We previously reported that the level of microRNA (miR)-145 is attenuated in human bladder cancer cells. In this current study, we investigated whether intravesical administration of miR-145 could be a potential therapeutic strategy for controlling bladder cancer by using an orthotopic human bladder cancer xenograft model. Following transfection of 253J B-V cells with miR-145, the effects of the ectopic expression of miR-145 were examined by performing MTT, Western blotting analysis, Hoechst33342 staining, and wound healing assay in vitro. Also, a mouse orthotopic human bladder cancer model was established by inoculating 253J B-V cells into the bladder wall of mice. The anti-cancer effects of intravesical injections of miR-145 into these mice were then assessed. Transfection of 253J B-V cells with miR-145 induced apoptosis and suppression of cell migration in vitro. Western blotting showed that the levels of c-Myc, socs7, FSCN1, E-cadherin, β-catenin, and catenin δ-1 were decreased and that the PI3K/Akt and Erk1/2 signaling pathways were increased in compensatory fashion. In vivo, mice treated with miR-145 showed 76% inhibition of tumor growth, with a significant prolongation of animal survival (p = 0.0183 vs. control). Western blotting showed that both apoptosis and cell motility-related genes were significantly decreased as seen in vitro. Furthermore, PI3k/Akt and Erk1/2 signaling pathways, which were activated in a compensatory manner in vitro, were decreased in vivo. Intravesical administration of exogenous miR-145 was thus concluded to be a valid therapy for bladder cancer in this human bladder cancer xenograft model.

Human DEAD-box RNA helicase gene DDX6 was cloned from B-cell lymphoma cell line RC-K8. Previously, we reported that DDX6 acts as oncogene in several cancers such as colorectal cancer and hepatocellular carcinoma. However, the detailed mechanism of DDX6 action in carcinogenesis is largely unknown. In this study, we examined the functions of DDX6 in clinical gastric cancer (GC) samples and GC cells. DDX6 protein expression levels of cancer samples were higher than those of the adjacent normal tissues in 25 clinical GC samples (median value: 1.4 times higher). Also, the results of an RNA immunoprecipitation-assay (RIP-assay) showed that DDX6 associated with c-Myc mRNA. Moreover, enforced overexpression of DDX6 promoted both mRNA and protein expression of c-Myc in GC cells. On the other hand, the gene silencing of DDX6 induced growth suppression through down-regulation of c-Myc in GC cells grown in either two or three dimensions. Furthermore, c-Myc mRNA expression levels of cancer samples were higher than those of the adjacent normal tissues in DDX6 up-regulated-GC clinical samples. Our findings in this study suggested that DDX6 acted as oncogene in GC cells through promotion of c-Myc expression by association with the mRNA of c-Myc.

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INTRODUCTION AND OBJECTIVES: Cancer cells generate a significant proportion of ATP by glycolysis, even in the presence of oxygen (Warburg effect). Lactate transporters are of utmost importance for regulation of metabolism. Recently, the significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma (UC) remains mostly unknown. The aim of the present study was to investigate the functional importance of the lactate transporters MCT1 and MCT4 and their chaperone CD147 (Basigin) in preclinical models of UC.METHODS: MCT1, MCT4 and CD147 protein expression was assessed in 12 UC cell lines, and in benign and malignant tissue from 180 patients with muscle invasive UC of the bladder. MCT1, MCT4 and CD147 expression were correlated with molecular subtypes in The Cancer Genome Atlas (TCGA) dataset and validated using gene expression microarray data from an independent cohort of 65 patients with muscle invasive UC. Transient silencing of MCT1, MCT4 and CD147 was performed using small-interfering RNAs (siRNAs) in three UC cell lines. Cell viability in varying concentrations of oxygen and glucose was assessed using MTS assay. We measured apoptosis and production of reactive oxygen species by flow cytometry. Moreover, intra-and extracellular lactate, extracellular acidification and the oxygenconsumption rate were determined. In vivo effects of MCT4 silencing were assessed in an orthotopic xenograft model. RESULTS: Protein expression of MCT1, MCT4 and CD147 was significantly increased in cancer tissue. A high expression of MCT1, MCT4 and CD147 was associated with poor disease-specific survival after radical cystectomy. Analysis of the TCGA data as well as our own cohort of 65 UC revealed a significantly higher expression of MCT1 and MCT4 in the basal subtype. In vitro silencing of MCT4 led to a significant reduction in cell growth, a significant induction of apoptosis and an increased synthesis of reactive oxygen species. Moreover, silencing of MCT4 resulted in intracellular accumulation of lactate and reduced extracellular acidification. In vivo, stable knockdown of MCT4 significantly reduced tumor growth.CONCLUSIONS: The expression of lactate transporters in UC is associated with features of aggressive tumor biology and portends a poor prognosis. Inhibition of MCT4 induces intracellular accumulation of lactate and results in decreased tumor growth in vitro and in vivo. Targeting lactate metabolism via MCT4 therefore provides a promising therapeutic approach for invasive UC.