RT4 Cell Research Articles

Importin-11 overexpression promotes the migration, invasion, and progression of bladder cancer associated with the deregulation of CDKN1A and THBS1

ObjectivesWe recently determined that a novel oncogene, IPO11 from 5q12, participates in bladder cancer (BCa) progression. However, the biological function of IPO11 and the molecular mechanisms through which it contributes to BCa progression remain unclear. The aim of this study was to investigate the role of IPO11 in BCa aggressiveness and elucidate the molecular mechanisms underlying its effects in BCa. Materials and methodsThe mRNA expression levels of IPO11 in BIU-87, RT4, UMUC3, EJ, 5637, T24, J82, and HT-1376 cell lines were determined using quantitative real-time polymerase chain reaction. Expression of importin-11 was detected in 134 formalin-fixed and paraffin-embedded (FFPE) BCa tissues and 10 paired nonneoplastic bladder tissue specimens by immunohistochemistry. The copy number of IPO11 was examined in 25 FFPE BCa specimens using fluorescent in situ hybridization. The effects of IPO11 on migration, invasion, and cell proliferation were investigated in EJ and 5637 cell lines using RNA interference. Potential molecular mechanisms were investigated using whole transcriptome sequencing and bioinformatic approaches in EJ cells and IPO11-silenced EJ cells and verified using quantitative real-time polymerase chain reaction. ResultsEndogenous IPO11 mRNA was highly expressed in 6 invasive BCa cell lines (EJ, HT-1376, UMUC3, 5637, J82, and T24) but had a low expression in the noninvasive BCa cell line BIU-87 and the papillary BCa cell line RT4. Immunohistochemical staining revealed that 87 (64.9%) of 134 FFPE BCa tissues displayed importin-11 overexpression. Moreover, importin-11 overexpression was positively associated with increased tumor stages and tumor grades, lymphatic invasion, and lymph node metastasis. Furthermore, importin-11 overexpression was detected in 100% (14/14) of BCa tissues with IPO11 amplification, and IPO11 amplification was not observed in 2 additional BCa tissues with importin-11 overexpression. Small interfering RNA-mediated knockdown of IPO11 is sufficient to inhibit the motility and invasiveness of EJ and 5637 cells. IPO11 knockdown also inhibited cell proliferation in EJ cells, whereas this was not observed in 5637 cells or the in vivo experiments. Using whole transcriptome sequencing, we found that 22 genes (including IPO11) were differentially expressed in IPO11-silenced EJ cells compared with wild-type EJ cells, 4 of which were upregulated, and 18 of which were downregulated. KEGG pathway enrichment analysis of the significantly differentially expressed genes showed that the proteoglycans in cancer pathway (pathway Id: hsa05205) was most significantly enriched among 10 genetically altered pathways and referred to 6 significantly altered genes (CDKN1A, HBEGF, PTK2, THBS1, CCNG2, and EGR1). The next 3 most significantly enriched pathways in order were the p53, ErbB, and BCa pathways. CDKN1A and THBS1 were the most 2 frequently covered genes and were involved in 9 and 6 pathways, respectively. They were also 2 key proteins in the BCa pathway (pathway Id: hsa05219) that were downregulated in IPO11-knockdown EJ cells compared with wild-type EJ cells. ConclusionsImportin-11 overexpression can promote BCa cell invasiveness, probably associated with the deregulation of CDKN1A and THBS1 primarily through the activation of the proteoglycans in cancer pathway and the classical BCa pathway. Importin-11 may be a useful target through which the progression of noninvasive BCa to invasive BCa can be blocked.

CXCL5 promotes mitomycin C resistance in non-muscle invasive bladder cancer by activating EMT and NF-κB pathway

The emergence of chemoresistance greatly increases the recurrence risk for non-muscle invasive bladder cancer (NMIBC) patients, which is still a big concern of clinicians. Understanding the mechanisms of drug resistance is of great significance for preventing and reversing it. We showed here that CXC motif chemokine ligand 5 (CXCL5) was overexpressed in mitomycin C-resistant bladder cancer cell line M-RT4. Meanwhile, parental RT4 cell treated with recombinant human CXCL5 (rhCXCL5) reduced its sensitivity to mitomycin C. Conversely, knockdown CXCL5 sensitized M-RT4 cell. We further investigated the molecular mechanisms finding that epithelial mesenchymal transition (EMT) and NF-κB pathway were activated in M-RT4 cell, which could be attenuated by knockdown CXCL5. All these data indicated that CXCL5 may promote mitomycin resistance by activating EMT and NF-κB pathway. Thus, our study identifies CXCL5 as a novel chemoresistance-related marker in NMIBC, thereby providing new strategies to overcome chemoresistance for NMIBC patients.

Determination of Apoptotic Effect of Juglone on Human Bladder Cancer TCC-SUP and RT-4 Cells: An In Vitro Study.

This study aimed to investigate the effects of juglone on the human bladder carcinoma cell lines TCC-SUB and RT-4 in monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. The activity of caspase-3 was detected in vitro using a caspase-3 colorimetric assay kit according to the manufacturer's instructions. The bromodeoxyuridine (BrdU) labeling index was used to determine the cells of the synthesis phase. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay was used to determine the death of cells in both the monolayer and spheroid cultures. The control group had a large S-phase fraction and many of the TCC-SUB and RT-4 cells nuclei were observed to be positive for BrdU. The dead cell count was higher in the TCC-SUB and RT-4 cell lines with juglone applied than in the controls. We conclude that juglone significantly inhibits the proliferation and induces the apoptosis of TCC-SUB and RT-4 cells in vitro.

Novel Application of Cell Penetrating R11 Peptide for Diagnosis of Bladder Cancer.

As a novel cell-permeable peptide, polyarginine (R11) showed great potential as contrast agent to target bladder cancer (BCa) for therapeutic applications. However, its diagnostic ability and uptake efficiency between BCa and normal bladder tissues is unknown. In this study, we investigated the feasibility of R11 conjugated with fluorescein isothiocyanate (FITC-R11) for detecting BCa in clinical practice. FITC-R11 was synthesized and incubated with BCa cell lines (T24, 5637, RT4), normal immortalized human bladder epithelial cell line (SVHUC) and clinical specimens from BCa and benign prostate hyperplasia patients. The uptake efficiency was determined by the mean values of relative FITC intensity. Furthermore, FITC-R11 was intravesically injected into the athymic nude mice bearing orthotopic T24-t tumors and pulmonary metastasis of bladder tumor mice models, the fluorescence intensity of bladder tumors and normal bladder tissues was examined, as well as lung tissues. After incubation with FITC-R11, the fluorescence intensity of T24, 5637, RT4 and SVHUC cell line was 64678.56 ± 9699.27, 46456.22 ± 2588.06, 33802.02 ± 429.72 and 17785.01 ± 1704.36, respectively (P < 0.05). In the athymic nude mice bearing orthotopic T24-t tumors, FITC-R11 showed elevated accumulation in the bladder tumors compared with normal bladder tissues, FITC-R11 was also accumulated in the lung of pulmonary metastasis mice. Moreover, the uptake efficiency of FITC-R11 in patients' BCa tissues was much higher than in normal bladder tissues (6441.95 vs. 1196.92, P < 0.05), as well as the urine samples of BCa patients and benign prostate hyperplasia patients (30250.37 vs. 4948.42, P < 0.05). As compared with normal bladder tissue, FITC-R11 is a more specific molecular probe for BCa, and has the potential application in clinical practice.

Hybrid FePt/SiO2/Au nanoparticles as a theranostic tool: in vitro photo-thermal treatment and MRI imaging.

We have produced an innovative, theranostic material based on FePt/SiO2/Au hybrid nanoparticles (NPs) for both, photo-thermal therapy and magnetic resonance imaging (MRI). Furthermore, a new synthesis approach, i.e., Au double seeding, for the preparation of Au nanoshells around the FePt/SiO2 cores, is proposed. The photo-thermal and the MRI response were first demonstrated on an aqueous suspension of hybrid FePt/SiO2/Au NPs. The cytotoxicity together with the internalization mechanism and the intracellular fate of the hybrid NPs were evaluated in vitro on a normal (NPU) and a half-differentiated cancerous cell line (RT4). The control samples as well as the normal cell line incubated with the NPs showed no significant temperature increase during the in vitro photo-thermal treatment (ΔT < 0.8 °C) and thus the cell viability remained high (∼90%). In contrast, due to the high NP uptake by the cancerous RT4 cell line, significant heating of the sample was observed (ΔT = 4 °C) and, consequently, after laser irradiation the cell viability dropped significantly to ∼60%. These results further confirm that the hybrid FePt/SiO2/Au NPs developed in the scope of this work were not only efficient but also highly selective photo-thermal agents. Furthermore, the improvement in the contrast and the easier distinction between the healthy and the cancerous tissues were clearly demonstrated with in vitro MRI experiments, proving that hybrid NPs have an excellent potential to be used as contrast agents.

Abstract 5941: Integrin signaling modulation demonstrates potential therapeutic strategy in bladder cancer using three-dimensional organoid culture

Abstract Introduction: Integrin signaling plays an important role in cellular proliferation and migration via interactions with extracellular matrix proteins. Prior studies indicate that integrin signaling facilitates tumor invasion and metastasis, and there are several ongoing clinical trials using agents that modulate this pathway. We recently identified clonal enrichment in missense mutations in the integrin cell surface interactions pathways in advanced chemotherapy-resistant urothelial carcinoma. An ideal strategy for investigating integrin signaling is via 3D organoid culture, maintaining intercellular interactions that replicate the epithelial microenvironment. We hypothesize that pharmacologic integrin signaling modulation will impair organoid growth in human bladder cancer cells and demonstrate a potential therapeutic utility for this approach. Methods: RT4 human bladder cancer cell line was used as well as a second cell line established from a patient-derived bladder cancer sample (PM748). Cells were grown in 3D organoid culture as previously described. For in vitro integrin modulation, defactinib (VS-6063), an orally-bioavailable selective inhibitor of focal adhesion kinase (FAK, a convergent and conserved enzyme activated by integrin ligand binding), was used. SDS-PAGE and immunoblotting were performed to show in vitro FAK inhibition. Single-cell suspensions and formed organoids were plated in the presence of various concentrations of defactinib to determine the impact on organoid formation and regression. Results: Both RT4 and PM748 bladder cancer cell lines demonstrated consistent organoid growth in three-dimensional culturing conditions. Addition of defactinib to cultured cells showed a dose-dependent decrease in autophosphorylation of FAK for both cell lines, demonstrating effective FAK inhibition. 3D culture of single cells in the presence of defactinib produced a dose-dependent decrease in organoid size after 96 hours (mean size for DMSO only, 100nM, 1uM, and 10uM were 128um, 75um, 48um, and 26um, respectively; p&amp;lt;0.0001 versus DMSO for all dilutions). Established bladder cancer organoids showed a dose-dependent regression in size after 72 hours of defactinib exposure (mean size for DMSO, 100nM, 1uM, and 10uM were 225um, 96um, 70um, and 34um, respectively; p&amp;lt;0.0001 versus DMSO). Experiments utilizing Crispr-Cas9-mediated FAK knock-out as well as in vivo studies with FAK inhibitors in mouse xenograft models are currently underway. Conclusions: Integrin modulation via FAK inhibition with defactinib causes both inhibition of organoid formation as well as regression of formed organoids, and the effects are seen at concentrations well below the cytotoxic range for the drug. This study suggests a utility for these agents in bladder cancer treatment. Citation Format: LaMont Barlow, Rebecca Meyer, Ethan Shelkey, Bishoy Faltas, Mark Rubin. Integrin signaling modulation demonstrates potential therapeutic strategy in bladder cancer using three-dimensional organoid culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5941. doi:10.1158/1538-7445.AM2017-5941

Abstract 5791: 3D tissue engineering bladder model for cancer invasion study

Abstract INTRODUCTION: Our understanding of the biological processes involved in bladder cancer (BCa) is greatly limited by the models currently available. In fact, the combination of in vitro and in vivo models of BCa has failed to elucidate all the fundamental aspects of the disease. The eighth most commonly diagnosed cancer in Western societies, BCa has become a growing public health concern, and more realistic models are needed to reveal the mechanisms involved in tumor initiation and progression. METHODS: Bladder substitutes have been constructed by tissue engineering with healthy human fibroblasts and urothelial cells, using the self-assembly method. Meanwhile, spheroids have been produced from non-invasive (RT4) and invasive (T24) BCa cell lines expressing DsRed fluorescent protein. The invasive potential of these spheroids was characterized in a type-I collagen gel (2.5mg/mL). Then, the spheroids were implanted on the surface of bladder substitutes, after which their development was followed by fluorescence microscopy. RESULTS: Both of the cancer cell lines used were able to form compact spheroids and grow on bladder equivalents. The invasive behaviour of spheroids varied depending on the nature of the cells used. The non-invasive RT4 cell line was unable to cross the basal lamina whereas the invasive T24 cell line was able to do so. CONCLUSION: The establishment of such a model for studying cancer biology in a physiological environment will help bridge the gap between overly simple cell culture models and more complex transgenic mice models. This new model offers a unique opportunity to study separately the players involved in the development of BCa and thus represents a powerful tool for the mechanistic analysis of this complex pathology. Citation Format: Cassandra Ringuette Goulet, Geneviève Bernard, Stéphane Chabaud, Frédéric Pouliot, Stéphane Bolduc. 3D tissue engineering bladder model for cancer invasion study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5791. doi:10.1158/1538-7445.AM2017-5791

MP48-12 EXTRAORDINARY ELEVATION OF SERPINB2 GENE AFTER ANTI-CANCER DRUG TREATMENT IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2017MP48-12 EXTRAORDINARY ELEVATION OF SERPINB2 GENE AFTER ANTI-CANCER DRUG TREATMENT IN BLADDER CANCER Miho Hiraki, Naoko Arichi, Hirofumi Kishi, Haruki Anjiki, Hiroaki Yasumoto, and Hiroaki Shiina Miho HirakiMiho Hiraki More articles by this author , Naoko ArichiNaoko Arichi More articles by this author , Hirofumi KishiHirofumi Kishi More articles by this author , Haruki AnjikiHaruki Anjiki More articles by this author , Hiroaki YasumotoHiroaki Yasumoto More articles by this author , and Hiroaki ShiinaHiroaki Shiina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1493AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The second line chemotherapy against advanced bladder cancer has not been established. We hypothesize that highly activated genes after anti-cancer drug treatment is related to the key for predicting the anti-tumor effect. For this purpose, a comprehensive analysis using cDNA microarray was performed to identify the common target genes after treatment of chemotherapeutic agent. METHODS Bladder cancer cell lines of T24, RT4, UMUC3, and prostate cancer cell line of PC3 were used as control. Clinical samples from 26 cases with muscle invasive bladder cancer who underwent radical cystectomy were also used. Anti-cancer drugs used were docetaxel (DTX), gemcitabine (GEM) and Cis-platinum (CDDP). The cell viability was evaluated by an MTT assay. Gene expression profiles in T24 cells were identified using cDNA microarray, which showed significant difference before and after treamnent of 1.1mM docetaxel (DTX). Individual gene expression was validated by quantitative RT-PCR. Functional study of the candidate genes was performed using specific siRNA transfection. RESULTS We identified SERPINB2 gene with the GO term of “negative regulation of apoptosis” as extraordinarily activated gene after DTX treatment (Z score=7.509) . In T24 and UMUC3 cells, SERPINB2 expression was significantly increased in a stepwise manner of DTX concentration, while no significant elevation of SERPINB2 was found in RT4 cells (Table). Likewise, CDDP extraordinarily enhanced the SERPINB2 expression in T24 and UMUC3, but not in RT4. On other hand, GEM significantly accelerated the SERPINB2 expression in T24, while it did not in UMUC3 and RT4. UMUC3 cells, in which high SERPINB2 expression was found before treatment, showed significant reduction of cell viability after transfection of SERPINB2 siRNA, suggesting that SERPINB2 might function as oncogene. In addition, in PC3 where SERPINB2 was highly expressed before treatment, SERPINB2 knockdown by siRNA transfection showed significant reduction of cancer cell survival. In clinical samples, there was no significant difference in SERPINB2 expression between normal and cancer tissues. Interestingly, SERPINB2 expression was significantly higher in 4 cases who underwent both neoadjuvant chemotherapy (NAC) and cystectomy than in 22 cases who did cystectomy alone. CONCLUSIONS In certain bladder cancers, SERPINB2 might function as oncogene and is highly up-regulated. Activation of SERPINB2, which is frequently found after anti-cancer agent treatment, might provide the essential information regarding the chemotherapeutic strategy in bladder cancer. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e641 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Miho Hiraki More articles by this author Naoko Arichi More articles by this author Hirofumi Kishi More articles by this author Haruki Anjiki More articles by this author Hiroaki Yasumoto More articles by this author Hiroaki Shiina More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP48-06 3D TISSUE ENGINEERING BLADDER MODEL FOR CANCER INVASION STUDY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2017MP48-06 3D TISSUE ENGINEERING BLADDER MODEL FOR CANCER INVASION STUDY Cassandra R Goulet, Geneviéve Bernard, Stéphane Chabaud, Frédéric Pouliot, and Stéphane Bolduc Cassandra R GouletCassandra R Goulet More articles by this author , Geneviéve BernardGeneviéve Bernard More articles by this author , Stéphane ChabaudStéphane Chabaud More articles by this author , Frédéric PouliotFrédéric Pouliot More articles by this author , and Stéphane BolducStéphane Bolduc More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1487AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our understanding of the biological processes involved in bladder cancer (BCa) is greatly limited by the models currently available. In fact, the combination of in vitro and in vivo models of BCa has failed to elucidate all the fundamental aspects of the disease. The eighth most commonly diagnosed cancer in Western societies, BCa has become a growing public health concern, and more realistic models are needed to reveal the mechanisms involved in tumor initiation and progression. METHODS Bladder substitutes have been constructed by tissue engineering with healthy human fibroblasts and urothelial cells, using the self-assembly method. Meanwhile, spheroids have been produced from non-invasive (RT4) and invasive (T24) BCa cell lines expressing DsRed fluorescent protein. The invasive potential of these spheroids was characterized in a type-I collagen gel (2.5mg/mL). Then, the spheroids were implanted on the surface of bladder substitutes, after which their development was followed by fluorescence microscopy. RESULTS Both of the cancer cell lines used were able to form compact spheroids and grow on bladder equivalents. The invasive behaviour of spheroids varied depending on the nature of the cells used. The non-invasive RT4 cell line was unable to cross the basal lamina whereas the invasive T24 cell line was able to do so. CONCLUSIONS The establishment of such a model for studying cancer biology in a physiological environment will help bridge the gap between overly simple cell culture models and more complex transgenic mice models. This new model offers a unique opportunity to study separately the players involved in the development of BCa and thus represents a powerful tool for the mechanistic analysis of this complex pathology. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e639 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Cassandra R Goulet More articles by this author Geneviéve Bernard More articles by this author Stéphane Chabaud More articles by this author Frédéric Pouliot More articles by this author Stéphane Bolduc More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different TP53 status.

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.

Argininosuccinate Synthetase 1 Loss in Invasive Bladder Cancer Regulates Survival through General Control Nonderepressible 2 Kinase–Mediated Eukaryotic Initiation Factor 2α Activity and Is Targetable by Pegylated Arginine Deiminase

Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

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 3068: Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model

Abstract Purpose: Paclitaxel is an effective anti-neoplastic agent in many solid tumors including bladder cancer. With the development of new drug delivery strategies, novel formulations of Cremophor EL-free paclitaxel have been developed. Here, we investigated the in vitro and in vivo anti-tumor effect of a mucoadhesive oral formulation of paclitaxel (DHP107) in comparison with iv paclitaxel in a human bladder cancer model. Methods: Four human bladder cancer cell lines (RT4, T24, J82, HT1376) and one murine bladder cancer cell line (MBT-2) were used in this study. In vitro antitumor activity and underlying mechanisms were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell-cycle analysis and apoptosis assay. In the in vivo study, RT4 cell line was implanted subcutaneously in the flank of nu/nu mice, and treated with iv paclitaxel and 2 doses of DHP107. The effects were assessed by the tumor growth, body weight and Ki-67 staining in tumor tissues. Results: The IC50 values of DHP107 ranged from 4.15 to 34.72 μg/ml with MTT assay. In RT4 cell line, both drugs induced similar G2-M arrest and apoptosis. The xenograft experiments showed that both DHP107 (per os, 50 mg/kg, equivalent of paclitaxel 10 mg/kg) and iv paclitaxel (intraperitoneal, 10 mg/kg) attenuated the growth of RT4 tumors in mice, equivalently. The higher dose (per os, 100 mg/kg) of DHP107 resulted in increased efficacy compared with the lower dose (50 mg/kg) without weight loss in mice. However, significant weight loss was observed in iv paclitaxel (-20% at Day 11) treated group. We observed significantly decreased proliferation with Ki-67 staining in DHP107 (100 mg/kg) treated group than in DHP107 (50 mg/kg) or iv paclitaxel (10 mg/kg) treated groups. Conclusion: Our findings provide the evidence that DHP107 showed comparable antitumor effect with iv paclitaxel and is a promising anti-tumor agent in bladder cancer. Clinical studies in patients with bladder cancer warrant further investigation. Citation Format: Se Hyun Kim, Jung Min Kim, Tae Soo Kim, Won Suk Lee, Woo Sun Kwon, Seung Tak Lim, Joong Bae Ahn, Se-Kyu Kim, Yeong Woo Jo, Sun Young Rha. Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model. [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 3068.

MP61-20 PRIMARY APPLICATION STUDY IN EARLY DIAGNOSIS OF BLADDER CANCER BY CELL-PERMEABLE PEPTIDES R11

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2016MP61-20 PRIMARY APPLICATION STUDY IN EARLY DIAGNOSIS OF BLADDER CANCER BY CELL-PERMEABLE PEPTIDES R11 Jinhai Fan, Chen Ding, Yiqing Du, and Kaijie Wu Jinhai FanJinhai Fan More articles by this author , Chen DingChen Ding More articles by this author , Yiqing DuYiqing Du More articles by this author , and Kaijie WuKaijie Wu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.894AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The aim of this study is to investigate the feasibility of cell-permeable peptides R11 conjugated with fluorescein isothiocyanate (FITC-R11) for detecting bladder cancer. METHODS FITC-R11 was synthesized and incubated with bladder cancer cell lines (T24, 5637, RT4), normal immortalized human bladder epithelial cell line (SVHUC) and clinical specimens from bladder cancer and benign prostate hyperplasia patients. The uptake efficiency was determined by the mean values of relative FITC intensity. Furthermore, FITC-R11 was intravesically injected into the athymic nude mice bearing orthotopic T24-t tumors, and the fluorescence intensity of bladder tumors and normal bladder tissues was examined. RESULTS After incubation with FITC-R11, the fluorescence intensity in T24, 5637, RT4 and SVHUC cell line was 64678.56±9699.27, 46456.22±2588.06, 33802.02±429.72 and 17785.01±1704.36, respectively (P ≤ 0.05). The uptake efficiency of FITC-R11 in bladder cancer tissues was much higher than normal bladder tissues (6441.95 vs 1196.92, P ≤ 0.05), as well as the urine samples of bladder cancer patients and benign prostate hyperplasia patients (30250.37 vs 4948.42, P ≤ 0.05); In the athymic nude mice bearing orthotopic T24-t tumors, FITC-R11 also showed elevated accumulation in the bladder tumor compared with normal bladder. CONCLUSIONS Our results demonstrated that FITC-R11 is a specific molecular probe for detecting bladder cancer in vitro and in vivo. The results presented within this pilot study warrant further research to determine the potential use of FITC-R11 in diagnostic of bladder cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e811 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jinhai Fan More articles by this author Chen Ding More articles by this author Yiqing Du More articles by this author Kaijie Wu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP68-10 EXPRESSION OF TFAP2C IS ASSOCIATED WITH THE BASAL MOLECULAR SUBTYPE OF BLADDER CANCER AND INCREASED TUMOR AGGRESSIVENESS

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2015MP68-10 EXPRESSION OF TFAP2C IS ASSOCIATED WITH THE BASAL MOLECULAR SUBTYPE OF BLADDER CANCER AND INCREASED TUMOR AGGRESSIVENESS Hironobu Yamashita, Zongyu Zheng, Vasty Osei Amponsa, Jay Raman, and David DeGraff Hironobu YamashitaHironobu Yamashita More articles by this author , Zongyu ZhengZongyu Zheng More articles by this author , Vasty Osei AmponsaVasty Osei Amponsa More articles by this author , Jay RamanJay Raman More articles by this author , and David DeGraffDavid DeGraff More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2472AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Expression of the transcription factor, Forkhead Box A1 (FOXA1) is absent in 40% of invasive urothelial cell carcinoma and 80% of squamous cell carcinomas of the bladder, while it is expressed in normal urothelium and non-invasive bladder cancers. Recent work indicates that bladder tumors molecularly characterized as “basal” exhibit decreased levels of FOXA1 expression and are enriched for squamous differentiation. Therefore, we endeavored to identify molecular factors regulated by FOXA1 that may also be associated with a basal molecular phenotype. METHODS Knock-down (KD) and overexpression studies followed by microarray analysis were performed to identify FOXA1-regulated genes. Western blotting analysis and PCR confirmed microarray findings. Immunohistochemistry (IHC) of a tissue microarray consisting of 300 clinical bladder cancer samples was performed to determine the expression of FOXA1-regulated genes in human bladder cancer tissue. Crystal violet growth assays and tissue recombination xenografting determined the impact of altered TFAP2 family members on bladder cancer cell proliferation and invasion. RESULTS To identify FOXA1-regulated genes associated with a basal molecular phenotype and important in bladder cancer, FOXA1 was knocked down and overexpressed in RT4 and T24 cell lines, respectively, with subsequent microarray analysis. This approach identified TFAP2C as a novel FOXA1-repressed gene. Western blotting analysis and PCR confirmed our microarray findings, and also identified additional TFAP2 family members (TFAP2A, TFAP2B, TFAP2D and TFAP2E) significantly regulated by FOXA1. IHC of a tissue microarray consisting of over 300 patients revealed that increased TFAP2C expression is inversely correlated with FOXA1 expression (p=0.007; rho= -0.13). Additionally, IHC revealed that increased TFAP2C expression is associated with expression of CK14 (p<0.001; rho=0.36), a marker of a basal phenotype in human bladder cancer. Overexpression of TFAP2C in RT4, T24 and HTB9 cell lines resulted in significant increases in cell proliferation and in vivo tumorigenicity (p<0.01). CONCLUSIONS We have identified TFAP2C as FOXA1-regulated transcription factor Furthermore, our data suggest that increased expression of TFAP2 family members may be partly responsible for the aggressive nature of the basal molecular subtype of human bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e861 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hironobu Yamashita More articles by this author Zongyu Zheng More articles by this author Vasty Osei Amponsa More articles by this author Jay Raman More articles by this author David DeGraff More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 5436: Potential of asparagine depletion for the treatment of bladder cancer and other urological tumors

Abstract Due to asparagine synthetase (ASNS) deficiency, tumors cells auxotrophic for L-asparagine lack capability to synthesize this amino acid and depend to circulating L-asparagine for survival. Based on this rational, L-asparaginase (L-Aspa), an enzyme depleting L-asparagine, is an essential drug in the treatment of acute lymphoblastic leukemia (ALL), as these hematological cancer cells express low levels of ASNS. Recent evidence suggests that some solid tumors are also deficient in ASNS (Lorenzi, 2008; Dufour, 2012; Zhang, 2013), providing rationale for testing L-Aspa treatment in other contexts. In this study, we investigated the levels of ASNS expression in tumor tissues from patients with urological cancers (bladder, prostate and kidney) and we investigated the sensitivity to L-Aspa of bladder and prostate cell lines. Material &amp; Method: ASNS expression was evaluated by a validated immunohistochemistry (IHC) method on tissue biopsie microarrays (USBiomax, Rockville, MD) including 384 cases of bladder, 94 cases of prostate and 75 cases of kidney tumors. A score was attributed to each tumor based on ASNS IHC labeling intensity from 0 (no labeling) to 3 (strong labeling). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. Sensitivity to L-Aspa (expressed as an IC50) was assessed in vitro by measuring the cell viability of bladder cancer cell lines (RT-4 and UM-UC-3) and prostate cancer cell line PC-3 in the presence of various concentrations of L-Aspa. ASNS expression in cell lines was evaluated by western-blot. Results: ASNS expression was low/null in 77.3% of bladder tumors, 86.2% of prostate tumors and 72% of kidney tumors. In bladder tumors, no correlation was found between the grade of the tumor and its expression of ASNS. Bladder and prostate cancer cell lines were all sensitive to L-Aspa (IC50 = 0.31, 0.12 and 0.22 IU/mL for RT-4, UM-UC-3 and PC-3 cell lines, respectively) at a level similar to the sensitivity observed with the L-Aspa-sensitive MOLT-4 ALL cell line (IC50=0.19 IU/mL). All cell lines displayed low basal expression of ASNS. Conclusion: The sensitivity of bladder and prostate cancer cell lines to L-Aspa and the low/no expression of ASNS in the majority of urological tumor biopsies suggest that an extensive population of patients with urological tumors may respond to asparagine depletion. However, L-Aspa is accompanied with well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, liver failure) and this drug should be used very carefully especially in older or frail patients. A new safer formulation is requested to make easier the clinical development of L-Aspa in urological cancer. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Fabien Gay, Gautam Borthakur, Yann Godfrin. Potential of asparagine depletion for the treatment of bladder cancer and other urological tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5436. doi:10.1158/1538-7445.AM2014-5436

Downregulation of HIPK2 increases resistance of bladder cancer cell to cisplatin by regulating Wip1.

Cisplatin-based combination chemotherapy regimen is a reasonable alternative to cystectomy in advanced/metastatic bladder cancer, but acquisition of cisplatin resistance is common in patients with bladder cancer. Previous studies showed that loss of homeodomain-interacting protein kinase-2 (HIPK2) contributes to cell proliferation and tumorigenesis. However, the role of HIPK2 in regulating chemoresistance of cancer cell is not fully understood. In the present study, we found that HIPK2 mRNA and protein levels are significantly decreased in cisplatin-resistant bladder cancer cell in vivo and in vitro. Downregulation of HIPK2 increases the cell viability in a dose- and time-dependent manner during cisplatin treatment, whereas overexpression of HIPK2 reduces the cell viability. HIPK2 overexpression partially overcomes cisplatin resistance in RT4-CisR cell. Furthermore, we showed that Wip1 (wild-type p53-induced phosphatase 1) expression is upregulated in RT4-CisR cell compared with RT4 cell, and HIPK2 negatively regulates Wip1 expression in bladder cancer cell. HIPK2 and Wip1 expression is also negatively correlated after cisplatin-based combination chemotherapy in vivo. Finally, we demonstrated that overexpression of HIPK2 sensitizes chemoresistant bladder cancer cell to cisplatin by regulating Wip1 expression.ConclusionsThese data suggest that HIPK2/Wip1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of bladder cancer.

Quinovic acid glycosides purified fraction from Uncaria tomentosa induces cell death by apoptosis in the T24 human bladder cancer cell line

Bladder cancer is the second most prevalent malignancy in the genitourinary tract and remains a therapeutic challenge. In the search for new treatments, researchers have attempted to find compounds with low toxicity. With this goal in mind, Uncaria tomentosa is noteworthy because the bark and root of this species are widely used in traditional medicine and in adjuvant therapy for the treatment of numerous diseases. The objective of this study was to investigate the antitumor effect of one purified bioactive fraction of U.tomentosa bark on cell proliferation in two human bladder cancer cell lines, T24 and RT4. Quinovic acid glycosides purified fraction (QAPF) of U.tomentosa decreased the growth and viability of both T24 and RT4 cell lines. In T24 cells, QAPF induced apoptosis by activating caspase-3 and NF-κB. Further study showed that this fraction does not induce cell cycle arrest and does not alter PTEN and ERK levels. In conclusion, we demonstrated that QAPF of U.tomentosa has a potent inhibitory effect on the growth of human bladder cancer cell lines by inducing apoptosis through modulation of NF-κB, and we suggest that QAPF may become a potential therapeutic agent for the prevention and/or treatment of this cancer.

Phosphorylated p70S6K in noninvasive low-grade urothelial carcinoma of the bladder: correlation with tumor recurrence

We investigated whether inhibiting phosphorylated p70S6K (p-p70S6K) suppresses the proliferation and growth of noninvasive low-grade urothelial carcinoma (LG-URCa) in vitro and whether p-p70S6K can serve as a predictive biomarker for the recurrence of noninvasive LG-URCa of the bladder in patients. We constructed a tissue microarray (TMA) for 95 LG-URCa and 35 benign urothelium samples and performed immunohistochemical staining for p-p70S6K and p-4E-BP1. A Cox regression model was used to investigate the predictive factors for recurrence of LG-URCa. We investigated the dose-dependent antiproliferative effect of rapamycin, its antiproliferative effect and the growth-inhibition effect of p70S6K siRNA transfection in RT4 and 253J cell lines. The pT1 staged group (P < 0.05; hazard ratio (HR), 2.415) and the high p-p70S6K staining group (P < 0.05; HR, 2.249) were independent factors for predicting recurrence. Rapamycin inhibited RT4 and 253J cell proliferation in a dose-dependent manner (r = −0.850, P < 0.001 in RT4 cells; r = −0.835, P < 0.001 in 253J cells). RT4 and 253J cell proliferation and growth were inhibited by the transfection of p70S6K siRNA and rapamycin, respectively (P < 0.05). Transfection of p70S6K siRNA resulted in inhibitory effects on cell proliferation and growth that were similar to those of rapamycin. Our results suggest that inhibiting p70S6K phosphorylation is important to prevent recurrence and that p70S6K phosphorylation can be used as a molecular biomarker to predict recurrence of certain LG-URCa of the bladder.

The effect of photochemical internalization of bleomycin in the treatment of urothelial carcinoma of the bladder: An in vitro study

ObjectivesIn this in vitro study, we determined whether meso-tetraphenyl chlorin disulphonate (TPCS2a)-based photochemical delivery of bleomycin was able to potentiate the cytotoxicity of bleomycin on bladder cancer cells. Materials and methodsThe human RT4, RT112, 253J, T24, and rat AY-27 urothelial carcinoma cell lines were used. Cells were seeded in 96-well plates. TPCS2a was added to the growth medium and the plates were incubated overnight. Cells were then resuspended in TPCS2a-free culture medium and incubated for 3 hours. Subsequently, cells were treated for 60 minutes with increasing doses of epirubicin, gemcitabine, mitomycin C, or bleomycin followed by illumination for different periods. Cell viability was measured with a colorimetric assay after 72 hours. ResultsFor the single treatments, in all 5 cell lines a dose-dependent inhibition of cell proliferation was observed. This was seen both after treatment with TPCS2a-based photodynamic therapy (PDT), as well as after treatment with either bleomycin or one of the control chemotherapeutic agents.After treatment with PDT (240-s illumination), bleomycin 9.0µM, and the combination of these treatments, relative survival percentages were 89.2±13.0, 70.2±8.9, and 30.5±6.1, respectively, in the T24 cell line. After treatment with PDT (120-s illumination), bleomycin 27µM and the combination of these treatments, relative survival percentages were 93.6±15.7, 74.7±9.6, and 30.0±11.1, respectively, in the AY-27 cell line. In both cell lines, PDT combined with bleomycin showed significantly (P<0.001) higher cell kill than the sum of the single treatments, suggesting a photochemical internalization effect. ConclusionsTPCS2a-based photochemical internalization of bleomycin showed a significant, at least, additive antiproliferative activity against human and rat urothelial carcinoma cells in vitro. Thus, photochemical internalization may have therapeutic potential as an intravesical strategy against bladder cancer. As the effect is heterogeneous, biomarker studies are warranted to be able to predict the effects of a photochemical internalization–based treatment.