Toru Kanno

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.

SPA-1 controls the invasion and metastasis of human prostate cancer.

Recent studies suggest that SIPA1 encoding a Rap GTPase‐activating protein SPA‐1 is a candidate metastasis efficiency‐modifying gene in human breast cancer. In this study, we investigated the expression and function of SPA‐1 in human prostate cancer (CaP). Immunohistochemical studies of tumor specimens from CaP patients revealed a positive correlation of SPA‐1 expression with disease progression and metastasis. The correlation was recapitulated in human CaP cell lines; LNCaP that rarely showed metastasis in SCID mice expressed an undetectable level of SPA‐1, whereas highly metastatic PC3 showed abundant SPA‐1 expression. Moreover, SIPA1 transduction in LNCaP caused prominent abdominal lymph node metastasis without affecting primary tumor size, whereas shRNA‐mediated SIPA1 knockdown or expression of a dominant‐active Rap1 mutant (Rap1V12) in PC3 suppressed metastasis. LNCaP transduced with SPA‐1 (LNCaP/SPA‐1) showed attenuated adhesion to the precoated extracellular matrices (ECM) including collagens and fibronectin, due to defective ECM‐medicated Rap1 activation. In addition, LNCaP/SPA‐1 showed a diminished level of nuclear Brd4, which is known to bind SPA‐1, resulting in reduced expression of a series of ECM‐related genes. These results suggest that SPA‐1 plays an important role in controlling metastasis efficiency of human CaP by regulating the expression of and interaction with ECM in the primary sites. (Cancer Sci 2011; 102: 828–836)

Complications and the learning curve for a laparoscopic nephrectomy at a single institution.

Background:u2002 We assessed our experiences in performing a laparoscopic nephrectomy, with regard to complications and the learning curve, during a 4‐year period.

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 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

Abstractu2003 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.

ENDOTHELIAL FENESTRATIONS IN TUMOR MICROVASCULATURE AS A POTENT PREDICTIVE MARKER OF ANTI-VEGF THERAPY IN CLEAR CELL RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVE: Vascular endothelial growth factor (VEGF)-targeted therapies show significant anti-tumor effects for advanced clear cell renal cell carcinomas (CC-RCCs) and kinds of surrogate markers that can successfully predict tumor responses, are highly required. Previously, experimental studies using VEGF neutralization in mice tumor model revealed that VEGF-dependent capillaries in tumor vessels were characterized by the existence of fenestrations in endothelium on electron microscopy study. Based on those results, we examined if endothelial fenestrations were associated with VHL status and tumor responses to anti-VEGF therapy in RCC. METHODS: Twenty-four primary CC-RCCs were examined for the status of VHL, microvessel density and existence of endothelial fenestrations. In mice xenograft models, VHL-/786-O RCC subclones stably transfected with plasmid empty (pRC3) or wild type VHL (WT8) were injected subcutaneously in nude mice to determine the correlation between phenotypes of tumor capillaries and their responses to anti-VEGF therapy. RESULTS: The number of endothelial fenestrations was significantly increased in CC-RCC specimens harboring VHL mutation comparing those without. This finding was also reproducible in mice xenograft models in that capillaries established from VHL null pRC3 cells exhibited more abundant endothelial fenestrations compared to those from VHL transfected WT8 cells. Treatment with bevacizumab resulted in the significant decrease in the mean tumor size of pRC3 but not WT8 cells. In bevacizumab sensitive pRC3 xenografts, a significant reduction of the number of endothelial fenestrations and microvessel density was observed after the treatment. In contrast, a xenograft established from WT8/HIF2 P531A cells which overexpressed an activated form of HIF2 constitutively, exhibited no significant increase of the number of endothelial fenestrations nor reduction of tumor size against bevacizumab treatment compared to that from WT8/mock cells. CONCLUSIONS: Our results suggest that sporadic CCRCCs with VHL mutation harbor VEGF-dependent tumor vessels and those capillaries are potent target for anti-VEGF therapy. Therefore, endothelial fenestration could be a useful surrogate marker in predicting susceptibilities of CC-RCCs to that therapy. Our results also indicated that an increase of VEGF dependent capillaries in VHL -/RCC is not merely caused by the subsequent VEGF overproduction followed by HIF accumulation.