Bladder Cancer Xenografts Research Articles

Evaluation of Su Fu'ning Lotion's Inhibitory Effects on Bladder Cancer Cells In Vitro and In Vivo by Intravesical Instillation.

Bladder cancer is a common malignant tumor with a very high recurrence rate after surgery. Intravesical instillation can help clear up the residual tumor cells after surgery and thereby reduce the recurrence rate. To establish a bladder tumor transplantation animal model and to evaluate the inhibitory effects of a novel perfusate, Su Fu'ning Lotion (SFN), on bladder tumor. SFN was compared with several commonly used chemotherapy drugs, including mitomycin (MMC) and pirarubicin (THP) for anticancer effects on the bladder cancer cell lines T24, BTT, and BIU-87 and SFN half inhibitory concentrations (IC50) were determined after 48 hours of treatment. In addition, bladder cancer orthotopic transplantation tumor models were established in BALB/C nude mice and T739 mice, and SFN anticancer effects were assessed in vivo, with normal saline and MMC as negative and positive controls, respectively. SFN, MMC, and THP were all lethal to bladder cancer cells, in vitro, with SFN and THP significantly superior to MMC. IC50 values for SFN were 13.22, 11.22, and 12.5 µg/mL on T24, BTT, and BIU-87 cells, respectively. In vivo, SFN significantly reduced the mouse bladder wet weight and prolonged the animal survival compared with controls (P < .05), suggesting that SFN significantly inhibited T24/BTT cell growth in mice. SFN inhibited the bladder cancer cell proliferation in vitro and in vivo and significantly prolonged the survival of mice with bladder cancer xenografts, indicating that SFN could be used as a perfusate after surgery for removal of residual bladder cancers cells.

Development and Characterization of Bladder Cancer Patient-Derived Xenografts for Molecularly Guided Targeted Therapy

BackgroundThe overarching goal of this project is to establish a patient-derived bladder cancer xenograft (PDX) platform, annotated with deep sequencing and patient clinical information, to accelerate the development of new treatment options for bladder cancer patients. Herein, we describe the creation, initial characterization and use of the platform for this purpose.Methods and FindingsTwenty-two PDXs with annotated clinical information were established from uncultured unselected clinical bladder cancer specimens in immunodeficient NSG mice. The morphological fidelity was maintained in PDXs. Whole exome sequencing revealed that PDXs and parental patient cancers shared 92–97% of genetic aberrations, including multiple druggable targets. For drug repurposing, an EGFR/HER2 dual inhibitor lapatinib was effective in PDX BL0440 (progression-free survival or PFS of 25.4 days versus 18.4 days in the control, p = 0.007), but not in PDX BL0269 (12 days versus 13 days in the control, p = 0.16) although both expressed HER2. To screen for the most effective MTT, we evaluated three drugs (lapatinib, ponatinib, and BEZ235) matched with aberrations in PDX BL0269; but only a PIK3CA inhibitor BEZ235 was effective (p<0.0001). To study the mechanisms of secondary resistance, a fibroblast growth factor receptor 3 inhibitor BGJ398 prolonged PFS of PDX BL0293 from 9.5 days of the control to 18.5 days (p<0.0001), and serial biopsies revealed that the MAPK/ERK and PIK3CA-AKT pathways were activated upon resistance. Inhibition of these pathways significantly prolonged PFS from 12 day of the control to 22 days (p = 0.001). To screen for effective chemotherapeutic drugs, four of the first six PDXs were sensitive to the cisplatin/gemcitabine combination, and chemoresistance to one drug could be overcome by the other drug.ConclusionThe PDX models described here show good correlation with the patient at the genomic level and known patient response to treatment. This supports further evaluation of the PDXs for their ability to accurately predict a patient’s response to new targeted and combination strategies for bladder cancer.

Abstract 1462: Characterization of patient-derived bladder cancer xenografts: role of xCT in response to cisplatin

Abstract Background: A major challenge in cancer drug development has been largely attributed to the inability of cell lines to recapitulate the heterogeneity of human tumors. Patient derived xenografts (PDX) represent a major advance as they are more representative of the clinical setting. However, appropriate characterization of PDXs is necessary to guide biomarker driven drug discovery research. Methods: H&amp;E staining was used to compare the original patient tumors with PDX developed from cystectomy specimens to validate morphological similarity. Immunohistochemical (IHC) staining for cytokeratins (CK) 5/6 &amp; 20 and EMT markers E cadherin and vimentin were used to identify whether these tumors represent a basal or a luminal subtype. In addition, Western blot and RT-PCR analysis were performed to assess markers of epithelial-to-mesenchymal transition (EMT). Therapeutic response to cisplatin was evaluated both in vitro and in vivo. Furthermore, expression of xCT (cystine transporter), one of the known cisplatin resistance markers, was determined by RT-PCR, Western blot and IHC in PDXs and cells. Results: Morphologically, we observed that our PDX models (RP-B-01 and RP-B-02) did not drift from the original patient tumors and they still retained their characteristic stromal features. Based on cytokeratin (CK) staining, we found both models to be CK 5/6 positive and CK 20 negative, suggesting a basal subtype of bladder cancer. Both PDXs, similarly to the original tumors, were E cadherin positive. However, cells derived from these tumors and cultured in vitro lost E cadherin and acquired vimentin expression, suggesting an EMT phenotype. When cells from PDX were transplanted back into mice they re-expressed E cadherin and lost vimentin expression, suggesting a reversion to MET. Despite no difference in cytokeratins and EMT markers between the two models, both PDX and cells isolated from PDXs had differential expression of xCT and possessed differential response to cisplatin treatment. RP-B01 expressed higher levels of xCT and was less responsive to cisplatin as compared to RP-B-02 which expressed lower levels of xCT. Conclusion: Differential expression of xCT in bladder cancer PDXs and cells correlated with cisplatin response, suggesting that pharmacological targeting of xCT in combination therapies will enhance cisplatin efficacy in bladder cancer. Citation Format: May F. Elbanna, Eric Ciamporcero, Swathi Ramakrishnan, Remi Adelaiye, Li Shen, Ashley Orillion, Sheng-Yu Ku, Sreenivasulu Chintala, Roberto Pili. Characterization of patient-derived bladder cancer xenografts: role of xCT in response to cisplatin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2015-1462

Targeting HER2 with T-DM1, an Antibody Cytotoxic Drug Conjugate, is Effective in HER2 Over Expressing Bladder Cancer

Systemic therapy for advanced bladder cancer has not changed substantially in more than 2 decades and mortality rates remain high. The recognition of HER2 over expression in bladder cancer has made HER2 a promising therapeutic target. T-DM1, a new drug consisting of the HER2 antibody trastuzumab conjugated with a cytotoxic agent, has been shown in breast cancer to be superior to trastuzumab. We tested T-DM1 in preclinical models of bladder cancer. We evaluated the effect of T-DM1 compared to trastuzumab in different in vitro and in vivo models of HER2 over expressing bladder cancer. RT4V6 was the highest HER2 expressing bladder cancer cell line and it showed higher growth inhibition with T-DM1 compared to trastuzumab. T-DM1 but not trastuzumab induced apoptosis of RT4V6 cells after G2/M arrest on cell cycle analysis. HER2 expression was higher in cell lines with acquired cisplatin resistance compared to the corresponding parental cell lines. Resistant cells showed higher sensitivity to T-DM1 by the induction of apoptosis. In addition, cells cultured in anchorage independent conditions increased HER2 expression compared to cells cultured in adherent conditions and T-DM1 significantly inhibited colony formation in soft agar compared to trastuzumab. In an orthotopic bladder cancer xenograft model tumor growth of cisplatin resistant RT112 was significantly inhibited by T-DM1 via the induction of apoptosis compared to treatment with control IgG or trastuzumab. T-DM1 has promising antitumor effects in preclinical models of HER2 over expressing bladder cancer.

Patient-derived bladder cancer xenografts in the preclinical development of novel targeted therapies

Optimal animal models of muscle invasive bladder cancer (MIBC) are necessary to overcome the current lack of novel targeted therapies for this malignancy. Here we report on the establishment and characterization of patient-derived primary xenografts (PDX). Patient tumors were grafted under the renal capsule of mice and subsequently transplanted over multiple generations. Patient tumor and PDX were processed for analysis of copy number variations by aCGH, gene expression by microarray, and expression of target pathways by immunohistochemistry (IHC). One PDX harbouring an FGFR3 mutation was treated with an inhibitory monoclonal antibody targeting FGFR3. Five PDX were successfully established. Tumor doubling time ranged from 5 to 11 days. Array CGH revealed shared chromosomal aberrations in the patient tumors and PDX. Gene expression microarray and IHC confirmed that PDXs maintain similar patterns to the parental tumors. Tumor growth in the PDX with an FGFR3 mutation was inhibited by the FGFR3 inhibitor. PDXs recapitulate the tumor biology of the patients' primary tumors from which they are derived. Investigations related to tumor biology and drug testing in these models are therefore more likely to be relevant to the disease state in patients. They represent a valuable tool for developing precision therapy in MIBC.

Development and characterization of patient-derived xenografts to guide precision medicine in bladder cancer.

e15522 Background: The objective of this project was to establish a patient-derived bladder cancer xenograft (PDX) platform annotated with genomic and patient clinical information to guide precisio...

Patient-derived bladder cancer xenografts as a platform for drug development in bladder cancer.

e15528 Background: The goal of this project is to develop and characterize a bladder cancer patient-derived xenograft (PDX) platform, and use this platform to determine the efficacy of bladder canc...

Intravesical administration of exogenous microRNA-145 as a therapy for mouse orthotopic human bladder cancer xenograft.

We previously reported that the level of microRNA (miR)-145 is attenuated in human bladder cancer cells. In this current study, we investigated whether intravesical administration of miR-145 could be a potential therapeutic strategy for controlling bladder cancer by using an orthotopic human bladder cancer xenograft model. Following transfection of 253J B-V cells with miR-145, the effects of the ectopic expression of miR-145 were examined by performing MTT, Western blotting analysis, Hoechst33342 staining, and wound healing assay in vitro. Also, a mouse orthotopic human bladder cancer model was established by inoculating 253J B-V cells into the bladder wall of mice. The anti-cancer effects of intravesical injections of miR-145 into these mice were then assessed. Transfection of 253J B-V cells with miR-145 induced apoptosis and suppression of cell migration in vitro. Western blotting showed that the levels of c-Myc, socs7, FSCN1, E-cadherin, β-catenin, and catenin δ-1 were decreased and that the PI3K/Akt and Erk1/2 signaling pathways were increased in compensatory fashion. In vivo, mice treated with miR-145 showed 76% inhibition of tumor growth, with a significant prolongation of animal survival (p = 0.0183 vs. control). Western blotting showed that both apoptosis and cell motility-related genes were significantly decreased as seen in vitro. Furthermore, PI3k/Akt and Erk1/2 signaling pathways, which were activated in a compensatory manner in vitro, were decreased in vivo. Intravesical administration of exogenous miR-145 was thus concluded to be a valid therapy for bladder cancer in this human bladder cancer xenograft model.

A microdosing study to identify chemoresistance in bladder cancer.

356 Background: DNA adduct formation and incorporation of gemcitabine into genomic DNA are critical steps in cancer cell response to platinum (Pt) and gemcitabine chemotherapy, respectively. We hypothesize that levels of Pt-DNA adducts and gemcitabine in genomic DNA below a threshold are predictive of chemoresistance. Accelerator mass spectrometry (AMS) is an ultrasensitive method for measuring radiocarbon. By measuring 14C bound to DNA, AMS was used to quantify carboplatin-DNA damage and gemcitabine incorporation into DNA after mice or patients received nontoxic “microdoses” of 14C-labeled carboplatin or gemcitabine. Methods: Cancer cells and mice bearing tumor xenografts were treated with one microdose (1% of the therapeutic dose) or therapeutic dose of [14C]carboplatin or [14C]gemcitabine. Carboplatin-DNA adducts and gemcitabine incorporation in DNA were correlated with cell/tumor response to chemotherapy. In the Phase 0 trial, patients with advanced bladder or non-small cell lung cancer were treated with one microdose of [14C]carboplatin followed by tumor sampling 24 hours later. Carboplatin-DNA adducts and other relevant parameters, such as pharmacokinetics and repair of DNA damage, were measured and correlated with cancer response to chemotherapy. Results: The levels of microdose-induced carboplatin-DNA damage were linearly proportional to that caused by the therapeutic dose (R2=0.92, p&lt;0.001); and correlated with chemoresistance to carboplatin. Low gemcitabine incorporation into DNA correlated to gemcitabine resistance in patient-derived bladder cancer xenografts (p&lt;0.001). In the Phase 0 trial, 18 patients have been enrolled. DNA damage induced by carboplatin was measured by AMS in peripheral blood mononuclear cells and, in some patients, in tumor tissues; and will be correlated with cancer response to chemotherapy. The pharmacokinetics and DNA adduct levels were linear between microdose and therapeutic doses. No microdose-related toxicity was observed. The radiation exposure was less than that of one chest X-ray per microdose. Conclusions: The levels of DNA damage induced by nontoxic microdosing carboplatin can potentially predict chemoresistance. The current status of the Phase 0 trial will be presented. Clinical trial information: NCT01261299.

Establishment of a Novel Bladder Cancer Xenograft Model in Humanized Immunodeficient Mice

Background/Aims: The aim of this study was to develop a novel model by transplanting human bladder cancer xenografts into humanized immunodeficient mice (SCID). Methods: The animals first underwent sublethal irradiation and then were subjected to simultaneous transplantation of human lymphocytes (5 × 10⁷ cells/mouse i.p.) and human bladder cancer cells (3 × 10⁶ cells/mouse s.c.). Results: The xenografts developed in all 12 mice that had received bladder cancer BIU-87 cells, and the tumor specimens were evaluated histologically. All 6 model mice expressed human CD3 mRNA and/or protein in the peripheral blood, spleens and xenografts. The mean proportion of human CD3⁺ cells was 19% with a level of human IgG 532.4µ/ml in the peripheral blood at Week 6 after transplant inoculation. The re-constructed human immune system in these mice was confirmed to be functional by individual in vitro testing of their proliferative, secretory and cytotoxic responses. Conclusion: The successful engraftment of the human bladder cancer xenografts and the establishment of the human immune system in our in vivo model described here may provide a useful tool for the development of novel therapeutic strategies targeting at bladder cancer.

MMP-1 and Pro-MMP-10 as potential urinary pharmacodynamic biomarkers of FGFR3-targeted therapy in patients with bladder cancer.

The aim of this study was to identify noninvasive pharmacodynamic biomarkers of FGFR3-targeted therapies in bladder cancer to facilitate the clinical development of experimental agent targeting FGFR3. Potential soluble pharmacodynamic biomarkers of FGFR3 were identified using a combination of transcriptional profiling and biochemical analyses in preclinical models. Two matrix metalloproteinases (MMP), MMP-1 and MMP-10, were selected for further studies in human bladder cancer xenograft models treated with a specific anti-FGFR3 monoclonal antibody, R3Mab. Serum and urinary levels of MMP-1 and MMP-10 were determined in healthy donors and patients with bladder cancer. The modulation of MMP-1 and MMP-10 by R3Mab in patients with bladder cancer was further evaluated in a phase I dose-escalation study. MMP-1 and MMP-10 mRNA and protein were downmodulated by FGFR3 shRNA and R3Mab in bladder cancer cell lines. FGFR3 signaling promoted the expression and secretion of MMP-1 and pro-MMP-10 in a MEK-dependent fashion. In bladder cancer xenograft models, R3Mab substantially blocked tumor progression and reduced the protein levels of human MMP-1 and pro-MMP-10 in tumor tissues as well as in mouse serum. Furthermore, both MMP-1 and pro-MMP-10 were elevated in the urine of patients with advanced bladder cancer. In a phase I dose-escalation trial, R3Mab administration resulted in an acute reduction of urinary MMP-1 and pro-MMP-10 levels in patients with bladder cancer. These findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic biomarkers for R3Mab in patients with bladder cancer.

Blocking PGE2-induced tumour repopulation abrogates bladder cancer chemoresistance.

Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.

Abstract 1928: Reporter construct for functional and real-time evaluation of cytokeratin 14+ bladder cancer stem cells

Abstract Bladder cancer is the fifth most common malignancy in the US. We recently reported that bladder cancer stem cells isolated by cell surface markers (i.e. CD44/CD90/CD49f) express a higher level of the intermediate filament cytokeratin 14 (CK14). Importantly, patients with a higher fraction of CK14+ cancer cells correlated with worse survival outcomes. Therefore, this CK14+ cancer cell subpopulation warrants further functional evaluation and characterization. In the current study, we report the generation and characterization of a lentiviral reporter construct that carries the gene promoter region of human KRT14 gene upstream to a red fluorescent protein (tdTomato; Tm). With this reporter stably integrated into the genome of bladder cancer cells, the viable CK14+ cancer cell subpopulation could be isolated by FACS and visualized by fluorescent microscopy. We first validated the reporter by demonstrating that Tm+ cancer cells indeed express a higher level of KRT14 mRNA and relatively lower levels of differentiated cell markers (i.e. KRT18 and UPK1B) by qPCR. Next, we verified both in vitro and in vivo that CK14+/Tm+ cancer cells have functional properties of cancer stem cells by demonstrating their enriched sphere-forming ability and proficient engraftment as xenograft tumors in immunocompromised mice. Since neoadjuvant chemotherapy is a well-established treatment approach in patients with high-risk bladder cancer, we investigated CK14+ and CK14- cancer cells' response to the cytotoxic chemotherapy, gemcitabine/cisplatin (GC). FACS-purified CK14+/Tm+ cancer cells were more resistant to GC chemotherapy in vitro than CK14-/Tm- cancer cells. These findings were confirmed with in vivo studies using both patient-derived xenografts and immortalized bladder cancer xenografts. GC treated xenografts demonstrated a greater expansion of CK14+ cancer cells than vehicle treated controls. Additionally, we obtained a panel of human bladder cancer specimens from patients before and after neoadjuvant GC chemotherapy (n=15) and evaluated the CK14 status. An expansion or persistence of an infiltrating pattern of CK14 in post-neoadjuvant GC chemotherapy tumor specimens was associated with worse overall survival. Collectively, these findings verified the unique intrinsic biological properties of CK14+/Tm+ bladder cancer cells and their response to GC chemotherapy. Ongoing experiments using fluorescence live imaging will evaluate Tm+/- bladder cancer cells' response to chemotherapy. The capacity to observe this CK14+/Tm+ subpopulation and their trace response to cytotoxic chemotherapy in these studies will open up new avenues to study the mechanisms of chemoresistance. Citation Format: Philip L. Ho, Antonina Kurtova, Jing Xiao, Ross Krasnow, Erica Lay, Senthil Pazhanisamy, Seth P. Lerner, Keith S. Chan. Reporter construct for functional and real-time evaluation of cytokeratin 14+ bladder cancer stem cells. [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 1928. doi:10.1158/1538-7445.AM2014-1928

Abstract 3690: MMP-1 and pro-MMP-10 as potential urinary pharmacodynamic biomarkers of FGFR3-targeted therapy in patients with bladder cancer

Abstract Aberrant activation of fibroblast growth factor receptor 3 (FGFR3) via overexpression or mutation occurs frequently in bladder cancer, and experimental therapeutic agents have entered into clinical trials to evaluate the potential activity of FGFR3-targeted therapy in this disease. However, a convenient accessible biomarker indicative of targeted pathway modulation in vivo drug activity is urgently needed. Through transcriptional profiling of bladder carcinoma cells expressing inducible FGFR3 short hairpin RNAs, we identified two matrix metalloproteinases (MMPs), MMP-1 and MMP-10, as potential downstream targets of FGFR3. We found that FGFR3 signaling promotes expression and secretion of MMP-1 and pro-MMP-10 in a MEK-dependent fashion. FGFR3 inhibition by an investigational anti-FGFR3 human monoclonal antibody, R3Mab, in bladder cancer xenograft models substantially blocked tumor progression and reduced the protein levels of human MMP-1 and pro-MMP-10 in tumor tissues as well as in mouse serum. Furthermore, we detected elevated levels of both MMP-1 and pro-MMP-10 in the urine of patients with advanced bladder cancer. In a phase 1 dose-escalation trial, R3Mab administration resulted in an acute reduction of urinary MMP-1 and pro-MMP-10 levels in bladder cancer patients. Together, these findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic biomarkers for FGFR3-targeted therapy in bladder cancer. Citation Format: Benjamin C. Lin, Xiangnan Du, Qian-Rena Wang, Hao Li, Ellen Ingalla, Janet Tien, Isabelle Rooney, Avi Ashkenazi, Elicia Penuel, Jing Qing. MMP-1 and pro-MMP-10 as potential urinary pharmacodynamic biomarkers of FGFR3-targeted therapy in patients with bladder cancer. [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 3690. doi:10.1158/1538-7445.AM2014-3690

Abstract 387: Activating transcription factor 3 (ATF-3) expression is a potential marker of tumor response to the HDAC inhibitor Pracinostat

Abstract BACKGROUND: Bladder cancer is the fifth most common cancer affecting the Western world. About 90% of bladder cancer is superficial and the remaining 10% is muscle invasive. Surgery followed by platinum based chemotherapy is the standard treatment for high grade tumors, and results in &amp;lt;50% 5 year survival rate following surgery. ATF3 can play a key role as either an oncogenic suppressor or an inducer depending on the degree of malignancy. Decreased ATF3 expression in bladder cancer has been associated with tumor progression and a reduced survival rate [1]. Pracinostat (SB939) is an orally available highly potent histone deacetylase inhibitor with a sustained inhibitory effect on tumor tissues [2]. This study aimed to determine whether ATF3 expression which is silenced in bladder cancer could be induced by Pracinostat and used as a potential marker of response in HDACi mediated cancer therapy. METHODS: A series of human bladder cancer cell lines of different grades and origin were utilized to characterize ATF3 expression. Proliferation, clonogenecity and migratory properties of Pracinostat treated cells were assayed by vialight, soft agar and scratch wound methods respectively. Athymic balb/c mice were used to study in vivo anti tumor effect. RESULTS: Treatment with Pracinostat activated the expression of ATF3 in a time and dose depended manner coincident with acetylation of histone 3 and 4. Pracinostat treated cells exhibited a dose depended effect on migratory properties that was abrogated in cells where ATF3 was depleted using short hairpin siRNA knockdown. Treatment with Pracinostat also showed a significant effect on proliferative and colony forming capacity of bladder cancer lines and induced growth arrest at the G1/G0 phase of the cell cycle and retinoblastoma (Rb) activation. In vivo experiments demonstrated a significant reduction in the tumor volume of the Pracinostat treated mice compared to the control group. Furthermore, reactivation of ATF3 expression was detectable in xenograft tumor cells which were in non - proliferating state. CONCLUSION: HDAC inhibitor Pracinostat activated expression of ATF3 has a significant impact on the malignant behavior of bladder cancer cell lines in vitro including activation of tumor suppressor genes and cell growth arrest. Pracinostat shows efficacy as an anti tumor agent in bladder cancer xenografts accompanied by reactivation of the expression of ATF3 suggesting its potential as a biomarker of response. 1.Yuan, X., et al., ATF3 Suppresses Metastasis of Bladder Cancer by Regulating Gelsolin-Mediated Remodeling of the Actin Cytoskeleton. Cancer Research, 2013. 73(12): p. 3625-3637. 2.Novotny-Diermayr,V.,et al.,SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer. Molecular Cancer Therapeutics, 2010. 9(3): p. 642-652. Citation Format: Dhanya Sooraj, Dakang Xu, Jason Cain, Daniel Gold, Bryan R. Williams. Activating transcription factor 3 (ATF-3) expression is a potential marker of tumor response to the HDAC inhibitor Pracinostat. [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 387. doi:10.1158/1538-7445.AM2014-387

Fibulin-1 is epigenetically down-regulated and related with bladder cancer recurrence.

BackgroundBladder cancer is one of the most common cancers worldwide. Fibulin-1, a multi-functional extracellular matrix protein, has been demonstrated to be involved in many kinds of cancers, while its function in bladder cancer remains unclear. So here we investigated the expression and function of fibulin-1 in Bladder cancer.MethodsWe used real-time PCR, Western blot analysis and immunohistochemistry to determine the expression of fibulin-1 in Bladder cancer cells and patient tissues respectively. Methylation-specific PCR and quantitative sequencing were used to examine the methylation status of FBLN1 gene promoter. Eukaryotic expression plasmid and lentiviral vector were used to overexpress fibulin-1 in Bladder cancer cells 5637, HT-1376 to investigate its function in vitro and in vivo.ResultsWe identified that fibulin-1 was significantly down-regulated in bladder cancer, and its dysregulation was associated with non-muscle-invasive bladder cancer (NMIBC) grade and recurrence. The promoter region of FBLN1 was generally methylated in bladder cancer cell lines and tissues, further investigation in patient tissues showed that the methylation status was associated with the fibulin-1 expression. Overexpression of fibulin-1 significantly suppressed tumor growth, induced tumor cell apoptosis, decreased cell motility, and inhibited angiogenesis in cultured bladder cancer cells and xenograft tumor in nude mice.ConclusionsAltogether, our results indicated that fibulin-1 expression is associated with NMIBC grade and recurrence, it is epigenetically down-regulated and functions as a tumor suppressor gene and angiogenesis inhibitor in bladder cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-677) contains supplementary material, which is available to authorized users.

Interleukin-4 receptor alpha overexpression in human bladder cancer correlates with the pathological grade and stage of the disease.

Previously, we have demonstrated that interleukin-4 receptor α (IL-4Rα) is overexpressed on a variety of human cancers and can serve as target for IL-4 immunotoxin comprised of IL-4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL-4Rα expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real-time RT-PCR and IHC/ISH techniques. We also examined the effect of IL-4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two-sided P-values. We used Cochran–Armitage Statistics with exact two-sided P-values to examine the trend analysis of IL-4Rα over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL-4Rα is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL-4 receptor complex, IL-2RγC and IL-13Rα1, were absent or weakly expressed. IL-4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration-dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL-4Rα overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL-4Rα immunostaining (≥2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ≤ 0.0001). Similarly, 9% stage I tumors were positive for IL-4Rα (≥2+) compared to 84% stage II (P ≤ 0.0001) and 100% stages III–IV tumors (P ≤ 0.0001). IL-13Rα1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL-2RγC was not expressed. Ten normal bladder specimens demonstrated ≤1+ staining for IL-4Rα and IL-13Rα1 and no staining for IL-2RγC. These results demonstrate that IL-4Rα is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL-4Rα may be a bladder tumor-associated protein and a prognostic biomarker.

Role in Tumor Growth of a Glycogen Debranching Enzyme Lost in Glycogen Storage Disease

Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.

518 Patient-derived sialyl-Tn positive invasive bladder cancer xenografts in nude mice: An exploratory model study

518 Patient-derived sialyl-Tn positive invasive bladder cancer xenografts in nude mice: An exploratory model study

MP39-14 METFORMIN IS A NEW THERAPEUTIC STRATEGY FOR TARGETING CD44 VARIANT IN BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Metformin is an oral biguanide drug widely used to treat type 2 diabetes mellitus and has recently been shown to have an antitumor effect through selectively targeting chemotherapy-resistant cancer stem cells, characterized by CD44+CD24phenotype. We focused particularly on variant isoforms of CD44 (CD44v) which have been recently reported as new markers for targeting cancer stem cells and evaluated whether metformin could have a therapeutic role for treating bladder cancer cells with a positive CD44v phenotype. METHODS: The efficacy of metformin alone and in combination with cisplatin was examined in T24 and HT1376 bladder cancer cell lines. WST assay was used to evaluate the cytotoxicity. The expressions of CD44v were evaluated by Western blotting analysis. The percentage of CD44v-positive cells in T24 cells treated with CDDP, metformin, or both were assessed by flow cytometry. A xenograft model was used to study the effects of metformin alone and in combination with cisplatin on bladder cancer tumor growth. Mice were divided into 4 groups; (1) no treatment, (2) metformin 200 ml/ml daily p.o., (3) cisplatin 2.5 mg/kg intraperitoneally for 1 day, or (4) combined metformin (200 ml/ml) and cisplatin (2.5 mg/kg). RESULTS: CD44v was positive in T24 cells, but not in HT1376 cells and significant dose-dependent growth inhibition by metformin was observed in T24 cells but not in HT1376 cells. Cell growth inhibition relative to vehicle control was significantly higher in the combination therapy (52.1%) compared to 1mM metformin alone (77.5%) or 1mM cisplatin alone (90.2%) in T24 cells. Flow cytometry showed that the percentage of CD44v-positive cells was 29.4%, 21.7%, 45.6%, and 42.6% in vehicle control, 1mM metformin, 1mM cisplatin, and the combination, respectively. The increased CD44v-positive cell population by cisplatin was reduced by metformin. In the T24 bladder cancer xenograft model, tumor volume at day 30 for the combination therapy was 467 75 mm3, which was significantly smaller than the vehicle control (1139 136 mm3), cisplatin monotherapy (1072 160 mm3), and metformin monotherapy (857 188 mm3) (p<0.001, p1⁄40.002 and p1⁄40.006, respectively). CONCLUSIONS: Metformin targeting CD44v-positive cells showed significant antitumor effects when given in combination with cisplatin for bladder cancer. Combined metformin and cisplatin therapy might be a novel therapeutic modality for treating bladder cancer.