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Exosomes are 40-100 nm nano-sized extracellular vesicles and are receiving increasing attention as novel structures that participate in intracellular communication. We previously found that miRNA-1 (miR-1) functions as a tumor suppressor in renal cell carcinoma (RCC). In this study, we investigated the function of exosomal miR-1 and the possibility that the exosome constitutes a tumor maker in RCC. First, we established the method to collect exosomes from cell lysates and human serum by a spin column-based method. Next, we assessed exosomes using Nanosight nanoparticle tracking analysis and Western blot analysis with exosome marker CD63. We confirmed that exosomes labeled with PKH26 fused with recipient cells. Moreover, miR-1 expression was elevated in RCC cells treated with exosomes derived from miR-1-transfected cells. Functional analyses showed that exosomal miR-1 significantly inhibited cell proliferation, migration and invasion compared to control treatment. Our analyses with TCGA database of RCCs showed that miR-1 expression was significantly downregulated in clinical RCC samples compared to that in normal kidney samples, and patients with low miR-1 expression had poorer overall survival in comparison to patients with high expression. Furthermore, RNA sequence analyses showed that expression levels of several genes were altered by exposure to exosomal miR-1. The analyses with TCGA database indicated that high expression of MYO15A was associated with a poorer outcome in RCC. In addition, RT-qPCR analysis of exosomes from clinical patients' sera showed that MYO15A was significantly upregulated in RCC patients compared to that in healthy controls. This study showed that treatment with exosomal miR-1 might be an effective approach to treating RCCs. In addition, exosomal MYO15A could be a diagnostic tumor marker in RCCs.

Background Combination chemotherapy with gemcitabine and cisplatin (GC) is recommended as the primary treatment for advanced bladder cancer (BC). However, the benefits of this approach are limited owing to the acquisition of drug resistance. Therefore, we characterized GC resistance in BCs and evaluated the effects of a pan-RAS inhibitor on these cells. Methods We examined cross-resistance between gemcitabine-resistant and cisplatin-resistant BCs. To overcome drug resistance, we used the newly developed pan-RAS inhibitor Compound 3144 in vitro and investigated genes and pathways. Results The half-maximal inhibitory concentration (IC50) of cisplatin in gemcitabine-resistant BCs was the same as that in parental cells, and the IC50 of gemcitabine in cisplatin-resistant BCs was also the same as that in parental cells. Furthermore, different mRNA patterns were observed between gemcitabine- and cisplatin-resistant BCs using RNA sequence analysis. Compound 3144 inhibited cell viabilities through suppression of RAS-Dependent Signaling in gemcitabine- and cisplatin-resistant BCs. RNA sequencing revealed that several genes and pathways, particularly those related to the cell cycle, were significantly downregulated in Compound 3144-treated BCs. Conclusion Cross-resistance was not observed, and the pan-RAS inhibitor exhibited antitumor effects in gemcitabine- and cisplatin-resistant BCs. These findings provide insights into potential therapeutic strategies for treating BC.

In recent years, cancer metabolism has attracted attention as a therapeutic target, and glutamine metabolism is considered one of the most important metabolic processes in cancer. Solute carrier family 1 member 5 (SLC1A5) is a sodium channel that functions as a glutamine transporter. In various cancer types, SLC1A5 gene expression is enhanced, and cancer cell growth is suppressed by inhibition of SLC1A5. However, the involvement of SLC1A5 in clear cell renal cell carcinoma (ccRCC) is unclear. Therefore, in this study, we evaluated the clinical importance of SLC1A5 in ccRCC using The Cancer Genome Atlas database. Our findings confirmed that SLC1A5 was a prognosis factor for poor survival in ccRCC. Furthermore, loss-of-function assays using small interfering RNAs or an SLC1A5 inhibitor (V9302) in human ccRCC cell lines (A498 and Caki1) showed that inhibition of SLC1A5 significantly suppressed tumor growth, invasion, and migration. Additionally, inhibition of SLC1A5 by V9302 in vivo significantly suppressed tumor growth, and the antitumor effects of SLC1A5 inhibition were related to cellular senescence. Our findings may improve our understanding of ccRCC and the development of new treatment strategies for ccRCC.

We encountered a 9-year-old boy with a small bladder who had previously undergone multiple ureteroneocystostomies for unilateral refluxing megaureter. He underwent excision of the affected non-functioning kidney and ureterocystoplasty used the dilated regional ureter, in which the loop shaped urinary bladder was reconstructed without detubularization of the dilated ureter. The long-term postoperative course has been satisfactory. There have been no reports of ureterocystoplasty used a dilated ureter after multiple ureteroneocystostomies and none describing ureterocystoplasty in which the ureter was looped. This case is presented herein.

Shigeru Nakamura,1 Kazuya Tanabe,1 Taiju Hyuga,1,2 Taro Kubo,1 Satoru Inoguchi,1 Shina Kawai,1 Hideo Nakai1 1Department of Pediatric Urology, Jichi Medical University, Children’s Medical Center Tochigi, Shimotsuke, Japan; 2Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, JapanCorrespondence: Shigeru NakamuraDepartment of Pediatric Urology, Jichi Medical University, Children’s Medical Center, 3311-1, Yakushiji, Shimotsuke, Tochigi 329-0498, JapanTel +81-285-44-2111Fax +81-285-44-8452Email naka251148@gmail.comPurpose: To evaluate the treatment outcomes and postoperative complications associated with the systematic multi-site hydrodistention implantation technique (SMHIT) for primary vesicoureteral reflux (VUR) and to determine its mid-term efficacy and safety.Patients and Methods: We retrospectively reviewed the data for 17 ureters from 12 consecutive children, aged ≥ 1 year, with grade II–IV reflux and a history of febrile urinary tract infections (FUTI), who underwent a single-session of SMHIT. The primary outcome was the absence of postoperative FUTI (clinical success). The secondary outcome was improvement in reflux to grade 0–I on postoperative voiding cystourethrography (radiographic success).Results: Five and 7 children had bilateral and unilateral reflux, respectively. Reflux was categorized as grade II, III, and IV reflux in 2, 12, and 3 ureters, respectively. Seven of 10 (70%) toilet-trained children had bladder-bowel dysfunction (BBD) preoperatively. The SMHIT was performed for all patients, after which BBD improved. The mean postoperative follow-up period was 6 years and 9 months. The clinical success rate was 100%. Radiographic success was achieved in 16/17 ureters (94%) at 3– 4 months, 17/17 (100%) ureters at 1 year, and 17/17 (100%) ureters at 3 years postoperatively. Major complications did not develop postoperatively.Conclusion: When prioritizing treatment of concomitant BBD in children with primary VUR and avoiding dextranomer/hyaluronic acid injection therapy in contraindicated children according to the Food and Drug Administration recommendations, a single-session of SMHIT may be as effective and safe in the mid-term as performing open anti-reflux surgery.Keywords: dextranomer hyaluronic acid, Dx/HA, endoscopic treatment, vesicoureteral reflux

Background: Analysis of a microRNA (miRNA) expression signature of bladder cancer (BC) by deep-sequencing revealed that clustered miRNAs microRNA (miR)-451a, miR-144-3p, and miR-144-5p were significantly downregulated in BC tissues. We hypothesised that these miRNAs function as tumour suppressors in BC. The aim of this study was to investigate the functional roles of these miRNAs and their modulation of cancer networks in BC cells. Methods: The functional studies of BC cells were performed using transfection of mature miRNAs. Genome-wide gene expression analysis, in silico analysis, and dual-luciferase reporter assays were applied to identify miRNA targets. The association between miR-144-5p levels and expression of the target genes was determined, and overall patient survival as a function of target gene expression was estimated by the Kaplan–Meier method. Results: Gain-of-function studies showed that miR-144-5p significantly inhibited cell proliferation by BC cells. Four cell cycle-related genes (CCNE1, CCNE2, CDC25A, and PKMYT1) were identified as direct targets of miR-144-5p. The patients with high CCNE1 or CCNE2 expression had lower overall survival probabilities than those with low expression (P=0.025 and P=0.032). Conclusion: miR-144-5p functions as tumour suppressor in BC cells. CCNE1 and CCNE2 were directly regulated by miR-144-5p and might be good prognostic markers for survival of BC patients.

Recent clinical trials of chemotherapeutics for advanced bladder cancer (BC) have shown limited benefits. Therefore, new prognostic markers and more effective treatment strategies are required. One approach to achieve these goals is through the analysis of RNA networks. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-23b/27b (miR-23b/27b) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR-23b/27b cluster in BC cells is still unknown. Thus, the aim of the present study was to investigate the functional significance of the miR-23b/27b cluster and its regulated molecular targets, with an emphasis on its contributions to BC oncogenesis and metastasis. The expression levels of the miR-23b/27b cluster were significantly reduced in BC clinical specimens. Restoration of mature miR-23b or miR-27b miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs function as tumor suppressors. Gene expression data and in silico analysis demonstrated that the genes coding for the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) were potential targets of the miR-23b/27b cluster. Luciferase reporter assays and western blotting demonstrated that EGFR and c-Met receptor trypsine kinases were directly regulated by these clustered miRNAs. We conclude that the decreased expression of the tumor-suppressive miR-23b/27b cluster enhanced cancer cell proliferation, migration and invasion in BC through direct regulation of EGFR and c-Met signaling pathways. Our data on RNA networks regulated by tumor-suppressive miR-23b/27b provide new insights into the potential mechanisms of BC oncogenesis and metastasis.

Our recent study of the microRNA expression signature of prostate cancer (PCa) revealed that microRNA-224 (miR-224) is significantly downregulated in PCa tissues. Here, we found that restoration of miR-224 significantly inhibits PCa cell migration and invasion. Additionally, we found that oncogenic TPD52 is a direct target of miR-224 regulation. Silencing of the TPD52 gene significantly inhibits cancer cell migration and invasion. Moreover, TPD52 expression is upregulated in cancer tissues and negatively correlates with miR-224 expression. We conclude that loss of tumour-suppressive miR-224 enhances cancer cell migration and invasion in PCa through direct regulation of oncogenic TPD52.

// Ryosuke Matsushita 1 , Hirofumi Yoshino 1 , Hideki Enokida 1 , Yusuke Goto 2 , Kazutaka Miyamoto 1 , Masaya Yonemori 1 , Satoru Inoguchi 1 , Masayuki Nakagawa 1 , Naohiko Seki 2 1 Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan 2 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chuo-ku, Chiba, Japan Correspondence to: Naohiko Seki, e-mail: naoseki@faculty.chiba-u.jp Keywords: miR-145-5p, miR-145-3p, tumor-suppressor, UHRF1, bladder cancer Received: February 22, 2016 Accepted: March 28, 2016 Published: April 09, 2016 ABSTRACT In microRNA (miRNA) biogenesis, the guide-strand of miRNA integrates into the RNA induced silencing complex (RISC), whereas the passenger-strand is inactivated through degradation. Analysis of our miRNA expression signature of bladder cancer (BC) by deep-sequencing revealed that microRNA ( miR ) -145-5p (guide-strand) and miR-145-3p (passenger-strand) were significantly downregulated in BC tissues. It is well known that miR-145-5p functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p on cancer cells is still ambiguous. The aim of the present study was to investigate the functional significance of miR-145-3p and BC oncogenic pathways and targets regulated by miR-145-5p/miR-145-3p . Ectopic expression of either miR-145-5p or miR-145-3p in BC cells significantly suppressed cancer cell growth, migration and invasion and it also induced apoptosis. The gene encoding ubiquitin-like with PHD and ring finger domains 1 ( UHRF1 ) was a direct target of these miRNAs. Silencing of UHRF1 induced apoptosis and inhibited cancer cell proliferation, migration, and invasion in BC cells. In addition, overexpressed UHRF1 was confirmed in BC clinical specimens, and the high UHRF1 expression group showed a significantly poorer cause specific survival rate in comparison with the low expression group. Taken together, our present data demonstrated that both strands of miR-145 ( miR-145-5p : guide-strand and miR-145-3p : passenger-strand) play pivotal roles in BC cells by regulating UHRF1 . The identification of the molecular target of a tumor suppressive miRNAs provides novel insights into the potential mechanisms of BC oncogenesis and suggests novel therapeutic strategies.

insulin signaling in the progression of a murine RCC model using metformin to treat hyperinsulinemia induced by a high-carbohydrate diet. METHODS: pT1-T4 tumor specimens from 99 patients with RCC were evaluated for IR expression by immunohistochemistry. Serum insulin and C-peptide levels were measured. The association between biomarker expression and serum concentration, prognostic factors, and clinical outcomes were assessed. C57BL/6 mice were randomized into four groups: low-carbohydrate diet, high-carbohydrate diet, low-carbohydrate diet þ metformin, and high-carbohydrate diet þ metformin groups. RENCA cells were injected subcutaneously. Body weight, glucose tolerance test, serum insulin level, and tumor volume were evaluated. Immunoblotting was performed to assess the insulin signaling pathway in the murine RCC model. RESULTS: The IR expression level was significantly lower in patients with tumor stage pT2-4 and with a preoperative serum Cpeptide level more than 5.14 ng/mL. High IR expression was an independent predictor of favorable progression-free survival after radical nephrectomy. In vivo progression of RENCA in the murine model was not significantly stimulated by hyperinsulinemia induced by a high-carbohydrate diet. However, in vivo progression of RENCA was significantly inhibited by metformin in both the lowand highcarbohydrate diet groups. IR expression was significantly higher in the tumors from the low-carbohydrate diet group and high-carbohydrate diet plus metformin group than that from the high-carbohydrate diet group. CONCLUSIONS: IR expression in RCC was inversely associated with cancer progression and serum insulin levels in both clinical and murine models. The anticancer effect of metformin in the murine RCC model might be derived from the direct effect of the agent on cancer cells rather than the effect of the lower insulin level. The lower expression of IR in the tumor from patients with a poor outcome might be the result of a higher serum insulin level, which is not strong cause of cancer progression.

INTRODUCTION AND OBJECTIVES: Generally in microRNA (miRNA) biogenesis, one strand (guide strand) of the duplex may potentially act as a functional miRNA and incorporated into the RNA-induced silencing complex and regulating target genes. In contrast, other strand (passenger strand: miRNA*) is disintegrated quickly. Our miRNA expression signature of bladder cancer (BC) by using deep-sequencing revealed that tumor-suppressive miRNA-145 and its passenger strand of miRNA-145* were significantly reduced in cancer tissues, suggesting these function as tumor suppressors in BC cells. The aim of the study was to investigate functional significance of miRNA-145* and its regulated molecular targets/ pathways in BC. METHODS: We evaluated the expression levels of miR-145/ miR-145* in 43 BC clinical specimens and 12 normal bladder epithelia by real-time PCR. The functional analysis of the miRNA and molecular targets/pathways searching strategies were described as our previous studies (J Urol,192:1822-1830,2014; FEBS Lett,588:3170-3179,2014; PLoS One,9:e84311,2014). RESULTS: Expression levels of miR-145 and miR-145* were significantly lower than that in normal bladder epithelia (P < 0.0001). Gain-of-function studies revealed that cell proliferation, migration, and invasion were significantly inhibited in both miR-145 and miRNA-145* transfection into BC cell lines (each, P < 0.001). Combination of gene expression analysis of miR-145*-transfectants and in silico analysis showed that several oncogenic genes (HK2, YES1, FOSL1 etc.) were identified as miR-145* regulation in BC cells. CONCLUSIONS: Our genome-wide miRNA expression signature showed that miR-145* as a tumor-suppressive miR-145 passenger strand frequently reduced in BC cells and acts as a tumorsuppressor. Elucidation of miR-145/145*-mediated novel cancer pathways provide new insights of the potential mechanisms of BC oncogenesis. Furthermore, tumor-suppressive miR-145/145* might be contributed to development of miRNA-based cancer therapeutics in the disease.

Here, we found that members of the microRNA-29 family (miR-29a/b/c; “miR-29s”) were significantly reduced in clear cell renal cell carcinoma (ccRCC) tissues, suggesting that they functioned as tumour suppressors. Restoration of all mature members of the miR-29 family inhibited cancer cell proliferation, migration and invasion. LOXL2 was a direct target gene of miR-29s, as shown by genome-wide gene expression analysis and luciferase reporter assay. Overexpressed LOXL2 was confirmed in ccRCC clinical specimens, and silencing of LOXL2 inhibited cancer cell migration and invasion in ccRCC cell lines. Our data demonstrated that the miR-29s-LOXL2 axis contributed to cancer cell migration and invasion in ccRCC.

We observed abnormal expression of the microRNA-23b/27b (miR-23b/27b) cluster in our previous study of miRNA expression signatures. However, the relationship between aberrant miRNA expression and clear cell renal cell carcinoma is not well established. We investigated the functional significance of the miR-23b/27b cluster in clear cell renal cell carcinoma cells and evaluated these miRNAs as biomarkers to predict the risk of clear cell renal cell carcinoma.Expression levels of miR-23b and miR-27b were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. The association between miRNA expression and overall survival was estimated by the Kaplan-Meier method. Gain of function assays were performed using mature miR-23b and miR-27b in the 786-O and A498 renal cell carcinoma cell lines. Targets regulated by these miRNAs were predicted by in silico analysis.Expression of the miR-23b/27b cluster was significantly decreased in clear cell renal cell carcinoma tissue specimens and associated with pathological grade and stage. Significantly shorter overall survival was observed in patients with lower expression of the miR-23b/27b cluster. Restoration of miR-23b and miR-27b significantly inhibited cancer cell proliferation, migration and invasion.Expression of the miR-23b/27b cluster was frequently decreased in clear cell renal cell carcinoma tissue. Reduced expression of these miRNAs increased the risk of disease progression and predicted poor survival. Thus, miR-23b and miR-27b function as tumor suppressors, targeting several oncogenic genes in clear cell renal cell carcinoma cells.

METHODS: We sequenced the whole genomes of the tumor and normal tissues from 60 RCC patients (T4N0M1) with well-documented clinical data to a mean depth of 30i A or greater. RESULTS: From the whole genome data, we identified somatic mutations and copy number alterations that may serve as the markers for and (or) possibly drive cancer progression. RCCs display two classes of structural variation: monogenomic and polygenomic tumors. We further deep-sequenced the whole genomes of several representative cases with polygenomic tumors to 120i A and more. Our studies identified the early genomic events that occur in the founder cells and shed light on the genome evolution of RCCs. By using the somatic mutations as the lineage markers, we traced the genetic record of the emergence of different sub-clones over time and found the genomic events that drive divergence of the founder cells into different subgroups. CONCLUSIONS: This study deciphers the fine genetic architecture of the RCC genomes and allows us to determine the extent of sub-clonal diversities within the bulk tumor mass of RCC, to build a polygenetic tree of the cancer to define when the divergence occurs, and to reveal what mutational processes are involved in the different stages of tumor evolution.

INTRODUCTION AND OBJECTIVES: Genomic characterization of urothelial carcinoma of the bladder (UCB) has begun to reveal significant intertumor heterogeneity when comparing samples from different subjects. As in other malignancies, intratumor heterogeneity, which may allow for tumor evolution and adaption, poses a significant challenge to personalized-medicine strategies. METHODS: To examine UCB tumor evolution and heterogeneity, we performed next-generation targeted sequencing on multiple temporally and spatially separated bladder tumors obtained at time of transurethral resection (TUR) and radical cystectomy (RC). Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 300 cancer-associated genes. RESULTS: Phylogenetic reconstruction revealed evidence of branched evolutionary growth. Evaluation of multiple tumors from individual subjects identified both shared and unique potential driver mutations. Evidence of convergent phenotypic evolution was detected through analysis of multiple distinct tumors from several subjects. For example, three separate tumors in one subject (see Figure) shared a common PIK3CA mutation (E453K) and had unique second mutations in PIK3CA (E542V, E545K, and E545Q, respectively). In another subject, distinct inactivatingmutations of EP300were identified in two temporally separated tumor samples. Macrodissection of single tumors into non-invasive and invasive components revealed significant intratumor heterogeneity; one case illustrates howanalysis of amuscleinvasive TURspecimen could result in undersampling and therebymiss the tumor clone that persisted at time of RC. CONCLUSIONS: We demonstrate branched evolution of UCB through genomic analyses of multiple temporally and spatially distinct bladder tumors from individual subjects. Macrodissection of individual tumor samples identified significant intratumor heterogeneity. These concepts may present major challenges to personalized-medicine approaches that rely on sampling of a single tumor at a specific timepoint in the evolution of a patient’s UCB.

INTRODUCTION AND OBJECTIVES: Cancer-stromal interaction is critical for cancer biology. Bladder cancer develops within the mucosa, and invades into muscle layer surrounded by abundant adipose tissue. Adipose tissue has stromal cell types, such as preadipocytes and mesenchymal stem cells (MSCs). Thus, the adipose tissue stromal cells (ATSCs) seem critical for bladder cancer biology. The aim of this study is to address the effects of ATSCs on the apoptosis, growth and invasion of the superficial and invasive types. METHODS: RT4 and RT112 were used as human superficial urothelial carcinoma type of bladder, while EJ and HT1376 were done as invasive type. ATSCs were isolated from subcutaneous adipose tissue of 1-week old male Wister rats. We examined the apoptosis, growth and invasion of cancer cells, using collagen gel invasion assay system, in which the tumor cells were co-cultured on ATSC-embedded gel layer. The cellular behavior, and the apoptosis-, growthand ivasion-related molecules were analyzed by morphometry, immunohistochemistry, Western blot and real-time RT-PCR. RESULTS: ATSCs promoted and inhibited the apoptosis and growth of superficial type cancer cells, respectively. In contrast, ATSCs inhibited and promoted the apoptosis and growth of invasive type cancer cells. ATSCs had no effect on the invasion of superficial type, whereas they promoted the invasion of invasive type. ATSCs promoted the expression of MAPK proteins (raf-1, MEK-1 and ERK1/ERK2) in all cancer cell types. In turn, superficial type promoted [alpha]-SMA-positive myofibroblastic differentiation in ATSCs, while invasive type did lipid dropletand S-100 protein-positive adipogenic differentiation in ATSCs. CONCLUSIONS: The data suggest the following issues: 1) ATSCs suppress the progression of superficial type urothelial carcinoma through the growth inhibition and apoptosis promotion, whereas ATSCs promote that of invasive type through the enhancement of the growth and invasion, and the apoptosis prohibition; 2) ATSCinduced MAPK pathway activation plays differential roles in biological behavior of superficial and invasive types; and 3) Superficial type induces myofibroblastic differentiation in ATSCs as cancer-associated phenotype, while invasive type does adipogenic differentiation in ATSCs. Source of Funding: none

Here, we found that microRNA-24-1 (miR-24-1) is significantly reduced in bladder cancer (BC) tissues, suggesting that it functions as a tumour suppressor. Restoration of mature miR-24-1 inhibits cancer cell proliferation and induces apoptosis. Forkhead box protein M1 (FOXM1) is a direct target gene of miR-24-1, as shown by genome-wide gene expression analysis and luciferase reporter assay. Overexpressed FOXM1 is confirmed in BC clinical specimens, and silencing of FOXM1 induces apoptosis in cancer cell lines. Our data demonstrate that the miR-24-1–FOXM1 axis contributes to cancer cell proliferation in BC, and elucidation of downstream signalling will provide new insights into the molecular mechanisms of BC oncogenesis.