Hiromasa Sakamoto

An EP4 Antagonist ONO-AE3-208 Suppresses Cell Invasion, Migration, and Metastasis of Prostate Cancer

EP4 is one of the prostaglandin E2 receptors, which is the most common prostanoid and is associated with inflammatory disease and cancer. We previously reported that over-expression of EP4 was one of the mechanisms responsible for progression to castration-resistant prostate cancer, and an EP4 antagonist ONO-AE3-208 in vivo suppressed the castration-resistant progression regulating the activation of androgen receptor. The aim of this study was to analyze the association of EP4 with prostate cancer metastasis and the efficacy of ONO-AE3-208 for suppressing the metastasis. The expression levels of EP4 mRNA were evaluated in prostate cancer cell lines, LNCaP, and PC3. EP4 over-expressing LNCaP was established, and their cell invasiveness was compared with the control LNCaP (LNCaP/mock). The in vitro cell proliferation, invasion, and migration of these cells were examined under different concentrations of ONO-AE3-208. An in vivo bone metastatic mouse model was constructed by inoculating luciferase expressing PC3 cells into left ventricle of nude mice. Their bone metastasis was observed by bioluminescent imaging with or without ONO-AE3-208 administration. The EP4 mRNA expression levels were higher in PC3 than in LNCaP, and EP4 over-expression of LNCaP cells enhanced their cell invasiveness. The in vitro cell invasion and migration were suppressed by ONO-AE3-208 in a dose-dependent manner without affecting cell proliferation. The in vivo bone metastasis of PC3 was also suppressed by ONO-AE3-208 treatment. EP4 expression levels were correlated with prostate cancer cell invasiveness and EP4 specific antagonist ONO-AE3-208 suppressed cell invasion, migration, and bone metastasis, indicating that it is a potential novel therapeutic modality for the treatment of metastatic prostate cancer.

CCL2 as a potential therapeutic target for clear cell renal cell carcinoma

We previously reported that the pVHL‐atypical PKC‐JunB pathway contributed to promotion of cell invasiveness and angiogenesis in clear cell renal cell carcinoma (ccRCC), and we detected chemokine (C‐C motif) ligand‐2 (CCL2) as one of downstream effectors of JunB. CCL2 plays a critical role in tumorigenesis in other types of cancer, but its role in ccRCC remains unclear. In this study, we investigated the roles and therapeutic potential of CCL2 in ccRCC. Immunohistochemical analysis of CCL2 expression for ccRCC specimens showed that upregulation of CCL2 expression correlated with clinical stage, overall survival, and macrophage infiltration. For functional analysis of CCL2 in ccRCC cells, we generated subclones of WT8 cells that overexpressed CCL2 and subclones 786‐O cells in which CCL2 expression was knocked down. Although CCL2 expression did not affect cell proliferation in vitro, CCL2 overexpression enhanced and CCL2 knockdown suppressed tumor growth, angiogenesis, and macrophage infiltration in vivo. We then depleted macrophages from tumor xenografts by administration of clodronate liposomes to confirm the role of macrophages in ccRCC. Depletion of macrophages suppressed tumor growth and angiogenesis. To examine the effect of inhibiting CCL2 activity in ccRCC, we administered CCL2 neutralizing antibody to primary RCC xenografts established from patient surgical specimens. Inhibition of CCL2 activity resulted in significant suppression of tumor growth, angiogenesis, and macrophage infiltration. These results suggest that CCL2 is involved in angiogenesis and macrophage infiltration in ccRCC, and that CCL2 could be a potential therapeutic target for ccRCC.

Determining the Efficacy of Ultrasonography for the Detection of Ureteral Stone

OBJECTIVE To assess the efficacy of ultrasonography (US) for the detection of ureteral stone using non-contrast-enhanced computed tomography (NCCT) as a standard reference. MATERIALS AND METHODS From January 2009 to September 2011, 428 patients underwent both NCCT and US on the same day. The sensitivity and specificity of US to detect ureteral stone was evaluated. The detection rates using US imaging were examined according to location and stone size. The sizes of stones determined in the longest axis of NCCT and US were compared. We also performed group classification based on size to examine whether stone sizes measured by NCCT and US were similar. Moreover, the factors that may affect the detection of ureteral stone by US were analyzed. RESULTS Out of 856 ureters, NCCT could detect 171 stones in 169 patients, whereas US could detect 98 stones, yielding a sensitivity of 57.3% and a specificity of 97.5%. Expectedly, detection rate of US increased with stone size but was lower for distal ureter. With hydronephrosis, the sensitivity of US improved from 57.3% to 81.3%. Stone sizes measured by US correlated positively with those by computed tomography, and were concordant with those of NCCT in 68 of 98 patients (69.4%). Interestingly, stone size and the presence of hydronephrosis were factors that independently affected ureteral stone detection by US. CONCLUSION These results indicate that US may be useful as an initial imaging modality for detecting ureteral stone.

Safety and efficacy of ureteroscopy after obstructive pyelonephritis treatment

An obstructed, infected kidney combined with ureteral stones can be lethal, and requires urgent drainage and complete stone removal. However, the optimal method of stone removal, and its safety and efficacy have yet to be conclusively established. The aim of this study was to determine the safety and efficacy of carrying out ureteroscopy after kidney drainage for septic patients with obstructing stones.

Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin are well-known therapeutic targets for renal cell carcinoma (RCC). Sunitinib is an agent that targets VEGF receptors and is considered to be a standard treatment for metastatic or unresectable clear cell RCC (ccRCC). However, ccRCC eventually develops resistance to sunitinib in most cases, and the mechanisms underlying this resistance are not fully elucidated. In the present study, we established unique primary xenograft models, KURC1 (Kyoto University Renal Cancer 1) and KURC2, from freshly isolated ccRCC specimens. The KURC1 xenograft initially responded to sunitinib treatment, however finally acquired resistance. KURC2 retained sensitivity to sunitinib for over 6 months. Comparing gene expression profiles between the two xenograft models with different sensitivity to sunitinib, we identified interleukin 13 receptor alpha 2 (IL13RA2) as a candidate molecule associated with the acquired sunitinib-resistance in ccRCC. And patients with high IL13RA2 expression in immunohistochemistry in primary ccRCC tumor tends to have sunitinib-resistant metastatic site. Next, we showed that sunitinib-sensitive 786-O cells acquired resistance in vivo when IL13RA2 was overexpressed. Conversely, shRNA-mediated knockdown of IL13RA2 successfully overcame the sunitinib-resistance in Caki-1 cells. Histopathological analyses revealed that IL13RA2 repressed sunitinib-induced apoptosis without increasing tumor vasculature in vivo. To our knowledge, this is a novel mechanism of developing resistance to sunitinib in a certain population of ccRCC, and these results indicate that IL13RA2 could be one of potential target to overcome sunitinib resistance.

Amino-terminal enhancer of split gene AES encodes a tumor and metastasis suppressor of prostate cancer

A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration‐resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR‐defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium‐specific inactivation of Aes in Ptenflox/flox mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways.

A family case with germline TSC1 and mtDNA mutations developing bilateral eosinophilic chromophobe renal cell carcinomas without other typical phenotype of tuberous sclerosis

Aim We examined the genetic alterations in a mother and son with multiple eosinophilic chromophobe renal cell carcinomas (chRCCs) showing no other features. Methods Germline DNA and bilateral renal cell carcinoma DNA were genetically analysed by whole-exome sequencing. Candidate gene alterations in the first patient’s germline were investigated in her child’s germline and the chRCCs. Results We detected several germline gene alterations in the mother. Among the identified alterations, TSC1 and mitochondrial DNA mutations were also confirmed in her son. Regarding somatic alterations in bilateral chRCCs, no common candidate gene alteration was found. Conclusion To the best of our knowledge, this is the first report of whole-exome sequencing revealing bilateral eosinophilic chRCCs associated with tuberous sclerosis complex in a family case without classical phenotype. These results suggest that germline TSC1 and mitochondrial DNA gene mutations may be involved in the development of chRCCs in some cases.

Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system.

THE FACTORS THAT AFFECT THE DECISION TO PERFORM LAPAROSCOPIC PARTIAL NEPHRECTOMY FOR SMALL RENAL TUMOR

(Objectives) Nephron sparing surgery (NSS) is strongly recommended for patients with T1a renal cell carcinoma (RCC) whenever surgically feasible. However, partial nephrectomy, particularly laparoscopic approach, remains underutilized in Japan compared to laparoscopic radical nephrectomy (LRN). In this study, we examined the safety and efficacy of laparoscopic partial nephrectomy (LPN) for T1a RCC compared to LRN. We also assessed the factors that affect the decision to perform LPN or LRN. (Patients and methods) From March 2001 to September 2014, 112 patients with T1a renal tumors received renal surgery at our institution. Of these, 100 patients (LPN: 36 patients, LRN: 64 patients) underwent laparoscopic surgery. Treatment outcomes including surgical and oncological outcomes among each approach were compared. In addition, multivariate analysis was performed to reveal the factors that affect the decision on surgical approach. (Results) The ratio of patients more than 75 years old and the RENAL nephrometry score were higher in LRN group than those in LPN group. Operating time was longer but renal function was well preserved in LPN group. Importantly, blood loss, intraoperative and postoperative complication rate, and oncological outcome (recurrence-free survival and overall survival) were similar in both groups. Multivariate analysis revealed that age (≥75 years old), high RENAL nephrometry score, operation period (before 2011), and the absence of Endoscopic Surgical Skill Qualification (ESSQ) in surgeon were independent predictive factors that select LRN. (Conclusions) Our data suggests that LPN for T1a renal tumor could be performed safely and the decision whether LPN or LRN were performed were associated with technical factors such as the presence of ESSQ or operation period, as well as patients factor such as age and tumor factor such as tumor complexity.

LAPAROSCOPIC RADICAL NEPHRECTOMY FOR RENAL TUMOR>7CM

(Objectives) Laparoscopic radical nephrectomy (LRN) is now a standard care for the treatment of renal tumors, but the limitation of LRN for large tumors remains to be elucidated. In this study, we examined the safety and efficacy of LRN for >7 cm renal tumors including tumors >10 cm. (Patients and methods) From March 2001 to September 2014, 167 patients received laparoscopic surgery for renal tumors at our institution. Of these, 126 patients (≤4.0 cm: 64 cases, 4.1-7.0 cm: 40 cases, 7.1-10.0 cm: 12 cases, >10.0 cm: 10 cases) underwent LRN. Treatment outcomes including surgical and oncological outcomes among each stage were compared. (Results) Operating time for 7.1-10.0 cm tumors were similar to that <7 cm tumors but that for >10 cm tumors was significantly longer than that <10 cm tumors. There was no significant difference among each stage in terms of complication rate. As expected, recurrence-free survival rate for >10 cm tumors were worse than <10 cm tumors. (Conclusions) Our data suggests that LRN for large tumors >7 cm can be performed safely, but LRN for >10 cm tumors are technically demanding and require longer operation time.