Ryo Tanji

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.

Comprehensive approach for post-prostatectomy incontinence in the era of robot-assisted radical prostatectomy

Robot-assisted radical prostatectomy (RARP) has enabled steady and stable surgical procedures due to both meticulous maneuvers and magnified, clear, 3-dimensional vision. Therefore, better surgical outcomes have been expected with RARP than with other surgical modalities. However, even in the RARP era, post-prostatectomy incontinence has a relatively high incidence as a bothersome complication. To overcome post-prostatectomy incontinence, it goes without saying that meticulous surgical procedures and creative surgical procedures, i.e., Preservation, Reconstruction, and Reinforcement of the anatomical structures of the pelvis, are most important. In addition, medication and appropriate pad usage might sometimes be helpful for patients with post-prostatectomy incontinence. However, patients who have 1) BMI > 26 kg/m2, 2) prostate volume > 70 mL, 3) eGFR < 60 mL/min, or a 4) Charlson comorbidity index > 2 have a tendency to develop post-prostatectomy incontinence despite undergoing the same surgical procedures. It is important for patients who have a high risk for post-prostatectomy incontinence to be given information about delayed recovery of post-prostatectomy incontinence. Thus, not only the surgical procedures, but also a comprehensive approach, as mentioned above, are important for post-prostatectomy incontinence.