Hidenori Kanno

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.

Levels of 4EBP1/eIF4E Activation in Renal Cell Carcinoma Could Differentially Predict Its Early and Late Recurrence

Micro‐Abstract A subset of patients who undergo curative surgery for localized clear cell renal cell carcinoma (ccRCC) will experience early or late recurrence. Tumor viability depends on protein synthesis via the eukaryotic initiation factor (eIF)4E‐binding protein 1 (4EBP1/eIF4E) axis. Activation levels of the axis in ccRCC tissues could differentially affect tumor recurrence and the timing of recurrence after curative nephrectomy. Background The objective was to explore the predictive markers of late recurrence (LR) > 5 years after curative nephrectomy for renal cell carcinoma (RCC). Patients and Methods We retrospectively examined the data from 303 patients with localized clear cell RCC treated surgically at our institution from 1993 to 2011. Activation of the eukaryotic initiation factor (eIF)4E‐binding protein 1 (4EBP1)/eIF4E axis at the mammalian target of rapamycin complex 1 (mTORC1) was evaluated in the tumor specimens. Weak, intermediate, and strong immunohistochemistry staining grades were defined for 4EBP1, phosphorylated 4EBP1, and eIF4E. The effects of clinicopathologic factors and activation level grades on tumor recurrence were analyzed using multivariate Cox regression models. To validate the present findings, we investigated clinical data from The Cancer Genome Atlas and protein/phosphoprotein data from corresponding patients from The Cancer Proteome Atlas. Results Of the 303 patients, 31 and 16 patients developed early recurrence (ER, ≤ 5 years) and LR, respectively. The activation levels were comparable among the subcategories of pathologic TN stage, Fuhrman grade, and microvascular and capsular invasion. Pathologic stage ≥ T1b, Fuhrman grade 3/4, and an intermediate or strong activation level correlated significantly with overall recurrence and ER. Strong activation of the axis and pathologic stage ≥ T1b were identified as independent predictors of LR. Only 2 patients with weak activation experienced recurrence (1 each with ER and LR). Similar results were confirmed by the analyses of The Cancer Genome Atlas and The Cancer Proteome Atlas data. Conclusion The activation level of the axis in RCC tissues could independently predict for recurrence and differentially affect the timing of recurrence.

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.

LONG-TERM OUTCOME IN PATIENTS WITH RETROPERITONEAL LIPOSARCOMA, A SINGLE INSTITUTION STUDY

(Objectives) To evaluate the outcomes of patients who were surgically treated for retroperitoneal liposarcoma in our hospital from February 2002 to August 2015. (Methods) Fifteen patients were surgically treated for retroperitoneal liposarcoma in our hospital during the study period. All patients were diagnosed with liposarcoma on pathological examination. The mean follow-up period was 46.7 months (range, 1-126 months). (Results) There was no difference in the sex distribution of the patients (7 men and 8 women). The median age was 67 years (range, 33-78 years). The median tumor diameter was 24 cm (range, 7.5-45 cm) and the median tumor weight was 1,959 g (range, 545-15,400 g). One patients operation was unsuccessful, with incomplete tumor resection. The surgical margin was positive in two patients. The 5- and 10-year survival rates were 67% and 50%, respectively. There was a significant difference in the survival rate between complete resection and incomplete resection, including surgical margin-positive patients (p=0.0019). Moreover, there was a significant difference in the recurrence-free rate between complete resection and surgical margin-positive patients (p=0.013). There was no significant difference according to whether removal of the tumor with adjacent viscera or removal of the tumor only had been performed (p=0.09 and 0.90, respectively). (Conclusions) Surgery is the mainstay of treatment for retroperitoneal liposarcoma, and complete resection is necessary.

Updated recommendation on molecular-targeted therapy for metastatic renal cell cancer

Molecular-targeted therapy was recommended for the systemic therapy of renal cell cancer (RCC) in the RCC guidelines, but these guidelines do not address the order of administration of the multiple presently available agents. There are several aspects that remain unknown regarding the optimal administration order and combination of molecular-targeted drugs. Until the optimal treatment sequence is determined by clinical trials, treatment individualization is required for each patient based on patient and disease characteristics. We herein investigate 12 cases of RCC patients who received axitinib. Axitinib was used as the first-line drug in 4 cases, second-line in 5 cases, third-line in 1 case and as a fourth-line drug in 2 cases. Partial response (PR) was observed in 4 cases (30%) and stable disease in 4 cases (30%) during axitinib treatment, with an overall response rate of 60%. The duration of PR ranged from 6 to 19 months. Based on our cases, axitinib exhibited reasonable therapeutic efficacy as first- as well as second-line treatment. However, more cases are required to draw firm conclusions.

Abstract 2381: The effect of ERK5 inhibition in clear cell renal cell carcinoma

Introduction Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and is characterized by von Hippel-Lindau tumor suppressor gene (VHL) mutation. VHL protein acts in a ubiquitin ligase complex, mediating the proteasomal degradation of ubiquitinized proteins, including hypoxia-inducible factor (HIF). Therefore, HIF accumulates in VHL-defective cell lines. A recent study revealed that extracellular signal-regulated kinase 5 (ERK5) is degraded through the ubiquitin-proteasome system, in a process mediated by VHL and HIF. Our objectives were to examine the effects of ERK5 in ccRCC and to investigate ERK5 as a potential therapeutic target. Methods ERK5 expression was investigated in surgically resected ccRCC specimens using immunohistochemistry. To confirm that ERK5 is degraded by the VHL pathway, ERK5 levels were examined by western blotting in Caki-1 VHL wildtype renal cell carcinoma (RCC) cells and in A498 and A704 VHL-mutant RCC cells with or without the MG132 proteasome inhibitor. Furthermore, VHL-mutant cell lines were examined, with or without siRNA and XMD8-92-mediated ERK5 inhibition, to investigate the role of ERK5 in RCC cells using flow cytometry, MTS assays, and western blotting . Results In surgical specimens, RCC cells showed higher levels of ERK5 expression than did normal cells. In addition, MG132 enhanced ERK5 expression in wildtype cell lines, but not in VHL-mutant cell lines. Therefore, ERK5 degradation involves the VHL pathway. Furthermore, ERK5 inhibition resulted in increase of the sub-G1 population as observed by flow cytometry and cleaved RARP expression as seen by western blotting, indicating an increase in apoptotic cells. ERK5 inhibition also suppressed cell viability and downregulated p16 and Bcl-2 expression. Conclusion Our results show that ERK5 inhibition contributes to the downregulation of anti-apoptotic and cell cycle proteins and suggest that ERK5 is a promising therapeutic target for ccRCC. Citation Format: Hidenori Kanno, Sei Naito, Osamu Ichiyanagi, Norihiko Tsuchiya. The effect of ERK5 inhibition in clear cell renal cell carcinoma [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 2381. doi:10.1158/1538-7445.AM2017-2381

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

Abstract 4120: Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients

Background: PI3K/Akt/mTORC1 signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated that glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. Herein, we aimed to investigate how the mTORC1 downstream substrate 4EBP1 is directly regulated by GSK-3β leading to acquisition of clinical chemoresistance to mTORC1 inhibitor (mTORi). Methods: The expression and phosphorylation of molecules in the subcellular pathways were examined by immunoblotting in surgically resected RCC tissues and human RCC cell lines including ACHN. Rapamycin-resistant ACHN cells (ACHN/RR) were generated by chronic exposure to an mTORi, rapamycin. Cell viability was investigated by MTS assay. The direct role of GSK-3β in regulating 4EBP1 was evaluated by an in vitro kinase assay. Pharmacological inhibition of GSK-3β, PI3K/Akt/mTORC1 pathway, and mTORC1 was achieved by treatment with AR-A014418, LY294002, and rapamycin, respectively. The effects of drug combination were determined using CompuSyn software. GSK-3β expression was evaluated in pathological RCC tissues obtained by nephrectomy from patients with metastatic RCC (mRCC), nine of whom were treated with mTORi as systemic therapies. GSK-3β expression was immunohistochemically graded as high and low expression using a semi-quantitative method based on positive staining intensity. Survival intervals were evaluated using the Kaplan-Meier method and log-rank test. Significance level was set as 0.05 in statistical analysis using a freeware R. Results: Inhibition of PI3K/Akt/mTORC1 pathway and mTORi treatment sufficiently decreased pS6RP, but only moderately decreased p4EBP1. In contrast to the effect of rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 in ACHN and ACHN/RR. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1 at Thr37, Thr46, Thr70 and Ser65, and these phosphorylations were blocked by GSK-3 inhibitor. A combination of AR- A014418 and rapamycin produced an additive effect at lower concentrations, and were antagonistic at higher concentrations. Immunoblotting demonstrated that 4EBP1 and p4EBP1 expression in RCC tissues was positively correlated with GSK-3β expression. Overall survival was significantly short in patients with high GSK-3β expression, compared with those having low expression (n = 25 and 14, respectively, p = 0.04). In addition, high GSK-3β expression in tumors tended to shorten progression-free intervals in mRCC patients treated with mTORi. Conclusions: High GSK-3ββ expression and direct GSK-3β/4EBP1 pathway can be a key mechanism for RCC acquiring clinical chemoresistance to mTORCi. Activation of the direct pathway might be predictive of poor prognosis of mRCC patients. Citation Format: Hiromi Ito, Sei Naito, Osamu Ichiyanagi, Hidenori Kanno, Yuta Kurota, Atushi Yamagishi, Vladimir Bilim, Yoshihiko Tomita, Tomoyuki Kato, Akira Nagaoka, Norihiko Tsuchiya. Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients [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 4120. doi:10.1158/1538-7445.AM2017-4120