Transitional Cell Carcinoma Cell Research Articles

Ablation of Red Stable Transfected Claudin Expressing Canine Prostate Adenocarcinoma and Transitional Cell Carcinoma Cell Lines by C-CPE Gold-Nanoparticle-Mediated Laser Intervention.

Claudin (CLDN) proteins are commonly expressed in cancers and targeted in novel therapeutic approaches. The C-terminal of Clostridium perfringens enterotoxin (C-CPE) efficiently binds several claudins. In this study, recombinant C-CPE conjugated to gold nanoparticles (AuNPs) has been used for prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) cell killing in vitro using gold-nanoparticle-mediated laser perforation (GNOME-LP). A PAC and TCC cell lines, as well as red fluorescence variants, allowing deep tissue imaging, were used. CLDN-3, -4, and -7 expression was confirmed by qPCR and immunofluorescences. The binding of C-CPE-AuNPs complexes on the cell surface was examined by scanning electron microscopy (SEM). Further, transcriptome analysis was carried out to evaluate the effect of C-CPE binder on the biological response of treated cells. Directed C-CPE-AuNP binding verified the capability to target CLDN receptors. Transcriptome analysis showed that C-CPE binding may activate immune and inflammatory responses but does not directly affect cell survival. Cancer cells ablation was demonstrated using a combination of GNOME-LP and C-CPE-AuNPs treatment reducing tumor cell viability to less than 10% depending on cell line. The fluorescent cell lines and the verified proof of concept in vitro provide the basis for perspective xenograft studies in an animal model.

Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target.

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.

Reduction in mitochondrial oxidative stress mediates hypoxia-induced resistance to cisplatin in human transitional cell carcinoma cells

Tumor hypoxia is known to promote the acquisition of more aggressive phenotypes in human transitional cell carcinoma (TCC), including drug resistance. Accumulating evidence suggests that mitochondria play a central role in the chemoresistance of TCC. However, the role of mitochondria in the hypoxia-induced drug resistance in TCC remains elusive. The present study investigated the function of mitochondria in the drug resistance using a TCC cell line under hypoxic conditions. In vitro hypoxia (0.1% O2, 48 h) was achieved by incubating TCC cells in air chamber. Mitochondrial events involving hypoxia-induced drug resistance were assessed. Hypoxia significantly reduced the cisplatin-induced apoptosis of TCC cells. Additionally, hypoxia substantially decreased the level of mitochondrial reactive oxygen species (ROS) generated by cisplatin treatment. Analogously, elimination of mitochondrial ROS significantly rescued cells from cisplatin-induced apoptosis. Hypoxia enhanced mitochondrial hyperpolarization, which was not related to ATP production or the reversal of ATP synthase activity. The mitochondrial DNA (mtDNA) amplification efficiency data illustrated that hypoxia significantly prevented oxidative damage to the mitogenome. Moreover, transmission electron microscopy revealed that cisplatin-induced disruption of the mitochondrial ultrastructure was abated under hypoxic conditions. Notably, depletion of mtDNA by ethidium bromide abrogated hypoxia-induced resistance to cisplatin. Taken together, the present study demonstrated that TCC cells exposed to hypoxic conditions rendered mitochondria less sensitive to oxidative stress induced by cisplatin treatment, leading to enhanced drug resistance.

Antitumor activity of an oncolytic measles virus against canine urinary bladder transitional cell carcinoma cells

The prognosis of canine transitional cell carcinoma (TCC) of urinary bladder is generally poor because it is difficult to diagnose at early stages and conventional therapies, such as surgical resection and/or chemotherapy, are often not curative treatments. Based on our previous report that recombinant measles virus (rMV-SLAMblind) therapy could be a new treatment for canine mammary tumor, the applicability of rMV-SLAMblind in canine urinary bladder TCC was examined in this study. A canine TCC cell line was established from a TCC patient dog by transplanting a piece of the tumor mass into an immunodeficient mouse and then isolating the primary TCC cells from the grown tumor mass. The primary cultured cells, named TCC-NU1, express nectin-4, a receptor for rMV-SLAMblind infection. The rMV-SLAMblind infected TCC-NU1 cells, and dose-dependently showed cell cytotoxicity. Moreover, intratumoral administration of rMV-SLAMblind in a xenograft model bearing TCC-NU1 cells significantly suppressed the tumor growth reducing the endpoint mass of tumors in treated mice compared to control mice. These results suggest that virotherapy with rMV-SLAMblind be a new candidate therapy for canine TCC.

Characterization of six canine prostate adenocarcinoma and three transitional cell carcinoma cell lines derived from primary tumor tissues as well as metastasis.

Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) of prostate and urinary bladder are highly invasive and metastatic tumors of closely neighbored organs. Cell lines are valuable tools to investigate tumor mechanisms and therapeutic approaches in vitro. PAC in dogs is infrequent, difficult to differentiate from TCC and usually characterized by poor prognosis, enhancing the value of the few available cell lines. However, as cell lines adapt to culturing conditions, a thorough characterization, ideally compared to original tissue, is indispensable. Herein, six canine PAC cell lines and three TCC cell lines were profiled by immunophenotype in comparison to respective original tumor tissues. Three of the six PAC cell lines were derived from primary tumor and metastases of the same patient. Further, two of the three TCC cell lines were derived from TCCs invading into or originating from the prostate. Cell biologic parameters as doubling times and chemoresistances to commonly used drugs in cancer treatment (doxorubicin, carboplatin and meloxicam) were assessed. All cell lines were immunohistochemically close to the respective original tissue. Compared to primary tumor cell lines, metastasis-derived cell lines were more chemoresistant to doxorubicin, but equally susceptive to carboplatin treatment. Two cell lines were multiresistant. COX-2 enzyme activity was demonstrated in all cell lines. However, meloxicam inhibited prostaglandin E2 production in only seven of nine cell lines and did neither influence metabolic activity, nor proliferation. The characterized nine cell lines represent excellent tools to investigate PAC as well as TCC in prostate and urinary bladder of the dog. Furthermore, the profiled paired cell lines from PAC primary tumor and metastasis provide the unique opportunity to investigate metastasis-associated changes PAC cells undergo in tumor progression. The combination of nine differently chemoresistant PAC and TCC cell lines resembles the heterogeneity of canine lower urinary tract cancer.

Effects of Epigallocatechin Gallate (EGCG) on Urinary Bladder Urothelial Carcinoma-Next-Generation Sequencing and Bioinformatics Approaches.

Background and objectives: Bladder urothelial carcinoma is the most common type of genitourinary cancer. Patients with bladder cancer may have limited treatment efficacy related to drug toxicity, resistance or adverse effects, and novel therapeutic strategies to enhance treatment efficacy or increase sensitivity to drugs are of high clinical importance. Epigallocatechin gallate (EGCG) is a polyphenolic compound found in green tea leaves, and a potential anti-cancer agent in various cancer types through modulating and regulating multiple signaling pathways. The current study aimed to explore the role and novel therapeutic targets of EGCG on bladder urothelial carcinoma. Materials and Methods: The BFTC-905 cells, human urinary bladder transitional cell carcinoma (TCC) cell line, were treated with EGCG or water for 24 hours, and the expression profiles of mRNAs and microRNAs were analyzed using next generation sequencing (NGS). The enriched biological functions were determined using different bioinformatics databases. Results: A total of 108 differentially expressed genes in EGCG-treated bladder TCC cells were identified, which were mainly involved in nicotinamide adenine dinucleotide (NAD) biogenesis, inflammatory response and oxidation-reduction metabolism. Moreover, several microRNA-mRNA interactions that potentially participated in the response of bladder TCC to EGCG treatment, including miR-185-3p- ARRB1 (arrestin beta 1), miR-3116- MGAT5B (alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B), miR-31-5p-TNS1 (tensin 1), miR-642a-5p-TNS1, miR-1226-3p- DLG2 (discs large homolog 2), miR-484-DLG2, and miR-22-3p- PPM1K (protein phosphatase 1K). Conclusions: The current findings provide insights into novel therapeutic targets and underlying mechanisms of action of EGCG treatment in bladder cancer.

Identifying the ErbB/MAPK Signaling Cascade as a Therapeutic Target in Canine Bladder Cancer.

Transitional cell carcinoma (TCC) of the bladder comprises 2% of diagnosed canine cancers. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. BRAF, a kinase in the mitogen-activated protein kinase (MAPK) pathway, is mutated in 70% of canine TCCs. In this study, we use BRAF mutant and wild-type TCC cell lines to characterize the role of BRAF mutations in TCC pathogenesis and assess the efficacy of inhibition of the MAPK pathway alone and in combination with other gene targets as a treatment for canine TCC. Analysis of MAPK target gene expression and assessment of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation following serum starvation indicated constitutive MAPK activity in all TCC cell lines. BRAF mutant TCC cell lines were insensitive to the BRAF inhibitor vemurafenib, with IC50 values greater than 5 μM, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC50: 0.2-1 μM). All TCC cell lines had IC50 values less than 7 nM to the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor trametinib independent of their BRAF mutation status. ERK1/2 phosphorylation decreased after 6-hour treatments with MAPK inhibitors, but rebounded by 24 hours, suggesting the presence of resistance mechanisms. Microarray analysis identified elevated expression of the ErbB family of receptors and ligands in TCC cell lines. The pan-ErbB inhibitor sapitinib synergized with BRAF inhibition in BRAF mutant Bliley TCC cells and synergized with MEK1/2 inhibition in Bliley and BRAF wild-type Kinsey cells. These findings suggest the potential for combined MAPK and ErbB receptor inhibition as a therapy for canine TCC. SIGNIFICANCE STATEMENT: The results of this study (1) identify a novel combination strategy for canine bladder cancer treatment: targeting the ErbB/MAPK signaling cascade and (2) establish the utility of canine bladder cancer as a naturally-occurring model for human MAPK-driven cancers.

Targeting the ErbB/MAPK Signaling Cascade in Canine Bladder Cancer Cell Lines

Canine transitional cell carcinoma (TCC) of the bladder accounts for 2% of diagnosed canine cancers. Most TCCs are inoperable and unresponsive to traditional chemotherapy. Median survival time is typically less than a year for all treatments indicating a need for more effective therapies. Recent studies have discovered that approximately 70% of canine TCCs harbor mutations in the proto‐oncogene BRAF, a kinase involved in the mitogen‐activated protein kinase (MAPK) pathway that controls cell proliferation, differentiation, and apoptosis. BRAF mutations are present in several human cancers, most of which exhibit adaptive or intrinsic resistance to BRAF‐targeted inhibitors. The goals of this study were to (1) further characterize the role of mutant BRAF in canine TCC and (2) determine whether inhibition of the MAPK pathway alone or in combination with other gene targets may be an effective therapy for TCC treatment.Analysis of ERK1/2 phosphorylation following serum starvation indicates that TCC cell lines exhibit constitutive MAPK pathway activation independent of their BRAF mutation status. MAPK activity was further quantified using gene expression analysis of ten MAPK target genes, revealing that TCC cell lines have significantly higher MAPK pathway activity compared to other canine cancer cell lines. These data suggest a causative role for MAPK signaling in TCC pathogenesis. To determine whether the MAPK pathway could be a therapeutic target for TCC treatment we assessed the effect of BRAF and MEK inhibition on TCC cell proliferation and ERK1/2 phosphorylation. Four BRAF mutant human cell lines with varying degrees of sensitivity to BRAF‐targeted agents were used to determine the relative sensitivity of canine TCC cell lines. BRAF mutant TCC cell lines were sensitive to BRAF inhibition with the “paradox‐breaking” inhibitor PLX7904 (IC50: 0.2–1.2μM), but not vemurafenib (IC50: 7–21μM). Both BRAF wild type and mutant TCC cell lines were sensitive to MEK inhibition with selumetinib (IC50: 15–420nM) and trametinib (IC50: 0.4–8nM). ERK1/2 phosphorylation decreased after 6‐hour treatments with MAPK inhibitors, but rebounded by 24 hours suggesting the presence of resistance mechanisms. Microarray analysis indicated that the ErbB family of receptors and ligands are up‐regulated in TCC cell lines relative to other canine cancer cell lines (fold‐change > 2, q < 0.05). Combined BRAF and ErbB inhibition synergized in the BRAF mutant Bliley TCC cell line, while combined MEK and ErbB inhibition synergized in both Bliley and BRAF wild type Kinsey cells. These findings suggest that targeting ErbB receptors with MAPK inhibition is a potential therapy for canine TCC treatment. Additionally, our data indicate that canine TCC may serve as a naturally‐occurring model for the study of resistance mechanisms to MAPK inhibition in human cancers.Support or Funding InformationMorris Animal Foundation, Shipley University Chair in Comparative OncologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti-tumour effect of lapatinib in canine transitional cell carcinoma cell lines.

Transitional cell carcinoma (TCC) accounts for >90% of canine malignant tumours occurring in urinary bladder, and the prognosis is poor. Our previous study, using RNA sequencing, showed that human epidermal growth factor 2 (HER2) was the most activated upstream regulator related to carcinogenesis in canine TCC. The aim of this study was to examine the anti-tumour effect of lapatinib, a tyrosine kinase inhibitor of HER2, on canine TCC cell lines in vitro and in vivo. Five canine TCC cell lines (TCCUB, Love, Sora, LCTCC, and MCTCC) were used. Western blotting showed that HER2 protein expression was observed in all of the canine TCC cell lines. Lapatinib inhibited phosphorylation of HER2 and cell growth in a dose-dependent manner. Cell cycle analyses using flow cytometry showed that lapatinib significantly increased the sub-G1 and G0 /G1 phase fractions and significantly decreased the S and G2 /M phase fractions in the cell lines (Sora and TCCUB). For the in vivo experiments, the canine TCC cells (Sora) were subcutaneously injected into nude mice. Six days after inoculation, lapatinib (100 mg/kg) or vehicle was administered daily via intraperitoneal administration for 14 days. Tumour volume was significantly smaller in the lapatinib group compared with the vehicle control group. Histologically, lapatinib significantly increased necrotic areas in the tumour tissues. These findings suggest that lapatinib exerts anti-tumour effects on canine TCC cells by inhibiting HER2 signalling and inducing cell cycle arrest.

Abstract 885: Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells

Abstract Doxorubicin (Dox) is a chemotherapy drug widely used for treatment of several cancers including bladder cancer. However, cardiovascular side effects caused by doxorubicin-induced cardiotoxicity has necessitated the synthesis and validation of next generation chemotherapy drugs that are as effective but not cardiotoxic. AD 312 (N-Nitrosureidodaunorubicin; Daunomustine®, Paradox Pharmaceuticals, Inc.) and AD 198 (N- benzyladriamycin-14-valerate; Benzarubicin®, Paradox Pharmaceuticals, Inc.) are novel anthracyclines with confirmed non-cardiotoxicity in mice. Compared to Dox, AD 312 and AD 198 induce apoptosis in cells through distinct mechanisms. The tumor suppressor gene p53 is a key regulator of cell-cycle arrest and apoptosis. Dysregulation of p53 expression and function was identified in over 70% of the bladder cancers and reported as a potential target for therapy. As a result, small molecule compounds that can restore mutated p53 function to wild-type, including PRIMA-1, are under intensive investigation. In this study, we have compared the efficacy of AD 312 and AD 198 to Dox to induce apoptosis in human bladder transitional cell carcinoma (TCC) cells by MTT assay and assessed the possible mechanisms of anthracyclines-induced apoptosis in TCC cells expressing wild-type (wt) p53 (RT4, SW780) and mutated (mt) p53 (UM-UC-3, 5637, J82, and TCCSUP) protein. We have also investigated the effects of co-treatment of anthracyclines with PRIMA-1 in J82 cells, as a representative mt-p53 TCC cell line, and compared the co-treatment effects in RT4, as a representative wt-p53 cell line. Our results demonstrated that anthracyclines inhibited cell viability of all tested TCC cells in vitro, with AD 198 demonstrating highest efficacy. Furthermore, anthracyclines upregulated p53 protein expression in wt-p53 RT4 and SW780 cells, but not in tested mt-p53 UM-UC-3, 5637, J82, and TCCSUP cells. A strong correlation was observed between p53 mutation status and activation of caspase 3/7 and cleavage of PARP. The upregulated caspase 3/7 activity and PARP cleavage was observed in wt-p53, but not in mt-p53 TCC cells. AD198 blocked c-myc expression in mt-p53 TCC cells and no expression of c-myc was detected in wt-p53 TCC cells. Furthermore, sensitivity to AD 198 treatment in mt-p53 J82 cells was significantly improved by a co-treatment with PRIMA-1. However, PRIMA-1 had no effect on cell viability in RT4, the wt-p53 TCC cells. Overall, our results demonstrated that AD 312 and AD 198 are potent inhibitors of bladder cancer cells in vitro and are promising candidates for new therapeutic strategies to replace Dox for bladder cancer treatment. PRIMA-1 even further sensitized mt-p53 cells to AD 198 treatment and thereby this co-treatment improved the inhibition of bladder cancer in vitro. Citation Format: Sony Pandey, Leonard Lothstein, Maria Cekanova. Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 885.

Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro.

PurposeReceptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with highly expressed receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR) and c-Kit receptor. Resistance to targeted therapies is partially due to the activation of alternative pro-survival signaling pathways, including cyclooxygenase (COX)-2. In this study, we validated the effects of two RTKIs, axitinib and AB1010, in combination with COX inhibitors on the V-akt murine thymoma oncogene homolog 1 (Akt) and COX-2 signaling pathways in bladder cancer cells.MethodsThe expression of several RTKs and their downstream signaling targets was analyzed by Western blot (WB) analysis in human and canine bladder transitional cell carcinoma (TCC) cell lines. The effects of RTKIs and COX inhibitors in bladder TCC cells were assessed by MTS for cell viability, by Caspase-3/7 and Annexin V assay for apoptosis, by WB analysis for detection of COX-2 and Akt signaling pathways, and by enzyme-linked immunosorbent assay for detection of prostaglandin E2 (PGE2) levels.ResultsAll tested TCC cells expressed the c-Kit and PDGFRα receptors, except human 5637 cells that had low RTKs expression. In addition, all tested cells expressed COX-1, COX-2, Akt, extracellular signal regulated kinases 1/2, and nuclear factor kappa-light-chain-enhance of activated B cells proteins, except human UM-UC-3 cells, where no COX-2 expression was detected by WB analysis. Both RTKIs inhibited cell viability and increased apoptosis in a dose-dependent manner in tested bladder TCC cells, which positively correlated with their expression levels of the PDGFRα and c-Kit receptors. RTKIs increased the expression of COX-2 in h-5637 and K9TCC#1Lillie cells. Co-treatment of indomethacin inhibited AB1010-induced COX-2 expression leading to an additive effect in inhibition of cell viability and PGE2 production in tested TCC cells.ConclusionCo-treatment of RTKIs with indomethacin inhibited cell viability and AB1010-induced COX-2 expression resulting in decreased PGE2 production in tested TCC cells. Thus, COX inhibition may further potentiate RTKIs therapies in bladder cancer.

In vitro evaluation of a simulated pneumoperitoneum environment using carbon dioxide on canine transitional cell carcinoma.

To assess the impact of a simulated CO2 pneumoperitoneum environment on the viability and proliferation of canine transitional cell carcinoma (TCC) cells in vitro. In vitro study. A control Madin-Darby canine kidney (MDCK) cell line and 3 canine TCC cell lines were exposed to 100% CO2 at pressure of 0, 5, 10, or 15 mmHg for 2 hours by using an airtight chamber and a mechanical insufflator at 37°C. Culture media pH was measured. Viability and proliferation were assessed by using a resazurin assay and trypan blue dye, respectively. The pH in the media significantly decreased immediately after CO2 exposure but returned to normal within 1 hour. The viability of the cell lines was variably affected at the evaluated pressures. Insufflation pressure of 10 mmHg resulted in significantly decreased cell viability compared with control. The impact of 15 mmHg CO2 was comparable to 0 mmHg and control. CO2 insufflation pressure had no significant effects on proliferation up to 7 days postexposure. Conclusion/Clinical significance: A positive pressure CO2 environment significantly decreased the viability of TCC and MDCK cells under specific conditions without influencing their proliferation up to 7 days postexposure. Investigating these effects in clinical patients undergoing CO2 laparoscopy is essential to assess for port site metastasis or peritoneal carcinomatosis in order to translate these in vitro results to clinical recommendations.

60P - Understanding and targeting Met signalling in bladder cancer

Background: Bladder cancer affects 430,000 patients and leads to 165,000 deaths annually worldwide. With no major advances in the management of this disease in the last 2 decades, there is an urgent need to identify therapeutic targets with validated biomarkers. Overexpression of Met, a Receptor Tyrosine Kinase, was shown to correlate with poor prognosis in bladder cancer making it an attractive target. Cabozantinib, a Met inhibitor, showed clinical activity in patients with refractory bladder cancer in a clinical trial. However, little is known about how Met exactly signals in bladder cancer and there are no validated biomarkers. This study aims at unravelling Met signalling in bladder cancer. Methods: Western blots and confocal/low light microscopy were used to assess Met signalling and its role in wound healing in Transitional Cell Carcinoma (TCC) cells. Met expression was assessed by immunohistochemistry in tissue samples (n = 64). Results: Met is overexpressed in TCC cells and in 78% of invasive bladder cancer tissues. This was associated with a shorter median survival as compared to Low Met levels (12.97 Vs 18.05 months). Stimulation of TCC cells with Met ligand, Hepatocyte Growth Factor (HGF), triggered Met activation and downstream signalling as well as wound healing, all of which were reduced with Met pharmacological inhibitors including Cabozantinib. The PI3K downstream effector AKT was highly activated upon Met activation. Moreover, class I PI3K inhibition with GDC094 significantly inhibited HGF-dependent wound healing. Interestingly, HGF triggered rapid Met endocytosis in TCC cells. Furthermore, pharmacological inhibition of endocytosis reduced Met downstream signalling. Conclusions: We report that Met is a major target in invasive bladder cancer. Our results further suggest that PI3K may be considered as a co-target of Met to improve patients’ outcome. It may also be developed as a biomarker to help select patients who may respond to Met targeted therapy. Finally, we report for the first time that, upon HGF stimulation, Met gets rapidly endocytosed in TCC cells. Furthermore, inhibiting endocytosis reduced Met dependent signalling. All together, our results open the way for novel strategies to target invasive bladder cancer. Legal entity responsible for the study: Queen Mary University of London Funding: Barts Cancer Institute, Queen Mary University of London Disclosure: T. Powles: Honoraria: AstraZeneca, Bristol-Myers Squibb, Roche and Merck. Research Funding: AstraZeneca, Roche and Merck. L. Menard: Employee: AstraZeneca. All other authors have declared no conflicts of interest.

Bladder cancer and Th1 chemokines

Bladder cancer arises from the epithelial lining of the urinary bladder, and it is known as transitional cell carcinoma (TCC). Tobacco smoking is the main known contributor to urinary bladder cancer. However thirty percent of bladder tumors probably result from occupational exposure in the workplace to carcinogens. Immunotherapy by intravesicular delivery of Bacillus Calmette–Guérin (BCG) is used to treat and prevent the recurrence of superficial bladder cancer. Successful BCG immunotherapy for bladder cancer is associated with proper induction of T helper (Th)1 immunity. In bladder cancer patients after intravesicular BCG, urine was found to contain high levels of IP-10, and Interferon (IFN)-γ. TCC and endothelial cell lines were able to secrete IP-10 in response to BCG or IFN stimulation in vitro. Furthermore intravesicular BCG induces a cytokine-rich urinary microenvironment that is inhibitory to human endothelial cells and it is anti-angiogenetic by the induction of Th1 chemokines. Other studies suggest that therapeutic strategies involving Th1 induction and Th2 dampening may improve responses to immunotherapy. Further studies are needed to evaluate the IP-10 in circulation, and urine, as prognostic marker of bladder cancer patients, also in relation to BCG immunotherapy

The effect of dichloroacetate in canine prostate adenocarcinomas and transitional cell carcinomas in vitro.

The Warburg effect describes the ability of cancer cells to produce energy via aerobic glycolysis instead of oxidative phosphorylation of pyruvate. This deviation in mitochondrial metabolism inhibits apoptosis, allowing increased proliferation under conditions of reduced oxygen levels. Dichloroacetate (DCA) was successfully used in several human cancer cell lines to reactivate oxidative phosphorylation in mitochondria. The aim of this study was the characterization and response of canine cancer cell lines after DCA exposure. The effect of 10mM DCA was characterized in vitro on a set of six canine prostate adenocarcinoma and transitional cell carcinoma (TCC) derived cell lines. Cell counts, lactate levels, apoptosis, expression of apoptotic proteins, survival factors and different miRNAs were analyzed. Additionally, metabolic activity, mitochondrial activity and proliferation were investigated. DCA significantly decreased cell number of all but one utilized cell lines and leads to a significant reduction of lactate release. Decreased survivin levels were found in all cell lines, two of which presented a significant reduction in metabolic activity. Increased miR-375 levels were measured in all TCC cell lines. Reactivation of pyruvate dehydrogenase and an elevated mitochondrial activity appear to induce the transition from aerobic glycolysis back to oxidative phosphorylation. Further, these results display that DCA treatment has a suppressant effect on proliferation of canine cancer cells.

Targeting XBP1-mediated β-catenin expression associated with bladder cancer with newly synthetic Oridonin analogues.

Conventional chemotherapy is commonly used for advanced stages of transitional cell carcinoma (TCC) with modest success and high morbidity; however, TCC eventually develops resistance. Muscle invasive bladder cancer (MIBC) is recognized as a lethal disease due to its poor response to traditional chemotherapy. Numerous studies have implicated β-catenin, a critical effector in Wnt–mediated pathway associated with epithelial-mesenchymal transition and cancer stem cell, is involved in TCC progression, and furthermore closely associated with chemo-resistance. In this study, we discovered a novel natural product analogue CYD 6-17 that has a potent inhibitory effect on TCC cells exhibiting drug resistance to various chemotherapeutics, with an IC50 at nM range. Delivery of CYD 6-17 significantly inhibited the tumor growth using xenograft model but without detectable side effects. Mechanistically, it targeted β-catenin gene transcription by decreasing the binding of XBP1 to the promoter region, which appeared to be a new regulatory mechanism for β-catenin gene expression. Clinically, XBP1 expression correlated with the poor overall survival of patients. Overall, this study unveils unique mechanism of β-catenin gene regulation in advanced TCC and also offers a potential rational therapeutic regimen to MIBC.

Abstract 4739: The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro

Abstract Several types of cancer, including transitional cell carcinoma (TCC) overexpress several receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR), c-kit receptor, epidermal growth factor receptor (EGFR), as well as the vascular endothelial growth factor receptor (VEGFR). Receptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with high expression of RTKs. Cyclooxygenase-2 (COX-2) is highly expressed in bladder cancer as well other types of cancers and is one of the key proteins during tumorigenesis. In this study, we validated the effects of RTKIs, Masitinib® (AB1010) and Axitinib on cell proliferation of human (UMUC-3 and T24) and canine (K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) bladder TCC cells. Using WB analysis, tested human and canine TCC cells, except T24 cells, expressed PDGFR and c-kit receptors. There was no detectable EGFR and VEGFR expressions in any of tested cells by WB analysis. The K9TCC#1Lillie and K9TCC#5Lilly cells had a high expression of COX-2, however K9TCC#2Dakota and human T24 had moderate expression of COX-2, and no COX-2 expression was detected in UMUC3 cells by WB analysis. Both RTKIs, Masitinib® and Axitinib inhibited cell proliferation of the tested human and canine bladder TCC cells in a dose-dependent manner by MTS and apoptosis assays. Interestingly, both tested RTKIs, Masitinib® and Axitinib increased COX-2 protein expressions in tested canine TCC cells. Combined treatment of RTKIs with COX-2 inhibitors decreased cell proliferation of tested TCC cells more effectively as either treatments alone. Our results indicate a possible role of COX-2 signaling pathway in developed RTKIs-resistance in bladder cancer cells in vitro. Co-treatment of RTKIs with COX-2 inhibitors might indicate better clinical outcomes in treatments of patients diagnosed with bladder cancer. Citation Format: Jennifer Bourn, Maria Cekanova. The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro. [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 4739.

Abstract 617: Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib

Abstract Transitional cell carcinoma (TCC) is the most common bladder cancer in humans and their canine companions. Canine TCCs are usually papillary infiltrative TCCs of intermediate to high grade. Genetic defects identified in human TCCs aid in the diagnosis and therapy of human bladder cancer. We utilized whole exome sequencing to screen canine TCCs for gene mutations that contribute to pathogenesis. Genomic DNA was isolated from 11 canine TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines. Whole exome capture was conducted using the Canine Agilent Sure-select in-solution capture system, and captured fragments were sequenced using an Illumina HiSeq2000 platform. The sequences were mapped to the CanFam3.1 canine reference genome and somatic mutations were identified using Freebayes. Somatic mutations were characterized and compared to the Cancer Gene Census (COSMIC). Nonsense, missense, and insertion/deletion mutations were identified in 126 genes shown to be drivers or repressors in human cancer. Missense mutations were further screened using SIFT to identify alterations deleterious to protein function. The genes exhibiting in/dels, nonsense, or missense mutations with SIFT scores < 0.45 in at least 2 sites or 2 samples were: BRAF, RPL5, RANBP2, EWSR1, NONO, PTPRB, LYL1, JAK1, MSH2, PER1, PIM1, and WRN. In fact, an activating BRAF V to E mutation was identified in 5 of the tumors and both cell lines. The RPL5 and RANBP2 mutations were also hotspot mutations identified in 5 and 3 of the samples, respectively. Mutations were confirmed using Sanger sequencing of amplified genomic DNA. Interestingly, deleterious RANBP2 mutations were only observed in non-BRAF mutant samples. Drug sensitivity assays using the BRAF V600E targeting drug, Vemurafenib, were conducted in the BRAF mutant canine cell lines as well as the BRAF mutant human A375 melanoma cell line. As previously described, the IC50 for the sensitive A375 line was approximately 100 nM, while each of the BRAF mutant canine lines had an IC50 value ≥ 10 μM. Reverse transcriptase PCR was used to amplify the coding sequence for BRAF from the Bliley canine TCC cell line. The amplified transcript (2125 bp) was sequenced, confirming the heterozygous expression of the V548 to E mutant form of BRAF in this cell line. The sequence also indicated that the predicted expressed protein (AA 10 - 715 of XP_013975364.1, corresponding to predicted exons 1 through 20 of XM_014119889.1) exhibited 99% homology to human BRAF AA53 - 763 (NP_004324.2). BRAF protein expression in these cell lines was confirmed by Western blot analysis. Thus, insensitivity to Vemurafenib is not due to differences in canine BRAF, reduced expression, or alternative splicing of the expressed transcript as previously observed in some Vemurafenib resistant human melanomas. These data indicate that although constitutively active BRAF is expressed in canine TCC, other factors may contribute to pathogenesis. Citation Format: Belen Hernandez, Kathryn Cronise, James C. Costello, Rodney Page, Susan Lana, Kenneth L. Jones, Dawn L. Duval. Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib. [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 617.

MiR-130b, an onco-miRNA in bladder cancer, is directly regulated by NF-κB and sustains NF-κB activation by decreasing Cylindromatosis expression.

Persistent activation of NF-κB signaling is closely related to chronic inflammation and tumorigenesis. Commonly, NF-κB signaling is tightly controlled by multiple feedback loops and regulators, such as the deubiquitinases (DUBs). However, in cancer cells, NF-κB may override these feedbacks through special pathways and lead to the sustained activation. In the present study, we demonstrate that in transitional cell carcinoma (TCC) of bladder, miR-130b plays an oncogenesis role, it enhanced proliferation, invasion and migration of TCC cell, and was highly correlated with tumor progression. On the other hand, NF-κB directly regulated the transcription of miR-130b by binding with its promoter region. Importantly, we verify that, through deceasing the expression of Cylindromatosis (CYLD), a K63-specific DUB and endogenous blocker of NF-κB signaling, miR-130b can in return sustain the persistent activation of NF-κB, which may promote the malignant progression of TCC. Thus, the present study uncovers a potential signaling transduction in which NF-κB is continuously activated, and may provide a novel therapeutic approach for the clinical management of TCC.

Long noncoding RNA HOTAIR is a prognostic biomarker and inhibits chemosensitivity to doxorubicin in bladder transitional cell carcinoma.

In this study, whether HOTAIR is a prognostic biomarker will be detected, and its regulative effects of chemosensitivity to doxorubicin in TCC cells will be examined. The expression of HOTAIR was detected by quantitative real-time PCR. Overall survival rate was calculated by Kaplan-Meier method with the log-rank test for comparisons. MTT assay was used to detect cell proliferation ability and chemosensitivity. Dual-color flow cytometric method was used to detect cell apoptosis. HOTAIR was up-regulated in bladder transitional cell carcinoma (TCC) tissues and cell lines compared with normal bladder transitional cell (NBTC) tissues and bladder epithelial immortalized SV-HUC-1 cells, and its expression level had positive correlation with histological grades of TCC. Moreover, HOTAIR was an independent prognostic biomarker of overall survival for TCC patients. The expression and silence vector for HOTAIR were transfected into T24 and J82 cells to up-regulate and silence the HOTAIR expression, respectively. In T24 and J82 cells, HOTAIR over-expression promoted cell proliferation and inhibited chemosensitivity to doxorubicin and cell apoptosis induced by doxorubicin; silence of HOTAIR showed opposite regulative effects. In summary, lncRNA HOTAIR was an independent prognostic biomarker of overall survival in TCC patients and could regulate chemosensitivity to doxorubicin of human TCC cells. HOTAIR might provide a new potential therapeutic target and stratagem for TCC.