Hisashi Kawazoe

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma

Background:Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3β positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-κB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3β and to assess the anti-cancer effect of GSK-3β inhibition in RCC.Methods:Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3β in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT–PCR, BrDU incorporation and MTS assays to study the effect of GSK-3β inactivation on renal cancer cell proliferation and survival.Results:We detected aberrant nuclear accumulation of GSK-3β in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-κB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells.Conclusions:Our results show nuclear accumulation of GSK-3β as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

GSK-3 inhibition in vitro and in vivo enhances antitumor effect of sorafenib in renal cell carcinoma (RCC).

Sorafenib is a multikinase inhibitor approved for the systemic treatment of renal cell carcinoma (RCC). However, sorafenib treatment has a limited effect due to acquired chemoresistance of RCC. Previously, we identified glycogen synthase kinase-3 (GSK-3) as a new therapeutic target in RCC. Here, we observed that sorafenib inhibits proliferation and survival of RCC cells. Significantly, we revealed that sorafenib enhances GSK-3 activity in RCC cells, which could be a potential mechanism of acquired chemoresistance. We found that pharmacological inhibition of GSK-3 potentiates sorafenib antitumor effect in vitro and in vivo. Our results suggest that combining GSK-3 inhibitor and sorafenib might be a potential new therapeutic approach for RCC treatment.

Sequential molecularly targeted drug therapy including axitinib for a patient with end-stage renal failure and metastatic renal cell carcinoma

A 62‐year‐old male patient with end‐stage renal disease and metastatic renal cell carcinoma (RCC) was referred to our hospital. Sequential targeted therapy consisting of sorafenib, sunitinib, and everolimus was administered, but the patients disease gradually progressed. Axitinib was subsequently administered at a decreased dose of 6 mg/day for 2 weeks, after which the dose was escalated to 10 mg/day. Axitinib therapy was maintained for a total of 6 months without severe adverse effects. Sequential molecularly targeted drug therapy including axitinib, with careful monitoring, is one possible treatment option for patients with metastatic RCC with renal impairment.

MP88-18 ERK5 IS A PROMISING THERAPEUTIC TARGET FOR CLEAR CELL RENAL CELL CARCINOMA

RASAL2 methylation or c-FOS mRNA or VEGFA mRNA in RCC tissues. Overexpression of RASAL2 in 786-O cells could inhibit the recruitment and tube formation of HUVECs, while RASAL2 knockdown (KD) in ACHN cells enhanced the recruitment and tube formation of HUVECs in vitro. Also, overexpression of RASAL2 could inhibit tumorigenecity of xenografts. Mechanistically, RASAL2 KD could enhance the phosphorylation of GSK3 and upregulate the expression of c-FOS and VEGFA. Furthermore, RASAL2 was inversely correlated with VEGFA and CD31 in tissues from human RCC specimens and xenografts. CONCLUSIONS: RASAL2 was downregulated in RCC tissues, which could lead to tumor angiogenesis via p-GSK3/c-FOS/VEGFA signaling pathway. Therefore, RASAL2 could be a potential target to prevent patients with RCC from resistance to anti-vascular therapy.

Renoprotective Procedures with a Cold Ischemia Time of

Introduction: We evaluated whether nephron sparing surgery (NSS) combined with meticulous suturing of the cut stump under clamping with cooling is beneficial for oncological outcomes and also assessed the relationship between cold ischemia time and deterioration of renal function. Methods: One hundred and six patients with renal cell carcinoma (RCC) were subjected to this procedure. Oncological outcomes and renal function according to the estimated glomerular filtration rate (eGFR) and the tubular excretion rate on renoscintigraphy before and at 12 months after surgery were evaluated. Results: Cancer recurrences were observed in 2 patients with past history of RCC; however, no patient died of cancer. Renal function was evaluated depending on 4 different ischemia times. All groups did not show a remarkable decrease of renal function in terms of eGFR. Renoscintigraphy revealed the deterioration of the affected kidney in patients with >60 min ischemia. Conclusion: The renoprotective procedure of NSS provided maximum preservation of renal function until 60 min of cold ischemia time.

CEREBRAL VENOUS SINUS THROMBOSIS IN PATIENTS WITH METASTATIC TESTICULAR CANCER DURING CHEMOTHERAPY: REPORTS OF TWO CASES

Cerebral venous sinus thrombosis (CVT) is rare but sometimes develops in association with malignant neoplasm. We report two cases of CVT that occurred during cisplatin-based chemotherapy for testicular cancer. A 46-year-old man with stage IIA non-seminomatous germ cell tumour was treated with conventional doses of etoposide and cisplatin (EP). On day 11 of the third treatment course, he developed a systemic seizure. Brain computed tomography (CT) and magnetic resonance (MR) imaging could not detect the cause. Enhanced chest-pelvic CT revealed pelvic thrombosis. Administration of phenytoin for epilepsy of unknown cause and heparin for thrombosis was started. He had completed 4 courses of EP therapy without seizure recurrence. After re-evaluating the brain CT images retrospectively, we found high density of superior sagittal sinus (SSS) and strongly suspected CVT. Another patient was a 47-year-old man with stage IIIB seminomatous germ cell tumour treated with bleomycin, etoposide, and cisplatin (BEP) therapy. On day 11 of the second treatment course, he developed a systemic seizure. Brain CT revealed subarachnoid haemorrhage localised in the right parietal lobe. CT venography revealed a filling defect in the superior sagittal sinus (SSS). MR venography revealed a SSS stenosis. We diagnosed the cause of the seizure as CVT and started administration of anticoagulant therapy. After the thrombus had diminished, chemotherapy was restarted and another 2 courses of BEP therapy was completed.

Abstract 2782: The potential of p4EBP1 expression as predictive biomarker of mRCC

Introduction & objectives Activation of Akt/mTOR pathway induces 4EBP1 phosphorylation, and enhances cell proliferation, anti-apoptotic effect, and angiogenesis in many types of cancers including renal cell carcinoma (RCC). As mTOR and angiogenetic proteins are main targets in metastatic RCC (mRCC) treatment. We assessed the correlation with survivals and phosphorylated 4EBP1 (p4EBP1) expression. Materials & methods We enrolled 254 non-mRCC patients who underwent primary surgery in Yamagata University between 2003 and 2010, and 59 mRCC patients whose resected primary lesion was available. Immunohistochemistry for p4EBP1 was performed on their FFPE samples. We assessed correlations between p4EBP1 expression manners and clinical features (disease-free interval [DFI] for non-mRCC patients, cause-specific survival [CSS] and progression-free survival [PFS] for mRCC patients). The CSS was calculated from mRCC diagnosis to death or last follow-up date. The PFS was calculated based on the durations patients were medicated. Univariate analysis was calculated by log-rank test and multivariate analysis was calculated by Cox-regression analysis. Results Non-mRCC patients with highly p4EBP1 expression were shorter DFI than those without high expression (p = 0.036). Their 5-year disease-free rates were 83.4% and 93.4%, respectively. The independent poor DFI factors were high p4EBP1 expression (HR; 3.4, p = 0.0054), grade (p = 0.0055), and pT stage (p In contrast, mRCC patients with p4EBP1 expression was longer CSS than those without expression (median CSSs; 56.7 and 32.2 months, p = 0.0246). The independent poor CSS factors were no p4EBP1 expression (hazard ratio [HR]; 3.3, p = 0.0409), grade 4 (HR; 8.7, p = 0.0006), and poor prognostic group on MSKCC criteria (HR; 4.2, p = 0.02770). Expression of p4EBP1 showed statistically longer PFS in mRCC patients with axitinib (median PFS; 9.2 and 2.5 months, p = 0.0255). The similar trends were shown in patients with other TKIs and mTOR inhibitors. Conclusion Since non-mRCC patients with the highly p4EBP1 expression had shorter DFI, expression of p4EBP1 should indicate aggressive RCC in nature. Nevertheless, mRCC patients with p4EBP1 expression had longer survival. These results mean that expression of p4EBP1 might be a predictive biomarker for TKIs and mTOR inhibitors. Citation Format: Sei Naito, Osamu Ichiyanagi, Hiromi Ito, Hidenori Kanno, Tomoyuki kato, Yuuta Kurota, Atsushi Yamagishi, Mayu Yagi, Toshihiko Sakurai, Hayato Nishida, Hisashi Kawazoe, Tomohiro Shibasaki, Akira Nagaoka, Norihiko Tsuchiya. The potential of p4EBP1 expression as predictive biomarker of mRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2782. doi:10.1158/1538-7445.AM2017-2782

2007 ESTIMATED GFR AS A BIOMARKER OF EFFICACY IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA TREATED WITH SUNITINIB

INTRODUCTION AND OBJECTIVES: Renal dysfunction is frequent side effect of VEGFR-inhibitors, especially sunitinib. We investigated the association of renal function change during VEGFR-inhibitors administration and antitumor efficacy in patients with metastatic renal cell carcinoma (mRCC). METHODS: Retrospective data were collected for mRCC patients received VEGF-targeted therapy between January 2005 and October 2011. We investigated renal adverse events and clinically significant increased serum creatinine level in patients who received VEGF targeted therapy. GFR was estimated with the Modification of Diet in Renal Disease (MDRD) formula. RESULTS: Ninety three patients with mRCC who received sunitinib (n 46), sorafenib (n 38), axitnib (n 9) were included in this analysis. During administration, gradual and significant increase of serum creatinine was observed in 34 (73.9 %) sunitinib recipients compared with sorafenib (15.2 %) or axitinib (33.3 %) recipients (p 0.041). Significant decrease of estimated GFR (eGFR) compared to baseline correlated with increase of serum creatinine level developed in ‘on’ period of 6 sunitinib administration cycle (p 0.013). No significant change was observed in serum creatinine level and eGFR in patients received other VEGF-targeted agents. Decrease of eGFR in the first ‘on’ period was associated with less frequent tumor response to sunitinib and a short time to disease progression (p 0.028 and 0.042). CONCLUSIONS: Our data suggest that nephrotoxicity developed in a high percentage of patients on sunitinib compared for sorafenib and axitinib in mRCC patients. In patients with mRCC, sunitinib-associated renal function impairment in the first ‘on’ period may be an efficacy biomarker.

An analysis of low miR-199a expression in renal cell carcinoma (RCC) and and its association with regulation of GSK-3beta.

e15020 Background: Renal cell carcinoma (RCC) is highly resistant to chemotherapy due to a high apoptotic threshold. Recently, we have shown nuclear accumulation of GSK-3β as a new marker of human RCC and identified that GSK-3 positively regulates RCC cell survival and proliferation. It has been reported that GSK-3β is a miR-199a target. Our objectives were to determine the expression level of miR-199a in human RCC and renal cancer cell lines, and to assess the anticancer effect of reintroduction of pre-miR-199a in renal cancer cells in vitro. METHODS TaqMan MicroRNA assay was applied to examine miR-199a expression in 54 human RCCs and normal kidney counterpart as well as in 8 renal cancer cell lines. Immunohistochemical staining was performed to determine the expression pattern of GSK-3β in RCC tissues. Western blotting, quantitative RT-PCR and MTS cell viability assay were used to study the effect of pre-miR-199a reintroduction into renal cancer cells in vitro. RESULTS We detected low miR-199a expression in 57% (31 of 54) of RCCs. We found that 87% (27 of 31) of low miR-199a tumors were clear cell carcinomas. Downregulation of miR-199a was correlated with higher stage (Chi square for trend 4.605, p=0.0319) and nuclear accumulation of GSK3β (Fishers exact test, p=0.0249) in clear cell RCCs. We found high miR-199a expression in two benign renal hamartomas (angiomyolipoma). Reintroduction of pre-miR-199a into renal cancer cells led to decreased GSK-3β, XIAP, Bcl-2 expression and suppression of RCC cells proliferation and survival. CONCLUSIONS We found that miR-199a is downregulated in most RCCs and this tumor phenotype is strongly associated with nuclear accumulation of GSK-3β and malignant potential of clear cell tumors. Our results identify low miR-199a as a novel diagnostic marker in RCC, and suggest reintroduction of pre-miR-199a as a new treatment strategy in RCC.

Abstract 2419: Glycogen synthase kinase-3 inhibitor and cisplatin synergically inhibit tumor growth of Urothelial cancer in xenograft rodent model

Introduction: Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase, as a positive regulator of several cancer cell survival and proliferation. We previously showed that nuclear accumulation of GSK-3beta indicates high grade, high stage, and poor survival in bladder urothelial cancer (UC). And inhibition of GSK-3beta suppresses UC cell proliferation and induces apoptosis via NF-kappaB down-stream genes viz. BCl-2 and XIAP (Clin Cancer Res 2010). Here we were to identify the interaction between cisplatin and GSK-3 inhibitor, AR-A014418 in UC. Experimental Procedures: We added cisplatin (0, 1, 3, 6 μg/ml) and AR-A014418 (0, 10, 20 μM) to medium in T24 and HT1376 human UC cells and assessed their viability by MTS assay. Furthermore, we intraperitoneally administrated PBS 5 time per week, 2.5 mg/kg body weight cisplatin once per week, 20 mg/kg body weight AR-A014418 5 time per week, or cisplatin and AR-A014418 into each 6 mice with subcutaneous xenografts of HT1376 for 3 weeks. Result: Treatment of cisplatin and AR-A014418 showed synergistic effect in cell lines. Cisplatin did not change expression level of GSK-3beta, and AR-A014418 suppressed XIAP for cell lines with cisplatin. The average tumor volume of the mice treated by cisplatin was equivalent to one by PBS within treatment period. However, the tumors treated by cisplatin grew bigger than ones by PBS after withdrawal of treatment. AR-A014418 inhibited the growth of tumor compared to PBS and cisplatin. And combine use of cisplatin and AR-A014418 prevented the tumor from re-growth by cisplatin after treatment. Body weight did not vary between mice by each treatment methods. Conclusion: Cisplatin and AR-A014418 synergistically suppressed UC cells viability. And AR-A014418 cancelled re-growth of low-dose cisplatin in xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2419. doi:10.1158/1538-7445.AM2011-2419