Regulates Bladder Cancer Progression Research Articles

CircRNA TATA-box binding protein associated factor 15 acts as an oncogene to facilitate bladder cancer progression through targeting miR-502-5p/high mobility group box 3.

Circular RNAs (circRNAs) are key in regulating bladder cancer progression. This study explored the effects of circRNA TATA-box binding protein associated factor 15 (circTAF15) on bladder cancer progression. We enrolled 80 bladder cancer patients to examine the relationship between circTAF15 expression and clinical features. The function of circTAF15 on bladder cancer cell viability, proliferation, migration, invasion, and glycolysis was monitored by cell counting kit-8 assay, 5-Ethynyl-2'-deoxyuridine experiment, Transwell experiment, and glycolysis analysis. Dual luciferase reporter gene assay, RNA pull-down assay, and RNA immunoprecipitation assay were used to verify the binding between circTAF15 and miR-502-5p or between miR-502-5p and high mobility group box 3 (HMGB3). circTAF15 effect on in vivo growth of bladder cancer was investigated by xenograft tumor experiment. Quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry were implemented to investigate the expression levels of genes. circTAF15 was upregulated in bladder cancer patients, associated with unfavorable outcomes. circTAF15 knockdown attenuated bladder cancer cell viability, proliferation, migration, invasion, epithelial-mesenchymal transition, and glycolysis. circTAF15 suppressed miR-502-5p expression, and miR-502-5p inhibited HMGB3 expression. Low miR-502-5p expression was associated with unfavorable outcomes in bladder cancer patients. miR-502-5p silencing and HMGB3 overexpression counteracted the inhibition of circTAF15 knockdown on the malignant phenotype of bladder cancer cells. circTAF15 knockdown attenuated the in vivo growth of bladder cancer cells. circTAF15 enhanced the progression of bladder cancer through upregulating HMGB3 via suppressing miR-502-5p. circTAF15 may be a novel target to treat bladder cancer in the future.

Modification of lysine-260 2-hydroxyisobutyrylation destabilizes ALDH1A1 expression to regulate bladder cancer progression

ALDH1A1 is one of the classical stem cell markers for bladder cancer. Lysine 2-hydroxyisobutyrylation (Khib) is a newfound modification to modulate the protein expression, and the underlying mechanisms of how ALDH1A1 was regulated by Khib modification in bladder cancer remains unknown. Here, ALDH1A1 showed a decreased K260hib modification, as identified by protein modification omics in bladder cancer. Decreasing ALDH1A1 expression significantly suppressed the proliferation, migration and invasion of bladder cancer cells. Moreover, K260hib modification is responsible for the activity of ALDH1A1 in bladder cancer, which is regulated by HDAC2/3. Higher K260hib modification on ALDH1A1 promotes protein degradation through chaperone-mediated autophagy (CMA), and ALDH1A1 K260hib could sensitize bladder cancer cells to chemotherapeutic drugs. Higher ALDH1A1 expression with a lower K260hib modification indicates a poor prognosis in patients with bladder cancer. Overall, we demonstrated that K260hib of ALDH1A1 can be used as a potential therapeutic target for bladder cancer treatment.

METTL1 drives tumor progression of bladder cancer via degrading ATF3 mRNA in an m7G-modified miR-760-dependent manner

7-methylguanosine (m7G) modification is recently found to conservatively exist in RNA internal position besides mRNA caps and mediates the various RNA metabolisms. As the core confirmed transmethylase of m7G modification, METTL1 has been reported in certain human cancers. However, the role of internal m7G at miRNAs and its core writer METTL1 in bladder cancer (BCa) remains to be elucidated. Here, we demonstrated that METTL1 was indispensable for BCa proliferation and metastasis in vitro and in vivo. By combining miRNA sequencing, m7G methylated RNA immunoprecipitation (MeRIP) and RIP, we identified METTL1 promoted the processing of miR-760 in an m7G-dependent manner. Transcription sequencing suggested that METTL1 indirectly degrades tumor suppressor ATF3 mRNA mediated by miR-760. Together, we concluded a regulatory axis composed of METTL1/m7G/miR-760/ATF3 in regulating BCa progression and provided potential therapeutic targets for BCa.

LncRNA HEIH/miR-4500/IGF2BP1/c-Myc Feedback Loop Accelerates Bladder Cancer Cell Growth and Stemness.

Bladder cancer (BCa) is one of the most prevalent malignancies and more common in men. An aberrantly expressed long noncoding RNA (lncRNA) hepatocellular carcinoma up-regulated EZH2-associated lncRNA (HEIH) has been reported to be implicated in the progression of many cancers, but its role in BCa remains little known. Our study intended to uncover whether and how HEIH regulates BCa progression. Quantitative real-time polymerase chain reaction (RT-qPCR) was adopted to determine HEIH expression in BCa cell lines. Functional experiments were performed to examine the effects of HEIH on BCa cell proliferation, apoptosis, migration, invasion and stemness. Bioinformatics analysis and mechanism experiments were conducted to investigate the regulatory relationship between HEIH and related molecules in BCa. HEIH expression was observed to be significantly increased in BCa cell lines. HEIH depletion significantly hindered BCa cell proliferation, migration and invasion. Besides, HEIH up-regulated MYC proto-oncogene, and bHLH transcription factor (c-Myc) expression to promote BCa cell stemness. Moreover, HEIH served as a sponge for miR-4500 to modulate insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) expression, thereby stabilizing c-Myc mRNA level. Our study demonstrated a positive feedback loop of HEIH/miR-4500/IGF2BP1/c-Myc in BCa progression, offering a novel insight into a possible BCa therapy.

Circular RNA_0000326 promotes bladder cancer progression via microRNA-338-3p/ETS Proto-Oncogene 1/phosphoinositide-3 kinase/Akt pathway

ABSTRACT Circular RNAs (circRNAs) play a pivotal regulatory role in bladder cancer (BC) occurrence and progression. The expression level, role and mechanism of circ_0000326 in BC remain unknown. In the present study, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to evaluate the expressions of circ_0000326, microRNA-338-3p (miR-338-3p) and ETS Proto-Oncogene 1(ETS1) mRNA in BC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, wound healing assay and flow cytometry were used to detect the impacts of circ_0000326 on BC cell growth, migration and apoptosis. Western blot was used to detect the expressions of ETS1, phospho-phosphoinositide-3 kinase (p-PI3K), phospho-AKT, PI3K and AKT protein. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to analyze the biological function of ETS1 in BC. Here, we found that circ_0000326 expression was significantly elevated in BC cell lines and tissues, and circ_0000326 could promote BC cell growth and migration, and inhibit apoptosis. Dual-luciferase reporter gene assay confirmed that circ_0000326 and ETS1 could bind directly to miR-338-3p. Furthermore, circ_0000326 sponged miR-338-3p and up-regulated ETS1 expression. ETS1 was associated with the activation of PI3K/AKT pathway. Moreover, circ_0000326 could activate PI3K/AKT pathway by miR-338-3p/ETS1 axis. Collectively, circ_0000326/miR-338-3p/ETS1/PI3K/AKT pathway is involved in regulating BC progression.

Circular RNA circSETD3 hampers cell growth, migration, and stem cell properties in bladder cancer through sponging miR-641 to upregulate PTEN

ABSTRACT Bladder cancer (BLCA) is a common malignant urothelial cancer in the world. Although circular RNAs (circRNAs) involve in regulating BLCA progression, the role of a novel circular RNA circSETD3 in regulating BLCA pathogenesis has not been studied. The expression of circSETD3, miR-641, PTEN mRNA in BLCA tissues and cell lines were measured using RT-qPCR. The gain-of-function experiments were performed in vitro and in vivo to detect the effects of circSETD3 on cell proliferation, migration, EMT, and stemness maintenance. Besides, rescue experiments were performed to demonstrate the regulatory mechanism of circSETD3/miR-641/PTEN in BLCA cell malignant phenotypes in vitro. CircSETD3 was remarkably downregulated in the cancerous clinical tissues and cell lines, in contrast with their normal counterparts, and circSETD3 tended to be deficient in BLCA patients with larger tumor size, advanced clinical stages, positive lymph metastasis and worse prognosis. In addition, circular isoforms of circSETD3 were more resistant to RNase R+ and actinomycetes D treatment compared to their linear isoforms, and circSETD3 mainly distributed in the cytoplasm of the BLCA cells. Further gain-of-function experiments showed that circSETD3 acted as a tumor suppressor to suppress BLCA cell proliferation, migration, EMT and stemness, and the underlying mechanisms had also been elucidated. Mechanistically, circSETD3 sponged miR-641 to upregulate PTEN, resulting in the blockage of BLCA progression. Our findings indicated that circSETD3 acted as a vital tumor suppressor in BLCA via regulating the miR-641/PTEN axis.

Retraction.

Retraction: "LncRNA AWPPH inhibits SMAD4 via EZH2 to regulate bladder cancer progression," by Feng Zhu, Xinjun Zhang, Qinnan Yu, Guangye Han, Fengxia Diao, Chunlei Wu, Yan Zhang, J Cell Biochem. 2018; 4496-4505: The above article, published online on 12 December 2017 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/abs/10.1002/jcb.26556), has been retracted by agreement between the the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. Several flaws and inconsistencies between results presented and experimental methods described were found, the editors consider the conclusions of this article to be invalid. The authors collaborated in the investigation initially, but were not available for a final confirmation of the retraction.

MicroRNA‑34a‑5p serves as a tumor suppressor by regulating the cell motility of bladder cancer cells through matrix metalloproteinase‑2 silencing.

Bladder cancer (BC), a common urologic cancer, is the fifth most frequently diagnosed tumor worldwide. hsa-miR-34a displays antitumor activity in several types of cancer. However, the functional mechanisms underlying hsa-miR-34a in BC remains largely unknown. We observed that hsa-mir-34a levels were significantly and negatively associated with clinical disease stage as well as regional lymph node metastasis in human BC. In a series of in vitro investigations, overexpression of hsa-miR-34a inhibited cell migration and invasion in BC cell lines 5637 and UMUC3 as detected by Transwell assays. We further found that hsa-miR-34a inhibited cell migration and invasion by silencing matrix metalloproteinase-2 (MMP-2) expression and thus interrupting MMP-2-mediated cell motility. Our analysis of BC datasets from The Cancer Genome Atlas database revealed a negative correlation between hsa-miR-34a and MMP-2. Moreover, higher MMP-2 protein expression was observed in the BC tissues when compared with that noted in the normal tissue. MMP-2 levels were also significantly associated with clinical disease stage and poor survival rate in human BC. These findings indicate that MMP-2 plays a critical role in regulating BC progression. Therefore, hsa-miR-34a is a promising treatment to target MMP-2 for the prevention and inhibition of cell migration and invasion in BC.

LncRNA MAGI2-AS3 inhibits bladder cancer progression by targeting the miR-31-5p/TNS1 axis.

In this study, we performed bioinformatics analysis to identify the competing endogenous RNAs (ceRNAs) that regulate bladder cancer (BCa) progression. RNA-sequencing data analysis identified 2451 differentially expressed mRNAs, 174 differentially expressed lncRNAs, and 186 microRNAs (miRNAs) in BCa tissues (n=414) compared to the normal urothelial tissues (n=19) from the TGCA database. CeRNA network analysis of the differentially expressed lncRNAs and mRNAs showed strong positive correlation between lncRNA MAGI2-AS3 and Tensin 1 (TNS1) mRNA in BCa tissues. Bioinformatics analysis also showed that both MAGI2-AS3 and TNS1 mRNA sequences contain miR-31-5p binding sites. Furthermore, we observed significantly lower MAGI2-AS3 and TNS1 mRNA expression and higher miR-31-5p expression in the BCa tissues and cell lines (T24 and J82) compared with their corresponding controls. Functional and biochemical experiments in BCa cell lines including luciferase reporter assays showed that MAGI2-AS3 upregulated TNS1 by sponging miR-31-5p. Transwell assays showed that the MAGI2-AS3/miR-31-5p/TNS1 axis regulated migration and invasion ability of BCa cell lines. Moreover, immunohistochemical staining of paired BCa and normal urothelial tissues showed that low expression of TNS1 correlated with advanced tumor (T) stages and lymph node metastasis in BCa. In conclusion, our study demonstrates that the MAGI2-AS3/miR-31-5p/TNS1 axis regulates BCa progression.

MP17-06 CIRCRIP2 ACCELERATES BLADDER CANCER PROGRESSION VIA MIR-1305/TGF-β2/SMAD3 PATHWAY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-06 CIRCRIP2 ACCELERATES BLADDER CANCER PROGRESSION VIA MIR-1305/TGF-β2/SMAD3 PATHWAY Yinjie Su*, Guanglei Zhong, and Tianxin Lin Yinjie Su*Yinjie Su* More articles by this author , Guanglei ZhongGuanglei Zhong More articles by this author , and Tianxin LinTianxin Lin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Increasing evidences indicate that circular RNAs exert critical function in regulating bladder cancer progression. However, the expressive patterns and roles of circular RNAs in bladder cancer remain less investigated. METHODS: circRIP2 was identified and evaluated by RNA-sequencing and qPCR; in vitro effects of circRIP2 were determined by CCK8, clone forming, wound healing and trans-well assays; while mice subcutaneous tumor model was designed for in vivo analysis. Western blot, RNA pulldown assay, miRNA capture and dual luciferase assessment were applied for mechanistic studies. RESULTS: circRIP2 was identified as conserved and dramatically repressed circular RNAs in bladder cancer. Patients that displayed higher circRIP2 expression negatively associate with the grade, stage, metastasis as well as outcome of bladder cancer. In vitro and in vivo studies suggest that circRIP2 enables to promote bladder cancer progression via inducing EMT. Regarding the mechanism, we performed RNA-sequencing analysis, RNA pulldown with biotin-labeled circRIP2-specific probe, dual luciferase reporter assay. It was found that circRIP2 enables to sponge miR-1305 to elevate Tgf-β2 in bladder cancer, and inducing EMT via Tgf-β2/smad3 pathway. Blocking Tgf-β2 in bladder cancer deprives circRIP2 induced cancer progression and EMT. CONCLUSIONS: Taken together, our study provides the first evidence that circRIP2 expresses differentially in bladder cancer and negatively along with the cancer progression; effective circRIP2 activity accelerates bladder cancer progression via inducing EMT by activating miR-1305/Tgf-β2/smad3 pathway. The research implies that circRIP2 might be a potential biomarker and therapeutic target for bladder cancer patients. Source of Funding: No © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e227-e228 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yinjie Su* More articles by this author Guanglei Zhong More articles by this author Tianxin Lin More articles by this author Expand All Advertisement PDF downloadLoading ...

Long non-coding RNA MEG3 functions as a competing endogenous RNA of miR-93 to regulate bladder cancer progression via PI3K/AKT/mTOR pathway.

BackgroundMaternally expressed gene 3 (MEG3) is a long non-coding RNA (lncRNA) and involved in progression of various human tumors. However, its underlying regulatory mechanism in tumorigenesis of bladder cancer (BC) remains unclear. To demonstrate effects of MEG3 on BC cell proliferation and elaborate its regulatory mechanism in BC.MethodsAberrant expressions of MEG3 and miR-93-5p were induced by cell transfection. The mRNA and protein expression were analyzed using qRT-PCR and western blot. Cell proliferation was examined by CCK-8 assay and EdU staining. The targeted regulation effect of MEG3 on miR-93-5p was confirmed by luciferase reporter assay. The number of LC3 punctated cells was detected by immunofluorescence. Xeno-graft mouse model was constructed for in vivo validation.ResultsMEG3 was down-regulated with increased expression of miR-93-5p in BC cells and tissues. Luciferase reporter assay showed that miR-93-5p was a direct target of MEG3 and was negatively regulated by MEG3. MEG3 overexpression inhibited cell proliferation and the expression of proliferation-, apoptosis- and autophagy-related proteins. The activation of PI3K/AKT/mTOR pathway was also suppressed with elevated cell apoptosis. miR-93-5p overexpression counteracted these results. In vivo experiments, we confirmed that miR-93-5p overexpression reversed the MEG3 overexpression-mediated suppression on tumor growth and protein expression.ConclusionslncRNA MEG3 could function as a competing endogenous RNA of miR-93 to regulate the tumorigenesis of BC via PI3K/AKT/mTOR pathway. The present research provided a new perspective to understanding the pathogenic mechanism of BC, and an effective therapeutic target for BC.

CircRIP2 accelerates bladder cancer progression via miR-1305/Tgf-\u03b22/smad3 pathway

BackgroundIncreasing evidences indicate that circular RNAs exert critical function in regulating bladder cancer progression. However, the expressive patterns and roles of circular RNAs in bladder cancer remain less investigated.MethodscircRIP2 was identified and evaluated by RNA-sequencing and qPCR; in vitro effects of circRIP2 were determined by CCK8, clone forming, wound healing and trans-well assays; while mice subcutaneous tumor model was designed for in vivo analysis. Western blot, RNA pulldown assay, miRNA capture and dual luciferase assessment were applied for mechanistic studies.ResultscircRIP2 was identified as a conserved and dramatically repressed circular RNA in bladder cancer. Patients that displayed higher circRIP2 expression negatively associate with the grade, stage, metastasis as well as outcome of bladder cancer. In vitro and in vivo studies suggest that circRIP2 enables to promote bladder cancer progression via inducing EMT. Regarding the mechanism, we performed RNA-sequencing analysis, RNA pulldown with biotin-labeled circRIP2-specific probe, dual luciferase reporter assay. It was found that circRIP2 enables to sponge miR-1305 to elevate Tgf-β2 in bladder cancer, and inducing EMT via Tgf-β2/smad3 pathway. Blocking Tgf-β2 in bladder cancer deprives circRIP2 induced cancer progression and EMT.ConclusionsTaken together, our study provides the first evidence that circRIP2 expresses differentially in bladder cancer and negatively along with the cancer progression; effective circRIP2 activity accelerates bladder cancer progression via inducing EMT by activating miR-1305/Tgf-β2/smad3 pathway. The research implies that circRIP2 might be a potential biomarker and therapeutic target for bladder cancer patients.

Circ5912 suppresses cancer progression via inducing MET in bladder cancer.

Background: Increasing evidence suggests that circular RNAs play a key role in regulating bladder cancer progression. However, this remains to be fully elucidated.Results: In this study, we reanalyzed our previous RNA sequence, and circ5912 was found to downregulate significantly in bladder cancer tissues compared with normal control. Expression of circ5912 inversely correlates with bladder cancer grade, stage, metastasis, and better patient outcomes. In vitro and in vivo, circ5912 has been shown to repress transforming growth factor β signaling, which suppresses proliferation, invasion and migration of bladder cancer induced by mesenchymal-to epithelial transition.Conclusions: Our study firstly demonstrate that circ5912 regulates mesenchymal-to epithelial transition pathway to suppress bladder cancer progression and propose new therapeutic targets and biomarkers for bladder cancer.Materials and Methods: Clinical values of circ5912 in human bladder cancer were examined in a cohort of 58 patients by qPCR. 2 bladder cancer cell lines, T24 and SW780, were used for biological evaluation of circ5912. CCK8, clone formation, wound healing and trans-well assays were performed to determine the in vivo effect of circ5912; a mouse subcutaneous model was designed for in vivo analysis. Western blotting, RNA pulldown assays and florescent in situ hybridization were applied for mechanistic analysis.

Long noncoding RNA MAGI2-AS3 regulates CCDC19 expression by sponging miR-15b-5p and suppresses bladder cancer progression

Bladder cancer (BCa) belongs to a popular urological malignancy and leads to large numbers of deaths worldwide. Recently, emerging evidences indicate that long noncoding RNAs (lncRNAs) are closely related with BC occurrence and progression. However, the function of lncRNA MAGI2-AS3 remains poorly understood in BC. In this present study, we screened out a novel lncRNA MAGI2-AS3 whose expression was downregulated in BCa tissues. We showed that MAGI2-AS3 downregulation in BCa patients indicated a poor prognosis. Functionally, we showed that MAGI2-AS3 overexpression inhibits proliferation, migration and invasion of BCa cells. Moreover, ectopic expression of MAGI2-AS3 suppresses BCa growth invivo. Bioinformatics analysis revealed that MAGI2-AS3 could serve as a competing endogenous RNA (ceRNA) for miR-15b-5p. In the meantime, miR-15b-5p directly targeted CCDC19, a tumor suppressor in BCa. Rescue assays demonstrated that knockdown of CCDC19 restored the proliferation, migration and invasion of BCa cells suppressed by MAGI2-AS3 overexpression. In conclusion, this study identified a novel mechanism that MAGI2-AS3/miR-15b-5p/CCDC19 signaling pathway regulates BCa progression.

LncRNA AWPPH inhibits SMAD4 via EZH2 to regulate bladder cancer progression.

This study aimed to investigate the effect and underlying mechanism of lncRNA AWPPH in bladder cancer (BC). A total of 20 Ta-T1 stage BC tissues, 20 T2-T4 stage BC tissues, and 20 normal bladder tissues, as well as human bladder epithelial cell line SV-HUC-1, human BC cell lines RT4, and T24 were obtained to detect the levels of AWPPH, enhancer of zeste homolog 2 (EZH2) and SMAD4 using RT-qPCR or Western blotting. RT4 cells were transfected with pc-AWPPH, pc-EZH2, or pc-control and T24 cells were transfected with si-AWPPH, si-EZH2, si-control, or pc-AWPPH + pc-SMAD4, respectively. Then, cell proliferation, apoptosis, autophagy, and migration, were detected using MTT assay, colony formation assay, Annexin V-FITC/PI method, Western blotting, and Transwell analysis, respectively. The relationship of AWPPH and EZH2 or SMAD4 was evaluated by RNA immunoprecipitation (RIP) assay or Chromatin immunoprecipitation (ChIP) assay. Compared with normal bladder tissues or cells, the levels of AWPPH and EZH2 were overexpressed, while SMAD4 was down-regulated in BC tissues or cells (all P < 0.01). Cell viability, colony number, and migration were significantly increased, while cell apoptosis ratio was reduced in cells with pc-AWPPH compared with cells with pc-control (all P < 0.05), meanwhile, these effects were reversed by the treatment of pc-SMAD4. Then, RIP assay revealed that AWPPH could bind to EZH2 and ChIP assay showed SMAD4 was regulated by EZH2. LncRNA AWPPH can promote cell proliferation, autophagy, and migration, as well as inhibit cell apoptosis in BC by inhibiting SMAD4 via EZH2.

USP21 promotes cell proliferation and metastasis through suppressing EZH2 ubiquitination in bladder carcinoma.

Bladder cancer (BC) is the second most common malignant tumor of the urinary tract in the world. In this study, we found that ubiquitin-specific protease (USP21) was upregulated in BC and the ectopic expression of USP21 was closely associated with tumor size and metastasis. Moreover, patients with higher levels of USP21 had poorer survival rate. Multiple function analysis such as CCK-8, colony formation, wound healing, and transwell analysis indicated that USP21 regulated cell proliferation and metastasis in bladder carcinoma cell lines. We also found that USP21 could facilitate epithelial–mesenchymal transition. As EZH2 has been reported to promote cell metastasis in BC, our work identified that USP21 deubiquitinated EZH2 and stabilized it. Our data demonstrated that USP21 might play a crucial role in regulating BC progression and could provide a potential therapeutic strategy for BC.

Abstract 692: Identifying genes that regulate bladder cancer progression and invasion

Abstract Transitional cell carcinoma (TCC) is the most common type of bladder cancer and can be categorized as either non-muscle invasive (Ta-T1) or muscle invasive (≥T2). Approximately 50% of bladder cancers are T1 at initial diagnosis; however, the recurrence rate for these tumors is high and they may progress into T2. In this study, we aimed to determine if there are specific gene expression differences between T1 vs. T2 bladder cancer that can help identify key regulators in bladder cancer progression and invasion. T1 and T2 bladder cancer tissues were subjected to RNA-Seq to evaluate for differences among these stages. Additionally, the Oncomine database was examined to further narrow down potential candidates that differentiate T1 from T2. These efforts led to the identification of an extracellular matrix glycoprotein, fibulin-3 (FBLN3), as being highly expressed in T2 compared to T1 tissues. To validate these findings, FBLN3 expression was measured using qRT-PCR from formalin fixed and paraffin embedded tissues from patient bladder samples ranging from stages Ta-T4. These studies confirmed that FBLN3 expression was elevated in muscle-invasive compared to non-muscle invasive bladder cancer. Consistent with these findings, FBLN3 expression level correlated with the invasive ability of several bladder cancer cell lines. Specifically, FBLN3 expression was determined using both qRT-PCR and western blotting amongst the T24, UMUC-13, UMUC-3, RT4, and 5637 bladder cancer cell lines. The most invasive cell lines, T24 and UMUC-13, demonstrated the highest FBLN3 expression. In contrast, the least invasive cells, RT4 and 5637, demonstrated the least FBLN3 expression. To determine a functional role for FBLN3 in bladder cancer invasion, we knocked down or increased FBLN3 expression in bladder cancer cell lines using lentiviral transduction. Knockdown of FBLN3 expression in the T24 and UMUC-13 cells inhibited the invasion and migration of these bladder cancer cells; whereas, FBLN3 overexpression in the 5637 cells promoted the invasiveness of the bladder cancer cells. Furthermore, cell viability and growth rates were not affected by manipulation of FBLN3 expression. Our results indicate that FBLN3 serves as a pro-invasive factor in bladder cancer. These findings suggest that FBLN3 could serve as (1) a biomarker to differentiate T1 from T2 bladder cancers and (2) a promising therapeutic target. Citation Format: Amy L. Han, Brendan Veeneman, Scott A. Tomlins, Evan T. Keller. Identifying genes that regulate bladder cancer progression and invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 692.

Association of Itraconazole, a Hedgehog Inhibitor, and Bladder Cancer

Activation of Hedgehog (Hh) signaling has been implicated in early stages of bladder cancer development while loss of Hh signaling has been described during progression to more invasive disease. Itraconazole, an antifungal, is the only azole known to be a potent Hh pathway antagonist. We evaluated whether itraconazole use is associated with bladder cancer risk or progression. We performed a case-control study nested in a United Kingdom database in 13,440 bladder cancer cases and 52,421 matched controls between 1995 and 2013. The use of itraconazole and other azoles was measured as the number of prescriptions. Conditional logistic regression was used for estimated AORs and the 95% CI of the association of bladder cancer with ever use and an increasing number of itraconazole prescriptions. Logistic regression was done to determine whether itraconazole use in patients diagnosed with bladder cancer was associated with invasive bladder cancer requiring cystectomy relative to the use of other azoles. Itraconazole was not associated with the risk of bladder cancer relative to never use (ever use AOR 0.89, 95% CI 0.70-1.14 and 4 or more prescriptions AOR 0.87, 95% CI 0.42-1.81). However, among patients diagnosed with bladder cancer there was a significantly increased risk of bladder cancer requiring cystectomy with itraconazole use (ever use AOR 2.05, 95% CI 1.12-3.38 and 2 or more prescriptions AOR 2.30, 95% CI 1.12-4.72). Inhibition of the Hh pathway with itraconazole was not associated with a risk of bladder cancer overall but it was associated with a higher risk ofinvasive bladder cancer requiring cystectomy. These data provide clinical evidence supporting the role of Hh signaling in regulating bladder cancer progression.

Downregulation of GAS5 Promotes Bladder Cancer Cell Proliferation, Partly by Regulating CDK6

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as transcriptional regulation, cell growth and tumorigenesis. However, little is known about whether lncRNA-GAS5 (growth arrest-specific 5) regulates bladder cancer progression. In the present study, we found that the GAS5 expression is commonly downregulated in bladder cancer cell lines and human specimens. Knockdown of GAS5 promotes bladder cancer cell proliferation, whereas forced expression of GAS5 suppresses cell proliferation. We further demonstrated that knockdown of GAS5 increases CDK6 mRNA and protein levels in bladder cancer cells. Expectedly, GAS5 inhibition induces a significant decrease in G0/G1 phase and an obvious increase in S phase. Gain-of-function and loss-of-function studies showed that GAS5 inhibits bladder cancer cell proliferation, at least in part, by regulating CDK6 expression.ConclusionsDownregulated GAS5 promotes bladder cancer cell proliferation, partly by regulating CDK6, and thus may be helpful in the development of effective treatment strategies against bladder cancer.

Androgen-dependent CD24 regulates bladder cancer progression—a new role for antiandrogen therapy?

Bladder cancer: Androgen-dependent CD24 regulates bladder cancer progression—a new role for antiandrogen therapy?