Inhibits Bladder Cancer Research Articles

Abstract 5095: 1,25D3 inhibits migration and invasion through miR-101-3p in human bladder cancer cells

Abstract Bladder cancer is the fourth most common cancer diagnosed and eighth leading cause of cancer death in men. Metastasis is the primary reason for mortality. The 5-year survival rates for localized and metastatic bladder cancers are 94% and 6%, respectively. Therefore, more effective strategies to prevent and inhibit bladder cancer invasion and metastasis are needed to prolong survival. Epidemiological and experimental studies support a role for 1,25(OH) 2D3 (1,25D3) in cancer prevention and treatment. More recently, 1,25D3 has also been reported to inhibit tumor cell invasion and metastasis in several model systems. To study the anti-tumor effect of 1,25D3 in bladder cancer, we selected four human bladder cell lines including T24, 253J, 253J-BV and TCC-SUP cells. 253J-BV is a metastatic line generated from 253J cells through a series of orthotopic inoculation. Vitamin D receptor was expressed and induced by 1,25D3 in all four cell lines. However, treatment with 1,25D3 did not significantly affect the proliferation of the bladder cancer cell lines. To investigate the impact of 1,25D3 on bladder cancer cell migration and invasion, the cells were treated with control EtOH or 1,25D3 in the wound healing assay or chamber-based migration or invasion assays. 1,25D3 suppressed migration and invasion of 253J-BV and TCC-SUP cells but neither 253J nor T24 cells. MiRNAs play critical regulatory roles in cancer cell migration and invasion. We selected 253J and 253J-BV cells for further investigation of the role of miRNAs in 1,25D3 regulation of migration and invasion. Our previous PCR panel studies showed that 1,25D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells. From the markedly regulated miRNAs, we chose miR-101-3p and miR-126-3p to examine their potential contribution to the inhibition of migration and invasion by 1,25D3. 1,25D3 induced the expression of miR-101-3p and reduced miR-126-3p expression in 253J-BV cells but not 253J cells. To investigate the role of the two miRNAs, we employed pre-miR and miRNA inhibitors. Transfection with miR-101-3p inhibitor vector suppressed migration and invasion in 253J-BV cells. In addition, miR-101-3p inhibitor rescued 1,25D3-induced inhibition of migration and invasion in 253J-BV cells. On the other hand, pre-miR-101-3p transfection followed by 1,25D3 treatment resulted in further reduction of migration and invasion in 253J-BV cells. However, transfection with miR-126-3p inhibitor or pre-miR-126-3p did not alter 1,25D3-inhibition of migration and invasion in 253J-BV cells. These results indicate that 1,25D3 suppresses migration and invasion through the induction of miR-101-3p in human bladder cancer 253J-BV cells. Citation Format: Yingyu Ma, Wei Luo, Rachel Pratt, Donald L. Trump, Candace S. Johnson. 1,25D3 inhibits migration and invasion through miR-101-3p in human bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5095. doi:10.1158/1538-7445.AM2015-5095

Abstract 1169: Growth regulation of bladder cancer cells by compounds that affect glucose metabolism

Abstract The observation that cancer cells tend to exhibit enhanced rates of glycolysis presents a target for therapeutic regulation of cancer cell growth by molecules that regulate glucose metabolism. We have extended our earlier studies on colon cancer cells with an examination in bladder cancer cells of the action of an established inhibitor of PFKFB3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB-3) is an enzyme regulating glycolysis in many tumor cells. Cell growth was monitored by staining with sulforhodamine B. Of the 8 cell lines examined, growth was most rapid in the T24 and UM-UC-3 bladder cancer cells. The slowest growth was seen with RT4, TCCSUP and HT1197 cells while intermediate growth rates were observed with 5637, HT1376, and SW780 cells. Glucose uptake from the medium and acidification of the medium over a 72-hour incubation tended to correlate with growth rate. Thus the greatest changes were seen with the T24 and UM-UC-3 cell lines and the least change was seen with RT4 and TCCSUP cells. Growth was inhibited when glutamine was omitted from the medium but growth was blocked in the absence of glucose except for RT4 cells in which growth was inhibited more in the absence of glutamine. Treatment with 10 μM 3PO as a single agent for 72 hours caused average growth inhibition in the range of 25-50% without apparent relationship to the control growth rates of the cell lines. Additive inhibitory effects on growth were observed when cells were coincubated with 3PO and either 1 mM 2-deoxyglucose, 50 μM phenformin or 1 mM butyrate. In addition to being growth inhibitory, butyrate also caused significant induction of alkaline phosphatase activity in some cell lines. When cells were incubated with phenformin as a single agent, there was generally an increased acidification of the medium that was diminished by coincubation with 3PO. In conclusion, 3PO was an inhibitor of proliferation of human bladder cancer cells and may be a useful agent in combination with other drugs that inhibit bladder cancer cell proliferation. Citation Format: Michael A. Lea, Charles desBordes. Growth regulation of bladder cancer cells by compounds that affect glucose metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1169. doi:10.1158/1538-7445.AM2015-1169

XIAP inhibitor Embelin inhibits bladder cancer survival and invasion in vitro.

Bladder cancer is the second most common urological malignancy around the world and is by far the most frequent urological malignancy in China. Embelin is an active compound identified as a novel X-chromosome-linked IAP (XIAP) inhibitor from the Embelia ribes that exhibits various medicinal effects including anti-inflammatory and anti-cancer activities. However, therapeutic effect of Embelin to human bladder cancer is not yet determined. We evaluated the sensitizing potential of Embelin on inhibiting cell growth and migration of bladder cancer cell line by CCK8, Transwell, and Western Blot, and explored its related mechanism. We performed IHC staining of XIAP in 35 bladder cancer tissues and corresponding adjacent non-neoplastic tissues. XIAP was significantly upregulated in bladder cancer cases. When the concentration of Embelin was used respectively at 5, 10, 20, 25, and 35 µmol/l, the survival of both T24 and 5637 cells decreased in a dose-/time-dependent manner. Our study confirmed that with the increase of concentration of Embelin, the expression levels of PI3K and p-Akt decreased significantly which further confirmed that Embelin inhibits cell growth by inducing apoptosis via PI3K/Akt pathway. Embelin may be developed into a novel and potential chemotherapeutic drug for bladder cancer.

Expression of hepaCAM inhibits bladder cancer cell proliferation via a Wnt/β-catenin-dependent pathway in vitro and in vivo

We previously established that hepatocyte cell adhesion molecule (hepaCAM), a typical structure of immunoglobulin (Ig)-like adhesion molecules, inhibited the proliferation and the progression of cultured human bladder cancer cells. As increasing evidence reveals that aberrant activation of canonical Wnt pathway is involved in the pathogenesis of bladder cancer, and β-catenin serves as a pivotal molecule of Wnt pathway. Then, we explored whether the anti-proliferation effect of hepaCAM was associated with Wnt/β-catenin pathway in human bladder cancer cells. The negative correlation between hepaCAM and β-catenin in transitional cell carcinoma of bladder (TCCB) was found. Follow by, studied the effect of hepaCAM on the key elements of Wnt pathway. Here, Our researches showed that hepaCAM played a central role in modulating the Wnt/β-catenin signaling pathway by interfering nuclear protein levels of β-catenin, leading to down-regulate transcriptional activity of LEF/TCF and its target genes c-Myc and cyclinD1. Mechanistically, we demonstrated that hepaCAM-activated GSK3β led to elevate the phosphorylation of β-catenin, contributing to the aberrant translocation of β-catenin. In addition, Anti-proliferation and associated molecular mechanisms of hepaCAM were demonstrated by using vivo experiment. In conclusion, our reports uncover that expression of hepaCAM suppresses the proliferation of bladder cancer cells through a Wnt/β-catenin-dependent signaling pathway in vitro and in vivo.

MicroRNA-195 inhibits cell proliferation in bladder cancer via inhibition of cell division control protein 42 homolog/signal transducer and activator of transcription-3 signaling.

microRNA (miR)-195 acts as a suppressor in multiple types of malignant tumors, including bladder cancer; however, the detailed function of miR-195 in bladder cancer remains largely unknown. The aim of the present study was to investigate the role of miR-195 in the regulation of bladder cancer cell proliferation and to determine whether cell division control protein 42 homolog (Cdc42)/signal transducer and activator of transcription-3 (STAT3) signaling acts as a downstream effector of miR-195 in bladder cancer cells. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of miR-195 in bladder cancer tissues and normal adjacent tissue. The results revealed that the expression of miR-195 was significantly downregulated in bladder cancer tissues compared with that in the normal adjacent tissues. A luciferase reporter assay was then conducted, which identified Cdc42 as a direct target of miR-195, and the expression of Cdc42 was significantly upregulated in bladder cancer tissues, as determined by western blotting. Furthermore, miR-195 negatively regulated the protein expression of Cdc42 in bladder cancer cells. An MTT assay was also conducted to determine the rate of cell proliferation. Upregulation of miR-195 or the inhibition of Cdc42 could inhibit bladder cancer cell proliferation, possibly through activation of STAT3 signaling. In addition, restoration of Cdc42 could reverse the inhibitory effect of miR-195 upregulation on bladder cancer cell proliferation. In conclusion, the results of the present study suggest that miR-195 plays an inhibitory role in the regulation of bladder cancer cell proliferation by directly targeting Cdc42/STAT3 signaling.

Nonpathogenic Mycobacterium brumae Inhibits Bladder Cancer Growth In Vitro, Ex Vivo, and In Vivo

BackgroundBacillus Calmette-Guérin (BCG) prevents tumour recurrence and progression in non–muscle-invasive bladder cancer (BC). However, common adverse events occur, including BCG infections. ObjectiveTo find a mycobacterium with similar or superior antitumour activity to BCG but with greater safety. DesignIn vitro, ex vivo, and in vivo comparisons of the antitumour efficacy of nonpathogenic mycobacteria and BCG. InterventionThe in vitro antitumour activity of a broad set of mycobacteria was studied in seven different BC cell lines. The most efficacious was selected and its ex vivo capacity to activate immune cells and its in vivo antitumour activity in an orthotopic murine model of BC were investigated. Outcome measurements and statistical analysisGrowth inhibition of BC cells was the primary outcome measurement. Parametric and nonparametric tests were use to analyse the in vitro results, and a Kaplan-Meier test was applied to measure survival in mycobacteria-treated tumour-bearing mice. Results and limitationsMycobacterium brumae is superior to BCG in inhibiting low-grade BC cell growth, and has similar effects to BCG against high-grade cells. M. brumae triggers an indirect antitumour response by activating macrophages and the cytotoxic activity of peripheral blood cells against BC cells. Although no significant differences were observed between BCG and M. brumae treatments in mice, M. brumae treatment prolonged survival in comparison to BCG treatment in tumour-bearing mice. In contrast to BCG, M. brumae does not persist intracellularly or in tumour-bearing mice, so the risk of infection is lower. ConclusionsOur preclinical data suggest that M. brumae represents a safe and efficacious candidate as a therapeutic agent for non–muscle-invasive BC. Patient summaryWe investigated the antitumour activity of nonpathogenic mycobacteria in in vitro and in vivo models of non–muscle-invasive bladder cancer. We found that Mycobacterium brumae effectively inhibits bladder cancer growth and helps the host immune system to eradicate cancer cells, and is a promising agent for antitumour immunotherapy.

MicroRNA-218 inhibits bladder cancer cell proliferation, migration, and invasion by targeting BMI-1.

MicroRNAs (miRNAs) are recognized as important molecules and have emerged as important gene regulators in tumorigenesis. Growing evidence suggested that miR-218 was a tumor suppressor in many human cancers. However, its underlying role in bladder cancer (BCa) remains unclear. The aim of this study was to explore the effect of miR-218 on the proliferation, migration, and invasion of BCa cells. We found that miR-218 was frequently downregulated in BCa tissues compared with normal adjacent tissues. In vitro and in vivo assays demonstrated that miR-218 overexpression in the BCa cells inhibited cell proliferation, migration, and invasion. Luciferase reporter assay showed that BMI-1 was a direct target of miR-218. In addition, we found that miR-218 regulated the expression of BMI-1 and its downstream target (PTEN) and participated in the phosphorylation of AKT. Our findings indicate that miR-218 functions as tumor suppressor in BCa, and the miR-218/BMI-1 axis may provide novel diagnostic and therapeutic strategies for the treatment of BCa.

γ-Secretase inhibitor inhibits bladder cancer cell drug resistance and invasion by reducing epithelial-mesenchymal transition.

A previous study by our group demonstrated that the expression levels of Notch 1 and Jagged 1 in bladder cancer cells was significantly lower compared with those in normal bladder mucosa, while the expression levels of Notch 1 and Jagged 1 in invasive bladder cancer were higher compared with those in superficial bladder cancer. The present study investigated the effect of the Notch signaling pathway on the drug resistance and invasiveness of bladder cancer cells. It was demonstrated that complete inhibition of the Notch signaling pathway induced significant morphological changes and inhibited cell proliferation and migration (P<0.05). Reverse transcription quantitative polymerase chain reaction and western blot analyses revealed that the mRNA and protein expression levels of E-cadherin were upregulated (P<0.05) and the mRNA and protein expression levels of N-cadherin, vimentin and α-smooth muscle actin were downregulated (P<0.05). The present study concluded that complete inhibition of the Notch signaling pathway inhibited cell proliferation and invasion, and reduced drug resistance in bladder cancer cells, a phenomenon which may be associated with the inhibition of the epithelial-mesenchymal transition.

MP36-15 MICRORNA-218 INHIBITS BLADDER CANCER CELL PROLIFERATION, MIGRATION AND INVASION BY TARGETING BMI-1

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-15 MICRORNA-218 INHIBITS BLADDER CANCER CELL PROLIFERATION, MIGRATION AND INVASION BY TARGETING BMI-1 Pengchao Li, Qiang Lu, Jun Tao, and Chao Qin Pengchao LiPengchao Li More articles by this author , Qiang LuQiang Lu More articles by this author , Jun TaoJun Tao More articles by this author , and Chao QinChao Qin More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.743AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It is increasingly clear that microRNAs (miRNAs) play an important role in tumorigenesis. Growing evidence suggested that miR-218 was a tumor suppressor in many human cancers. However, its underlying role in bladder cance (BCa) remains unclear. The aim of this study was to explore the effect of miR-218 on the proliferation, migration and invasion of BCa cells. METHODS The expression of miR-218 and BMI-1 in tissues and cell lines were detected by qRT-PCR. The expression of BMI-1, PTEN, AKT and pAKT in cell lines were detected by western blot. Cell viability assay, colony formation assay, wound healing assay and transwell assay in vitro and tumour xenograft studies in vivo were performed to investigate the influence of miR-218 on the proliferation, invasion and migration of the BCa cell lines. Luciferase reporter assay was used to demonstrate whether BMI-1 was a direct target of miR-218. RESULTS The miR-218 expression level was frequently downregulated in BCa tissues compared with normal adjacent tissues. The overexpression of miR-218 significantly inhibited cell proliferation, migration and invasion in BCa cell lines. Luciferase reporter assay showed that BMI-1 was a direct target of miR-218. In addition, we found that miR-218 suppressed the expression of BMI-1 and its downstream target (PTEN) and participated in the regulation of phospho-AKT (pAKT). CONCLUSIONS Our findings indicated that miR-218 functions as tumour suppressor in BCa, and the miR-218/BMI-1/PTEN/AKT axis may provide novel diagnostic and therapeutic strategies for the treatment of BCa. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e434-e435 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pengchao Li More articles by this author Qiang Lu More articles by this author Jun Tao More articles by this author Chao Qin More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP46-01 AKR1C3 ACTIVATION AND INTRACRINE ANDROGENS CONFER RESISTANCE TO ENZALUTAMIDE

INTRODUCTION AND OBJECTIVES: The metabolism of cancer cells is fundamentally different from that of their normal counterparts. The M2 isoenzyme of pyruvate kinase (PKM2), expressed primarily during embryogenesis, is switched back on and exerts two major effects during oncogenesis: (i) slowing down glycolysis thus shunting glycolytic intermediates for anabolic process necessary for cell proliferation; and (ii) interacting with transcriptional factors thus upregulating growth-promoting genes. The role of PKM2 in bladder cancer formation and progression is presently unknown. METHODS: Human specimens and cultured cells from normal urothelia, low-gradenon-invasive and high-grademuscle-invasive bladder cancerwereassessed forPKM2expressionby immunohistochemistryand immunoblotting. Selected cancer cell lines over-expressing PKM2 were treated with shikonin, a naphthoquinone inhibitor of PKM2 found in medicinal plants, alone or in combination with rapamycin, an mTOR inhibitor, and their effects on cell proliferation, metabolism, apoptotic, autophagic and unfolded protein responses were determined using biochemical and molecular approaches. Target-specific effects of shikonin were verified by transfection with PKM2-specifc small interfering RNA (siRNA). RESULTS: Compared to normal urothelia, both low-grade noninvasive and high-grade muscle-invasive bladder cancer markedly over-express PKM2. Accompanying this is the hyper-phosphorylation of tyrosine residue at codon Y105 and nuclear translocation of PKM2. Cultured bladder cancer cell lines including UroTsa, RT4, RT112, J82, T24 and UMUC3 also express significantly more PKM2 than primarycultured urothelial cells. Exposure of RT112 and T24 to shikonin results in a dose-dependent inhibition of proliferation, reduced oxygen consumption and marked upregulation of signals indicative of apoptosis, autophage and unfolded protein response. A greater inhibition of cell proliferation is observed when shikonin is combined with rapamycin, suggesting a synergism between PKM2 and mTOR inhibition. Enforced expression of siRNA of PKM2 produces a similar inhibitory effect on cell line proliferation, establishing target-specific effects of shikonin. CONCLUSIONS: These data demonstrate for the first time an important role of PKM2 overexpression in bladder cancer formation and progression, and suggest a potentially unconventional way to therapeutically inhibit bladder cancer via specific alteration of tumor-promoting metabolism.

MP36-12 CLONALITY OF BLADDER TUMORS FOLLOWING RADICAL NEPHROURETERECTOMY – AGAINST THE FIELD DEFECT HYPOTHESIS

INTRODUCTION AND OBJECTIVES: Macroautophagy, also known as type II programmed cell death, is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here, we investigated the activated basal autophagy in bladder cancer cells and its roles in maintaining cancer cell growth. METHODS: Human immortalized uroepithelium (SV-Huc-1) and bladder cancer cells including RT-4 (grade I), 5637 (grade II), HT1376 and T24 (grade III) were used. We also included human prostate cancer cells (Rv1 and PC3), breast cancer cell (MCF-7) and embryonic kidney cancer cell (293T) as reference cells. The cell viability was accessed by WST-8. Autophagy was detected by the expression level of LC3-II protein. Human bladder tumor tissue array (TMA; 12 cases, paired with normal tissue) was used to detect the LC3 expression level. Detection of apoptosis level was measured by caspase 3/7 activation, caspase 3 cleavage and TUNEL assays. The ROS generation, mitochondria membrane potential in cells treated with autophagy inhibitors was assayed by staining with H2DCFCA and JC-1 dyes, respectively, and subsequently subjected to flow cytometry analysis. Disruption of lysomome membrane (LMP) by was detected by immunofluorescent of cathepsin-D in cells treated with CQ. RESULTS: We found bladder cancer cells exhibited high basal level of autophagy compared to prostate, breast or embryonic kidney cancer cells. Immunohistochemistry (IHC) detection of LC3 in human bladder TMA showed increased level of LC3 in bladder tumor tissues compared to their paired normal tissues. In the cell-based experiment, we found that autophagy inhibitors alone inhibited bladder cancer cell growth and increased apoptosis. The ROS generation and MMP disruption were not detected in cells treated with CQ or HCQ. Immunofluorescent detection of cathepsin-D in cells treated with CQ showed dispread pattern in the cytosol, suggesting the disruption of LMP in CQ-treated bladder cancer cells. CONCLUSIONS: Our results showed that human bladder cancer exhibits high basal level of autophagy. Autophagy inhibitors showed significant effects on the growth of bladder cancer cells by disruption of lysosome function and elevation of apoptotic cell death.

MP49-10 KNOCKDOWN OF GLYCOPROTEIN-130 INHIBITS BLADDER CANCER PROGRESSION AND MIGRATION

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-10 KNOCKDOWN OF GLYCOPROTEIN-130 INHIBITS BLADDER CANCER PROGRESSION AND MIGRATION Darryl T. Martin, Jill M. Steinbach, Marcia A. Wheeler, Cayce B. Nawaf, W. Mark Saltzman, and Robert M. Weiss Darryl T. MartinDarryl T. Martin More articles by this author , Jill M. SteinbachJill M. Steinbach More articles by this author , Marcia A. WheelerMarcia A. Wheeler More articles by this author , Cayce B. NawafCayce B. Nawaf More articles by this author , W. Mark SaltzmanW. Mark Saltzman More articles by this author , and Robert M. WeissRobert M. Weiss More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.514AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Seventy-five percent of bladder cancer patients in the United States have noninvasive disease at initial diagnosis. Although, Bacillus Calmette-Guerin (BCG) is the most effective adjuvant agent for treating non-muscle invasive bladder cancer, the response to BCG is unpredictable. Therefore, alternative treatments are needed. Glycoprotein-130 (GP130) is a transmembrane protein which is central to a number of signal transduction pathways involved in tumor growth/progression. Our goal is to target and degrade GP130 with small interfering RNAs (siRNAs). GP130 siRNA will be encapsulated in our previously developed chitosan functionalized nanoparticle (NP) system. Using a bladder cancer mouse model we will determine if knocking down GP130 expression has a potential role in decreasing tumor growth/progression. METHODS GP130 expression was determined in human bladder cancer specimens on a tissue microarray (TMA) by immunohistochemistry. In vitro, cell viability and cell migration, as affected by down-regulation of GP130, were assessed using a tetrazolium colorimetric assay and a scratch assay, respectively. Down-regulation of GP130 expression was achieved with chitosan-NP encapsulating GP130 siRNA (NP-siGP130-CH) and controls such as untreated (PBS), chitosan NP vehicle (NP-CH), and chitosan-NP encapsulating scrambled siRNA (NP-siSC-CH) were used. Foxn1 nu/nu mice were subcutaneously injected with 5X106 UM-UC-3 bladder cancer cells and then randomly divided into 3 groups: PBS, NP-CH or NP-siGP130-CH. RESULTS In a human bladder cancer TMA, GP130 expression was significantly higher in high grade bladder cancer specimens (2.40 +/- 0.18 relative units) than in low grade cancer specimens (1.67 +/- 0.16 relative units). Furthermore, more aggressive human bladder cancer cell lines (UM-UC-3 and TCC sup) had a higher GP130 expression than a more differentiated bladder cancer cell line (RT4). When human bladder cancer cells were treated with GP130 siRNA, there was a 60% decrease in cell viability. In addition, the cells treated with GP130 siRNA only migrated at half the rate as the cells that received the controls. Xenograft mouse bladder tumors treated with NP-siGP130-CH were significantly smaller than tumors receiving either PBS or NP-CH controls at day 7 (41%, 38%), 11 (65%, 60%), and 14 (75%, 60%), respectively. CONCLUSIONS We have shown that GP130 expression is higher in high grade than low grade human bladder cancers and that GP130 plays a role in bladder cancer migration in vitro and in cancer progression in an in vivo bladder cancer mouse model. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e606 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Darryl T. Martin More articles by this author Jill M. Steinbach More articles by this author Marcia A. Wheeler More articles by this author Cayce B. Nawaf More articles by this author W. Mark Saltzman More articles by this author Robert M. Weiss More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP36-11 AUTOPHAGY IS ACTIVATED IN HUMAN BLADDER CANCER AND REQUIRED FOR CANCER CELL GROWTH

INTRODUCTION AND OBJECTIVES: Macroautophagy, also known as type II programmed cell death, is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here, we investigated the activated basal autophagy in bladder cancer cells and its roles in maintaining cancer cell growth. METHODS: Human immortalized uroepithelium (SV-Huc-1) and bladder cancer cells including RT-4 (grade I), 5637 (grade II), HT1376 and T24 (grade III) were used. We also included human prostate cancer cells (Rv1 and PC3), breast cancer cell (MCF-7) and embryonic kidney cancer cell (293T) as reference cells. The cell viability was accessed by WST-8. Autophagy was detected by the expression level of LC3-II protein. Human bladder tumor tissue array (TMA; 12 cases, paired with normal tissue) was used to detect the LC3 expression level. Detection of apoptosis level was measured by caspase 3/7 activation, caspase 3 cleavage and TUNEL assays. The ROS generation, mitochondria membrane potential in cells treated with autophagy inhibitors was assayed by staining with H2DCFCA and JC-1 dyes, respectively, and subsequently subjected to flow cytometry analysis. Disruption of lysomome membrane (LMP) by was detected by immunofluorescent of cathepsin-D in cells treated with CQ. RESULTS: We found bladder cancer cells exhibited high basal level of autophagy compared to prostate, breast or embryonic kidney cancer cells. Immunohistochemistry (IHC) detection of LC3 in human bladder TMA showed increased level of LC3 in bladder tumor tissues compared to their paired normal tissues. In the cell-based experiment, we found that autophagy inhibitors alone inhibited bladder cancer cell growth and increased apoptosis. The ROS generation and MMP disruption were not detected in cells treated with CQ or HCQ. Immunofluorescent detection of cathepsin-D in cells treated with CQ showed dispread pattern in the cytosol, suggesting the disruption of LMP in CQ-treated bladder cancer cells. CONCLUSIONS: Our results showed that human bladder cancer exhibits high basal level of autophagy. Autophagy inhibitors showed significant effects on the growth of bladder cancer cells by disruption of lysosome function and elevation of apoptotic cell death.

MP45-19 UPREGULATION OF ISOENZYME M2 OF PYRUVATE KINASE IN BLADDER CANCER: IMPLICATION IN TUMOR INITIATION, PROGRESSION AND THERAPEUTIC TARGETING.

INTRODUCTION AND OBJECTIVES: The metabolism of cancer cells is fundamentally different from that of their normal counterparts. The M2 isoenzyme of pyruvate kinase (PKM2), expressed primarily during embryogenesis, is switched back on and exerts two major effects during oncogenesis: (i) slowing down glycolysis thus shunting glycolytic intermediates for anabolic process necessary for cell proliferation; and (ii) interacting with transcriptional factors thus upregulating growth-promoting genes. The role of PKM2 in bladder cancer formation and progression is presently unknown. METHODS: Human specimens and cultured cells from normal urothelia, low-gradenon-invasive and high-grademuscle-invasive bladder cancerwereassessed forPKM2expressionby immunohistochemistryand immunoblotting. Selected cancer cell lines over-expressing PKM2 were treated with shikonin, a naphthoquinone inhibitor of PKM2 found in medicinal plants, alone or in combination with rapamycin, an mTOR inhibitor, and their effects on cell proliferation, metabolism, apoptotic, autophagic and unfolded protein responses were determined using biochemical and molecular approaches. Target-specific effects of shikonin were verified by transfection with PKM2-specifc small interfering RNA (siRNA). RESULTS: Compared to normal urothelia, both low-grade noninvasive and high-grade muscle-invasive bladder cancer markedly over-express PKM2. Accompanying this is the hyper-phosphorylation of tyrosine residue at codon Y105 and nuclear translocation of PKM2. Cultured bladder cancer cell lines including UroTsa, RT4, RT112, J82, T24 and UMUC3 also express significantly more PKM2 than primarycultured urothelial cells. Exposure of RT112 and T24 to shikonin results in a dose-dependent inhibition of proliferation, reduced oxygen consumption and marked upregulation of signals indicative of apoptosis, autophage and unfolded protein response. A greater inhibition of cell proliferation is observed when shikonin is combined with rapamycin, suggesting a synergism between PKM2 and mTOR inhibition. Enforced expression of siRNA of PKM2 produces a similar inhibitory effect on cell line proliferation, establishing target-specific effects of shikonin. CONCLUSIONS: These data demonstrate for the first time an important role of PKM2 overexpression in bladder cancer formation and progression, and suggest a potentially unconventional way to therapeutically inhibit bladder cancer via specific alteration of tumor-promoting metabolism.

Small RNAs inhibit bladder cancer by up-regulating tumor suppressor genes

RNA activation (RNAa) is a newly discovered mechanism in which non-coding RNAs like small double-stranded RNAs (dsRNAs) or micro RNAs (miRNAs) induce sequence-specific gene activation by targeting promoter. Although its underlying mechanism remains unclear, we and others have demonstrated that Ago protein, RNA polymerase II (RNA Pol II) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) are required for RNAa. In addition, RNAa is conserved in mammalian cells. Increasing evidences indicated that dsRNAs or miRNAs can induce tumor suppressing genes expression and hold great capacity to inhibit bladder cancer cells. RNAa provides a novel method for gene manipulation and offers an exciting potential for therapeutic modality against bladder cancers. In this review, we will focus on the research advances in exploiting the mechanism of RNAa and its applications in bladder cancer therapeutics.

Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis.

Cyclosporine A and tacrolimus inhibit bladder cancer growth through down-regulation of NFATc1.

The functional role of nuclear factor of activated T-cells (NFAT), a key regulator of the immune response, in bladder cancer progression remains uncertain. In this study, we assessed biological significance of NFAT in human bladder cancer. Immunohistochemistry detected nuclear/cytoplasmic NFATc1 signals in 14 (21.5%)/34 (52.3%), respectively, of 65 muscle-invasive bladder carcinomas and showed that patients with nuclear NFATc1-positive tumor had a significantly higher risk of disease progression (P = 0.006). In bladder cancer cell lines, cyclosporine A (CsA) and tacrolimus (FK506), immunosuppressant drugs/non-selective NFAT inhibitors, attenuated NFATc1 expression and its nuclear translocation, NFAT transcriptional activity, and the expression of cyclooxygenase-2 and c-myc, downstream targets of NFATc1. NFAT inhibition via NFATc1-small interfering RNA (siRNA) or treatment with these NFAT inhibitors resulted in decreases in cell viability/colony formation, cell migration/invasion, and the expression/activity of MMP-2 and MMP-9, as well as an increase in apoptosis, in the parental/control lines. No significant additional inhibition in the viability and invasion of NFATc1-siRNA cells was seen. In xenograft-bearing mice, CsA and FK506 significantly retarded tumor growth. These results suggest that NFATc1 plays an important role in bladder cancer outgrowth. Thus, NFATc1 inactivation, especially using CsA and FK506, has the potential of being a therapeutic approach for bladder cancer.

A New Double Stranded RNA Suppresses Bladder Cancer Development by Upregulating p21 (Waf1/CIP1) Expression.

We have previously demonstrated that miR-1180-5p has potent ability to upregulate p21 expression by targeting promoter and inhibit bladder cancer. This prompted us to conjecture that a candidate dsRNA (dsP21-397) with perfect complementarity to the miR-1180-5p target site of p21 promoter may also trigger p21 expression. Transfection of dsP21-397 into T24 and EJ cells significantly activated p21 expression at 72 h and the activation presented in a time-course and dose-dependent manner. Moreover, the p21-activated activities of dsP21-397 and miR-1180-5p are not significantly different. Overexpression of p21 downregulated Cyclin D1, CDK4/6, and Cyclin A2 expression, and thereby induced cell cycle arrest and inhibited proliferation. Moreover, dsP21-397 suppressed bladder cancer largely depended on manipulating p21. In conclusion, our study identifies a pair of miRNA-dsRNA mediating endogenous p21 overexpression.

MicroRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44.

BackgroundMetastasis have considered as an important clinical obstacle in the treatment of human cancer including bladder cancer. Post-transcriptional regulation has emerged as robust effectors of metastasis. MiRNAs are involved in cancer development and progression, acting as tumor suppressors or oncogenes. In this study, we focus on it that microRNA-34a functions as an anti-metastatic microRNA and suppress angiogenesis in bladder cancer by directly targeting CD44.MethodsThe expression of mir-34a was detected by quantitative real-time PCR. Oligonucleotide and lentivirus were used to overexpress miR-34a. Tube formation assay and transwell assay were used to examine the effect on bladder cancer tube formation, migration and invasion in vitro. Animal models were used to examine the effect on metastasis and angiogenesis in vivo. Luciferase assay was carried out to verify the precise target of miR-34a.ResultsWe not only proved that mir-34a was significantly downregulated in bladder cancer tissues and cell lines but also that circulating miR-34a levels are reduced in bladder cancer, and their levels were positively relevance. Gain-of-function experiments investigated that increased mir-34a expression suppressed tube formation and reduced cell migration and invasion. In vivo metastasis, assays also demonstrated that overexpression of mir34a markedly inhibited bladder cancer metastasis. CD31, an endothelial cell–specific marker which stained in T24 tumors to evaluate for blood vessel density, the immunohistochemistry results showed that blood vessel quantification reduced dramatically in the T24 tumors over-expressing mir-34a. Combining with our previous studies and bioinformatics analysis, we expected that CD44 gene was a direct target of mir-34a, siRNA-mediated knockdown of CD44 partially phenocopied mir-34a overexpression suggesting that the pro-apoptotic role of mir-34a may be mediated primarily through CD44 regulation, whereas restoring the expression of CD44 attenuated the function of mir-34a in bladder cancer cells. Additionally, we identified that EMT (epithelial-mesenchymal transition) related proteins could be regulated by mir-34a which indicated that mir-34a could partially reserve EMT.ConclusionOur study defines a major metastasis and angiogenesis suppressive role for mir-34a, a microRNA functions as a tumor suppressor in bladder cancer by directly targeting CD44, which would be helpful as a therapeutic approach to block bladder cancer metastasis.

Synthesizing AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer cells.

The conventional strategy for cancer gene therapy offers limited control of specificity and efficacy. A possible way to overcome these limitations is to construct logic circuits. Here we present modular AND gate circuits based on CRISPR-Cas9 system. The circuits integrate cellular information from two promoters as inputs and activate the output gene only when both inputs are active in the tested cell lines. Using the luciferase reporter as the output gene, we show that the circuit specifically detects bladder cancer cells and significantly enhances luciferase expression in comparison to the human telomerase reverse transcriptase-renilla luciferase construct. We also test the modularity of the design by replacing the output with other cellular functional genes including hBAX, p21 and E-cadherin. The circuits effectively inhibit bladder cancer cell growth, induce apoptosis and decrease cell motility by regulating the corresponding gene. This approach provides a synthetic biology platform for targeting and controlling bladder cancer cells in vitro.