Ability Of Bladder Cancer Cells Research Articles

LINC00612 enhances the proliferation and invasion ability of bladder cancer cells as ceRNA by sponging miR-590 to elevate expression of PHF14

BackgroundBladder cancer (BC) is a common type of cancer that involves tumors of the urinary system and poses a serious threat to human health. Long noncoding RNAs (lncRNAs) have emerged as crucial biomarkers and regulators in many cancers. Novel lncRNA biomarkers in BC urgently need to be investigated in regard to its function and regulatory mechanisms.MethodsIdentification of differentially expressed lncRNAs in BC tissue was performed via microarray analysis. To investigate the biological functions of LINC00612, loss-of-function and gain-of-function experiments were performed in vitro and in vivo. Bioinformatics analysis, dual-luciferase reporter assays, AGO2-RIP assays, RNA pull-down assays, real-time quantitative PCR (RT-qPCR) arrays, fluorescence in situ hybridization assays, and western blot assays were conducted to explore the underlying mechanisms of competitive endogenous RNAs (ceRNAs).ResultsLINC00612 was upregulated in BC tissues and cell lines. Functionally, downregulation of LINC00612 inhibited cell proliferation and invasion in vitro and in vivo, whereas overexpression of LINC00612 resulted in the opposite effects. Bioinformatics analysis and luciferase assays revealed that miR-590 was a direct target of LINC0061, which was validated by dual-luciferase reporter assays, AGO2-RIP assays, RNA pull-down assays, RT-qPCR arrays, and rescue experiments. Additionally, miR-590 was shown to directly target the PHD finger protein 14 (PHF14) gene. LNIC00612 modulated the expression of E-cadherin and vimentin by competitively sponging miR-590 to elevate the expression of PHF14, thus affecting BC cellular epithelial-mesenchymal transition (EMT).ConclusionsOur results indicate that LINC00612 enhances the proliferation and invasion ability of BC cells by sponging miR-590 to upregulate PHF14 expression and promote BC cellular EMT, suggesting that LINC00612 may act as a potential biomarker and therapeutic target for BC.

MiR-3622a promotes proliferation and invasion of bladder cancer cells by downregulating LASS2

As a tumor metastasis suppressor gene, LASS2 has been found to be negatively associated with the stage of bladder cancer and overall survival of patients. However, the mechanisms regulating LASS2 in bladder cancer remain poorly understood. Here, we aim to identify a miRNA that targets LASS2 from bladder cancer-associated miRNAs and to reveal its potential functions in bladder cancer cells. Through miRNA microarray and bioinformatics analyses, we identified miR-3622a as a negative regulator of LASS2. The expression levels of miR-3622a in bladder cancer tissues were negatively correlated with the overall survival of patients. Overexpression of miR-3622a significantly increased the proliferation and invasion abilities of bladder cancer cells. In conclusion, our results indicate that miR-3622a promotes the proliferation and invasion of bladder cancer cells by downregulating LASS2.

Polypyrimidine tract binding protein 1 promotes lymphatic metastasis and proliferation of bladder cancer via alternative splicing of MEIS2 and PKM

Lymph node (LN) metastasis is the leading cause of bladder cancer-related mortality. Splicing factors facilitate cancer progression by modulating oncogenic variants, but it is unclear whether and how splicing factors regulate bladder cancer LN metastasis. In this study, Polypyrimidine tract binding protein 1 (PTBP1) expression was found to relate to bladder cancer LN metastasis, and was positively correlated with LN metastasis status, tumor stage, histological grade, and poor patient prognosis. Functional assays demonstrated that PTBP1 promoted bladder cancer cell migration, invasion, and proliferation in vitro, as well as LN metastasis and tumor growth in vivo. Mechanistic investigations revealed that PTBP1 upregulated MEIS2-L variant to promote metastasis and increased expression of PKM2 variant to enhance proliferation by modulating alternative mRNA splicing. Moreover, overexpression of MEIS2-L or PKM2 could rescue the oncogenic abilities of bladder cancer cells and the expression of MMP9 or CCND1 respectively after PTBP1 knockdown. In conclusion, our data demonstrate that PTBP1 induces bladder cancer LN metastasis and proliferation through an alternative splicing mechanism. PTBP1 may serve as a novel prognostic marker and therapeutic target for LN-metastatic bladder cancer.

MiR-99a-5p inhibits bladder cancer cell proliferation by directly targeting mammalian target of rapamycin and predicts patient survival.

Bladder cancer is a common malignancy and miR-99a-5p has been reported to be downregulated in bladder cancer, but its function and the underlying mechanism in bladder cancer development remains largely unclear. Here, we report that miR-99a-5p expression was decreased in bladder cancer compared with the adjacent normal tissues. Receiver operating characteristic curve revealed that miR-99a-5p expression signature had area under curve value of 0.7989 in differing bladder cancer from the adjacent normal tissues. Bladder cancer patients with low expression of miR-99a-5p had a poor survival rate. Gain-of-function and loss-of-function approaches demonstrated that miR-99a-5p inhibited bladder cell proliferation and cell cycle. Furthermore, we identified that mammalian target of rapamycin (mTOR) was a direct target of miR-99a-5p and mTOR restore could rescue the proliferative ability of bladder cancer cells. Moreover, miR-99a-5p/mTOR axis regulated S6K1 phosphorylation. These suggested that miR-99a-5p/mTOR axis might be a therapeutic target for bladder cancer.

Long term exposure to γ‑rays induces radioresistance and enhances the migration ability of bladder cancer cells.

In Western countries, bladder cancer (BCa) is one of the most common types of malignancy, with highest incidence rate among urinary system malignancies. At present, as a strategy to preserve the quality and function of the bladder tissues, external‑beam radiotherapy has been applied to an increasing number of patients with BCa, particularly the elderly and those in poor health. The onset of acquired radioresistance (ARR) during radiotherapy notably reduces the effectiveness of radiotherapy; however, the cause of ARR is unclear and remains untreatable. In order to investigate the mechanism of ARR, clinical fractionated irradiation was mimicked invitro to obtain radioresistant BCa cells, 5637R, in the present study. Compared with the parental cell line, 5637R cells exhibited an increased distribution of S phase cells, enhanced migration ability and elevated levels of phosphorylated‑STAT3, as determined using flow cytometry, cell migration analysis and western blotting, respectively. Furthermore, 5637R cells exhibited notable increases in extracellular signal‑regulated kinase activation in response to ionizing radiation (IR) compared with 5637cells. In addition, 5637R cells showed an early G2/M arrest within 8h post‑IR (18.23±3.85 vs. 6.42±3.82%, IR cells vs. control cells), whereas 5637cells were arrested in G2/M phase 24h post‑IR (19.26±2.21 vs. 12.36±4.45%, IR cells vs. control cells). Overall, the present study suggested that phosphorylated STAT3 may be a potential biomarker to predict radioresistance and tumor recurrence in patients with BCa following conventional radiotherapeutic intervention. In addition, co‑treatment with ERK inhibitor may be a viable approach to increase the anticancer efficacy of radiotherapy in patients with ARR; however, further investigation is required.

RSPO3 promotes the aggressiveness of bladder cancer via Wnt/β-catenin and Hedgehog signaling pathways.

R-spondin 3 (RSPO3) is a secreted protein that associates directly with Wnt/β-catenin signaling. However, its functional contribution and prognostic value in human bladder cancer remain unclear. Here, we showed that RSPO3 is upregulated in bladder cancer tissues and cells, and high expression of RSPO3 correlates with advanced clinicopathological features, poor prognosis and disease progression in bladder cancer patients. Furthermore, we observed that ectopic expression or knockdown of RSPO3 profoundly promoted or inhibited, respectively, the invasive ability of bladder cancer cells. Mechanistically, RSPO3 promoted bladder cancer progression via mediating the Wnt/β-catenin and Hedgehog signaling pathways. These findings demonstrate, for the first time, that RSPO3 exhibited a tumor-promoting effect in bladder cancer cells through activation of Wnt/β-catenin and Hedgehog signaling pathways. Thus, RSPO3 may be served as a potential therapeutic target for bladder cancer treatment.

PTBP1 Facilitates Lymphatic Metastasis and Proliferation of Bladder Cancer Via Alternative Splicing of MEIS2 and PKM

Background: Lymph node (LN) metastasis is the leading cause of bladder cancer-related mortality. Splicing factors facilitate cancer progression by modulating oncogenic variants, but it is unclear whether and how splicing factors regulate bladder cancer LN metastasis. Methods: In this study, qRT-PCR was used to identify differential expression of splicing factors between bladder cancer with and without LN metastasis. Associations between expression of the splicing factor polypyrimidine tract binding protein 1 (PTBP1) and clinicopathologic features and prognosis of bladder cancer were analyzed. In vitro and in vivo assays were performed to explore the biological role of PTBP1 in bladder cancer cells. RNA-sequencing, RNA immunoprecipitation, and RNA pull down assays were used to investigate the molecular mechanisms of PTBP1 in bladder cancer. Findings: PTBP1 expression was found to relate to bladder cancer LN metastasis, and was positively correlated with LN metastasis status, tumor stage, histological grade, and poor patient prognosis. Functional assays demonstrated that PTBP1 promoted bladder cancer cell migration, invasion, and proliferation in vitro, as well as LN metastasis and tumor growth in vivo. Mechanistic investigations revealed that PTBP1 upregulated MEIS2-L variant to promote metastasis and increased expression of a PKM2 variant to enhance proliferation by modulating alternative mRNA splicing. Moreover, overexpression of MEIS2-L or PKM2 could rescue the oncogenic abilities of bladder cancer cells after PTBP1 knockdown. Interpretation: Our data demonstrate that PTBP1 induces bladder cancer LN metastasis and proliferation through an alternative splicing mechanism. PTBP1 may serve as a novel prognostic marker and therapeutic target for LN-metastatic bladder cancer. Funding Statement: This study was supported by the National Natural Science Foundation of China (Grant No. 81702523, 81772719, 81772728, 81572514, U1301221, 81472384), National Natural Science Foundation of Guangdong (Grant No. 2016A030313321, 2016A030313244), Science and Technology Program of Guangzhou (Grant No. 201804010041, 201604020156, 201604020177), Project Supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (for Tianxin Lin) , the Fundamental Research Funds for the Central Universities (for Xu Chen, 18ykpy18), Yat-Sen Scholarship for Young Scientist (for Xu Chen), Sun Yat-sen Initiative Program for Scientific Research (for Xu Chen, YXQH201708), and National Clinical Key Specialty Construction Project for Department of Urology and Department of Oncology. Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, Sun-Yat-Sen University. Grant [2013]163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology. Declaration of Interests: We declare that we have no conflicts of interest. Ethics Approval Statement: Ethical consent was approved by Sun Yat-sen University’s Committees for Ethical Review of Research involving Human Subjects

LncRNA PVT1 regulates VEGFC through inhibiting miR-128 in bladder cancer cells.

Long noncoding RNA PVT1 is considered to be an oncogene in multiple cancers. Our previous studies indicated that PVT1 levels were higher in bladder cancer tissue and correlated with clinical progression and poor prognosis in bladder cancer patients. A bioinformatics analysis showed that PVT1 may regulate VEGFC expression through miR-128 as a competing endogenous RNA (ceRNA). In this study, we demonstrated that PVT1 expression levels affect the proliferation and migration ability of bladder cancer cells. Moreover, PVT1 knockdown significantly decreased the proliferation capacity of bladder cancer cells in nude mice. Luciferase assays and RNA-binding protein immunoprecipitation were performed to investigate the potential mechanism of ceRNAs in the regulation of PVT1 and VEGFC. The results showed that the increased number of PVT1 transcripts interacted directly with miR-128 to decrease miR-128 binding to the VEGFC 3'-untranslated region. This effect suppressed VEGFC mRNA degradation by miR-128. In conclusion, these results indicated that PVT1 might play a critical role in bladder cancer tumorigenesis via miR-218 and VEGFC. Therefore, PVT1 could be a new biomarker for bladder cancer diagnosis and therapy.

MP58-17 THE CONTRIBUTION OF HGF-MET-MMP1 SIGNALING IN BLADDER CANCER INVASION, AND MET INHIBITOR AS A POTENTIAL THERAPEUTIC OPTION FOR INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2018MP58-17 THE CONTRIBUTION OF HGF-MET-MMP1 SIGNALING IN BLADDER CANCER INVASION, AND MET INHIBITOR AS A POTENTIAL THERAPEUTIC OPTION FOR INVASIVE BLADDER CANCER Tomoya Fukawa, Kei Daizumoto, Tomoharu Fukumori, Masayuki Takahashi, and Hiro-omi Kanayama Tomoya FukawaTomoya Fukawa More articles by this author , Kei DaizumotoKei Daizumoto More articles by this author , Tomoharu FukumoriTomoharu Fukumori More articles by this author , Masayuki TakahashiMasayuki Takahashi More articles by this author , and Hiro-omi KanayamaHiro-omi Kanayama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1849AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Muscle-invasive bladder cancer (MIBC) has an especially poor prognosis, and clarification of the mechanism of MIBC invasion is urgent to discover appropriate treatment strategies and to improve the prognosis of patients with MIBC. Our previous report showed that co-culture of bladder cancer cells with mesenchymal cells which highly expressed HGF stimulated the invasion ability of bladder cancer cells. The aim of this study was to elucidate the underlying mechanisms of the increased invasion ability of bladder cancer cells by co-culture with mesenchymal cells, clarify the contribution of HGF-MET signaling pathway to bladder cancer progression and find a new therapeutic option for MIBC. METHODS We analyzed the expression of MET in the bladder cancer cell lines by Western blotting. Using the cell lines which highly expressed MET, we examined the effects of Cabozantinib on their proliferation and invasion abilities by using MTT and Matrigel invasion assays. To investigate the biological function of HGF-MET signaling, we analyzed gene expression profiles of bladder cancer cells that were cultivated with/without the effects of the MET inhibitor, Cabozantinib (XL184). We also examined the effects of MMP-1 depletion on invasion ability to clarify the importance of HGF-MET-MMP1 signaling in bladder cancer invasion. To verify our results with clinical samples, we checked the relationship between the expression of MET and MMP-1 by using TCGA data. RESULTS The expression of MET was high in four of five bladder cancer cell lines, and 5637 and T24 cells showed especially high protein expression of MET. The treatment with HGF stimulated cell proliferation and invasion of these cancer cells, whereas Cabozantinib inhibited those effects induced by HGF stimulation. To clarify the underlying mechanisms, we analyzed gene expression profiles by using microarray and revealed that the expression of MMP-1 was significantly elevated by HGF addition. We also confirmed that Cabozantinib suppressed the expression of MMP-1 which was induced by HGF addition in bladder cancer cells. Consistent with these results, we found the strong association between the expression of MET and MMP1 by using TCGA data. Furthermore, the depletion of MMP-1 dramatically suppressed the invasion ability of bladder cancer cells, suggesting that HGF contributes bladder cancer invasion by regulating the expressions of MMP-1 CONCLUSIONS The results of this study suggested that the blockade of HGF-MET-MMP1 signaling could be a potential therapeutic option and Cabozantinib is one of the potential molecules for the treatment of MIBC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e779 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Tomoya Fukawa More articles by this author Kei Daizumoto More articles by this author Tomoharu Fukumori More articles by this author Masayuki Takahashi More articles by this author Hiro-omi Kanayama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Pyruvate kinase M2 inhibits the progression of bladder cancer by targeting MAKP pathway.

Pyruvate kinase M2 (PKM2) was found overexpressed in tumor cells and played a significant role in tumor formation and growth. We sought to explore the correlation of PKM2 expression with cell proliferation, invasion, and apoptosis in bladder cancer cell line. Real-time polymerase chain reaction and Western blot were performed to determine the expression level of PKM2 at mRNA and protein level. MTT and flow cytometry were respectively used to explore the proliferation, invasion, and apoptosis in bladder cancer cell line in vitro. The results suggested that suppression of PKM2 significantly decreased the proliferation rate, invasive cell number, and migration ability of bladder cancer cells compared with blank group. Moreover, proteins such as MMP2 and MMP9 as well as P38 expression were also affected by the PKM2 expression changes. These results proved that PKM2 could be involved in the progression of bladder cancer by mitogen-activated protein kinases signaling pathway. The data presented in this study revealed that PKM2 up-regulation may promote the development and metastasis of bladder cancer through promoting cell proliferation, migration and invasion via MAPK signal pathway.

MP98-20 THE ROLE OF TOLL-LIKE RECEPTOR 4 IN BLADDER CANCER PROGRESSION.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology V1 Apr 2017MP98-20 THE ROLE OF TOLL-LIKE RECEPTOR 4 IN BLADDER CANCER PROGRESSION. Tomoya Fukawa, Terumichi Shintani, Kei Daizumoto, Tomoharu Fukumori, Masayuki Takahashi, and Hiro-omi Kanayama Tomoya FukawaTomoya Fukawa More articles by this author , Terumichi ShintaniTerumichi Shintani More articles by this author , Kei DaizumotoKei Daizumoto More articles by this author , Tomoharu FukumoriTomoharu Fukumori More articles by this author , Masayuki TakahashiMasayuki Takahashi More articles by this author , and Hiro-omi KanayamaHiro-omi Kanayama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3085AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Toll-like receptors (TLRs) play important roles in immune response and have been reported that expression levels of these molecules were related to prognoses of several kinds of cancer. It has been reported that the expression level of TLR4 is reduced in bladder cancer. However, the role of TLR4 in bladder cancer is still unclear. The aim of this study is to clarify the effects of TLR4 expression on prognosis of bladder cancer patients and to elucidate its underlying mechanisms. METHODS To evaluate the effects of TLR4 expression on prognosis, we analyzed the clinical outcomes of 95 patients with bladder cancer and also conducted immunohistochemistry for TLR4 in bladder cancer specimens. The cancer-specific survival was determined by Kaplan-Meier method and the statistical significance was examined by Log-rank test. We also performed multivariate analysis to evaluate the relationship between TLR4 expression and clinical outcomes. We checked the effects of TLR4 depletion on cell growth and invasive abilities of bladder cancer cells. To clarify the underlying mechanisms, we also analyzed the gene expression profiles in TLR4-depleted cells. These results were validated by quantitative RT-PCR. We also confirmed our finding by using data from public database. RESULTS The expression levels of TLR4 were inversely correlated with local invasion and worse prognosis (log-rank test, p=0.007). Although the effect of TLR4 depletion on cell growth was small, it could dramatically enhance invasion ability of cancer cells. We also observed morphologic changes in these cells. Both gene ontology analysis and gene set enrichment analysis of microarray data revealed that most of upregulated genes in TLR4-depleated cells were related to differentiation of epithelium cells. Among these genes, small proline-rich protein (SPRR) family genes which are related to poor prognosis of squamous cell carcinoma were upregulated in TLR4-depleated cells. By analyzing data from public database, we confirmed that expression levels of SPRRs family genes were inversely correlated with TLR4 expression and positively correlated with poor prognosis of bladder cancer patients. Furthermore, we found that expression of TLR4 diminished in the areas with squamous differentiation in bladder cancer. CONCLUSIONS Our finding suggested that TLR4 is related to bladder cancer aggressiveness through regulation of squamous differentiation. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1319 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Tomoya Fukawa More articles by this author Terumichi Shintani More articles by this author Kei Daizumoto More articles by this author Tomoharu Fukumori More articles by this author Masayuki Takahashi More articles by this author Hiro-omi Kanayama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-1-3p Suppresses the Proliferation, Invasion and Migration of Bladder Cancer Cells by Up-Regulating SFRP1 Expression

Bladder cancer is of compelling morbidity and mortality due to its high recurrence rate. Little development has been made in the last decades in the therapy methods. Thus, the mechanism of its growth and invasiveness involving novel molecular targets are needed. Our research objective is to confirm the hypothesis that miR-1-3p suppresses the proliferation, invasion and migration of bladder cancer cells. The expression levels of miR-1-3p and SFRP1 were evaluated using RT-qPCR in bladder cancer tissues and cells as well as in normal tissues and cells. J82 cell lines were selected as experiment subjects due to their low expression levels of miR-1-3p. Plasmids carrying miR-1-3p mimics, miR-1-3p inhibitors and SFRP1 were transfected into the J82 cell lines. Subsequently, the protein expression of SFRP1 was detected using Western Blot analysis, and cell proliferation, apoptosis, invasion and migration ability was measured using MTT, the flow cytometry, the Transwell test and wound healing assays, respectively Results: Bladder cancer tissues and cells exhibited significant decrease in the expression of miR-1-3p and SFRP1 compared to normal tissues and cells, and human bladder cancer cell line J82 exhibited the most significant decrease in these expressions (P < 0.05). MiR-1-3p up-regulates SFRP1 expression in bladder cancer cells, and the over-expression of miR-1-3p can suppress the proliferation, invasion and migration ability of bladder cancer cells. This mechanism is similar to the effect of SFRP1 over-expression on bladder cancer cells. MiR-1-3p suppresses the proliferation, invasion and migration of bladder cancer cells by up-regulating SFRP1 expression.

MiR-24-3p regulates bladder cancer cell proliferation, migration, invasion and autophagy by targeting DEDD.

microRNAs (miRNAs), a class of small non-coding RNA molecules, can regulate gene expression by interacting with the 3'-untranslated regions (3'UTR) of target genes and influence various biological processes. We investigated the potential role of miR-24-3p in the development of bladder cancer by regulating DEDD, a member of the death effector domain-containing protein family. First, we found that miR-24-3p was highly expressed and that DEDD was expressed at a low level in bladder cancer tissues compared with that in adjacent bladder tissues by qRT-PCR (P<0.0001). Second, we found that miR-24-3p promoted the proliferation ability of bladder cancer cells using the MTT assay and colony forming assay; and showed that miR-24-3p accelerated the migration and invasion of bladder cancer cells using migration and invasion assays (P<0.05). Moreover, miR-24-3p inhibited apoptosis of bladder cancer cells, as shown by flow cytometry (P<0.05). Western blot results demonstrated that miR-24-3p participated in autophagy of bladder cancer cells by DEDD. In addition, the tumor formation assay showed that miR-24-3p promoted the growth of bladder tumor invivo. Furthermore, the luciferase reporter gene assay indicated that miR-24-3p suppressed DEDD gene transcription. Therefore, our study indicated that miR-24-3p promoted bladder cancer progression by inhibiting DEDD.

Expression of PAK1 in bladder cancer and its influence on invasion of bladder cancer cells

Objective: To investigate the expression of p21-activated kinase 1 (PAK1) in bladder cancer and its biological influence on invasion ability of bladder cancer cells. Methods: A total of 54 paraffin-embedded bladder cancer tissue samples and 12 normal bladder tissue specimens were retrieved in Jinshan Hospital Affiliated to Fudan University between January 2009 and December 2012. The expression of PAK1 in these tissues was detected by immunohistochemical staining. The PAK1 mRNA and protein levels were measured in bladder cancer cell lines and human normal bladder epithelial cell line using real-time, fluorescence-based quantitative polymerase chain reaction (RT-PCR) and Western blot, respectively. A stable PAK1 gene silencing bladder cancer cell strain 5637 were successfully constructed. After treatment with PAK1 RNA interference(RNAi), the ability of migration and invasion of the 5637 cell was evaluated using a Transwell system. Results: The expression of PAK1 proteins was significantly higher in bladder cancer tissues than in normal bladder tissues (28/54 vs 1/12, P<0.05). The overexpression of PAK1 was positively correlated with high histological grade, lymph node metastasis, and tumor size (all P<0.05). The mRNA level and protein level of PAK1 was much higher in bladder cancer cell lines T24, 5637 than human normal urothelial cell line SV-HUC-1.PCR and Western blot showed satisfactory inhibitory effect of PAK1 short hairpin RNA (shRNA) on PAK1 expression in 5637 bladder cancer cells. The number of PAK1 RNAi-treated 5637 cells traversed the membrane was decreased compared with the control group in migration and invasion assays. Conclusions: Overexpression of PAK1 in bladder cancer tissues may be an important feature of bladder cancer and related with the metastasis and invasion of bladder cancer. The molecular mechanisms involved in regulation of PAK1 expression needs further research.

Abstract 3408: Oncogenic SOX2 signaling in bladder cancer

Abstract The intriguing phenomenon that cancer cells have biological features similar to stem cells suggests that cancer and stem cells may share the same regulatory signaling pathway. We found that SOX2, a master transcriptional factor controlling self-renewal of stem cells, was highly expressed in a subgroup of transitional cell carcinoma of bladder cancer, correlating with the advanced pathological grade. Moreover, SOX2 expression was associated with poor overall survival and recurrence-free survival outcomes in patients with bladder cancer. Knockdown of SOX2 in SOX2-high 5637 bladder cancer cells attenuated cell growth. Consistently, ectopic expression of SOX2 in SOX2-low T24 bladder cancer cells endowed cells with increased cell proliferation. Spheroid assay showed that SOX2 expression encouraged spheroid-forming ability of bladder cancer cells in low serum condition, demonstrating the involvement of SOX2 signaling in the maintenance of stemness in bladder cancer cells. Immunoblotting revealed that the expression of SOX2 induced phosphorylation of AKT in the serum-free condition, supporting that SOX2 regulates bladder cancer cell survival. Gene expression microarray analysis showed that SOX2 expression induced IGF2, which was further confirmed by Q-PCR analysis in bladder cancer cells. Chi-square test assay showed that IGF2 expression correlated with the advanced tumor stages in bladder cancer. Pharmacological inhibition of IGF2 signaling attenuated cell growth in SOX2-postive bladder cancer cells. Our findings support the notion that SOX2-IGF2 signaling axis confers aggressiveness in bladder cancer cells with potentials as biomarkers and therapeutic targets for bladder cancer intervention. Citation Format: Chia-Chang Wu, Yu-Fan Chiu, Yu-Ting Chou, Yuan-Hung Wang. Oncogenic SOX2 signaling in bladder cancer. [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 3408.

C14orf166 is a high-risk biomarker for bladder cancer and promotes bladder cancer cell proliferation.

BackgroundC14orf166 (chromosome 14 open reading frame 166) plays a crucial role in some tumors, but its role in bladder cancer hasn’t been explored.MethodWe determined C14orf166 expression in uroepithelial cell, bladder cancer cells, normal bladder tissues and bladder cancer tissues using quantitative RT-PCR and western blot, we then analyzed the correlation between C14orf166 expression and clinicopathologic characteristics in a cohort of 149 patients with bladder cancer. Finally we downregulated C14orf166 and determined its role in the proliferation of bladder cancer cell lines using MTT assay, colony formation assay and cell cycle assay.ResultsWe demonstrated C14orf166 was upregulated in bladder cancer cells and tissues, C14orf166 expression was significantly correlated with larger tumor size (P = 0.001), lymph node involvement (P < 0.001), histological differentiation (P < 0.001), survival time and vital states, and high C14orf166 expression correlated with poor survival, these results suggested C14orf166 served as a high-risk marker for bladder cancer. Knockdown of C14orf166 decreased the proliferation rate and colony formation ability of bladder cancer cells, and arrested cell cycle in G1/S transition. Further analysis showed that C14orf166 knockdown caused abnormal expression of key proteins for G1/S transition, such as Cyclin D1, P21, P27 and Rb phosphorylation.ConclusionsThis study demonstrates that C14orf166 promotes bladder cancer cell proliferation and can be a novel prognostic biomarker for patients with bladder cancer.

Long non-coding RNA urothelial cancer-associated 1 promotes bladder cancer cell migration and invasion by way of the hsa-miR-145-ZEB1/2-FSCN1 pathway.

Numerous studies suggest that several long non-coding RNAs (lncRNAs) play critical roles in bladder cancer development and progression. Long non-coding RNA urothelial cancer-associated 1 (lncRNA-UCA1) is highly expressed in bladder cancer tissues and cells, and it has been shown to play an important role in regulating aggressive phenotypes of bladder cancer cells. However, little is known about the molecular mechanism of lncRNA-UCA1-mediated bladder cancer cell migration and invasion. Here, we show that overexpression of lncRNA-UCA1 could induce EMT and increase the migratory and invasive abilities of bladder cancer cells. Mechanistically, lncRNA-UCA1 induced EMT of bladder cancer cells by upregulating the expression levels of zinc finger E-box binding homeobox 1 and 2 (ZEB1 and ZEB2), and regulated bladder cancer cell migration and invasion by tumor suppressive hsa-miR-145 and its target gene the actin-binding protein fascin homologue 1 (FSCN1). Furthermore, we also observed a positive correlation between lncRNA-UCA1 and ZEB1/2 expression, and a negative correlation between lncRNA-UCA1 and hsa-miR-145 expression in bladder cancer specimens. Importantly, we found that lncRNA-UCA1 repressed hsa-miR-145 expression to upregulate ZEB1/2, whereas the suppression of hsa-miR-145 could upregulate lncRNA-UCA1 expression in bladder cancer cells. Moreover, the binding site for hsa-miR-145 within exons 2 and 3 of lncRNA-UCA1 contributed to the reciprocal negative regulation of lncRNA-UCA1 and hsa-miR-145. Taken together, our results identified that lncRNA-UCA1 enhances bladder cancer cell migration and invasion in part through the hsa-miR-145/ZEB1/2/FSCN1 pathway. Therefore, lncRNA-UCA1 might act as a promising therapeutic target for the invasion and metastasis of bladder cancer.

PP242 suppresses bladder cancer cell proliferation and migration through deactivating the mammalian target of rapamycin complex 2/AKT1 signaling pathway.

While most cancer types are resistant to mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin, recent studies have identified mTORC2 as an important prospective therapeutic target for cancer. The present study assessed the effects of mTORC2 inhibitor PP242 on the proliferation and migration of bladder cancer cells by using Cell Counting Kit‑8, 5‑ethynyl‑2'‑deoxyuridine incorporation, wound healing and Transwell assays. Furthermore, the phosphorylation status of downstream signaling proteins of mTORC1 and mTORC2 was assessed using western blot analysis. The results demonstrated that PP242 concentration‑dependently inhibited the proliferation of bladder cancer cells. Simultaneously, the migration ability of bladder cancer cells was suppressed by PP242. In addition, PP242 markedly restrained the phosphorylation of AKT1 and mTORC2, while the phosphorylation status of S6K1 and mTORC1 was not affected. These results suggested that PP242 exerts potent inhibitory effects on bladder cancer cells by modulating the activity of the mTORC2/AKT1 pathway.

Beyond proliferation: KLF5 promotes angiogenesis of bladder cancer through directly regulating VEGFA transcription.

Abundant evidence has demonstrated critical roles of KLF5 in regulating cell proliferation in various cancers, however, its additional roles in other aspects of cancer development remain to be further clarified. In this study, we found that KLF5 was essential for cancer cell-endothelial cell interaction in vitro and tumor angiogenesis in nude mice based on lentivirus-mediated KLF5 knockdown bladder cancer cell models. Moreover, KLF5 insufficiency abolished the ability of bladder cancer cells to induce neovascularization in rabbit cornea. Mechanistically, the pro-angiogenic factor VEGFA was identified as a direct downstream target of KLF5, which bound to GC-boxes and CACCC elements of VEGFA promoter and regulated its transcriptional activity. In addition, there was a positive correlation between KLF5 and VEGFA expression in human bladder cancer tissues by immunohistochemistry assay and statistical analysis from TCGA and GEO data. Furthermore, we found that two pivotal pathways in bladder cancer, RTKs/RAS/MAPK and PI3K/Akt, might convey their oncogenic signaling through KLF5-VEGFA axis. Taken together, our results indicate that KLF5 promotes angiogenesis of bladder cancer through directly regulating VEGFA transcription and suggest that KLF5 could be a novel therapeutic target for angiogenesis inhibition in bladder cancer.

A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT. Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay. ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration. ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.