Potential Of Bladder Cancer Cells Research Articles

20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

Bladder cancer is one of the most common malignancies of the urinary tract and a prevalent cancer worldwide, still requiring efficient therapeutic agents and approaches. 20-Hydroxyecdysone (20-HE), a steroid hormone, can be found in insects and few plants and mediate numerous biological events to control the progression of varying diseases; however, its impacts on bladder cancer remain unclear. In the study, we found that 20-HE treatments effectively inhibited the viability and proliferation of bladder cancer cells and induced apoptosis by activating Caspase-3. The migratory and invasive potential of bladder cancer cells was markedly repressed by 20-HE in a dose-dependent manner. The inhibitory effects of 20-HE on bladder cancer were confirmed in an established xenograft mouse model, as indicated by the markedly reduced tumor growth rates and limited lung and lymph node metastasis. High-throughput RNA sequencing was performed to explore dysregulated genes in bladder cancer cells after 20-HE treatment. We identified ubiquitin-specific protease 21 (USP21) as a key deubiquitinating enzyme for bladder cancer progression and a positive correlation between USP21 and nuclear factor-κB (NF-κB)/p65 in patients. Furthermore, 20-HE treatments markedly reduced USP21 expression, NF-κB/p65 mRNA, stability and phosphorylated NF-κB/p65 expression levels in bladder cancer cells, which were validated in animal tumor tissues. Mechanistic studies showed that USP21 directly interacted with and stabilized p65 by deubiquitinating its K48-linked polyubiquitination in bladder cancer cells, which could be abolished by 20-HE treatment, contributing to p65 degradation. Finally, we found that USP21 overexpression could not only facilitate the proliferation, migration, and invasion of bladder cancer cells, but also significantly eliminated the suppressive effects of 20-HE on bladder cancer. Notably, 20-HE could still perform its anti-tumor role in bladder cancer when USP21 was knocked down with decreased NF-κB/p65 expression and activation, revealing that USP21 suppression might not be the only way for 20-HE during bladder cancer treatment. Collectively, all our results clearly demonstrated that 20-HE may function as a promising therapeutic strategy for bladder cancer treatment mainly through reducing USP21/p65 signaling expression.

Abstract LB437: Development of a bladder cancer-on-a-chip assay to assess the invasive potential of bladder cancer cells

Abstract Goals: The primary goal of this study was to develop a 3D functional bioassay to assess the relative invasiveness of bladder cancer cells. This model will support the testing of migrastatic drugs and investigation of the molecular mechanisms which allow for bladder cancer cell invasion to occur. Methods: Our model uses an organ-on-a-chip platform to assess bladder cancer cell invasiveness. This platform supports 3D culture of cells as well as pharmacokinetic studies. Our chips were designed to have a center channel to hold cells supported in Matrigel®, 2 adjacent side channels filled with Matrigel® only, and 2 outer media channels. Total cell and Matrigel® side channel length is 3 cm, channel height is 150 µm, and each channel width is 1 mm. Four bladder cancer cell lines (MB49, T24, J82, RT4) and cells isolated from a mouse bladder were used to develop our model. Cells were mixed with 1:1 media and Matrigel® and a total of 5,000 cells/µl introduced into the center channel. The 2 adjacent side channels were then filled with Matrigel®. The upper media channel was filled with 10% FBS cell culture media while the lower media channel was filled with 1% charcoal stripped cell culture media to thereby create a nutrient and growth factor gradient. Invasion of cells into the Matrigel®-containing side channels was assessed every 24 hours. Using the ImageXpress machine (Molecular Dynamics), we measured the furthest distance travelled by the cells. The chips were then fixed in 4% paraformaldehyde and stained using FITC-phalloidin and DAPI. Results: J82 and T24 cell lines, derived from patients with muscle invasive bladder cancer, and the MB49 cell line, a highly invasive mouse bladder cancer cell line, started invading into the Matrigel® side channels within 24 hours. T24, J82, and MB49 cells moved a maximum of 110, 87, and 170 µm by 48 hours, respectively. In contrast, RT4 cells, derived from a patient with non-invasive bladder cancer, took 48 hours to invade the Matrigel® channels and only invaded a maximum of 3 µm. Cells isolated from a mouse bladder did not invade into the Matrigel® side channels but survived within the chips for over 20 days. Staining of chips with DAPI and FITC-phalloidin allowed for clear visualization of cells and the actin cytoskeleton and demonstrated that immunostaining of cells within chips is possible. Conclusions: Our chip model allows for studying the invasiveness of bladder cancer cells and immunostaining experiments which can be used to assess the molecular mechanisms which drive invasion. It can also support the growth and assessment of primary cells. This model has the potential to improve bladder cancer patient treatments and outcomes by providing a physiologically relevant system to test new and existing drug combinations and to better understand the molecular mechanisms which drive bladder cancer cell invasion. Citation Format: Desiree Ewell, Nita Vue, Sakib Moinuddin, Tanoy Sarkar, Fakhrul Ahsan, Ruth L. Vinall. Development of a bladder cancer-on-a-chip assay to assess the invasive potential of bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB437.

Glycoprotein nonmetastatic melanoma protein B impacts the malignant potential of bladder cancer cells through its hem-immunoreceptor tyrosine-based activation motif.

Bladder cancer is one of the most common cancers among men worldwide. Although multiple genomic mutations and epigenetic alterations have been identified, an efficacious molecularly targeted therapy has yet to be established. Therefore, a novel approach is anticipated. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that is highly expressed in various cancers. In this study, we evaluated bladder cancer patient samples and found that GPNMB protein abundance is associated with high-grade tumors, and both univariate and multivariate analyses showed that GPNMB is a prognostic factor. Furthermore, the prognosis of patients with high GPNMB levels was significantly poorer in those with nonmuscle invasive bladder cancer (NMIBC) than in those with muscle invasive bladder cancer (MIBC). We then demonstrated that knockdown of GPNMB in MIBC cell lines with high GPNMB inhibits cellular migration and invasion, whereas overexpression of GPNMB further enhances cellular migration and invasion in MIBC cell lines with originally low GPNMB. Therefore, we propose that GPNMB is one of multiple driver molecules in the acquisition of cellular migratory and invasive potential in bladder cancers. Moreover, we revealed that the tyrosine residue in the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) is required for GPNMB-induced cellular motility.

Network pharmacology and in vitro experiments reveal that Noscapine induces ROS-mediated apoptosis and cell cycle arrest via PI3K/Akt/FoxO3a signaling pathway in human bladder cancer cells.

Noscapine (NA) has been demonstrated to have antitussive and antitumoral activities. Nonetheless, the potential mechanism of action on Bladder Cancer (BLCA) is yet to be completely grasped. The targets of NA action and bladder cancer disease targets were found by the database. Construct the PPI network. Subsequently, conduct pathway enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) on core targets. A "drug-disease-target-pathway" network map was made. Cytotoxicity was examined via CCK-8 and colony formation assays. Both a scratch test and a transwell assay confirmed that NA was capable of suppressing the invasiveness and migratory potential of bladder cancer cells. Hoechst 33342 staining was used to visualize NA-induced apoptosis in bladder cancer cells. Flow cytometry was employed to investigate the induction of apoptosis, the distribution of the cell cycle, the production of Reactive Oxygen Species (ROS), and the Mitochondrial Membrane Potential (MMP). The Western blot was applied to show the expression of proteins that are implicated in the pathway, cell cycle, apoptotic process, and proliferation. 198 Noscapine-BLCA-related targets were obtained. GO functional enrichment analysis yielded 428 entries (P < 0.05 and FDR < 0.05). KEGG pathway enrichment analysis identified 138 representative signaling pathways (P < 0.01 and FDR < 0.01). NA concentration-dependently suppressed cell growth and colony formation, along with the invasiveness and migratory potential of bladder cancer cells, by promoting apoptosis, a cell cycle arrest in the G2/M phase, generation of ROS, and depolarization of MMPs. In addition, Western blotting illustrated that NA down-regulated the protein levels associated with pathway, anti-apoptotic proteins, proliferation-related proteins, and cell cycle promoters but up-regulated pro-apoptotic proteins, cell cycle modulators, and Endoplasmic Reticulum (ER) stress expression. Pretreatment with Acetylcysteine N-acetyl-L-cysteine (NAC) and YS-49 counteracted the influence of NA on ROS induction and apoptosis. Noscapine induces ROS-mediated apoptosis and cell cycle arrest via PI3K/Akt/FoxO3a signaling pathway in human BLCA cells.

Complement Regulatory Protein CD46 Manifests a Unique Role in Promoting the Migration of Bladder Cancer Cells.

CD46 is a membrane-bound complement regulatory protein (mCRP) possessing a regulatory role with the complement system. CD46 protects the host cells from damage by complement. Expression of CD46 is also highly maintained in many cancers, including bladder cancers, and thus functions as a receptor for many cancer therapeutic viruses. In this study we report a unique role of CD46 as a progression factor of cancer cells in bladder cancers. Resulting data from a DNA microarray using CD46-altered HT1376 bladder cancers demonstrated a pool of target genes, including complement C3 α chain (C3α), matrix Gla protein (MGP), AFAP-AS1, follicular dendritic cell secreted protein (FDCSP), MAM domain containing 2 (MAMDC2), gamma-aminobutyric acid A receptor pi (GABRP), transforming growth factor, beta-induced (TGFBI), a family of cytochrome P450 (CYP24A1), sialic acid binding Ig-like lectin 6 (SIGLEC6), metallothionein 1E (MT1E), and several members of cytokeratins. Subsequent studies using quantitative RT-PCR and Western blot analyses confirmed CD46-mediated regulation of C3α, MGP, and keratin 13 (KRT13). MGP and KRT13 are known to be involved in cell migration and cancer cell metastasis. A cell migration assay demonstrated that CD46 enhanced migratory potential of bladder cancer cells. Taken all together, this report demonstrated that CD46 is generally overexpressed in bladder cancers and plays a unique role in the promotion of cancer cell migration. Further detailed studies are needed to be performed to clarify the action mechanism of CD46 and its application to cancer therapeutics.

Tumor-Derived C-C Motif Ligand 2 Induces the Recruitment and Polarization of Tumor-Associated Macrophages and Increases the Metastatic Potential of Bladder Cancer Cells in the Postirradiated Microenvironment

Radiation therapy (RT) is mainly used for bladder preservation in patients with muscle-invasive bladder cancer. The response of urothelial tumors to RT remains unsatisfactory. We investigated the interaction of RT and tumor-associated macrophages (TAMs) in the context of bladder cancer radioresistance. We evaluated the therapeutic effects of RT and TAM distribution by establishing an ectopic allograft mouse model. A Transwell coculture system was used to simulate the interaction between TAMs and MB49 bladder cancer cells in the tumor microenvironment. Cytokines and chemokines were analyzed in irradiated MB49 cells. Colony formation and Boyden chamber assays were used to assess the cytotoxic effects and the effects of TAMs on MB49 cell invasion, respectively. Local RT delayed primary tumor growth but promoted pulmonary metastases in C57BL/6 mice. Increased secretion of C-C motif chemokine ligand (CCL2) by irradiated MB49 cells, especially in the presence of M1-type TAMs, contributed to the infiltration of bone marrow-derived C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and the polarization of M1-type TAMs toward the M2 type to promote MB49 cell invasion. Blockade of CCL2-CCR2 activation by a CCR2 antagonist reversed the phenotypic TAM transformation and suppressed pulmonary metastases. Bladder cancer cells responded to RT by producing CCL2, which recruited TAM precursors from bone marrow and polarized M1-type TAMs toward the M2 type. This phenotypic TAM transformation promoted the pulmonary metastasis of bladder cancer cells after RT. Disrupting the CCL2-CCR2 signaling axis in combination with RT holds promise for improving RT efficacy in bladder cancer.

NF-κB inhibitor, BAY11-7082, suppresses M2 tumor-associated macrophage induced EMT potential via miR-30a/NF-κB/Snail signaling in bladder cancer cells

BackgroundChronic inflammatory microenvironment has been shown to play a key role in initiating tumorigenesis and facilitating malignant progression. Primary tumors surrounded with and infiltrated by tumor-associated macrophages (TAMs) significantly promote the epithelial-to-mesenchymal transition (EMT) and distant metastasis in urothelial bladder cancer. MethodsIn this study, we aimed to explore the potential of targeting TAMs for the treatment of malignant bladder cancer. ResultsFirst, we found a higher number of TAMs, CD68 (pan-macrophage marker), and clever-1 (M2 macrophage marker) was associated with a higher pT category and grade in a cohort of 108 patients. In vitro assays showed that the co-culture of TAMs promoted the metastatic potential in HTB-1 and T24 by up-regulating EMT markers including Snail, VEGF and Vimentin, as well as oncogenic markers such as β-catenin and NF-κB. More importantly, M2 co-cultured HTB-1 and T24 showed an increased level of metastatic microRNA, miR-30. Silencing of miR-30 resulted in the reduced metastatic potential, migration/invasion, in association with the decreased expression of Twist1 and Vimentin. The addition of BAY11-7082 into the TAM/cancer co-culture system significantly reduced the M2 phenotype and tumorigenic properties. Coincidentally, miR-30a level was significantly lowered in the presence of BAY11-7082. ConclusionOur study demonstrated that AMs promoted metastatic potential of bladder cancer cells via promoting EMT through the increase of miR-30a. BAY11-7082 treatment suppressed both oncogenic and metastatic potential in bladder cancer cells while preventing the M2 polarization of TAMs.

MKAD-21 Suppresses the Oncogenic Activity of the miR-21/PPP2R2A/ERK Molecular Network in Bladder Cancer.

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21-induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A-ERK network in mice. Mol Cancer Ther; 17(7); 1430-40. ©2018 AACR.

Abstract 4481: Tumor suppressive role of aquaglyceroporin-3 and PTPN13 in muscle invasive bladder cancer

Abstract High mortality rates in bladder cancer (BCa) and lack of effective systemic therapies warrant identifying novel oncogenic pathways and therapeutic strategies. The cell-cell adhesion protein E-cadherin (Cdh1) is an important component of the transitional epithelium of the bladder wall, the cellular origin of most BCa. Cdh1 loss is crucial in epithelial-to-mesenchymal transition (EMT) and subsequent tumor metastasis in multiple cancers, including BCa. Cdh1 reportedly accumulates at cell junctions with the transmembrane aquaglyceroporin-3 channel (AQP3). Reports suggest that AQP3 expression is reduced in BCa of higher grade and stage. Although AQP3 has not been implicated in BCa metastasis, these observations led us to hypothesize that loss of AQP3 contributes to Cdh1 loss, thereby promoting EMT and systemic BCa invasion. Our previous data suggested that AQP3 expression is lower in higher grades of BCa cell lines. Reduced levels of Cdh1 and increased levels of Cdh1 repressors TWIST1, SNAIL1 and ZEB1 were noted in higher grade BCa cells. Our results indicate that AQP3 co-localizes with Cdh1 in lower grade BCa cell lines. This co-localization is not noted in higher grade BCa cells. Silencing of AQP3 by a specific siRNA in lower grade BCa cell lines resulted in reduced Cdh1 localization on the cell membrane. It remains to be deciphered if there is a physical association between Cdh1 and AQP3 and if reduced expression of AQP3 in higher grade BCa cell lines contributes to the loss of Cdh1 and thus facilitate increased EMT progression. Using tools at c-BioPortal to analyze The Cancer Genome Atlas (TCGA) BCa provisional dataset (n=413) revealed a direct correlation between AQP3 and tyrosine phosphatase PTPN13 expression, and similar to other cancers, PTPN13 deletion was associated with significantly reduced overall survival (p&amp;lt;0.04). It is known that PTPN13 negatively regulates Src activation. Furthermore, Src-mediated Cdh1 phosphorylation leading to Cdh1 degradation is known. We hypothesize that PTPN13 loss may promote BCa progression by increased Src-mediated Cdh1 phosphorylation resulting in Cdh1 degradation. Our data indicate that transcript and protein levels of PTPN13 are significantly lower in BCa cell lines derived from higher grade tumors relative to lower grade tumors. Silencing PTPN13 in low grade BCa cell lines significantly increased Src phosphorylation at Tyr416, the activation loop of the kinase domain. These results suggest that loss of PTPN13 in higher grade BCa cells result in increased Src-activation. PTPN13 has a novel tumor suppressive function in BCa: loss of PTPN13 results in sequential Src activation, Cdh1 degradation and increased EMT and metastatic potential of BCa cells. Further defining these mechanisms may help identify new strategies to effectively control BCa progression. Citation Format: Arpita A. Roy, Dinuka De Silva, Molly M. Lee, Donald P. Bottaro. Tumor suppressive role of aquaglyceroporin-3 and PTPN13 in muscle invasive bladder cancer [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 4481.

MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9.

Background:The miRNA deregulation is commonly observed in human malignancies, where they act as tumour suppressors or oncogenes. Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs that contribute to bladder cancer progression from non-muscle invasive (NMI) to muscle-invasive (MI) disease.Methods:We first profiled the expression of miRNAs and mRNAs in a cohort of urothelial carcinomas and further characterised the role of miR-126 in invasion, as it emerged as the most downregulated miRNA between MI and NMI tumours.Results:We found that restoration of miR-126 levels attenuated the invasive potential of bladder cancer cells. Mechanistically, we identified the role of miR-126 in invasion through its ability to target ADAM9. Notably, a significant inverse correlation between miR-126 and ADAM9 expression was observed, where ADAM9 was upregulated in MI bladder cancer cells. While knockdown of ADAM9 attenuated the invasiveness of cells with low miR-126 levels, experimental upregulation of ADAM9 recapitulated the invasive phenotype. Furthermore, ADAM9 expression assessed by immunohistochemistry significantly correlated with poor prognosis in patients with urothelial carcinoma.Conclusions:In this study we describe the role of miR-126 in bladder cancer progression, identifying miR-126 and ADAM9 as potential clinical biomarkers of disease aggressiveness.

MP21-08 GLYCAN MODIFICATION OF PODOPLANIN ENHANCES GROWTH FACTOR PRODUCTION THROUGH STABILIZATION OF PLATELET AGGREGATION IN BLADDER CANCER CELL

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2014MP21-08 GLYCAN MODIFICATION OF PODOPLANIN ENHANCES GROWTH FACTOR PRODUCTION THROUGH STABILIZATION OF PLATELET AGGREGATION IN BLADDER CANCER CELL Yuki Tobisawa, Shingo Hatakeyama, Tohru Yoneyama, Teppei Okamoto, Hayato Yamamoto, Kazuyuki Mori, Akiko Okamoto, Atsushi Imai, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, and Chikara Ohyama Yuki TobisawaYuki Tobisawa More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Tohru YoneyamaTohru Yoneyama More articles by this author , Teppei OkamotoTeppei Okamoto More articles by this author , Hayato YamamotoHayato Yamamoto More articles by this author , Kazuyuki MoriKazuyuki Mori More articles by this author , Akiko OkamotoAkiko Okamoto More articles by this author , Atsushi ImaiAtsushi Imai More articles by this author , Takahiro YoneyamaTakahiro Yoneyama More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , Takuya KoieTakuya Koie More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.836AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Platelet aggregation together with cancer cell is one of the key factors for cancer progression and metastasis. Podoplanin (PDPN), which is known as a lymphatic endothelial marker, expressed on cancer cell surface has a potential of platelet aggregation through interaction with CLEC-2 expressed on Platelet. Core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) is a key enzyme for O-glycosylation of membrane proteins. Although PDPN has a core2 O-glycosylation site on their mucin domain, its roll in cancer progression is still unclear. Here, we examine the roles of PDPN and their O-glycosylation in bladder cancer (BCa) progression and invasion. METHODS YTS-1 is a BCa cell line established from muscle invasive BCa and highly positive for PDPN and C2GnT. KK-47 is a BCa cell line established from non-muscle invasive BCa and expresses a faint level of PDPN and C2GnT. YTS-1 cells were transfected with PDPN-siRNA or C2GnT-siRNA to silence of PDPN or C2GnT expression. KK47 cells were transfected with pcDNA3.1-PDPN and/or pcDNA3.1-C2GnT to make KK47-PDPN, KK47-C2GnT and KK47-C2PDPN which are highly positive for PDPN, C2GnT or both of them. These cells were subjected to growth factor assays and invasion assay. Co-culture system of BCa cells and platelet was used to evaluate growth kinetics of the BCa cells and production of growth factors. In vitro invasion assay was used to evaluate invasion potential of the BCa cells. RESULTS In the co-culture system of BCa cells and platelet, BCa cells culturing with platelet had higher growth activity than without platelet (p<0.05). YTS-1 produced significantly higher amounts of bFGF, EGF, NGF beta, PDGF-BB, TGF-beta and VEGF when they are co-cultured with platelet than they are cultured without platelet (p<0.05). O-glycosylation of PDPN on BCa cells significantly increased the production of growth factors (p<0.05). When PDPN is knocked-down, invasive potential of YTS-1 was markedly reduced. (p=0.005). CONCLUSIONS These results suggest that expression of PDPN and its O-glycosylation enhance growth factors production, proliferation activity, and invasion potential of bladder cancer cells through interaction with platelet. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e228 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yuki Tobisawa More articles by this author Shingo Hatakeyama More articles by this author Tohru Yoneyama More articles by this author Teppei Okamoto More articles by this author Hayato Yamamoto More articles by this author Kazuyuki Mori More articles by this author Akiko Okamoto More articles by this author Atsushi Imai More articles by this author Takahiro Yoneyama More articles by this author Yasuhiro Hashimoto More articles by this author Takuya Koie More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The role of inflammation in bladder cancer.

The aim of this book chapter is to present the latest basic research developments on the role of inflammation in bladder cancer and provide insights into their future clinical significance in preventing bladder carcinogenesis and progression. Bladder cancer is a highly immunogenic malignancy. Urothelial cancer cells aim to manipulate the immune system by inhibiting its cytotoxic function while stimulating the secretion of growth promoting factors. Cytokine-induced imbalances in the distribution and differentiation of tumor-infiltrating cytotoxic cells can boost bladder cancer cell proliferation. Tumor-induced release of excessive amount of cytokines causes an "inflammatory storm" which drives metastasis formation via degradation of extracellular matrix proteins. Tumor-related selective cyclooxygenase-2 (COX-2) upregulation suppresses the cell-mediated immune response via aberrant prostaglandin metabolism resulting in failure of differentiation of myeloid cell progenitors into mature antigen-presenting cells. T cells are capable of increasing the oxidative stress on bladder cancer cells via induction of COX-2 and STEAP expression. Some evidence also suggests that COX-2 activation may be also involved in inflammation-mediated cancer stem cell proliferation. Antibodies against the VEGF-co-receptor neuropilin decrease the angiogenetic potential of bladder cancer cells. Inflammation-based predictive bladder cancer models have demonstrated to accurately predict response to treatment both in the curative and palliative setting. While randomized trials do not support a clinical benefit for the use of anti-inflammatory drugs (i.e., celecoxib, atorvastatin) in preventing recurrence of low-grade bladder cancer, further investigations are warranted in the setting of high-grade tumors since the immune response to cancer stimuli is most probably more pronounced in advanced stages.

Endogenous BTG2 expression stimulates migration of bladder cancer cells and correlates with poor clinical prognosis for bladder cancer patients

Background:The B-cell translocation gene 2 (BTG2) is considered to act as a tumour-suppressor gene because of its antiproliferative and antimigratory activities. Higher levels of BTG2 expression in tumour cells have been linked to a better clinical outcome for several cancer entities. Here, we investigated the expression and function of BTG2 in bladder cancer.Methods:The expression of BTG2 in bladder cancer cells was silenced by RNA interference. Cell motility was investigated by wound healing and Boyden chamber assays. The protein expression of BTG2 in bladder cancer was studied by immunohistochemistry.Results:We observed that targeted suppression of BTG2 by RNA interference did not result in growth stimulation but led to a substantial inhibition of bladder cancer cell motility. Tissue microarray analyses of bladder cancer cystectomy specimens revealed that higher BTG2 expression levels within the tumours correlated strongly with a decreased cancer-specific survival for bladder cancer patients.Conclusion:These results indicate that endogenous BTG2 expression contributes to the migratory potential of bladder cancer cells. Moreover, high levels of BTG2 in bladder cancers are linked to decreased cancer-specific survival. These findings question the conception that BTG2 generally acts as a tumour suppressor and typically represents a favourable clinical marker for cancer patients.

FAVL impairment of the Fanconi anemia pathway promotes the development of human bladder cancer

Effectiveness of DNA cross-linking drugs in the treatment of bladder cancer suggests that bladder cancer cells may have harbored an insufficient cellular response to DNA cross-link damage, which will sensitize cells to DNA cross-linking agents. Cell sensitivity benefits from deficient DNA damage responses, which, on the other hand, can cause cancer. Many changed cellular signaling pathways are known to be involved in bladder tumorigenesis; however, DNA cross-link damage response pathway [Fanconi anemia (FA) pathway], whose alterations appear to be a plausible cause of the development of bladder cancer, remains an under-investigated area in bladder cancer research. In this study, we found FAVL (variant of FA protein L—FANCL) was elevated substantially in bladder cancer tissues examined. Ectopic expression of FAVL in bladder cancer cells as well as normal human cells confer an impaired FA pathway and hypersensitivity to Mitomycin C, similar to those found in FA cells, indicating that FAVL elevation may possess the same tumor promotion potential as an impaired FA pathway harbored in FA cells. Indeed, a higher level of FAVL expression can promote the growth of bladder cancer cells in vitro and in vivo, which, at least partly, results from FAVL perturbation of FANCL expression, an essential factor for the activation of the FA pathway. Moreover, a higher level of FAVL expression was found to be associated with chromosomal instability and the invasiveness of bladder cancer cells. Collectively, FAVL elevation can increase the tumorigenic potential of bladder cancer cells, including the invasive potential that confers the development of advanced bladder cancer. These results enhance our understanding the pathogenesis of human bladder cancer, holding a promise to develop additional effective tools to fight human bladder cancer.

Abstract 74: Differential regulation of Erk signaling by mitochondrial superoxide controls the metastatic potential of bladder cancer cells

Abstract Microarray profiling and analysis of in vivo-selected metastatic bladder cancer has revealed a strong association between manganese superoxide dismutase (Sod2) expression and invasiveness. However, whether increased Sod2 expression contributes to progression from transitional cell carcinoma (TCC) to invasive bladder cancer has not been established. To test this hypothesis we evaluated the impact of Sod2 overexpression on invasive and migratory properties of two distinct TCC cell lines (UC3 and UC6). Sod2 overexpression increased wound healing and matrigel transwell migration of UC6 while inhibiting these activities in the UC3 cell line. Increases or decreases in invasiveness were accompanied by parallel shifts in the levels of matrix metalloproteinases and VEGF. The opposing effects of Sod2 on TCC invasion and the secretory phenotype were associated with differential Erk activation. Sod2-dependent suppression of Erk activation and MMP-1 expression was reversed by EGF activation in UC3 cells while Erk inhibition blocked the Sod2-mediated elevation in ERK activity, MMP-1 and VEGF levels. Activation or inhibition of Erk by Sod2 in the UC6 and UC3 cell lines, respectively, was driven by superoxide (O2−.) and not H2O2. Increasing mitochondrial superoxide by treatments with complex I or III inhibitors reversed the effects of Sod2 overexpression in both cell lines. Sod2-dependent inhibition of invasion in vitro was associated with impairment in growth of orthotopically implanted tumors. These findings unveil distinct redox regulated attributes of TCC that participate in controlling their invasiveness both in vitro and in vivo. In addition, we have identified the ERK signaling network as a primary target for controlling divergent tumorigenic responses to Sod2 overexpression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 74.

Overexpression of claudin-11 decreases the invasive potential of bladder cancer cells in vitro, but has no effect on their electrical resistance

21038 Background: An increasing body of evidence suggests that tight junctions (TJ) play an important role in invasion and metastasis. Claudins (Cl), a group of integral membrane proteins play an important role in maintaining TJ structure and function. Hepatocyte growth factor (HGF) has been shown to disrupt TJ structure. We investigated the effect of forced overexpression of Cl-11 on TJ function and invasive potential of T24 bladder cancer cells, in response to HGF. Methods: Expression of Cl-11 in bladder cancer cells was determined by RT-PCR. Cl-11 expression construct was made from full length human Cl-11 obtained from normal human cDNA bank, using pcDNA 3.1/ NT-GFP-TOPO expression vector. T24 cells were transfected with the resultant expression cassette (T24Cl-11) or an empty plasmid control (T24GFP). Change in TJ function of stably transfected cells was assessed by trans-urothelial resistance (TUR). In- vitro matrigel adhesion and invasion assays were used to determine the malignant potential of the cells. Cells were treated with HGF at 50 ng/ml for TUR and invasion assay. Immunofluorescence was used to localise Cl-11 within the cell. Results: Successful transfection of T24 cells was confirmed by endogenous GFP fluorescence and by an enhanced Cl-11 mRNA signal in T24Cl-11 compared to wild- type (T24WT) or T24GFP. There was no difference in TUR between the three cell types. Adhesion was significantly higher in T24Cl-11 than in T24GFP [median (IQR); 0.11 (0.096–0.137) vs 0.095 (0.089- 0.115), p=0.045]. T24Cl-11 cells had significantly reduced invasiveness in response to HGF, compared to T24GFP [mean (95% CI); 2.4 (1.8–3.01) vs 3.31 (2.89–3.71), p=0.032]. Immunoflourescence staining with Cl-11 showed redistribution of Cl-11 protein to the cytoplasm and perinuclear regions. Conclusion: Cl-11does not appear to be involved in the TJ function in T24 bladder cancer cells; however, its cytoplasmic location and its effect on the metastatic potential of these cells suggests that it may have a role in modulating the cytoskeletal or signalling network of the cell. Further studies are required to elucidate the exact mechanisms involved. No significant financial relationships to disclose.

Bladder Cancer Cell Invasion Is Enhanced by Cross-Talk with Fibroblasts Through Hepatocyte Growth Factor

To examine the effects of fibroblast-derived humoral factors, especially hepatocyte growth factor (HGF), on the invasive potential of bladder cancer cells. Stromal cells in cancer tissue are thought to play an important role in the transformation and invasion of cancer cells. The influence of fibroblast cells (TIG-1 cells) and HGF on the invasive potential of bladder cancer cells (5637, T24, J82, HT1376, and MGHU-1 cells) was evaluated by in vitro cell invasion assay. The expression of HGF and c-Met, which is the receptor of HGF, was examined by reverse transcriptase-polymerase chain reaction. To clarify the relationship between the serum HGF level and invasive bladder cancer, we measured the serum concentrations of HGF in patients with bladder cancer without metastatic disease and normal controls, using an enzyme-linked immunosorbent assay. The in vitro cell invasion assay showed that the number of invading bladder cancer cells was significantly increased by the conditioned medium (CM) of the fibroblast cells. HGF neutralization antibody partially inhibited the enhancement of invasiveness by fibroblast CM. The CM of fibroblasts cultured with bladder cancer CM stimulated cancer cell invasion more strongly (with increased HGF secretion) than did the CM of fibroblasts cultured without bladder cancer CM. The serum HGF levels were significantly greater in patients with muscle-invasive bladder cancer (regardless of tumor size) than in patients with non-muscle-invasive bladder cancer. The present results have suggested that bladder cancer cell invasion is enhanced by cross-talk with fibroblasts through humoral factors, including HGF. Elucidation of this mechanism could lead to novel therapeutic strategies for bladder cancer.

792: Role of Oxidative Stress in Tumorigenic Potential of Bladder Cancer Cells

792: Role of Oxidative Stress in Tumorigenic Potential of Bladder Cancer Cells

Evaluation of Nucleolar Organizer Regions in Human Bladder Cancers by Light- and Electron-Microscopic Morphometry

The number of nucleolar organizer regions (NOR) of human bladder cancers was evaluated at the light- and electron-microscopic level. The average number of argyrophilic NOR (AgNOR), stained by the one-step silver colloid method, was measured in benign and malignant urothelial cells in the human urinary bladder using a light microscope. The average number of nucleolar fibrillar centers (FC) per nucleus was also calculated by quantitative ultrastructural morphometry in the specimens from the same patients. Statistical evaluations revealed that the average number of AgNOR per nucleus was significantly correlated with the elevation of tumor grade and stage (p < 0.05). An average FC number per nucleus also increased in association with tumor grade and stage (p < 0.05). Although the average number of FC was 5.6 times higher than that of AgNOR, the correlation between the average number of FC and AgNOR was statistically significant. In conclusion, these results suggested that the silver staining method was a useful and convenient tool for the evaluation of the differentiation and invasive potential of bladder cancer cells at the light-microscopic level.

Bacillus Calmette-Guérin enhances production and secretion of type IV collagenases in peripheral blood mononuclear cells.

Intravesical administration of bacillus Calmette‐Guérin (BCG) is an effective and widely accepted treatment for superficial bladder cancer. Rapid progression of the disease after BCG therapy, however, has been reported in some cases refractory to the treatment. We examined whether BCG treatment and coexistence of peripheral blood mononuclear cells (PBMCs) alter the invasive potential of bladder cancer cells. Production and secretion of two type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP 9, by PBMCs from five healthy donors or bladder cancer cells (T24, JTC 30, and JTC 32) were evaluated by gelatin zymography, western blot analysis, and northern blot analysis. Invasion of bladder cancer cells was also examined using reconstituted basement membrane (Matrigel). BCG (5, 50, and 500 μg/ml) had no effect on secretion of MMP 2 and MMP 9 by bladder cancer cells, but increased the production and secretion of MMP 9 by PBMCs in a dose‐dependent manner. The coexistence of PBMCs increased invasion of T24 cells and BCG further enhanced the invasion. Thus, BCG promotes invasion of bladder cancer cells under certain conditions. An increase in the secretion of MMP 9 by PBMCs may account in part for the effect.