Progression Of Human Bladder Cancer Research Articles

Down-regulation of Polo-like kinase 4 (PLK4) induces G1 arrest via activation of the p38/p53/p21 signaling pathway in bladder cancer.

Polo‐like kinase 4 (PLK4) has been reported to contribute to tumor growth, invasion, and metastasis. However, the role of PLK4 in human bladder cancer (BC) remains unclear. Here, we demonstrate the regulatory function of PLK4 in human BC progression. PLK4 is overexpressed in BC cell lines and tissues, and its overexpression correlated with poor prognosis. Our transcriptome analysis combined with subsequent functional assays indicated that PLK4 inhibition can suppress BC cell growth and induce cell cycle arrest at G1 phase via activation of the p38/p53/p21 pathway in vitro and in vivo. Overall, our data suggest that PLK4 is a critical regulator of BC cell proliferation, and thus, it may have potential as a novel molecular target for BC treatment.

NCR3LG1 (B7-H6) is a potential prognostic factor for bladder cancer patients

Background NCR3LG1 (B7-H6) protein is selectively overexpressed on tumour and is associated with fatal disease progression of various cancer. However, its prognostic value in bladder cancer (BCa) has not been well elaborated. Methods We examined the expression of NCR3LG1 in human BCa and analysed its clinical significance and prognostic value. Meanwhile, the expression of NCR3LG1 was intervened in human BCa cell line 253JBV to analyse subsequent effects on tumour. Results According to TCGA data, the disease-free survival rate was statistically significant between the NCR3LG1 high expression group and the low expression group (Log Rank p = 0.006). Immunohistochemical staining showed that the expression of NCR3LG1 in BCa tissue was significantly higher than that in adjacent tissues (p < 0.0001), which was positively correlated with TNM staging (p = 0.008), histological grade (p = 0.022), and lymphoma metastasis of BCa (p = 0.032). The proliferation (p < 0.0001), invasion (p < 0.001) and migration ability (p < 0.001) of 253JBV cells are significantly inhibited by knocking down the expression of NCR3LG1, and the cell cycle arrest is induced at the G1 phase, which accelerates the apoptosis of BCa cells (p < 0.005). Conclusion Our findings indicate that NCR3LG1 is involved in the progression of human BCa and may become a potential prognostic biomarker for BCa.

Corrigendum] Mutation of TGF‑β receptorII facilitates human bladder cancer progression through altered TGF‑β1 signaling pathway.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that, in Fig.1B on p.1552, the MCF‑7 andT24, and the A549 andScaBER data panels, respectively, appeared to be strikingly similar. After having re‑examined the original data, the authors have realized that these pairings of data panels were indeed duplicates of each other. Essentially, errors were made in the labelling of the data panels pertaining to the separate experiments, and in the compilation of the published version of Fig.1. The authors, however, were willing to repeat the affected experiments, and obtained results that were consistent with those of the experiments that had been originally performed. Consequently, the revised version of Fig.1 is shown below, showing the new data for Fig.1B. The results from the flow cytometric analysis demonstrated the abnormally high expression level of TGF‑β receptorII in T24cells. The authors confirm that these data support the main conclusions presented in their paper, and are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum. They also apologise to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 43: 1549‑1559, 2013; DOI: 10.3892/ijo.2013.2065].

IGF2BP3 facilitates cell proliferation and tumorigenesis via modulation of JAK/STAT signalling pathway in human bladder cancer.

Insulin‐like growth factor‐2 messenger RNA‐binding protein 3 (IGF2BP3) has been reported to contribute to tumorigenesis in several human cancers. However, the biological functions of IGF2BP3 in bladder cancer are poorly understood. We investigated the relation between IGF2BP3 expression and prognosis of bladder cancer patients. Cell proliferation, cell cycle and cell apoptosis assays were performed to assess IGF2BP3 functions. The results showed that IGF2BP3 was overexpressed in bladder cancer tissues compared with that in normal bladder tissues, and its higher expression was closely correlated with poor prognosis in bladder cancer patients. Overexpression of IGF2BP3 markedly promoted cell proliferation and cell cycle progression and inhibited cell apoptosis, while knockdown of IGF2BP3 notably suppressed the proliferation, promoted cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Mechanistically, we revealed that IGF2BP3 promotes the activation of the JAK/STAT pathway in bladder cancer cells. Moreover, the JAK/STAT inhibitor dramatically blocked the tumour‐promoting activity of IGF2BP3. Tumour growth in vivo was also suppressed by knocking down of IGF2BP3. Hence, IGF2BP3 facilitated bladder cancer cell proliferation by activating the JAK/STAT signalling pathway. These findings suggest that IGF2BP3 exhibits an oncogenic effect in human bladder cancer progression.

An outcome model for human bladder cancer: A comprehensive study based on weighted gene co-expression network analysis.

The precision evaluation of prognosis is crucial for clinical treatment decision of bladder cancer (BCa). Therefore, establishing an effective prognostic model for BCa has significant clinical implications. We performed WGCNA and DEG screening to initially identify the candidate genes. The candidate genes were applied to construct a LASSO Cox regression analysis model. The effectiveness and accuracy of the prognostic model were tested by internal/external validation and pan‐cancer validation and time‐dependent ROC. Additionally, a nomogram based on the parameter selected from univariate and multivariate cox regression analysis was constructed. Eight genes were eventually screened out as progression‐related differentially expressed candidates in BCa. LASSO Cox regression analysis identified 3 genes to build up the outcome model in E‐MTAB‐4321 and the outcome model had good performance in predicting patient progress free survival of BCa patients in discovery and test set. Subsequently, another three datasets also have a good predictive value for BCa patients' OS and DFS. Time‐dependent ROC indicated an ideal predictive accuracy of the outcome model. Meanwhile, the nomogram showed a good performance and clinical utility. In addition, the prognostic model also exhibits good performance in pan‐cancer patients. Our outcome model was the first prognosis model for human bladder cancer progression prediction via integrative bioinformatics analysis, which may aid in clinical decision‐making.

MicroRNA-124-3p suppresses cell migration and invasion by targeting ITGA3 signaling in bladder cancer

A growing body of studies have demonstrated the aberrant expression of microRNAs (miRNAs) contributes to human tumor metastasis. MicroRNA-124-3p (miR-124-3p), which is down-regulated in various cancers, has been found to be involved in several signaling pathways relevant to tumor cell migration and invasion. However, the roles of miR-124-3p in human bladder cancer remain unclear. This study aims to investigate the functional significance of miR-124-3p and to understand how it targets the integrin receptor, and thus affects the progression of human bladder cancer. Clinical specimens from 36 patients and three human bladder cancer cell lines were analyzed for miR-124-3p and integrin α3 (ITGA3) . To investigate the effects of miR-124-3p and ITGA3 on proliferation of bladder cancer cells, the MTT assay, colon-formation assay and flow cytometry were performed. In addition, wound healing assay and transwell assay were carried out to examine the migration and invasion of the bladder cancer cells transfected with miR-124-3p mimics or si-ITGA3. The luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied to validate the miR-124-3p directly binding with ITGA3. Finally, western blot was used to examine the expression level of the proteins involved in FAK/PI3K/AKT and FAK/Src signal pathway as well as epithelial-mesenchymal transition (EMT) process. The down-regulation of miR-124-3p and up-regulation of ITGA3 were observed in clinical specimens and bladder cancer cell lines. Overexpression of miR-124-3p or silencing ITGA3 inhibited tumor cell migration and invasion. Luciferase assay confirmed miR-124-3p directly targets ITGA3, and western blot suggested that miR-124-3p plays a crucial role in the EMT and metastasis of human bladder cancer through FAK/PI3K/AKT and FAK/Src signaling mechanism. Also, by targeting ITGA3, miR-124-3p can modulate the expression of N- and E-cadherin, and thus inhibit the EMT. By targeting ITGA3 and downstream FAK/PI3K/AKT and FAK/Src signaling pathways, miR-124-3p suppresses cell migration and invasion in bladder cancer. Our study reasonably speculates that miR-124-3p can be potentially developed as a therapeutic target and prognostic biomarker for bladder cancer.

Cancer-associated fibroblasts induce epithelial\u2013mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling

BackgroundCancer-associated fibroblasts (CAFs), activated by tumour cells, are the predominant type of stromal cells in cancer tissue and play an important role in interacting with neoplastic cells to promote cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. However, the mechanism by which CAFs induce EMT program in bladder cancer cells remains unclear.MethodsTo investigate the role of CAFs in bladder cancer progression, healthy primary bladder fibroblasts (HFs) were induced into CAFs (iCAFs) by bladder cancer-derived exosomes. Effect of conditioned medium from iCAFs (CM iCAF) on EMT markers expression of non-invasive RT4 bladder cancer cell line was determined by qPCR and Western blot. IL6 expression in iCAFs was evaluated by ELISA and Western blot. RT4 cell proliferation, migration and invasion were assessed in CM iCAF +/− anti-IL6 neutralizing antibody using cyQUANT assay, scratch test and transwell chamber respectively. We investigated IL6 expression relevance for bladder cancer progression by querying gene expression datasets of human bladder cancer specimens from TCGA and GEO genomic data platforms.ResultsCancer exosome-treated HFs showed CAFs characteristics with high expression levels of αSMA and FAP. We showed that the CM iCAF induces the upregulation of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ß-catenin expression in non-invasive RT4 cells. Moreover, EMT transcription factors SNAIL1, TWIST1 and ZEB1 were upregulated in CM iCAF-cultured RT4 cells compared to control. We also showed that the IL-6 cytokine was highly expressed by CAFs, and its receptor IL-6R was found on RT4 bladder cancer cells. The culture of RT4 bladder cancer cells with CM iCAF resulted in markedly promoted cell growth, migration and invasion. Importantly, inhibition of CAFs-secreted IL-6 by neutralizing antibody significantly reversed the IL-6-induced EMT phenotype, suggesting that this cytokine is necessary for CAF-induced EMT in the progression of human bladder cancer. Finally, we observed that IL6 expression is up-regulated in aggressive bladder cancer and correlate with CAF marker ACTA2.ConclusionsWe conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6.

A surgical orthotopic approach for studying the invasive progression of human bladder cancer.

The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice. This technique consists of a small laparotomy and direct implantation of human cancer cells into the bladder lumen. Unlike other protocols, it does not require debriding of the urothelial lining, injection into the bladder wall, specialized imaging equipment, bladder catheterization or costly surgical equipment. With minimal practice, the procedure can be executed in <10 min. Tumors develop with a high take rate, and most cell lines exhibit local invasion within 4 weeks of implantation.

Hypoxia-elevated circELP3 contributes to bladder cancer progression and cisplatin resistance

Hypoxia plays a critical role in cancer biology. It induces genomic instability, which in turn helps cancer cells respond adaptively to meet the needs of carcinogenesis, cancer progression and relapse. Circular RNA has not been reported among the variety of downstream factors in this adaptive response. Although a few studies have demonstrated the important role of circular RNAs in driving human bladder cancer progression, their carcinogenic roles are still under investigated. Here, we identified a hypoxia-elevated circular RNA, circELP3, that contributes to bladder cancer progression and cisplatin resistance. Decreasing the level of circELP3 via siRNA clearly reduced the in vitro proliferation and cisplatin resistance of bladder cancer cells and promoted apoptosis. Interfering with circELP3 suppressed tumor xenograft growth in nude mice in vivo. In addition, lower circELP3-expressing bladder cancer cells displayed poorer self-renewal capacity, as demonstrated by lower levels of sphere formation and stem cell marker expression. Furthermore, in human bladder cancer patients, strong correlations between a high circELP3 level and advanced tumor grade and lymph node metastasis were observed. In summary, we provide the first direct evidence that circular RNA participates in the adaptive response to hypoxia and may play a role in the progression and drug resistance of bladder cancer.

Programmed cell death 4 overexpression enhances sensitivity to cisplatin via the JNK/c-Jun signaling pathway in bladder cancer.

The aim of the present study was to evaluate the effects of programmed cell death4 (PDCD4) on cell proliferation and apoptosis, and to elucidate the potential role of the Jun N-terminal kinase(JNK)/c-Jun pathway in human bladder cancer (BCa) cells. Mixed BCa cells were transfected with plasmids containing PDCD4 (PDCD4-pcDNA3). The sensitivity to cisplatin was analyzed using cell viability, invasion/migration, apoptosis, flow cytometry, wound healing and Transwell assays at different transfection times. Furthermore, epithelial-to-mesenchymal transition (EMT) markers were detected by immunofluorescence staining, and the protein expression of c-Jun, and phosphorylated Jun N-terminal kinase(p-JNK) and c-Jun (p-c-Jun, Ser-73) were also tested using western blotting. It was observed that BCa cell proliferation and invasion and tumor growth were significantly inhibited, whereas apoptosis was enhanced in PDCD4-transfected cells treated with cisplatin compared with controls. Moreover, the western blotting and immunofluorescence results demonstrated that PDCD4 upregulated the expression of epithelial cell markers, but downregulated the expression of mesenchymal cell markers. Furthermore, overexpression of PDCD4 reduced the protein levels of p-JNK and p-c-Jun. Taken together, the findings of the present study indicate that PDCD4 enhances the sensitivity of BCa cells to cisplatin, partially via regulation of the JNK/c-Jun pathway, and reverses EMT. In conclusion, the results of the present study suggested that PDCD4, a nuclear/cytoplasmic shuttling protein with multiple functions, plays an important role in the development and progression of human BCa.

Expression of aromatase in tumor related stroma is associated with human bladder cancer progression

ABSTRACT Putative gender differences in bladder cancer (BCa) have been proposed to result from sex hormone influence. Aromatase is the key enzyme catalyzing the conversion of androgens to estrogens which may result in an intratumoral microenviroment with increased estrogen production. In this study, we investigated the expression pattern of aromatase and its association with BCa progression. Tissue samples from 88 BCa patients who underwent cystectomy were obtained. Using immunohistochemistry (IHC), expression of aromatase in tumor epithelium (TE) and tumor related stroma (TS) were evaluated separately, and the association of aromatase expression status with pathologic variables and overall survival (OS) outcome was examined. High aromatase expression was found in 33/88 (37.5%) of TE and in 65/88 (73.9%) of TS. Increased aromatase expression in TE had a trend to correlate with male gender. Increased aromatase in TS was significantly associated with adverse pathologic variables including higher pathologic pT, positive lymph node metastasis (pN), lymphovascular invasion (LVI), and distant metastasis. In univariate analysis, high aromatase expression in TS was significantly associated with poorer overall survival (p = 0.014), but this association was not significant (p = 0.163) in multivariate cox analysis adjusted for independent factors including age at surgery and pN. These results demonstrate that aromatase expression in TS but not TE may play a critical role in BCa progression. Our findings provide direct evidence of aromatase involvement in BCa and suggest endocrine therapy may have a potential role in the treatment of BCa.

Abstract LB-B08: Validation of ADAM15 as a therapeutic target in invasive bladder cancer cells

Abstract Introduction: Advanced bladder cancer is a deadly and costly disease with locally invasive tumors carrying the worst prognosis for progression and mortality. The ADAM15 membrane disintegrin is key regulator in the invasive processes. Our previous studies demonstrated that increased expression of ADAM15 mRNA and protein is significantly associated with the local submucosal and lymphovascular invasion (LVI) and metastatic progression of human bladder cancer. Additionally, ADAM15 mediates integrin binding and has specifically been linked to the tumor-associated β1 integrins. This study aims to understand the ADAM15 disintegrin function and its potential as target in invasive bladder cancer. Methods: Collagen I represents the bulk of the submucosal matrix in the bladder and likely contributes to bladder cancer progression. Here, we developed a 3-dimensional (3D) culture system in collagen I to evaluate the phenotype of a panel of invasive bladder cancer cells. Furthermore, we developed a cyclic hexapeptide based on ADAM15 structure (adamatide), which can bind to the β1 integrin binding site; a rabbit polyclonal antibody against ADAM15 disintegrin domain (anti-A15DD); and, human recombinant ADAM15 disintegrin domain (hrA15DD) to compete with the endogenous protein. The effect of these inhibitors was evaluated on our 3D systems and compared to previously stablished ADAM15 knockdowns (shADAM15) in bladder cancer cell lines. Results: We assessed ADAM15 expression by Western blotting and immunofluorescence imaging in a panel of invasive bladder cancer cells. ADAM15 was highly expressed in the invasive cell lines, including UM-UC6 and UM-UC9. However, when cells were grown on plastic, ADAM15 was exclusively localized in cytoplasmic vesicles. On the contrary, when using 3D collagen cultures, ADAM15 translocated to filopodium-like protrusions (FLPs) on the cell membrane. Confocal imaging showed both co-expression and localization of ADAM15 and integrin β1, at the cellular level in collagen systems. These findings were validated by IHC of novel orthotopic models of bladder cancer, showing that both molecules localized at the muscle invasive front of the tumors as well as tumor emboli (LVI). Cells grown in collagen also presented increased FLP formation, invasion and proliferation rate when compared with those in plastic. Pharmacological inhibition of ADAM15 disintegrin activity or shADAM15 decreased cell attachment to collagen, invasion and FLPs in our 3D systems. Cells proliferation was also inhibited. Decreased cell viability was observed after treatment with the inhibitors on the 3D cultures. Early intracellular signaling events associated with these observations included inhibition of the FAK-Akt pathway. Conslusions: Our results show that ADAM15 has a key role in the invasive phenotype of bladder cancer cells via integrin β1 and FAK-Akt pathway. Thus, inhibition of ADAM15 activity represents a novel in vitro targeting strategy for bladder cancer Citation Format: Guadalupe Lorenzatti Hiles, Angelica L. Cates, John R. Rubin, Matthew C. Winkler, Hannah L. Briggs, Mark L. Day. Validation of ADAM15 as a therapeutic target in invasive bladder cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B08.

The impact of cruciferous vegetable isothiocyanates on histone acetylation and histone phosphorylation in bladder cancer

Cruciferous vegetable intake is associated with reduced risk of bladder cancer, yet mechanisms remain unclear. Cruciferous vegetable isothiocyanates (ITCs), namely sulforaphane (SFN) and erucin (ECN), significantly inhibit histone deacetylase (HDAC) activity in human bladder cancer cells representing superficial to invasive biology (59–83% inhibition with 20μM, 48h treatment), and in bladder cancer xenografts (59±3% ECN inhibition). Individual HDACs inhibited by SFN and ECN include HDACs 1, 2, 4 and 6. Interestingly, global acetylation status of histones H3 or H4 remain unaltered. The interplay between HDAC inhibition and modest modulation of AcH3 and AcH4 status is partially explained by decreased histone acetyl transferase activity (48.8±5.3%). In contrast, a significant decrease in phosphorylation status of all isoforms of histone H1 was observed, concomitant with increased phosphatase PP1β and PP2A activity. Together, these findings suggest that ITCs modulate histone status via HDAC inhibition and phosphatase enhancement. This allows for reduced levels of histone H1 phosphorylation, a marker correlated with human bladder cancer progression. Therefore, ITC-mediated inhibition of histone H1 phosphorylation presents a novel direction of research in elucidating epidemiological relationships and supports future food-based prevention strategies. SignificanceCollectively, our findings suggest that the cruciferous vegetable isothiocyanates: sulforaphane (SFN) and erucin (ECN), impact histones status in bladder cancer cells by modulating specific HDACs and HATs, and enhancing phosphatase activity, resulting in reduction of histone H1 phosphorylation. These findings are significant due to the fact that our previous work positively correlated histone H1 phosphorylation with bladder cancer carcinogenesis and progression. Therefore, we propose that SFN and ECN may inhibit bladder carcinogenesis via epigenetic modulation of gene expression associated with histone H1 phosphorylation. These efforts may elucidate biomarkers useful in epidemiologic studies related to cruciferous vegetable intake and cancer risk or provide intermediate biomarkers for food-based clinical intervention studies in high-risk cohorts.

Study on the significance of Cofilin 1 overexpression in human bladder cancer.

Cofilin 1 is a type of cytoskeletal protein. The overexpression of this gene has been regarded to hold a special relationship with the development and progress of some cancers. However, the detailed position of Cofilin 1 in human bladder cancer has not been investigated intensively. In this study, we mainly explored the relationship between human bladder cancer and the expression of Cofilin 1. The expression of Cofilin 1 in bladder cancer tissues and paracancerous tissues of patients was evaluated with quantitative polymerase chain reaction, Western blot, and immunohistochemical staining. Downregulation of Cofilin 1 expression model was established with siRNA in human RT4 bladder cancer cell line, and the changing cell viability was analyzed to determine the role of Cofilin 1 in human bladder cancer. Our results showed that the expression of Cofilin 1 was much higher in both RNA level and protein level in human bladder cancer tissues than paracancerous tissues for 3 patients. Downregulation of Cofilin 1 expression could inhibit cell proliferation, cell migration, cell adhesion, and colony formation ability, and increase the percentage of cell apoptosis in RT4 cells. Our study indicates that Cofilin 1 holds an important position in the development and progression of human bladder cancer, and this gene might become a novel target in the diagnosis and treatment of human bladder cancer.

Regulation of SLD5 gene expression by miR-370 during acute growth of cancer cells.

SLD5 is a member of the GINS complex, essential for DNA replication in eukaryotes. It has been reported that SLD5 is involved in early embryogenesis in the mouse, and cell cycle progression and genome integrity in Drosophila. SLD5 may be involved in malignant tumor progression, but its relevance in human cancer has not been determined. Here, we found strong SLD5 expression in both human bladder cancer tissues from patients and cell lines. Knockdown of SLD5 using small interfering RNA resulted in reduction of cell growth both in vitro and an in vivo xenograft model. Moreover, we found that high levels of SLD5 in bladder cancer cells result from downregulation of microRNA (miR)-370 that otherwise suppresses its expression. High level expression of DNA-methyltransferase (DNMT) 1 and IL-6 were also observed in bladder cancer cells. Knockdown of IL-6 led to downregulation of DNMT1 and SLD5 expression, suggesting that IL-6-induced overexpression of DNMT1 suppresses miR-370, resulting in high SLD5 expression. Our findings could contribute to understanding tumorigenic processes and progression of human bladder cancer, whereby inhibition of SLD5 could represent a novel strategy to prevent tumor growth.

Silencing of RTKN2 by siRNA suppresses proliferation, and induces G1 arrest and apoptosis in human bladder cancer cells.

Human bladder cancer is the most common urological malignancy in China. One of the causes of carcinogenesis in the cancer may be gene mutation. Therefore, the present study investigated the expression levels of Rhotekin2 (RTKN2), a Rho effector protein, in human bladder cancer tissues and cell lines, and examined the effect of RTKN2 on the proliferation, cell cycle, apoptosis and invasion of human bladder cancer cell lines. The mRNA expression levels of RTKN2 in 30human bladder cancer tissue samples were significantly higher, compared with those in 30 normal human bladder tissue samples. The protein expression levels of RTKN2 was markedly higher in T24 and 5637 cells, compared with those in four other human bladder cancer cell lines. The silencing of RTKN2 by small interfering (si)RNA inhibited cell proliferation and arrested cell cycle at the G1 phase, via reducing the expression levels of the MCM10, CDK2, CDC24A and CDC6 cell cycle‑associated proteins in the T24 and 5637 cells. Furthermore, RTKN2 knockdown in the cells led to cell apoptosis and the suppression of invasion. These results suggested that RTKN2 is involved in the carcinogenesis and progression of human bladder cancer, indicating that RTKN2 may be a molecular target in cancer therapy.

DNA Methyltransferase 1(DNMT1) Represses Regulator of G Protein Signaling 6 (RGS6) Expression in BBN‐induced Bladder Carcinogenesis

Urinary bladder cancer, the fifth most common cancer in the United States, is largely caused by exposure to toxic chemicals including those from smoking. Polymorphisms in the RGS6 gene leading to increased RGS6 expression are associated with a pronounced reduction in the risk of bladder cancer, especially among smokers. Recent studies in our laboratory indicate that RGS6 functions as a tumor suppressor in bladder as RGS6−/− mice exhibit a dramatic acceleration of bladder carcinogenesis induced by 4‐Hydroxybutyl(butyl)nitrosamine (BBN), a model that closely resembles human invasive bladder cancer. We also found that BBN‐induced bladder carcinogenesis in wild‐type (WT) mice is associated with loss of RGS6 expression. Given the tumor suppressor role of RGS6 in bladder we undertook studies to investigate the mechanisms underlying its loss during bladder carcinogenesis. In silico analyses of the RGS6 gene promoter revealed a highly conserved CpG island, located at −491/+398 or −751/+299 in the human and mouse RGS6 gene, respectively. Given that CpGs are substrates of DNMT1, whose increased expression is correlated with human bladder cancer progression, we investigated the role of DNMT1 in modulating RGS6 expression during bladder carcinogenesis. BBN treatment of mice led to a reciprocal change in expression of DNMT1 and RGS6 in bladder epithelium, where transitional carcinoma originates. The normally high level of RGS6 and low level of DNMT1 expression in bladder epithelium was reversed within 4 days of BBN treatment. Genomic DNA isolated from BBN‐treated mouse bladder epithelium revealed a time‐dependent methylation of the RGS6 gene promoter, consistent with the observed upregulation of DNMT1 and downregulation of RGS6 during carcinogenesis. Given that DNMT1 possesses transcriptional repressor activity independent of its methylation activity, we next examined whether DNMT1 repressed RGS6 promoter activity using a mouse RGS6 promoter driven luciferase (Rgs6‐luc) construct. In PC6 cells which exhibit a high level of RGS6 promoter activity, consistent with high neuronal expression of RGS6, transient transfection with DNMT1 markedly repressed Rgs6‐luc reporter activity. In contrast, RGS6 promoter activity was barely detectable in T24 bladder cancer cells, possibly reflecting DNMT1 mediated silencing. To test this possibility we examined the effects of treating cells with DNMT1 inhibitor 5‐Aza‐2′‐deoxycytidine (5‐Aza) on RGS6 promoter activity. Consistently, we observed that RGS6 promoter activity in T24 stable cell lines was induced by treatment with 5‐Aza. In conclusion, our data demonstrates that DNMT1 negatively regulates RGS6 gene promoter activity during bladder carcinogenesis, likely via a combination of DNMT1 effects on the RGS6 promoter including direct repression and methylation of a highly conserved CpG island. Given our findings that RGS6 promotes Tip60‐mediated DNMT1 degradation, silencing RGS6 by DNMT1 would represent a feed forward loop in which loss of RGS6 further promotes cellular accumulation of DNMT1 and bladder carcinogenesis.Support or Funding InformationSupported by AHA 11SDG7580008 (JY) and NIH CA161882 (RAF)

Tetracycline-inducible shRNA targeting antisense long non-coding RNA HIF1A-AS2 represses the malignant phenotypes of bladder cancer

Various studies have indicated that long non-coding RNAs (lncRNAs) play vital roles in the cancer development and progression. LncRNA hypoxia inducible factor 1alpha antisense RNA-2 (HIF1A-AS2) is upregulated in gastric carcinomas and knockdown of HIF1A-AS2 expression by siRNA could inhibit cell proliferation in vitro and tumorigenesis in vivo. Inspired by these observations, we hypothesized that HIF1A-AS2 possibly plays the analogous roles in bladder cancer. In our study, we first reported that HIF1A-AS2 was up-regulated in bladder cancer tissues and cells, and HIF1A-AS2 expression level in bladder cancer tissues is positively associated with advanced clinical pathologic grade and TNM phase. Cell proliferation inhibition, cell migration suppression and apoptosis induction were observed by silencing HIF1A-AS2 in bladder cancer T24 and 5637 cells. Overexpression of HIF1A-AS2 in SV-HUC-1 cells could promote cell proliferation, cell migration and anti-apoptosis. Besides, we utilized the emerging technology of medical synthetic biology to design tetracycline-inducible small hairpin RNA (shRNA) vector which specifically silenced HIF1A-AS2 in a dosage-dependent manner to inhibit the progression of human bladder cancer. In conclusion, our data suggested that HIF1A-AS2 plays oncogenic roles and can be used as a therapeutic target for treating human bladder cancer. Synthetic “tetracycline-on” switch system that quantitatively controlled the expression of HIF1A-AS2 in bladder cancer can inhibit the progression of bladder cancer cells in a dosage-dependent manner. Our findings provide new insights into the role of the lncRNA HIF1A-AS2 in the bladder cancer.

ADAM15 Is Functionally Associated with the Metastatic Progression of Human Bladder Cancer.

ADAM15 is a member of a family of catalytically active disintegrin membrane metalloproteinases that function as molecular signaling switches, shed membrane bound growth factors and/or cleave and inactivate cell adhesion molecules. Aberrant metalloproteinase function of ADAM15 may contribute to tumor progression through the release of growth factors or disruption of cell adhesion. In this study, we utilized human bladder cancer tissues and cell lines to evaluate the expression and function of ADAM15 in the progression of human bladder cancer. Examination of genome and transcriptome databases revealed that ADAM15 ranked in the top 5% of amplified genes and its mRNA was significantly overexpressed in invasive and metastatic bladder cancer compared to noninvasive disease. Immunostaining of a bladder tumor tissue array designed to evaluate disease progression revealed increased ADAM15 immunoreactivity associated with increasing cancer stage and exhibited significantly stronger staining in metastatic samples. About half of the invasive tumors and the majority of the metastatic cases exhibited high ADAM15 staining index, while all low grade and noninvasive cases exhibited negative or low staining. The knockdown of ADAM15 mRNA expression significantly inhibited bladder tumor cell migration and reduced the invasive capacity of bladder tumor cells through MatrigelTM and monolayers of vascular endothelium. The knockdown of ADAM15 in a human xenograft model of bladder cancer inhibited tumor growth by 45% compared to controls. Structural modeling of the catalytic domain led to the design of a novel ADAM15-specific sulfonamide inhibitor that demonstrated bioactivity and significantly reduced the viability of bladder cancer cells in vitro and in human bladder cancer xenografts. Taken together, the results revealed an undescribed role of ADAM15 in the invasion of human bladder cancer and suggested that the ADAM15 catalytic domain may represent a viable therapeutic target in patients with advanced disease.

MicroRNA-542-3p suppresses cellular proliferation of bladder cancer cells through post-transcriptionally regulating survivin

AimTo investigate the clinical significance of microRNA-542-3p (miR-542-3p) and its target gene survivin in human bladder cancer, and to determine their functions in malignant phenotypes of this disease. MethodsExpression levels of miR-542-3p and survivin mRNA in bladder cancer and adjacent normal tissues were detected by quantitative RT-PCR. Cell proliferation and cell cycle were assessed by cell viability assay, flow cytometry and colony formation assay based on two human bladder cancer cell lines. ResultsMiR-542-3p expression was downregulated, while survivin mRNA expression was upregulated in bladder cancer tissues, compared to adjacent normal tissues (both P<0.001). Importantly, the expression level of miR-542-3p in bladder cancer tissues was negatively correlated with that of survivin mRNA. MiR-542-3p-low and/or survivin-high expression were all significantly associated with tumor stage (all P<0.05) and tumor recurrence (all P<0.05) of patients with bladder cancer. Moreover, the enforced expression of miR-542-3p remarkably inhibited cell proliferation by inducing G1 phase arrest in both T24 and J82 cells, and decreased the expression level of survivin protein. In contrast, the knock-down of miR-542-3p dramatically promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression level of survivin protein. ConclusionMiR-542-3p and its target gene survivin may play crucial roles in the aggressive progression of human bladder cancer. More interestingly, miR-542-3p may function as a tumor suppressor and inhibit the proliferation of bladder cancer cells, implying that miR-542-3p-survivin signal axis might be a novel therapeutic target of this disease.