Hiromi Ito

ERK5 induces ankrd1 for catecholamine biosynthesis and homeostasis in adrenal medullary cells

Extracellular signal-regulated kinases (ERKs) play important roles in proliferation, differentiation and gene expression. In our previous study, we demonstrated that both ERK5 and ERK1/2 were responsible for neurite outgrowth and tyrosine hydroxylase (TH) expression in rat pheochromocytoma cells (PC12) (J Biol Chem 284, 23,564-23,573, 2009). However, the functional differences between ERK5 and ERK1/2 signaling in neural differentiation remain unclear. In the present study, we show that ERK5, but not ERK1/2 regulates TH levels in rat sympathetic neurons. Furthermore, microarray analysis performed in PC12 cells using ERK5 and ERK1/2-specific inhibitors, identified ankyrin repeat domain 1 (ankrd1) as an ERK5-dependent and ERK1/2-independent gene. Here, we report a novel role of the ERK5/ankrd1 signaling in regulating TH levels and catecholamine biosynthesis. Ankrd1 mRNA was induced by nerve growth factor in time- and concentration-dependent manners. TH levels were reduced by ankrd1 knockdown with no changes in the mRNA levels, suggesting that ankrd1 was involved in stabilization of TH protein. Interestingly, ubiquitination of TH was enhanced and catecholamine biosynthesis was reduced by ankrd1 knockdown. Finally, we examined the relationship of ERK5 to TH levels in human adrenal pheochromocytomas. Whereas TH levels were correlated with ERK5 levels in normal adrenal medullas, ERK5 was down-regulated and TH was up-regulated in pheochromocytomas, indicating that TH levels are regulated by alternative mechanisms in tumors. Taken together, ERK5 signaling is required for catecholamine biosynthesis during neural differentiation, in part to induce ankrd1, and to maintain appropriate TH levels. This pathway is disrupted in pathological conditions.

A diagnostic marker for superficial urothelial bladder carcinoma: lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression.

BackgroundPathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma.MethodsSeven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation.ResultsATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P = 0.021) and intravesical recurrence-free survival (P = 0.013) were detected between ATBF1+ (n = 110) and ATBF1− (n = 7) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P = 0.008).ConclusionsCleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.

The pattern of GPI-80 expression is a useful marker for unusual myeloid maturation in peripheral blood

Myeloid‐derived suppressor cells (MDSCs) have a wide spectrum of immunosuppressive activity; control of these cells is a new target for improving clinical outcomes in cancer patients. MDSCs originate from unusual differentiation of neutrophils or monocytes induced by inflammatory cytokines, including granulocyte‐colony stimulating factor (G‐CSF) and granulocyte–macrophage (GM)‐CSF. However, MDSCs are difficult to detect in neutrophil or monocyte populations because they are not uniform cells, resembling both neutrophils and monocytes; thus, they exist in a heterogeneous population. In this study, we investigated GPI‐80, a known regulator of Mac‐1 (CD11b/CD18) and associated closely with neutrophil maturation, to clarify this unusual differentiation. First, we demonstrated that the mean fluorescence intensity (MFI) of GPI‐80 and coefficient of variation (CV) of GPI‐80 were increased by treatment with G‐CSF and GM‐CSF, respectively, using a human promyelocytic leukaemia (HL60) cell differentiation model. To confirm the value of GPI‐80 as a marker of unusual differentiation, we measured GPI‐80 expression and MDSC functions using peripheral blood cells from metastatic renal cell carcinoma patients. The GPI‐80 CV was augmented significantly in the CD16hi neutrophil cell population, and GPI‐80 MFI was increased significantly in the CD33hi monocyte cell population. Furthermore, the GPI‐80 CV in the CD16hi population was correlated inversely with the proliferative ability of T cells and the GPI‐80 MFI of the CD33hi population was correlated with reactive oxygen species production. These results led us to propose that the pattern of GPI‐80 expression in these populations is a simple and useful marker for unusual differentiation, which is related to MDSC functions.

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.

Rise in ambient temperature predisposes aging, male Japanese patients to renal colic episodes due to upper urolithiasis

Abstract Objective: Urolithiasis is a common urological problem, and its incidence has been increasing worldwide, including in Japan. Relationships between stone etiology and rise in ambient temperature have been reported, but it remains unclear how age and gender affect these relationships. Materials and methods: A retrospective examination was conducted of the medical archives of 1005 patients (aged ≥15 years) with acute renal colic diagnosed with urolithiasis upon image examination who consecutively visited emergency departments in three hospitals. The patients were categorized into six groups according to age: younger than 30, 30–39, 40–49, 50–59, 60–69, and 70 years and older. The net difference and fold increase in the number of patients in summer (July to September) versus in winter (December to February) were calculated. Results: Overall, the actual number of the patients varied according to the temperature rise throughout the year and among the age groups. Net increases in the number of patients were observed in all age groups for both genders, apart from 30–39-year-old women. The age group of 50–59 years considerably outnumbered all other groups. A significant statistical correlation was detected between the fold increase and male aging using Spearman’s rank correlation analysis (ρ = 0.94, p = 0.017), but not in females (ρ = –0.03, p = 1). Conclusions: These results support a positive association between ambient temperature rise and increase in the incidence of renal colic due to urolithiasis in Japan, and indicate that aging and gender affect the association differently.

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