Tomoyuki Koguchi

Regulation of myo-inositol biosynthesis by p53-ISYNA1 pathway

In response to various cellular stresses, p53 exerts its tumor suppressive effects such as apoptosis, cell cycle arrest, and senescence through the induction of its target genes. Recently, p53 was shown to control cellular homeostasis by regulating energy metabolism, glycolysis, antioxidant effect, and autophagy. However, its function in inositol synthesis was not reported. Through a microarray screening, we found that five genes related with myo-inositol metabolism were induced by p53. DNA damage enhanced intracellular myo-inositol content in HCT116 p53+/+ cells, but not in HCT116 p53-/- cells. We also indicated that inositol 3-phosphate synthase (ISYNA1) which encodes an enzyme essential for myo-inositol biosynthesis as a direct target of p53. Activated p53 regulated ISYNA1 expression through p53 response element in the seventh exon. Ectopic ISYNA1 expression increased myo-inositol levels in the cells and suppressed tumor cell growth. Knockdown of ISYNA1 caused resistance to adriamycin treatment, demonstrating the role of ISYNA1 in p53-mediated growth suppression. Furthermore, ISYNA1 expression was significantly associated with p53 mutation in bladder, breast cancer, head and neck squamous cell carcinoma, lung squamous cell carcinoma, and pancreatic adenocarcinoma. Our findings revealed a novel role of p53 in myo-inositol biosynthesis which could be a potential therapeutic target.

Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade

Metastatic renal cell carcinoma (RCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKI) represent the standard of care for RCCs, however a significant proportion of RCC patients develop resistance to this therapy. Interleukin-6 (IL-6) is considered to be associated with poor prognosis in RCCs. We therefore hypothesized that TKI resistance and IL-6 secretion are causally connected. We first analyzed IL-6 expression after TKI treatment in RCC cells and RCC tumor specimens. Cell proliferation and signal transduction activity were then quantified after co-treatment with tocilizumab, an IL-6R inhibitor, in vitro and in vivo. 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. Using a mouse xenograft model we can show that a combination therapy with tocilizumab and low dosage of sorafenib suppresses 786-O tumor growth, reduces AKT-mTOR pathway and inhibits angiogenesis in vivo more efficient than sorafenib alone. Furthermore FDG-PET imaging detected early decrease of maximum standardized uptake values prior to extended central necrosis.Our findings suggest that a combination therapy of IL-6R inhibitors and TKIs may represent a novel therapeutic approach for RCC treatment.Metastatic renal cell carcinoma (RCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKI) represent the standard of care for RCCs, however a significant proportion of RCC patients develop resistance to this therapy. Interleukin-6 (IL-6) is considered to be associated with poor prognosis in RCCs. We therefore hypothesized that TKI resistance and IL-6 secretion are causally connected. We first analyzed IL-6 expression after TKI treatment in RCC cells and RCC tumor specimens. Cell proliferation and signal transduction activity were then quantified after co-treatment with tocilizumab, an IL-6R inhibitor, in vitro and in vivo. 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. Using a mouse xenograft model we can show that a combination therapy with tocilizumab and low dosage of sorafenib suppresses 786-O tumor growth, reduces AKT-mTOR pathway and inhibits angiogenesis in vivo more efficient than sorafenib alone. Furthermore FDG-PET imaging detected early decrease of maximum standardized uptake values prior to extended central necrosis. Our findings suggest that a combination therapy of IL-6R inhibitors and TKIs may represent a novel therapeutic approach for RCC treatment.

Case of possible multiple system atrophy with a characteristic imaging finding of open bladder neck during storage phase as an initial sign

Multiple system atrophy is a neurodegenerative disease that affects autonomic and motor systems. Patients with multiple system atrophy usually experience lower urinary tract symptoms, which sometimes appear as an initial symptom before the emergence of the generalized symptoms. An open bladder neck during the filling phase on video urodynamic study is one characteristic imaging finding after the diagnosis of multiple system atrophy, but has not previously been reported at an early phase of the disease. We report a case in which an open bladder neck was observed on several imaging modalities before generalized symptoms emerged. Because occult neurogenic bladder might exist in patients whose lower urinary tract symptoms are resistant to pharmacotherapy, we report this case to raise awareness of the importance of sufficient imaging evaluations. An open bladder neck might be an important imaging finding for diagnosing multiple system atrophy, irrespective of the presence of generalized symptoms. This finding could help avoid false diagnosis and unnecessary treatment.

The impact of nerve‐sparing robot‐assisted radical prostatectomy on lower urinary tract function: Prospective assessment of patient‐reported outcomes and frequency volume charts

To elucidate the effects of a nerve‐sparing (NS) procedure on lower urinary tract symptoms (LUTS) and urinary function after robot‐assisted radical prostatectomy (RARP), the associations between the NS procedure and LUTS and urinary function were investigated.

Protective Effect of a Rho-kinase Inhibitor on Bladder Dysfunction in a Rat Model of Chronic Bladder Ischemia

OBJECTIVE To investigate the effect of fasudil, a Rho-kinase inhibitor, on chronic ischemia-related bladder dysfunction. MATERIALS AND METHODS Male Sprague-Dawley rats (16 weeks old) were divided into control, chronic bladder ischemia (CBI), and CBI with fasudil treatment (CBI-Fa) groups. The CBI and CBI-Fa groups underwent balloon endothelial injury of bilateral iliac arteries and received a 2% cholesterol diet for 8 weeks after the procedure to induce CBI. The CBI-Fa group was given oral fasudil (30 mg/kg/day) using zonde for 8 weeks after the procedure. The control group received a regular diet for 8 weeks. After cystometry in a conscious state, rats from each group were euthanized, and the bladders and common iliac arteries were harvested for pharmacologic and histologic examination. RESULTS Mean wall thickness of the common iliac arteries was significantly greater in the CBI group than in controls. Contractile responses of muscle strips were significantly lower in CBI group rats than in controls. In the CBI group, micturition interval was significantly shorter, and bladder capacity was significantly lower compared with those in controls. In the CBI-Fa group, arterial wall thickening was significantly suppressed compared with the CBI group. Significant improvements in muscle strip contractility and cystometric parameters were seen in the CBI-Fa group compared with the CBI group. CONCLUSION Our results suggest that chronic treatment with fasudil could prevent neointimal formation in arteries and bladder dysfunction in this rat model. Fasudil may be therapeutically useful in protecting bladder function in chronically ischemic bladders.

The association between local atherosclerosis of the prostatic artery and benign prostatic enlargement in humans: Putative mechanism of chronic ischemia for prostatic enlargement

To investigate the possible pathogenesis of the benign prostatic enlargement (BPE) induced by local atherosclerosis, the association between local atherosclerosis and prostatic enlargement was investigated, and molecular biological analyses were performed using human prostatectomy specimens.

Single nucleotide polymorphisms of HAAO and IRX 6 genes as risk factors for hypospadias

Purpose: We evaluated the association of hypospadias and 17 susceptibility loci previously identified by a European genome-wide association study in a cohort of Japanese patients. We also examined the expression of candidate genes in male mouse embryos to determine the possible underlying mechanisms of this disease. Materials and Methods: We enrolled 169 Japanese patients (mean age at surgery 3.7 years) who underwent repair of hypospadias. Genotyping of 17 single nucleotide polymorphisms was performed using a multiplex polymerase chain reaction invader assay. We also performed in situ hybridization to determine whether candidate genes were expressed in the male genital tubercle during embryonic development of the external genitalia in mice. Results: Single nucleotide polymorphism rs3816183 of HAAO was significantly associated with susceptibility to hypospadias in general (p = 0.0019) and to anterior/middle hypospadias (p = 0.0283) and posterior hypospadias (p = 0.0226), while single nucleotide polymorphism rs6499755 of IRX6 showed an association with susceptibility to anterior/middle hypospadias (p = 0.0472). In mouse embryos there was no significant upregulation of Haao expression in the developing male external genitalia. Irx3 and Irx5, which are linked to Irx6 within the IrxB cluster, were expressed in the mesenchyme remote from the urethral plate epithelium during the critical embryonic period for masculinization. Irx6 was expressed in the ectodermal epithelium, demonstrating prominent dorsal ectodermal expression without expression in the ventral ectoderm adjacent to the urethral plate during the same period. Conclusions: Genetic variations of HAAO and IRX6 influence susceptibility to hypospadias in the Japanese population. Further research is needed to clarify the mechanism by which variations in these genes contribute to the pathogenesis of hypospadias.

MP88-07 THE ISYNA1 EXPRESSION IN PRIMARY RENAL CANCER AS A POTENTIAL PROGNOSTIC BIOMARKER FOR THE POST-OPERATIVE METASTASIS.

INTRODUCTION AND OBJECTIVES: In response to cellular stress, p53 exerts its tumor suppressive effects such as apoptosis, cell cycle arrest, and cellular homeostasis through the induction of its target genes. On the other hand, Various tumors had p53 mutations, nevertheless renal cell carcinoma(RCC) had few p53 mutations. Previous study reported that p53 inactivated pathways affected the renal carcinogenesis. Other report showed that p53 expression pattern in immunohistochemistry analysis of tumor was associated prognosis of RCC. Therefore, we considered a possibility that p53 target gene might affect prognosis of RCC. We identified a novel p53 function, and examined whether the p53 target was useful as a prognostic factor of RCC. METHODS: To screen novel p53 target genes, we conducted cDNA microarray analysis using mRNAs isolated from HCT116 p53þ/þ and HCT116 p53-/cells that were treated with 2 mg/ml of adriamycin (ADR). To investigate whether mRNA transcription was regulated by p53, we performed a reporter assay and a chromatin immunoprecipitation (ChIP) assay using U373MG cells. To evaluate the biosynthesis of myo-inositol by a novel p53 target, we performed myo-inositol (MI) assay using HEK293T cells that were transfected with plasmid expressing the p53 target, and using HCT116 p53þ/þ and HCT116 p53-/cells that were treated with ADR. To examined whether it was useful as a prognosis of RCC, we performed the overall survival (OS) analysis and progression free survival (PFS) analysis using mRNA expression of the p53 target in primary RCC. RESULTS: The result of cDNA microarray analysis indicated ISYNA1 as a novel candidate gene. We found p53 response elements in the promoter region and the seventh exon by results of reporter assay and ChIP assay. Therefore, we identified ISYNA1 as a novel p53 target. The results of MI assay showed that intracellular myo-inositol content in cells expressing ISYNA1. In addition, DNA damage significantly increased intracellular myo-inositol content in HCT116 p53þ/þ cells, but did not affect the myo-inositol content in HCT116 p53-/cells. ISYNA1 mRNA expression in primary RCC were significantly correlated to OS and PFS ( p < 0.01 ). CONCLUSIONS: We identified ISYNA1 as a novel p53 target gene, and unveiled to regulate intracellular myo-inositol content by p53ISYNA1 pathway. Our findings shew that ISYNA1 mRNA expression in primary renal cancer was correlated to overall survival and progression free survival. Therefore, we considered that ISYNA1 might be useful as a novel prognostic factor for the post-operative metastasis.

Suppression of SOCS3 enhances TRAIL-induced cell growth inhibition through the upregulation of DR4 expression in renal cell carcinoma cells

Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumor-selective apoptosis inducer that is expressed in natural killer cells, whose cytotoxicity is activated by interferon (IFN). We investigated the effect of suppressor of cytokine signaling (SOCS) 3 on the expression of TRAIL receptors (DR4) and on TRAIL sensitivity in renal cell carcinoma (RCC) cells. Methods Vector expression, RNA interference and IL-6 receptor antibody tocilizumab were used to investigate the functional role of SOCS3 in DR4 expression. Immunoprecipitation was employed to detect the biochemical interaction between SOCS3 and DR4. The expression of DR4 induced by combination with IFN-α and tocilizumab was also examined by immunohistochemical staining using mice xenograft model. Results DR4 expression was up-regulated by IFN stimulation in RCC cells. 786-O cells were resistant to TRAIL and showed higher SOCS3 expression. ACHN cells showed higher DR4 expression and lower SOCS3 expression. Suppression of SOCS3 up-regulated DR4 expression and enhanced the TRAIL sensitivity in 786-O cells. In ACHN cells, DR4 expression was down-regulated by transfection with pCI-SOCS3, and the cells became resistant to TRAIL. Immunoprecipitation revealed the biochemical interaction between SOCS3 and DR4. A marked increase in IFN-induced DR4 protein expression after tocilizumab treatment was observed by immunohistochemical staining in the tumor from the mice xenograft model. Conclusions Our results indicate that IFN and SOCS3 regulate DR4 expression in RCC cells. Combination therapy with IFN-α, tocilizumab and an anti-DR4 agonistic ligand appears to effectively inhibit advanced RCC cell growth.

Interleukin-6 induces drug resistance in renal cell carcinoma

Metastatic renal cell carcinoma (mRCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKIs), the novel targeted agents have been used for the treatment of mRCC and have shown efficacy. Interferon (IFN)-α is also one of the most frequently used agents in immunotherapy. However, drug resistance needs to be overcome to achieve a sufficiently positive effect. Interleukin-6 (IL-6), which induce suppressor of cytokine signaling-3 (SOCS3) expression, is one of the factors associated with poor prognosis of patients with renal cell carcinoma (RCC). To analyze the influence of IL-6 in drug resistance of RCC, anti-IL-6 receptor antibody was used in combination with IFN or TKIs. The SOCS3 mRNA expression level was significantly increased by IFN-α stimulation in 786-O RCC cells which were resistant to IFN, but not in ACHN cells that were sensitive to IFN. The overexpression of SOCS3 by gene transfection in ACHN significantly inhibited the growth-inhibitory effect of IFN-α. An in vivo study demonstrated that co-administration of SOCS3-targeted siRNA promoted INF-α-induced cell death and growth suppression in 786-O cell xenograft. SOCS3 could be a key component in the resistance to interferon treatment of renal cell carcinoma. Because SOCS3 is rapidly up-regulated by IL-6 and a negative regulator of cytokine signaling, IL-6 expression on RCC cells was also analyzed and the 786-O cells showed the high level of IL-6 mRNA expression under the condition of interferon stimulation. IL-6R antibody, tocilizumab, significantly suppressed cell proliferation in 786-O cells by interferon stimulation accompanied with phosphorylation of STAT1 and inhibited SOCS3 expression. The in vivo effects of combination therapy with tocilizumab and interferon showed significant suppression of 786-O tumor growth in a xenograft model. We also hypothesized that TKI resistance and IL-6 secretion are causally connected. And we found that 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. A combination therapy with tocilizumab and TKI suppresses 786-O tumor growth and inhibits angiogenesis in vivo more efficient than TKI alone. Our findings suggest that IL-6 could induce drug resistance on RCC, and combination therapy of IL-6R inhibitors and IFN/TKIs may represent a novel therapeutic approach for RCC treatment.