Noboru Shibasaki

JunB promotes cell invasion and angiogenesis in VHL -defective renal cell carcinoma

Inactivation of the von Hippel–Lindau (VHL) tumor-suppressor gene causes both hereditary and sporadic clear-cell renal-cell carcinoma (ccRCC). Although the best-characterized function of the VHL protein (pVHL) is regulation of hypoxia-inducible factor-α (HIFα), pVHL also controls the development of pheochromocytoma through HIF-independent pathways by regulating JunB. However, it is largely unknown how these pathways contribute to the development and progression of ccRCC. In the present study, we confirmed that JunB was upregulated in VHL-defective ccRCC specimens by immunostaining. Short-hairpin RNA (shRNA)-mediated knockdown of JunB in 786-O and A498 VHL null ccRCC cells suppressed their invasiveness. In addition, JunB knockdown significantly repressed tumor growth and microvessel density in xenograft tumor assays. Conversely, forced expression of wild-type, but not dimerization-defective, JunB in a VHL-restored 786-O subclone promoted invasion in vitro and tumor growth and vessel formation in vivo. Quantitative PCR array analysis revealed that JunB regulated multiple genes relating to tumor invasion and angiogenesis such as matrix metalloproteinase-2 (MMP-2), MMP-9 and chemokine (C-C motif) ligand-2 (CCL2) in 786-O cells. JunB knockdown in these cells reduced the proteolytic activity of both MMPs in gelatin zymography and the amount of CCL2 in the culture supernatant. Moreover, shRNA-mediated knockdown of MMP-2 or inhibition of CCL2 activity with a neutralizing antibody repressed xenograft tumor growth and angiogenesis. Collectively, these results suggest that JunB promotes tumor invasiveness and enhances angiogenesis in VHL-defective ccRCCs.

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.

Tumor microvasculature with endothelial fenestrations in VHL null clear cell renal cell carcinomas as a potent target of anti-angiogenic therapy.

Vascular endothelial growth factor (VEGF)‐targeted therapies show significant antitumor effects for advanced clear cell renal cell carcinomas (CC‐RCCs). Previous studies using VEGF inhibitors in mice models revealed that VEGF‐dependent capillaries were characterized by the existence of endothelial fenestrations (EFs). In this study, we revealed that capillaries with abundant EFs did exist, particularly in CC‐RCCs harboring VHL mutation. This finding was recapitulated in mice xenograft models, in which tumors from VHL null cells showed more abundant EFs compared to those from VHL wild‐type cells. Importantly, treatment with bevacizumab resulted in a significant decrease of tumor size established from VHL null cells. Additionally, a significant reduction of EFs and microvessel density was observed in VHL null tumors. Indeed, xenograft from 786‐O/mock (pRC3) cells developed four times more abundant EFs than that from 786‐O/VHL (WT8). However, introduction of the constitutively active form of hypoxia‐inducible factor (HIF)‐2α to WT8 cells failed to either augment the number of EFs or restore the sensitivity to bevacizumab in mice xenograft, irrespective of the equivalent production of VEGF to 786‐O/mock cells. These results indicated that HIF‐2α independent factors also play significant roles in the development of abundant EFs. In fact, several angiogenesis‐related genes including CCL2 were upregulated in 786‐O cells in a HIF‐2α independent manner. Treatment with CCL2 neutralizing antibody caused significant reduction of capillaries with EFs in 786‐O xenograft, indicating that they were also sensitive to CCL2 inhibition as well as VEGF. Collectively, these results strongly indicated that capillaries with distinctive phenotype developed in VHL null CC‐RCCs are potent targets for anti‐angiogenic therapy.

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.

Role of mammalian target of rapamycin inhibitor in the treatment of metastatic epithelioid angiomyolipoma: A case report

Epithelioid angiomyolipoma has malignant potential; however, no effective therapy has been established for advanced cases. A 50‐year‐old woman with a history of right nephrectomy for epithelioid angiomyolipoma was referred to our institution. Computed tomography and magnetic resonance imaging showed multiple tumors in her lung, liver and pelvic cavity. The liver and pelvic tumor specimens obtained by needle biopsy confirmed the diagnosis of epithelioid angiomyolipoma recurrence. The patient was treated with everolimus (10 mg/day). Three months later, pulmonary lesions disappeared; liver and pelvic tumors significantly shrank in size, but the pelvic tumor gradually enlarged again. We carried out surgical resection of the residual liver and pelvic cavity tumors. Although the mammalian target of rapamycin inhibitor seems to be effective for treating epithelioid angiomyolipoma, its long‐term effects remain unknown. Thus, aggressive administration of a multidisciplinary treatment including molecular target therapy and surgical resection is required to improve the prognosis of epithelioid angiomyolipoma.

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.

Infection-induced urethral defect treated by urethral reconstruction with a radial forearm flap

Abstract  A 47‐year‐old man was admitted with the chief complaint of a urethral defect. An approximately 17‐cm defect of the urethra seemed to have been occurred by the infection of implanted foreign bodies in the penile skin. Reconstruction of the urethra and the ventral skin was performed with a free radial forearm flap. A fistula formed at the proximal anastomosis after the operation, but was controlled conservatively. Urethral stricture at the proximal anastomosis subsequently developed. A urethral stent made of shape memory alloy was placed with the preservation of voiding function.

Laparoscopic intracorporeal ileal conduit after laparoscopic radical cystectomy: A modified technique to facilitate ureteroenteric anastomosis

DOI: 10.1111/iju.13782 ICUD has the potential benefits of a smaller incision, reduced pain, decreased bowel exposure, reduced risk of fluid imbalance and early postoperative recovery. Indeed, ICUD is gaining popularity during RARC, but purely laparoscopic ICUD has seldom been reported because of the technical difficulty, particularly precise intracorporeal suturing. We herein describe our modified technique to facilitate ureteroenteric anastomosis during pure laparoscopic intracorporeal IC after LRC and evaluate perioperative outcomes. From 2007 to 2017, 66 patients underwent LRC with pelvic lymphadenectomy with IC diversion at three institutions (35 intracorporeal IC, 31 extracorporeal). Total intracorporeal IC began in May 2014. LRC started using a five-port, fan shaped, transperitoneal approach. After lymphadenectomy and radical cystectomy, the left ureter was delivered under the sigmoid mesocolon to the right side. A 15-cm ileal segment of the bowel was harvested as previously described during RARC. The schema of ureteroenteric anastomosis and intraoperative findings are shown in Figure 1 and Video S1. After inserting a guidewire from a 3-mm port placed just superior to the pubic symphysis, a 6-Fr single-J ureteral stent was placed in the ureter. A conduit was irrigated using a 22-Fr catheter to minimize spillage of bowel contents. A single-J ureteral stent attached to the ureter was passed through two enterotomies in the proximal conduit and 12-mm assistant port. Ureteroenteric anastomosis was carried out using a continuous running suture method comprising two running 4-0 synthetic absorbable polyglyconate sutures. The first stitch was placed on the tip of spatulation of the ureter and IC. The second stitch was placed on the end opposite to the first stitch, and both stitches were tied. Then, the short tails of the two stitches were pulled by two laparoscopic graspers inserted from the port above the pubis and assistant port. The traction of the short tails of the two sutures could adjust the suture line parallel to a laparoscopic needle holder in the surgeon’s right hand, which makes carrying out running suture easier. The long tail of the second stitch was used for the anterior part of ureteroenteric anastomosis with three or four stitches using the running suture method, and it was tied to the first stitch. The posterior part of anastomosis was inverted by pulling the short tail of both stitches, and running suture was carried out in the same manner. A 3–4-cm skin incision was made at the supraumbilical site, and the specimen was removed. As for extracorporeal IC, a 5–7-cm skin incision was made at the infraumbilical site. The isolated ileum for conduit was washed outside the incision, and ureteroenteric anastomosis was carried out using a continuous running suture method. Perioperative data were compared between the intracorporeal and extracorporeal IC groups using propensity score matching. The primary study end-point was postoperative early (within 90 days) complication rates. In total, 28 matched pairs were evaluated. No significant difference in preoperative data was found (Table S1). The operative time for intracorporeal IC diversion and ureteroenteric anastomosis was 187 31 and 74 17 min, respectively. The median total operative time in intracorporeal groups was significantly longer, but the total median blood loss significantly decreased compared with that in extracorporeal groups (560 vs 1165 mL, respectively, P < 0.001). Importantly, the postoperative early complication rate in the intracorporeal group was significantly lower than that in the extracorporeal group (39% vs 71%, P = 0.016; Table S2). Similarly, the postoperative early major complication rate (Clavien grade 3–5) tended to decrease in the intracorporeal group (7% vs 29%, respectively, P = 0.036). The details are summarized in Table S3. As for postoperative recovery of bowel function, the mean days to regular oral food intake were 4 and 7 days in the intracorporeal and extracorporeal groups, respectively (P < 0.001). The Kaplan–Meier method showed no significant difference in ureteroenteric strictures between both groups (P = 0.60; Fig. S1). Urological Notes

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.

MP35-13 IL13RA2 MEDIATES RESISTANCE TO SUNITINIB IN PRIMARY XENOGRAFT MODELS OF CLEAR CELL RENAL CELL CARCINOMA

METHODS: We tested efficacy of the MET TKI crizotinib, the HSP90 inhibitor ganetespib and their combination in MKN45, wild-type MET-overexpressed cell line and MET-Y1248H, TKI-resistant mutant MET-overexpressed cell line in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in xenograft models. RESULTS: MET is an HSP90-dependent kinase, and we show that HSP90 preferentially interacts with and stabilizes activated MET, whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET TKI show a preference for the inactive form of the kinase and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in TKI-sensitive METdriven tumor models. Unexpectedly, low-dose ganetespib also partially restores crizotinib sensitivity, in vitro and in vivo, to cells and tumors expressing TKI-resistant MET mutants. CONCLUSIONS: Our findings support the use of HSP90 inhibitors to overcome or delay the initiation of resistance to MET TKI, and they provide the basis for clinical evaluation of this combination in patients with MET-driven papillary renal cancer.