Yuki Yoshikawa

MiR-145 negatively regulates Warburg effect by silencing KLF4 and PTBP1 in bladder cancer cells

The Warburg effect is a well-known feature in cancer-specific metabolism. We previously reported on the role of microRNA (miR)-145 as a tumor-suppressor in human bladder cancer (BC) cells. In this study, we reveal that miR-145 decreases the Warburg effect by silencing KLF4 in BC cells. The expression levels of miR-145 were significantly lower in clinical BC samples and BC cell lines compared to those in normal tissues and HUC cells. Luciferase assay results showed that miR-145 directly bound to 3′UTR of KLF4, which was shown to be overexpressed in the clinical BC samples using Western blot analysis and immunohistochemistry. Remarkable growth inhibition and apoptosis were induced by the ectopic expression of miR-145 or by the gene silencing of KLF4 (siR-KLF4). Also, Warburg effect-related genes such as PTBP1/PKMs were regulated by the transfection of BC cells with miR-145 or siR-KLF4. These results thus indicate that the miR-145/KLF4/PTBP1/PKMs axis is one of the critical pathways that maintain the Warburg effect in BC carcinogenesis. MiR-145 perturbed the Warburg effect by suppressing the KLF4/PTBP1/PKMs pathway in BC cells, resulting in significant cell growth inhibition.

A Novel Combination RNAi toward Warburg Effect by Replacement with miR-145 and Silencing of PTBP1 Induces Apoptotic Cell Death in Bladder Cancer Cells

Bladder cancer is one of the most difficult malignancies to control. We explored the use of a novel RNA-interference method for a driver oncogene regulating cancer specific energy metabolism by the combination treatment with a small interfering RNA (siRNA) and a microRNA. After transfection of T24 and 253JB-V cells with miR-145 and/or siR-PTBP1, we examined the effects of cell growth and gene expression by performing the trypan blue dye exclusion test, Western blot, Hoechst 33342 staining, reverse transcription polymerase chain reaction (RT-PCR), and electron microscopy. The anti-cancer effects of xenograft model mice with miR-145 and/or siR-PTBP1 were then assessed. The combination treatment induced the deeper and longer growth inhibition and reduced the levels of both mRNA and protein expression of c-Myc and polypyrimidine tract-binding protein 1 (PTBP1) more than each single treatment. Notably, the combination treatment not only impaired the cancer specific energy metabolism by inhibiting c-Myc/PTBP1/PKMs axis but also inactivated MAPK/ERK and PI3K/AKT pathways examined in vitro and in vivo. Furthermore, the combination treatment induced apoptosis or autophagy; but, in some cells, apoptotic cell death was accompanied by autophagy, because the condensation of chromatin and many autophagosomes were coexistent. This combination treatment could be a novel RNA-interference strategy through the systemic silencing of the Warburg effect-promoting driver oncogene PTBP1 in bladder cancer cells.

The systemic inflammation-based Glasgow Prognostic Score as a powerful prognostic factor in patients with upper tract urothelial carcinoma

Introduction and Objective The combination of C-reactive protein and albumin, the Glasgow Prognostic Score (GPS), had independent prognostic value in patients with varying cancers, except for upper tract urothelial carcinoma (UTUC). The aim of this study was to describe the relationship between GPS and survival in patients with UTUC after adjustment for other prognostic factors. Materials and Methods We queried 2 UTUC databases. Retrospective clinical series on patients with localized UTUC managed by nephroureterectomy with bladder cuff, for whom data from the Yamaguchi Uro-Oncology Group and Osaka Medical College registry, including age, presence of bladder cancer, pT stage, lymphovascular invasion, C-reactive protein (CRP) and albumin, were analyzed. The GPS was constructed by combining CRP and albumin. Cancer specific survival (CSS) and overall survival (OS) and relative excess risk of death were estimated by GPS categories after adjusting for gender, age, ECOG performance status (PS), grade, and lymphovascular invasion (LVI). Results Seven hundred and twenty four UTUC patients were identified. Our final cohort included 574 patients; of these, 29.2% died during a maximum follow up of 16.7 years. The estimated mean 10-year CSS of patients with GPS of scre-0, -1, and -2 was 99.5, 95.1, and 75.9 months, respectively. Patients with GPS of score-2 had poorest 10-year estimated mean OS of 67.6 months (57.2–77.9). Raised GPS also had a significant association with excess risk of cancer death at 10 years (GPS 2: Relative Excess Risk = 1.74, 95% CI 1.20–2.54) after adjusting for gender, patients’ age, ECOG PS, and tumor focality. C-index of GPS both for CSS and OS were superior to patients’ age and tumor focality, and comparable to grade. Conclusions The GPS is an independent prognostic factor for CSS and OS after surgery with curative intent for localized UTUC. It significantly increases the accuracy of established prognostic factors. The GPS may provide a meaningful adjunct for patient counseling and clinical trial design.

The Anti-Proliferative Effect of Boron Neutron Capture Therapy in a Prostate Cancer Xenograft Model

Purpose Boron neutron capture therapy (BNCT) is a selective radiation treatment for tumors that preferentially accumulate drugs carrying the stable boron isotope, 10B. BNCT has been evaluated clinically as an alternative to conventional radiation therapy for the treatment of brain tumors, and more recently, recurrent advanced head and neck cancer. Here we investigated the effect of BNCT on prostate cancer (PCa) using an in vivo mouse xenograft model that we have developed. Materials and Methods Mice bearing the xenotransplanted androgen-independent human PCa cell line, PC3, were divided into four groups: Group 1: untreated controls; Group 2: Boronophenylalanine (BPA); Group 3: neutron; Group 4: BPA-mediated BNCT. We compared xenograft growth among these groups, and the body weight and any motility disturbance were recorded. Immunohistochemical (IHC) studies of the proliferation marker, Ki-67, and TUNEL staining were performed 9 weeks after treatment. Results The in vivo studies demonstrated that BPA-mediated BNCT significantly delayed tumor growth in comparison with the other groups, without any severe adverse events. There was a significant difference in the rate of freedom from gait abnormalities between the BPA-mediated BNCT group and the other groups. The IHC studies revealed that BNCT treatment significantly reduced the number of Ki-67-positive cells in comparison with the controls (mean±SD 6.9±1.5 vs 12.7±4.0, p<0.05), while there was no difference in the number of apoptotic cells, suggesting that BPA-mediated BNCT reduced PCa progression without affecting apoptosis at 9 weeks post-treatment. Conclusions This study has provided the first preclinical proof-of-principle data to indicate that BPA-mediated BNCT reduces the in vivo growth of PCa. Although further studies will be necessary, BNCT might be a novel potential treatment for PCa.

Feasibility of Photodynamic Diagnosis for Challenging TUR- Bt Cases Including Muscle Invasive Bladder Cancer, BCG Failure or 2 nd -TUR

BACKGROUND Despite widely adopted standard methods for follow-up including cystoscopy plus cytology, recurrence rates of non muscle-invasive bladder cancer (NMIBC) have not improved over the past decades, still ranging from 60% through 70%. Hence, widely acceptable surveillance strategies with excellent sensitivity are needed. Early recurrence has led to the development of a novel cystoscopy technique utilizing photodynamic diagnosis (PDD). Although, no studies have evaluated the efficacy of PDD for patients of MIBC, BCG failure or 2nd-transurethelial resection (TUR). MATERIALS AND METHODS The present study was performed from October 2012 through May 2013. IRB approved 25 patients initially underwent a cystoscopy examination of white light and blue light followed by the resection of tumors identified. Resections were performed from bladder mucosa areas considered suspicious at PDD, along with PDD negative normal bladder mucosa area resected by random biopsy. Specimens were divided into two groups, PDD positive and negative. Primary endpoints were sensitivity and specificity. RESULTS A total of 147 specimens extracted from 25 patients were included in the analysis. Some 45 out of 92 PDD-positive specimens were confirmed to have bladder cancer, and 51 out of PDD-negative 55 specimens were confirmed to be cancer negative. The sensitivity of PDD was 91.8% (45/49) and specificity was 52.0% (51/98). The sensitivity:specificity was 89.5% (17/19) : 47.6% (30/63) in 12 2nd-TUR patients, 90.5% (19/21) : 61.1% (11/18) in seven MIBC patients, and 95.0% (19/20) : 48.5% (16/33) in eight failed BCG cases. CONCLUSIONS PDD-TURBT has high sensitivity to diagnose BC even for 2nd-TUR, MIBC or BCG failure cases.

Dual Gas Treatment With Hydrogen and Carbon Monoxide Attenuates Oxidative Stress and Protects From Renal Ischemia-Reperfusion Injury

BACKGROUND Hydrogen (H2) and carbon monoxide (CO) gas are both reported to reduce reactive oxygen species and alleviate tissue ischemia-reperfusion (I-R) injury. The present study was conducted to evaluate the effects of a mixture of H2 gas and CO gas (dual gas) in comparison with hydrogen gas (H2: 2%) alone on I-R renal injury (composition of dual gas; N2: 77.8%; O2: 20.9%; H2: 1.30%; CO: 250 parts per million). METHODS Adult male Sprague-Dawley rats (body weight 250-280 g) were divided into 5 groups: (1) sham operation control, (2) dual gas inhalation (dual treatment) without I-R treatment, (3) I-R renal injury, (4) H2 gas alone inhalation (H2 treatment) with I-R renal injury, and (5) dual treatment with I-R renal injury. I-R renal injury was induced by clamping the left renal artery and vein for 45 minutes followed by reperfusion, and then contralateral nephrectomy was performed 2 weeks later. Renal function was markedly decreased at 24 hours after reperfusion, and thereafter the effects of dual gas were assessed by histologic examination and determination of the superoxide radical, together with functional and molecular analyses. RESULTS Pathologic examination of the kidney of I-R rats revealed severe renal damage. Importantly, cytoprotective effects of the dual treatment in comparison with H2 treatment and I-R renal injury were observed in terms of superoxide radical scavenging activity and histochemical features. Rats given dual treatment and I-R renal injury showed significant decreases in blood urea nitrogen. Increased expression of several inflammatory cytokines (tumor necrosis factor-α, interleukin-6, intracellular adhesion molecule-1, nuclear factor-κB, hypoxia inducible factor-1α, and heme oxygenase-1) was attenuated by the dual treatment. CONCLUSIONS Dual gas inhalation decreases oxidative stress and markedly improves I-R-induced renal injury.

ATR inhibition controls aggressive prostate tumors deficient in Y-linked histone demethylase KDM5D

&NA; Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male‐specific histone demethylase lysine‐specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP‐sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA‐replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress‐induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D‐deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.

MP88-19 MIR-145 MODULATES WARBURG EFFECT BY TARGETING KLF4/PTB1/PKMS AXIS IN BLADDER CANCER CELLS.

assay). A major limitation in the advancement of UTUC field is the lack of appropriate models. The objective of this study was to develop and evaluate preclinical models that would recapitulate treatment response observed in patients. METHODS: 35 surgical specimens from nephroureterectomy of patients with UTUC were implanted into immunocompromised NODSCID IL2Rg-/(NSG) mice. The histological and the genomic characterization of patient tumors and PDXs were examined by a board certified pathologist and MSK-IMPACTTM assay, respectively. Cell lines were also established to assess histologic and genetic fidelity. Chemosensitivity of PDX models was assessed using a 4-week cycle of gemcitabine/cisplatin (or carboplatin) administration and analysis of tumor growth was performed using a two-way ANOVA test. RESULTS: 12 patient-derived xenograft (PDX) models were established with a success rate of 34% (12/35) and a 14% (3/21) success in developing cell lines. Both models were highly reflective of their original tumors in terms of histology and genomic characteristics as noted in Figure 1 and 2. For a representative PDX, chemosensitivity experiments identified gemcitabine/carboplatin as a potentially effective combination, which was also used in the clinical scenario with a therapeutic response. CONCLUSIONS: We developed a cohort of stable PDX models and cell lines for UTUC that maintains the genetic characteristics of the patient’s initial tumor. The continued development of these models may facilitate personalized medicine strategies in the treatment of UTUC.

MP10-08 A PANEL OF MICRO-RNA SIGNATURE AS A TOOL FOR PREDICTING SURVIVAL OF PATIENTS WITH UROTHELIAL CARCINOMA OF THE BLADDER

99% of 23 million people in Taiwan. The National Health Insurance Research Database (NHIRD) was released for research purposes. A total of 868 CKD patients who received renal transplantation (RT) and 54,243 non-CKD controls matched for age, gender and index date were recruited from the NHIRD. The CKD patients with RT was also confirmed by the registry of catastrophic illness. The cancer incidence was identified through cross-referencing with the Cancer Registry Database. Risks were estimated as hazard ratios (HRs) and their 95% confidence intervals (CIs) by using a Cox proportional hazards model. RESULTS: For CKD patients with RT, a significant higher incidence rate ratio (IRR) of all cancer sites (IRR 1⁄4 3.79, 95% CI 1⁄4 3.12-4.62) was found. After the adjustment for age, sex and co-morbidities, we also observed a significantly increased cancer risk of 3.87 (HR 1⁄4 3.79, 95% CI 1⁄4 3.16-4.73). Especially, we found that CKD patients with RT have a significant increased IRR of bladder cancer (IRR 1⁄4 14.42, 95% CI 1⁄4 8.09-25.67). A greatly increased bladder cancer risk (HR 1⁄4 17.67, 95% CI 1⁄4 9.64-32.38) was found for CKD patients with RT after the adjustment for age, sex and co-morbidities. CONCLUSIONS: CKD patients have a higher risk of subsequent cancers, but the effect of therapeutic modalities such as RT on cancer risk is still unclear. Our finding is that CKD patients with RT have a significant increased risk of bladder cancer. Therefore, we should pay more attention to carry out effective treatments and implement an intensive follow-up to prevent CKD patients to progress to cancer.

The combination of preoperative platelet count and neutrophil lymphocyte ratio as a prognostic indicator in localized renal cell carcinoma

Introduction The combination of platelet count and neutrophil to lymphocyte ratio (COP-NLR) has been shown to provide prognostic information in several cancers, whereas its prognostic value in renal cell carcinoma (RCC) has not been reported. The objective of the present study was to examine the preoperative prognostic value of the COP-NLR in patients with localized RCC undergoing nephrectomy. Material and Methods The record of 268 patients, who underwent nephrectomy due to a diagnosis of RCC at our institute was analyzed in the study. The cut-off value of platelet count and NLR were defined by receive operating characteristic (ROC) analysis and the areas under the curve (AUC). Patients with both an increased platelet count (> 310×109/l) and an elevated NLR (> 3.85) were assigned to the score 2, and patients with one or neither of these indicators were assigned to the score 1 or 0, respectively. The impact of the COP-NLR and other clinicopathological characteristics on overall survival (OS) and recurrence-free survival (RFS) were evaluated using the univariate and multivariate Cox regression analysis. Result The median follow-up duration after surgical resection was 60 months. Multivariate analysis using the 10 clinicopathological findings selected by univariate analyses demonstrated that the preoperative COP-NLR was an independent prognostic factor for OS (HR: 2.32, 95%CI: 1.22 to 4.26, p=0.011) and RFS (HR: 1.91, 95%CI: 1.02 to 3.53, p=0.044). Conclusion The findings of the current study suggested that the preoperative COP-NLR is an independent prognostic indicator of OS and RFS for patients with localized RCC.