and cell lines. Luciferase reporter assay showed that miR-186 targets HMGN5 3'-untranslated region (UTR) directly and suppresses HMGN5 expression in human urothelial bladder cancer cells. HMGN5 siRNA- and miR-186-mediated HMGN5 knock-down experiments revealed that miR-186 suppresses cell proliferation and invasion through suppression of HMGN5 expression. Expression analysis of a set of epithelial-mesenchymal transition (EMT) markers showed that HMGN5 involves miR-186 suppressed EMT which reducing the expression of mesenchymal markers (vimentin and N-cadherin) and inducing the expression of epithelial marker (E-cadherin).ConclusionsMiR-186 is the upstream regulator of HMGN5. MiR-186-suppressed HMGN5 is a potential novel therapeutic approach for human urothelial bladder cancer.

MP45-05 UROTHELIUM-SPECIFIC AND TEMPORALLY CONTROLLED BI-ALLELIC INACTIVATION OF BOTH PTEN AND P53 TRIGGERS BASAL-SUBTYPE MUSCLE-INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: PTEN is a tumor suppressor whose inhibitory activity towards phosphoinositide 3 kinase replies on membrane association, a process that is tightly regulated by electrostatic charges of PTEN tail domain. Precisely how and to what extent this domain affects PTEN’s activity remains unclear and is the subject of the present study. METHODS: Urothelial carcinoma cell lines without PTEN (UMUC3 and J82), or that expressing a mutated PTEN (T24), were transiently transfected with vector only, vector containing a full-length PTEN or vector containing a tailless PTEN, and their effects on cell proliferation, AKT activity, cell cycle and apoptosis were assessed under different culture conditions. To circumvent the extreme deleterious effects of the tailless PTEN, stable transfectants of UMUC3 were created that only expressed the tailless PTEN (or the full-length PTEN as a control) upon doxycycline exposure (e.g., stable transfection of CMV/rtTA and TRE-tailless PTEN (or TRE/full-length PTEN). The effects of their inducible expression were assessed in vitro and in xenograft mouse models. RESULTS: Introduction of full-length PTEN and tailless PTEN into PTEN-deficient UMUC3 and J82 cells inhibited proliferation at comparable levels in serum free and low serum (2%) media. However, in high serum (10%) medium, tailless PTEN was three times more inhibitory than full-length PTEN. This was due primarily to the phosphorylation of the serine/threonine clusters of the full-length PTEN’s tail domain, an effect absent in the tailless PTEN. Similar results were observed with transiently transfected T24 cells which expressed an endogenous mutant PTEN. UMUC3, J82 and T24 transfected with tailless PTEN could not sustain long-term culture because of its extreme deleterious effects, prompting us to establish doxycycline-inducible expression cell lines. UMUC3 inducibly expressing tailless PTEN exhibited significantly greater inhibition of proliferation, AKT phosphorylation, G1/S transition and significantly more apoptosis than UMUC3 inducibly expressing full-length PTEN. Finally, xenografted UMUC3 cells inducibly expressing full-length PTEN established tumors, albeit 30% smaller than the vector only controls. In contrast, those inducibly expressing tailless PTEN were devoid of any tumor in nude mice. CONCLUSIONS: Our in vitro and in vivo data are the first demonstration of the high potency of tailless PTEN as a tumor suppressor. They suggest that delivering tailless PTEN via viral vectors or other vehicles can be an effective approach to tackle advanced urothelial carcinomas with wild-type, mutated or deleted PTEN.

MP88-16 ROLE OF GLUCOCORTICOID RECEPTOR (GR) SIGNALING IN UROTHELIAL TUMORIGENESIS: INHIBITION BY COMPOUND A (CPDA) VIA BOTH GR AND ANDROGEN RECEPTOR (AR) PATHWAYS

INTRODUCTION AND OBJECTIVES: Recent evidence not only has indicated a critical role of AR signals in bladder cancer development but has also suggested the involvement of GR signals in tumor outgrowth [e.g. stimulation of cell proliferation by dexamethasone (DEX), suppression of cell invasion by DEX, predonisone (PRED), or corticosterone]. Nonetheless, it remains unclear whether GR ligands affect urothelial tumorigenesis. Meanwhile, CpdA is known to function as not only a GR modulator inducing GR transrepression but also an AR antagonist. In this study, we assessed the effect of GR ligands including CpdA on neoplastic transformation of urothelial cells. METHODS: A control subline of bladder cancer UMUC3 [UMUC3-control-short hairpin RNA (shRNA)] and its GR knockdown subline (UMUC3-GR-shRNA) were implanted into mouse flanks. The SVHUC normal urothelial cell line and that stably expressing wild-type AR or GR-shRNA were exposed to a chemical carcinogen 3-methylcholanthrene (MCA) 3 times and subsequently cultured for 6 weeks in the presence or absence of 11 pure glucocorticoids, CpdA, an antiandrogen hydroxyflutamide (HF), and an GR antagosist RU486, followed by cell proliferation assay and plate/soft agar colony formation assays to assess neoplastic transformation. RESULTS: Tumor formation as an end point was significantly (P<0.001) delayed in control UMUC3 xenograft-bearing mice, compared with GR knockdown xenografts. In SVHUC cells (GR-positive/AR-negative) exposed to MCA, only PRED and CpdA significantly prevented neoplastic transformation, which was abolished by RU486. In MCA-SVHUC-AR cells (GR/AR-positive), HF treatment also resulted in inhbition of neoplastic transformation, but the effect of CpdA (67% decrease in colony formation) was significantly stronger than that of HF (42% decrease) or PRED (42% decrease). In MCA-SVHUC-GR-shRNA cells (GR/AR-negative), CpdA, HF, and all other glucocorticoids failed to inhibit neoplastic transformation. In addition, among the GR ligands tested, CpdA most significantly reduced the expression of oncogenes, such as c-fos and c-jun, and induced that of UGT1A known to prevent urothelial tumorigenesis via detoxifying carcinogens in MCA-SVHUC cells. CONCLUSIONS: GR signaling appears to play an inhibitory role in urothelial tumorigenesis. Our results also suggest that CpdA via both GR and AR pathways more strongly prevents neoplastic transformation of urothelial cells than other glucocorticoids/pure GR ligands or AR antagonists.

MP83-20 ENZALUTAMIDE AS AN ANDROGEN RECEPTOR INHIBITOR PREVENTS UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-20 ENZALUTAMIDE AS AN ANDROGEN RECEPTOR INHIBITOR PREVENTS UROTHELIAL TUMORIGENESIS Takashi Kawahara, Hiroki Ide, Eiji Kashiwagi, Yi Li, Satoshi Inoue, Yichun Zheng, and Hiroshi Miyamoto Takashi KawaharaTakashi Kawahara More articles by this author , Hiroki IdeHiroki Ide More articles by this author , Eiji KashiwagiEiji Kashiwagi More articles by this author , Yi LiYi Li More articles by this author , Satoshi InoueSatoshi Inoue More articles by this author , Yichun ZhengYichun Zheng More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2202AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Emerging preclinical evidence suggests the critical role of androgen-mediated androgen receptor (AR) signals in the development of bladder cancer. However, little is known about the efficacy of enzalutamide, an AR signaling inhibitor that not only blocks androgen binding to AR but also prevents AR nuclear translocation, DNA binding, and coactivator recruitment, in urothelial tumorigenesis. In the current study, we aimed to assess the effects of enzalutamide as well as other anti-androgens, such as flutamide and bicalutamide, on androgen-induced neoplastic transformation of urothelial cells. METHODS An immortalized normal urothelial cell line SVHUC stably expressing wild-type AR (SVHUC-AR) was exposed to a chemical carcinogen 3-methylcholanthrene (MCA) 3 times, to induce neoplastic transformation, and subsequently cultured for 6 weeks in the presence or absence of anti-androgens. Tumorigenesis was then monitored, using plate and soft agar colony formation assays as well as mouse xenograft models. In the SVHUC-AR cells with the carcinogen challenge, quantitative real-time polymerase chain reaction analysis was also performed to determine the expression levels of tumor suppressor genes and oncogenes. RESULTS In SVHUC-AR cells, each anti-androgen was found to inhibit AR-mediated transcriptional activity. In vitro transformation showed that treatment with each anti-androgen during the process of neoplastic transformation similarly reduced the efficiency of colony formation in vitro. Compared with mock treatment, culture with enzalutamide (P = 0.028), hydroxyflutamide (P = 0.033), or bicalutamide (P = 0.038) also resulted in prevention/retardation of tumor formation in male NOD-SCID mice. Additionally, anti-androgens up-regulated the expression of several molecules that play a protective role in bladder tumorigenesis, including p53, p21, p27, and PTEN, and down-regulated that of several oncogenic genes, such as c-myc, cyclin D1, cyclin D3, and cyclin E, in MCA-exposed SVHUC-AR cells. However, significant effects of anti-androgens on the expression of these genes were not observed in SVHUC-AR cells without the carcinogen challenge. CONCLUSIONS Enzalutamide, flutamide, and bicalutamide similarly prevent neoplastic transformation of urothelial cells. These findings offer a potential chemopreventive approach for urothelial tumors using AR antagonists. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1088-e1089 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takashi Kawahara More articles by this author Hiroki Ide More articles by this author Eiji Kashiwagi More articles by this author Yi Li More articles by this author Satoshi Inoue More articles by this author Yichun Zheng More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP88-17 IMMUNOHISTOCHEMISTRY OF ANDROGEN RECEPTOR AND ESTROGEN RECEPTOR-β IN BLADDER CANCER AS PREDICTORS OF CHEMOSENSITIVITY

INTRODUCTION AND OBJECTIVES: Recent evidence not only has indicated a critical role of AR signals in bladder cancer development but has also suggested the involvement of GR signals in tumor outgrowth [e.g. stimulation of cell proliferation by dexamethasone (DEX), suppression of cell invasion by DEX, predonisone (PRED), or corticosterone]. Nonetheless, it remains unclear whether GR ligands affect urothelial tumorigenesis. Meanwhile, CpdA is known to function as not only a GR modulator inducing GR transrepression but also an AR antagonist. In this study, we assessed the effect of GR ligands including CpdA on neoplastic transformation of urothelial cells. METHODS: A control subline of bladder cancer UMUC3 [UMUC3-control-short hairpin RNA (shRNA)] and its GR knockdown subline (UMUC3-GR-shRNA) were implanted into mouse flanks. The SVHUC normal urothelial cell line and that stably expressing wild-type AR or GR-shRNA were exposed to a chemical carcinogen 3-methylcholanthrene (MCA) 3 times and subsequently cultured for 6 weeks in the presence or absence of 11 pure glucocorticoids, CpdA, an antiandrogen hydroxyflutamide (HF), and an GR antagosist RU486, followed by cell proliferation assay and plate/soft agar colony formation assays to assess neoplastic transformation. RESULTS: Tumor formation as an end point was significantly (P<0.001) delayed in control UMUC3 xenograft-bearing mice, compared with GR knockdown xenografts. In SVHUC cells (GR-positive/AR-negative) exposed to MCA, only PRED and CpdA significantly prevented neoplastic transformation, which was abolished by RU486. In MCA-SVHUC-AR cells (GR/AR-positive), HF treatment also resulted in inhbition of neoplastic transformation, but the effect of CpdA (67% decrease in colony formation) was significantly stronger than that of HF (42% decrease) or PRED (42% decrease). In MCA-SVHUC-GR-shRNA cells (GR/AR-negative), CpdA, HF, and all other glucocorticoids failed to inhibit neoplastic transformation. In addition, among the GR ligands tested, CpdA most significantly reduced the expression of oncogenes, such as c-fos and c-jun, and induced that of UGT1A known to prevent urothelial tumorigenesis via detoxifying carcinogens in MCA-SVHUC cells. CONCLUSIONS: GR signaling appears to play an inhibitory role in urothelial tumorigenesis. Our results also suggest that CpdA via both GR and AR pathways more strongly prevents neoplastic transformation of urothelial cells than other glucocorticoids/pure GR ligands or AR antagonists.