Role In Bladder Cancer Progression Research Articles

Long non-coding RNA UCA1 promotes the tumorigenesis in pancreatic cancer.

The contribution of long non-coding RNAs (lncRNAs) to tumorigenesis and metastasis of pancreatic cancer (PC) remains largely unknown. Urothelial cancer-associated 1 (UCA1), which is an originally identified lncRNA in bladder cancer, has be proved to play a pivotal role in bladder cancer progression and embryonic development. In this study, we detected the mRNA expression of UCA1 in 128 PC patients by qRT-PCR, and found that UCA1 expression was significantly, up-regulated in tumor tissues than that in matched adjacent non-tumor tissues (p<0.05). Clinicopathological analysis demonstrated that UCA1 expression in PC significantly correlated with malignant potential factors such as tumor size (p=0.021), depth of invasion (p=0.033), CA19-9 level (p=0.034) and tumor stage (p=0.013). Cox proportional hazards regression analysis also confirmed that high UCA1 expression was an independent prognostic biomarker of PC (p=0.046), which led to an obviously shorter 5-year overall survival (OS) compared to those patients with low UCA1 expressions (p=0.018). Furthermore, we effectively down-regulated UCA1 mRNA expression by transfecting RNA interfere fragments into SW-1990 cells, and our results in vitro indicated that down-regulation of UCA1 could effectively inhibit the cell proliferative activities, induce apoptotic rate and cause cell cycle arrest in PC cells (p<0.05). Meanwhile, UCA1 expression negative-correlated with p27 in PC tissues (r2=0.46, p<0.01), and knockdown of p27 partly abrogated the cell proliferative activities caused by UCA1 (p<0.05). Our results raised the possibility of using UCA1 as a potential prognostic biomarker and therapy target of PC, and down-regulation of UCA1 might be considered to be a novel molecular treatment strategy for patients with PC.

PD38-12 BLADDER CANCER PROGRESSION IS ASSOCIATED WITH THE ACCUMULATION OF MDSC INDUCED BY DOWN-REGULATION OF IL6

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2016PD38-12 BLADDER CANCER PROGRESSION IS ASSOCIATED WITH THE ACCUMULATION OF MDSC INDUCED BY DOWN-REGULATION OF IL6 guoliang yang, lianhua zhang, ming cao, haige chen, and juanjie bo guoliang yangguoliang yang More articles by this author , lianhua zhanglianhua zhang More articles by this author , ming caoming cao More articles by this author , haige chenhaige chen More articles by this author , and juanjie bojuanjie bo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1492AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Myeloid-derived suppressor cells (MDSC) are widely implicated in immune suppression associated with tumor progression and chronic in?ammation. However, very little is known about their possible role in bladder cancer development. Here, we evaluated the role of MDSC in bladder cancer. METHODS Flow cytometry was performed on peripheral blood of 113 patients with bladder cancer and 20 healthy controls with a median follow-up of 43 months for overall survival (OS). We also assessed the expression of MDSC-inducing cytokines. RESULTS For the ?rst time, we characterized CD33-CD11b+HLA-DR- cells sorted from the peripheral blood of BC patients. CD33-CD11b+HLA-DR- cells suppressed autologous, antigen-independent T cell proliferation. The frequency of MDSC correlated with tumor grade and stage. We also showed that increased levels of circulating MDSCs correlate with reduced overall survival. Increases in the levels of pro-MDSC cytokines were observed for bladder cancer patients versus controls, and the pro-MDSC cytokine IL-6 was decreased in those patients, down-regulation of IL6 is sufficient to induce MDSC formation in vitro. CONCLUSIONS MDSC played a major role in bladder cancer progression and survival, which suggests that their targeting may provide clinical bene? ts in bladder cancer © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e929 Advertisement Copyright & Permissions© 2016MetricsAuthor Information guoliang yang More articles by this author lianhua zhang More articles by this author ming cao More articles by this author haige chen More articles by this author juanjie bo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of Sonic Hedgehog (Shh) Signaling in Bladder Cancer Stemness and Tumorigenesis.

Sonic hedgehog (Shh) signaling pathway has emerged as a critical component of bladder development, cancer initiation, and progression. While the role of Shh signaling in bladder development is well documented, its role in bladder cancer progression is uncertain. Additionally, epithelial-to-mesenchymal transition (EMT) has been identified to promote bladder cancer progression in the initial stages and also contribute to drug resistance in the later stage and ultimately metastasis. We speculate that epithelial-to-mesenchymal transitions (EMT) and Shh fuel the carcinogenesis process. This review presents the most recent studies focusing on the role of Shh signaling in bladder cancer progression.

GOLPH3 is a potential therapeutic target and a prognostic indicator of poor survival in bladder cancer treated by cystectomy.

Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development of several human cancers. However, its clinical significance and biological role in bladder cancer remains unclear. In this study, we sought to analyze the GOLPH3 expression in bladder cancer samples and cells, and explore its clinical significance and biological role. We found that GOLPH3 was significantly increased in bladder cancer tissues and cells. Overexpression of GOLPH3 had significant correlation with poorer survival for bladder cancer patients treated by cystectomy. Knockdown of GOLPH3 inhibited the proliferation, migration and invasion of cancer cells, and tumor growth in a xenograft mouse model. GOLPH3 silencing inhibited AKT/m-TOR signaling, increased the cyclin-dependent kinase (CDK) inhibitor p27 and decreased the CDK regulator cyclin D1 and matrix metallopeptidase 9 (MMP9). Thus, GOLPH3 is likely to play important roles in bladder cancer progression via modulating AKT/mTOR signaling, and it is a novel prognostic biomarker and promising therapeutic target for bladder cancer.

Involvement of the insulin-like growth factor I receptor and its downstream antiapoptotic signaling pathway is revealed by dysregulated microRNAs in bladder carcinoma

ObjectiveUrothelial carcinoma is one of the most common pathological types of bladder cancer. Several studies have shown that dysregulated microRNAs (miRNAs) play an important role in bladder cancer progression. We performed the present miRNA microarray analysis in samples of urothelial carcinoma of the bladder and adjacent normal bladder tissue from Taiwanese patients to investigate dysregulated miRNAs. Materials and methodsTo detect dysregulated miRNAs in urothelial carcinoma of the bladder, samples of tumor and adjacent normal tissues were collected from 10 patients. Tissue samples from three patients were subjected to miRNA microarray analysis, and the remaining tissue samples from the other seven patients were used to validate the results obtained from the microarray data. Potential targets of these dysregulated miRNAs were identified using online databases, including MicroCosm and TargetScan. ResultsA panel of 30 differentially expressed miRNAs with at least fourfold differences in expression compared with normal controls, including 19 upregulated and 11 downregulated miRNAs, was generated. The expression levels of miR-30a-5p, miR-30a-3p, miR-99a, miR-130b, miR-133b, miR-135b, miR-145, miR-195, miR-204, and miR-214 were experimentally verified using real-time RT-PCR analysis. Using an online miRNA target database, we discovered that these dysregulated miRNAs potentially control components of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway. ConclusionOur results indicate that dysregulated miRNAs may be involved in bladder cancer pathogenesis and are potential biomarkers.

Upregulation of long non-coding RNA urothelial carcinoma associated 1 by CCAAT/enhancer binding protein α contributes to bladder cancer cell growth and reduced apoptosis

Long non-coding RNA urothelial carcinoma associated 1 (lncRNA-UCA1) is upregulated in bladder cancer and plays a pivotal role in bladder cancer progression and metastasis. Recent studies and our research found that lncRNA-UCA1 may be an important biomarker and therapeutic target for bladder cancer. However, the molecular mechanism involved in the upregulation of lncRNA-UCA1 in bladder cancer is largely unknown. In the present study, we showed that lncRNA-UCA1 expression in bladder cancer cells was upregulated by transcription factor CCAAT/enhancer binding protein α (C/EBPα), which was the only candidate transcription factor simultaneously predicted by a total of five bioinformatical software programs. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay indicated that C/EBPα bound to the lncRNA-UCA1 core promoter region in vitro and in vivo. The luciferase assays further showed that there was a point mutation (A231G) in the C/EBPα binding site of the lncRNA-UCA1 core promoter in various bladder cancer cell lines, which in turn significantly increased the transcriptional activity of lncRNA-UCA1. We also demonstrated that C/EBPα siRNA treatment contributed to the downregulation of lncRNA-UCA1 expression, whereas overexpression of C/EBPα enhanced lncRNA-UCA1 expression. Furthermore, lncRNA-UCA1 transcriptional repression by C/EBPα siRNA sharply reduced cell viability and induced cell apoptosis in vitro. Collectively, our results provide a novel therapeutic strategy for bladder cancer by effectively interrupting the binding of the lncRNA-UCA1 promoter and certain transcription factors, so as to reverse the upregulation of lncRNA-UCA1 and prevent bladder cancer progression.

Suppression of ERβ signaling via ERβ knockout or antagonist protects against bladder cancer development

Epidemiological studies showed that women have a lower bladder cancer (BCa) incidence, yet higher muscle-invasive rates than men, suggesting that estrogen and the estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), may play critical roles in BCa progression. Using in vitro cell lines and an in vivo carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced mouse BCa model, we found that ERβ plays a positive role in promoting BCa progression. Knockdown of ERβ with ERβ-shRNA in ERβ-positive human BCa J82, 647v and T24 cell lines led to suppressed cell growth and invasion. Mice lacking ERβ have less cancer incidence with reduced expression of the proliferation marker Ki67 in BBN-induced BCa. Consistently, our results show that non-malignant urothelial cells with ERβ knockdown are more resistant to carcinogen-induced malignant transformation. Mechanism dissection found that targeting ERβ suppressed the expression of minichromosome maintenance complex component 5 (MCM5), a DNA replication licensing factor that is involved in tumor cell growth. Restoring MCM5 expression can partially reverse ERβ knockdown-mediated growth reduction. Supportively, treating cells with the ERβ-specific antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), reduced BCa cell growth and invasion, as well as MCM5 expression. Furthermore, we provide the first evidence that BCa burden and mortality can be controlled by PHTPP treatment in the carcinogen-induced BCa model. Together, these results demonstrate that ERβ could play positive roles in promoting BCa progression via MCM5 regulation. Targeting ERβ through ERβ-shRNA, PHTPP or via downstream targets, such as MCM5, could serve as potential therapeutic approaches to battle BCa.

Knockdown of regulator of cullins-1 (ROC1) expression induces bladder cancer cell cycle arrest at the G2 phase and senescence.

Regulator of Cullins-1 (ROC1) is a key subunit in the Cullin-RING ligase (CRL) protein complex. Overexpression of ROC1 protein is associated with tumor progression and poor prognosis of non-muscle invasive bladder transitional cell carcinoma (NMIBC). This study was designed to assess the effects of ROC1 knockdown in bladder cancer cells and to determine the potential mechanisms involved. A total of 112 bladder cancer tissue specimens were recruited for immunohistochemical analyses of ROC1 overexpression. Bladder cancer cell lines were used to knockdown ROC1 expression using ROC1 siRNA. Our data showed that ROC1 knockdown remarkably inhibited bladder cancer cell growth, arrested cells at the G2 phase of the cell cycle, and induced the p53-dependent cell senescence. Molecularly, G2 arrest was associated with upregulation of p21, p27, cyclin B1, and Cdc2 proteins. ROC1 knockdown induced-senescence functioned through p53/p21 pathway. Knockdown of p21 expression partially rescued ROC1 knockdown-induced growth inhibition in cancer cells. Furthermore, nude mouse xenograft analyses confirmed these in vitro data. In conclusion, data from the current study indicate that ROC1 plays an essential role in bladder cancer progression and could serve as a novel anticancer target for bladder transitional cell carcinoma (BTCC).

Abstract 310: Role of AMPKalpha isoforms in bladder tumorigenesis.

Abstract Bladder cancer is the fifth most diagnosed cancer and currently is one of the most expensive cancers to treat due to lifelong surveillance. Bladder cancer is marked by two distinct pathways that result in either noninvasive low grade or invasive high grade cancer. Although low and high grade bladder cancers are defined by driver mutations, similarities such as mTORC1 pathway activation exist. Along these lines rapamycin treatment, an mTORC1 inhibitor, has been shown to suppress tumorigenesis in a mouse model of bladder cancer. These data suggest that mTORC1 activation may be an early tumorigenic event in the bladder. In order to elucidate the mechanism by which the mTORC1 signaling is increased, AMPK (AMP-activated protein kinase), a critical negative regulator of the pathway, was further investigated. AMPK governs a cell's response to metabolic stressors by suppressing anabolic pathways and inhibiting cell growth. Furthermore, it has been observed that AMPK activation is diminished in primary breast cancer. In order to determine whether AMPK activation and/or levels are altered in bladder cancer, a pilot cohort of human samples was obtained and the expression of both AMPKa catalytic isoforms was assessed. Both AMPKa1 and AMPKa2 were significantly suppressed in bladder cancer. AMPKa isoform levels in adjacent non-tumor bladder epithelium and bladder cancer were evaluated in matched patient samples and this revealed cancer-specific AMPKa suppression does indeed exist. Additionally, AMPKa1 protein levels were partially suppressed in low grade cancer and further suppressed in high grade cancer. In contrast, AMPKa2 levels were uniformly suppressed in both high and low grade of cancer. Interestingly, it was observed that AMPKa1 is localized predominantly to the cytoplasm while AMPKa2 was present in cytoplasmic and nuclear compartments. This data suggests that the AMPKa isoforms may have distinct roles in the bladder. To test whether loss of AMPKa2 function promoted bladder tumorigenesis, wild-type and AMPKa2-/- mice received 0.05% BBN [n-butyl-n-(4 hydroxybutyl)] nitrosamine, a rodent bladder carcinogen, in drinking water over the course of 20 weeks. Significant tumors were observed at 20 weeks in both wild-type and AMPKa2-/- mice and bladder weights were significantly higher in AMPKa2-/- mice correlating with larger bladder tumor mass. Additionally, there was a significant increase in the amount of proliferating tumor cells as assessed by Ki67 in the AMPKa2-/- mice. This study suggests that AMPKa isoforms are suppressed in bladder cancer and that AMPKa may play a critical role in bladder cancer progression by loss of expression and tumor suppressive function. Citation Format: Stavros Kopsiaftis, Kathryn N. Phoenix, Katie L. Sullivan, Poornima Hegde, John A. Taylor, Kevin P. Claffey. Role of AMPKalpha isoforms in bladder tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2013-310

Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.

Abstract Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression. Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones. Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death. Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes. Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG). Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922

Evaluation of the Antitumor Activity of Dacomitinib in Models of Human Bladder Cancer

Members of the human epidermal growth factor receptor (HER) family play a significant role in bladder cancer progression and may underlie the development of chemotherapy resistance. Dacomitinib is an irreversible tyrosine kinase inhibitor with structural specificity for the catalytic domains of epidermal growth factor receptor (EGFR), HER2 and HER4 that has exhibited vigorous efficacy against other solid tumors. We evaluated the antitumor activity of dacomitinib in human bladder cancer cell lines expressing varying levels of HER family receptors. These cell lines also were established as bladder cancer xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to assess dacomitinib activity in vivo. Significant cytotoxic and cytostatic effects were noted in cells expressing elevated levels of the dacomitinib target receptors with apoptosis and cell cycle arrest being the predominant mechanisms of antitumor activity. Cells expressing lower levels of HER receptors were much less sensitive to dacomitinib. Interestingly, dacomitinib was more active than either trastuzumab or cetuximab in vitro, and exhibited increased growth inhibition of bladder tumor xenografts compared with lapatinib. Pharmacodynamic effects of dacomitinib included decreased E-cadherin (E-cad) expression, reduction of EGFR and extracellular signal-regulated kinase (ERK) phosphorylation and reduced mitotic count. Dacomitinib also inhibited tumor growth in a chemotherapy-resistant xenograft and, when combined with chemotherapy in a sensitive xenograft, exhibited superior antitumor effects compared with individual treatments. Evaluation in xenograft-bearing mice revealed that this combination was broadly feasible and well tolerated. In conclusion, dacomitinib exhibited pronounced activity both as a single agent and when combined with chemotherapy in human bladder cancer models. Further investigation of dacomitinib in the preclinical and clinical trial settings is being pursued.

Expression analysis and clinical significance of CXCL16/CXCR6 in patients with bladder cancer

The interactions between chemokines and their receptors are closely involved in the progression and metastasis of cancer. We hypothesized that the CXCL16-CXCR6 ligand-receptor system plays an important role in bladder cancer progression. To evaluate this hypothesis, the expression levels of CXCL16 and CXCR6 were evaluated in 160 patients, including 155 patients with bladder cancer and 5 patients with benign bladder disease. The tissues were analyzed by immunohistochemical (IHC) staining and real-time reverse-transcription polymerase chain reaction. We compared the expression of CXCL16/CXCR6 in bladder cancer and benign bladder disease. The expression of CXCR6 was increased in patients with bladder cancer compared with benign bladder disease in RT-PCR. The mRNA expression levels of CXCL16 and CXCR6 were 1.75×10(-2) and 1.99×10(-2) in benign bladder tissue and 1.39×10(-2) and 2.32×10(-2) in bladder cancer tissue, respectively. In IHC staining, the expression of CXCL16/CXCR6 in bladder cancer tissues was higher compared with benign bladder tissues. On multivariate analysis, the IHC staining of CXCL16 was correlated with the 2004 WHO grade and lymphovascular invasion (P=0.021 and P=0.011, respectively). CXCR6 was correlated with the 1973 WHO grade (P=0.001), 2004 WHO grade (P<0.001), pathological T stage (P=0.002) and perineural invasion (P=0.031). However, Cox regression analysis revealed that the expression of CXCL16 and CXCR6 was not correlated with cancer recurrence and cancer-specific survival (P=0.142 and P=0.324, respectively). The expression of CXCL16/CXCR6 was higher in bladder cancer compared to benign disease and correlated with aggressive cancer behavior. Based on our results, the CXCL16/CXCR6 axis appears to be important in the progression of bladder cancer. Thus, CXCL16 and CXCR6 serve as potential therapeutic targets.

The Role of the Androgen Receptor in the Development and Progression of Bladder Cancer

Men are at a higher risk of developing bladder cancer than women. Since bladder cancer cell lines and tissues were found to express the androgen receptor, efforts have been made to inspect whether androgen-mediated androgen receptor signals are implicated in bladder carcinogenesis as well as cancer progression. Mounting evidence supports the view that bladder cancer is a member of the endocrine-related tumors and may clearly explain the gender-specific difference in the incidence. However, the underlying mechanisms of how androgen receptor signals regulate bladder cancer growth are still far from fully characterized. Moreover, it remains controversial whether the androgen receptor pathway always plays a dominant role in bladder cancer progression. In this review, we summarize the available data on the involvement of androgen receptor signaling in bladder cancer. In particular, current evidence demonstrating the stimulatory effects of androgens on tumor progression or, more convincingly, tumorigenesis via the androgen receptor pathway may offer great potential for androgen deprivation as a therapeutic or chemopreventive option in patients with bladder cancer.

Long non-coding RNA UCA1a(CUDR) promotes proliferation and tumorigenesis of bladder cancer

Our previous studies identified UCA1 as a novel biomarker for bladder cancer and detected three variant transcripts of UCA1 in the human bladder TCC cell line BLZ-211 using northern blot analysis. One (1.4 kb) of the three transcripts has been shown to play a pivotal role in bladder cancer progression and embryonic development. In this study, we cloned a second transcript (2.2 kb), designated UCA1a, which was identical to previously reported cancer upregulated drug resistant gene (CUDR). Sequence comparison of UCA1 (1.4 kb transcript) and UCA1a(CUDR) cDNA revealed a 1,265 bp common region. Previous studies have demonstrated that CUDR is upregulated in various human tumors, including colon, cervical and lung cancer. However, the exact role of UCA1a(CUDR) in bladder cancer has not yet been reported. In this study, RT-PCR analysis indicated that UCA1a(CUDR) was also an embryonic development and bladder cancer-associated RNA. Overexpression of UCA1a(CUDR) significantly enhanced proliferation, migration and invasion of the bladder cancer cell line UM-UC-2. Moreover, microarray analysis demonstrated that overexpression of UCA1a(CUDR) was associated with signaling pathways regulating cell apoptosis and tumorigenesis. Furthermore, overexpression of UCA1a(CUDR) could antagonize cell apoptosis induced by cisplatin and promote the tumorigenicity of UM-UC-2 cells in vivo. Taken together, our data strongly suggest that similar to the 1.4 kb transcript of UCA1, UCA1a(CUDR) may also play an important role in the growth and tumorigenesis of human bladder cancer, and their common region may be critical for biological activity, thereby indicating that their common region may serve as a new therapeutic target for bladder cancer.

Abstract 2293: MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer.

Abstract Introduction and Objective: The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer progression. Tumor suppressive miRNAs targeting these cancer pathways may provide a new therapeutic approach for bladder cancer. Methods: We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC, MEK1 using several target scan algorithms. The expression of miR-1826 was confirmed in bladder cancer tissues and bladder cancer cell lines. Then we performed several functional analyses of miR-1826. Finally 3′UTR luciferase assay and Western blot were performed to look at relationship between miR-1826 and target genes. Results: MiR-1826 expression was significantly lower in bladder cancer tissues and bladder cancer cell lines (J82, T24, TCC-SUP) compared to normal bladder tissues and normal bladder cell lines (SV-HUC-1). The expression of miR-1826 was lower in bladder cancer tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. The functional analyses showed that miR-1826 inhibited bladder cancer cell viability, invasion and migration. Also we also found increased apoptosis and G1 cell cycle arrest in miR-1826 transfected bladder cancer cells. 3′UTR luciferase activity and protein expression of these target genes (CTNNB1, MEK1, VEGFC) were significantly down-regulated in miR-1826 transfected bladder cancer cells (J82, T24). To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knock down, we knocked down CTNNB1/MEK1 mRNA using a si-RNA technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in bladder cancer cells. Conclusions: Our data suggests that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC down-regulation in bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2293. doi:1538-7445.AM2012-2293

The tumor-suppressor gene Nkx2.8 suppresses bladder cancer proliferation through upregulation of FOXO3a and inhibition of the MEK/ERK signaling pathway

Invasive bladder cancer is a lethal disease for which effective prognostic markers as well as potential therapy targets are still lacking. Nkx2.8 (Nk2 homeobox 8), a novel member of the NK-2 gene family, was reported to play an important role in the development and progression of human cancer. Herein, we reported that Nkx2.8 was markedly reduced in bladder cancer tissues compared with matched adjacent normal urothelial tissues. Nkx2.8 levels were inversely correlated with advanced T classification, N classification, tumor multiplicity, high proliferation index (Ki-67) and poor survival of patients. Furthermore, we found that overexpression of Nkx2.8 in bladder cancer cells significantly inhibited cell proliferation in vitro and in vivo, whereas silencing Nkx2.8 dramatically enhanced cell proliferation. Moreover, we demonstrated that overexpression of Nkx2.8 resulted in G(1)/S phase arrest, accompanied by upregulation of p27(Kip1), downregulation of cyclin D1 and p-FOXO3a and inhibition of MEK/ERK pathway activity. Meanwhile, silencing Nkx2.8 led to acceleration of G(1)/S transition, downregulation of p27(Kip1), upregulation of cyclin D1 and p-FOXO3a and increase of MEK/ERK pathway activity. These findings suggest that Nkx2.8 plays a potential tumor suppressor role in bladder cancer progression and represents a valuable clinical prognostic marker of this disease.

Nuclear Hormone Receptor Signals as New Therapeutic Targets for Urothelial Carcinoma

Unlike prostate and breast cancers, urothelial carcinoma of the urinary bladder is not yet considered as an endocrine-related neoplasm, and hormonal therapy for bladder cancer remains experimental. Nonetheless, there is increasing evidence indicating that nuclear hormone receptor signals are implicated in the development and progression of bladder cancer. Androgen-mediated androgen receptor (AR) signals have been convincingly shown to induce bladder tumorigenesis. Androgens also promote the growth of AR-positive bladder cancer cells, although it is controversial whether AR plays a dominant role in bladder cancer progression. Both stimulatory and inhibitory functions of estrogen receptor signals in bladder cancer have been reported. Various studies have also demonstrated the involvement of other nuclear receptors, including progesterone receptor, glucocorticoid receptor, vitamin D receptor, and retinoid receptors, as well as some orphan receptors, in bladder cancer. This review summarizes and discusses available data suggesting the modulation of bladder carcinogenesis and cancer progression via nuclear hormone receptor signaling pathways. These pathways have the potential to be an extremely important area of bladder cancer research, leading to the development of effective chemopreventive/therapeutic approaches, using hormonal manipulation. Considerable uncertainty remains regarding the selection of patients who are likely to benefit from hormonal therapy and optimal options for the treatment.

Differential Protein Expression Profiling by iTRAQ-Two-dimensional LC-MS/MS of Human Bladder Cancer EJ138 Cells Transfected with the Metastasis Suppressor KiSS-1 Gene

KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.

Abstract 3196: The role of Ga12 and Thromboxane receptor beta interaction in bladder cancer progression

Abstract In the United States, it is predicted that there will be over 70,000 new cases of bladder cancer this year. Bladder cancer is the fourth most common cancer in men and eighth in women. Our lab has identified that Thromboxane receptor beta (TPβ) plays a role in bladder cancer progression. Our studies have previously shown that TPβ can induce malignant transformation in the immortalized non-transformed bladder cell line, SV-HUC. TPβ is a G-protein coupled receptor that is over-expressed in bladder cancer tumors and high levels of TPβ in bladder cancer patients correlate with decreased survival. G-proteins, such as Gα12, are involved in second messenger cascades that influence a variety of cellular functions, including transcription and motility. Constitutive activation of Gα12 has been shown to induce malignant transformation. Gα12 messenger RNA and protein are expressed in bladder cell lines and paraffin-embedded tissue obtained from bladder cancer patients. This study was designed to elucidate the TPβ and Gα12 dependent pathways that contribute to bladder cancer progression. We found that treatment with TP agonist, U46619, increases cell migration, while treatment with TP antagonist, GR32191, blocks agonist dependent increase in cell migration. We observe that knockdown of Gα12 with shRNA partially reduces U46619-induced cell migration and MAP kinase signaling. Together our results suggest that TPβ mediated malignant phenotypes are in part modulated by Gα12. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3196.

Abstract B26: The role of G alpha12 and thromboxane receptor beta interaction in bladder cancer progression

Abstract In the United States, it is predicted that there will be over 70,000 new cases of bladder cancer this year. Bladder cancer is the fourth most common cancer in men and eighth in women. Our lab has previously identified Thromboxane receptor-β (TP-β) as potentially playing a role in bladder cancer progression. TP-β is a G-protein coupled receptor that is over-expressed in bladder cancer tumors and high levels of TP-β in bladder cancer patients correlate with decreased survival. G-proteins are involved in second messenger cascades that influence a variety of cellular functions, including transcription and motility. In multiple cellular systems TP-β has been found to interact with multiple G proteins such as Gαi, Gαs, Gα12, and Gα13. We found high levels of mRNA and protein expression of the G-protein, Gα12, in bladder cancer cell lines. We also observe high levels of Gα12 expression in human bladder cancer tissues. Studies using a constitutively active Gα12 mutant, suggest that signaling through Gα12 can contribute to oncogenic transformation. In this study we aim to elucidate the TP-β associated Gα12 dependent pathways that contribute to bladder cancer progression. We found that TP agonist U46619 increases cell migration and that treatment with TP antagonist blocks this increase in cell migration induced by U46619. TP agonist stimulation also increases the phosphorylation of extracellular regulated kinase (ERK) and U46619-induced phosphorylation of ERK is blocked with TP agonist treatment. Using a shRNA to knockdown Gα12 we observe a reduction in U46619-induced cell migration and ERK phosphorylation. Together our results suggest that TP-β mediated cell migration and the MAPK signaling pathway is in part regulated through Gα12. This work could have clinical implications by targeting the TP-β and Gα12 interaction as a potential therapeutic approach for bladder cancer treatment. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B26