Language：English   Publishing type：Research paper (scientific journal)   Publisher：Spandidos Publications  

Identification and purification of cancer stem cells (CSCs) lead to the discovery of novel therapeutic targets
however, there has been no study on isolation of the CSC population among pancreatic neuroendocrine tumors (pNETs). This study aimed to identify pNET CSCs and to characterize a therapeutic candidate for pNET CSCs. We identified CSCs by aldehyde dehydrogenase (ALDH) activity in pNET clinical specimens and cell lines. We verified whether or not these cells have the stemness property in vivo and in vitro. ALDHhigh cells, but not control bulk cells, formed spheres, proliferated under hypoxic condition as well as normoxic condition and promoted cell motility, which are features of CSCs. Injection of as few as 10 ALDHhigh cells led to subcutaneous tumor formation, and 105 ALDHhigh c ells, b ut n ot c ontrol bulk cells, established metastases in mice. Comprehensive gene expression analysis revealed that genes associated with mesenchymal stem cells, including CD73, were overexpressed in ALDHhigh cells. Additionally, the in vitro and in vivo effects of an inhibitor of CD73 were investigated. The CD73 inhibitor APCP significantly attenuated in vitro sphere formation and cell motility, as well as in vivo tumor growth observed for ALDHhigh cells. Finally, its expression was evaluated using clinical pNET tissue samples. Immunohistochemical analysis of clinical tissue samples demonstrated CD73 expression was significantly correlated with the invasion into adjacent organs. Since recent studies revealed CD73 as a potential biomarker of anti-PD-1 immune checkpoint therapy, CD73 might be a promising therapeutic target for pNET CSCs.

DOI： 10.3892/ijo.2015.3299

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

Living cells are frequently exposed to various stresses. Hypoxic conditions induce endoplasmic reticulum (ER) stress, and activate the unfolded protein response (UPR) to maintain homeostasis. We previously reported that CREB has an important role in the proper response to prolonged hypoxia. To further understand the role of CREB in the hypoxic response, CREB stable knock-down (CREB-KD) cells were established from breast cancer MDA-MB231 cells and analyzed. CREB was activated by ER stress, and activation of CREB and the UPR pathway occurred in a coordinated manner in response to different stimuli, including ER stress-inducing chemicals, prolonged hypoxia, and oxygen-glucose deprivation (OGD). Depletion of CREB decreased the expression of IRE1α and PERK, two critical UPR signaling molecules. Promoter analysis and a chromatin immunoprecipitation assay indicated that CREB binds to the promoter region of these genes and regulates their expression. ER stress induced by hypoxia was reduced in CREB-KD cells, leading to reduced tumor metastasis to the lung. Finally, OGD strongly activated the UPR and induced cell death in control cells, whereas the UPR was moderately activated in CREB-KD cells, which were more resistant to cell death. This study demonstrates a new role for CREB as a regulator of ER stress, which is required to properly respond to stressful conditions, such as hypoxia.

DOI： 10.1016/j.bbrc.2015.11.113

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Cells are frequently exposed to hypoxia in physiological and pathophysiological conditions in organisms. Control of energy metabolism is one of the critical functions of the hypoxic response. Hypoxia-Inducible Factor (HIF) is a central transcription factor that regulates the hypoxic response. HIF prolyl-hydroxylase PHDs are the enzymes that hydroxylate the α subunit of HIF and negatively regulate its expression. To further understand the physiological role of PHD3, proteomics were used to identify PHD3-interacting proteins, and pyruvate dehydrogenase (PDH)-E1β was identified as such a protein. PDH catalyzes the conversion of pyruvate to acetyl-coA, thus playing a key role in cellular energy metabolism. PDH activity was significantly decreased in PHD3-depleted MCF7 breast cancer cells and PHD3(-/-) MEFs. PHD3 depletion did not affect the expression of the PDH-E1α, E1β, and E2 subunits, or the phosphorylation status of E1α, but destabilized the PDH complex (PDC), resulting in less functional PDC. Finally, PHD3(-/-) cells were resistant to cell death in prolonged hypoxia with decreased production of ROS. Taken together, the study reveals that PHD3 regulates PDH activity in cells by physically interacting with PDC.

DOI： 10.1016/j.bbrc.2014.07.114

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Background: Changes in gene expression mediate adaption to hypoxia. Results: MMP1 was induced during prolonged hypoxia, and this induction was blocked after depletion of CREB and/or NF-κB. Conclusion: CREB and NF-κB mediate the up-regulation of MMP1 during prolonged hypoxia. Significance: CREB-and NF-κB-mediated gene transcription may play a major role in the cellular response to prolonged hypoxia. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M112.421636

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Language：English   Publishing type：Research paper (scientific journal)  

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies because of recurrence and/or metastasis even after curative resection. Emerging evidence suggests that tumor metastasis and recurrence might be driven by a small subpopulation of stemness cells, so-called cancer stem cells (CSCs). Previous investigations have revealed that glioma and breast CSCs exhibit intrinsically low proteasome activity and that breast CSCs also reportedly contain a lower reactive oxygen species (ROS) level than corresponding nontumorigenic cells. Here we visualized two stem cell features, low proteasome activity and low intracellular ROS, in HCC cells using two-color fluorescence activated cell sorting to isolate cells with stem cell features. These cells were then analyzed for their division behavior in normoxia and hypoxia, expression of stem cell markers, tumorigenicity, metastatic potential, specific gene expression signatures, and their clinical implications. A visualized small subpopulation of HCC cells demonstrated asymmetric divisions. Their remarkable tumorigenicity in nonobese diabetic/severe combined immunodeficient mice suggested the cancer initiation potential of these HCC CSCs. Comprehensive gene expression analysis revealed that chemokine-related genes were up-regulated in the CSCs subpopulation. Our identified HCC CSCs facilitated the migration of macrophages in vitro and demonstrated metastatic potential by way of recruitment of macrophages in vivo. In patients who undergo curative operation for HCC, the CSC-specific gene signature in the liver microenvironment significantly correlates with recurrence. Conclusion: Based on these findings, the stem cell feature monitoring system proposed here is a promising tool to analyze the in vivo significance of CSC microenvironments in human HCCs. © 2013 American Association for the Study of Liver Diseases.

DOI： 10.1002/hep.26345

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Language：English   Publishing type：Research paper (scientific journal)  

Muscarinic acetylcholine receptors (mAchRs) are critical components of the cholinergic system, which is the key regulator of both the central and peripheral nervous systems in mammals. Interestingly, several components of the cholinergic system, including mAchRs and choline acetyltransferase (ChAT), have recently been found to be expressed in mouse embryonic stem (ES) cells and human placenta. These results raise the intriguing possibility that mAchRs play physiological roles in the regulation of early embryogenesis. Early embryogenesis can be mimicked in vitro using an ES cell-based culture system in which the cells form a primitive streak-like structure and efficiently develop into mesodermal progenitors. Here we report that chemical inhibitors specifically targeting mAchRs suppressed the expression of genes essential for primitive streak formation, including Wnt3, and thereby blocked mesodermal progenitor differentiation. Interestingly, mAchR inhibitors also reduced the expression of Cyp26a1, an enzyme involved in the catabolism of retinoic acid (RA). RA is an important regulator of Wnt3 signaling. Our study presents evidence indicating that mAchRs influence RA signaling necessary for the induction of the primitive streak. To our knowledge, this is the first report showing that mAchRs have important functions not only in adult mammals but also during early mammalian embryogenesis. © 2013 Elsevier Inc..

DOI： 10.1016/j.bbrc.2013.05.006

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Authorship：Lead author   Language：English   Publisher：Blackwell Publishing  

DOI： 10.1111/1440-1681.12071

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER INC  

Prolyl-hydroxylase PHDs are the key regulators of hypoxia-inducible factor (HIF) stability. PHD3 has been shown to form a large complex under hypoxic conditions. While attempting to characterize the complex by determining its components, we identified human PRP19. hPRP19 is a multi-functional protein that plays a role in splicing, ubiquitination, and cell growth. Here, we report that PHD3 efficiently forms a complex with hPRP19 under hypoxic conditions and prevents cell death under prolonged hypoxic conditions. hPRP19 interacts with PHD3 via its C-terminal WD40 region, and the interaction is enhanced under hypoxic conditions through the utilization of the N-terminal coiled-coil domain. Cell death observed under prolonged hypoxic conditions is suppressed by the forced expression of hPRP19 in PC12 and HEK293T cells. In contrast, hPRP19 silencing by siRNA increased the caspase activity and enhanced cell death under hypoxic conditions in HeLa cells. Further, silencing of both PHD3 and hPRP19 recovers the cell death induced by hPRP19 single siRNA. Taken together, the results of our study indicate that hPRP19 interacts with PHD3 to suppress the cell death under hypoxic conditions by limiting the function of PHD3 which leads to caspase activation. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2010.06.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Neuroendocrine (NE) phenotype, seen in >30% of prostate adenocarcinomas (PCa), and NE prostate tumors are implicated in aggressive prostate cancer. Formation of NE prostate tumors in the TRAMP mouse model was suppressed in mice lacking the ubiquitin ligase Siah2, which regulates HIF-1 alpha availability. Cooperation between HIF-1 alpha and FoxA2, a transcription factor expressed in NE tissue, promotes recruitment of p300 to transactivate select HIF-regulated genes, Hes6, Sox9, and Jmjd1a. These HIF-regulated genes are highly expressed in metastatic PCa and required for hypoxia-mediated NE phenotype, metastasis in PCa, and the formation of NE tumors. Tissue-specific expression of FoxA2 combined with Siah2-dependent HIF-1 alpha availability enables a transcriptional program required for NE prostate tumor development and NE phenotype in PCa.

DOI： 10.1016/j.ccr.2010.05.024

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Growing evidence indicates that ubiquitin ligases play a critical role in the hypoxia response. Among them, Siah2, a RING finger ligase, is an important regulator of pathways activated under hypoxia. Siah2 regulates prolyl hydroxylases PHD3 and 1 under oxygen concentration of 2% to 5%, thereby allowing accumulation of hypoxia-inducible factor (HIF)-1a, a master regulator of the hypoxia response within the range of physiological normoxic to mild hypoxic conditions. Growing evidence also indicates an important function for Siah2 in tumor development and progression based on pancreatic cancer, mammary tumor, and melanoma mouse models. This review summarizes our current understanding of Siah2 regulation and function with emphasis on hypoxia and tumorigenesis. (Mol Cancer Res 2009;7(4):443-51)

DOI： 10.1158/1541-7786.MCR-08-0458

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1 alpha availability. Because HIF-1 alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1 alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, our data establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms.

DOI： 10.1073/pnas.0804063105

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

PHD1-3 (prolyl hydroxylases 1-3) catalyse the hydroxylation of HIF (hypoxia-inducible factor)-alpha subunit that triggers the substrate ubiquitination and subsequent degradation. The RING (really interesting new gene) finger E3 ligase Siah2 preferentially targets PHD3 for degradation. Here, we identify the requirements for such selective targeting. Firstly, PHD3 lacks an N-terminal extension found in PHD1 and PHD2; deletion of this domain from PHD1 and PHD2 renders them susceptible to degradation by Siah2. Secondly, PHD3 can homo- and hetero-multimerize with other PHDs. Consequently, PHD3 is found in high-molecular-mass fractions that were enriched in hypoxia. Interestingly, within the lower-molecular-mass complex, PHD3 exhibits higher specific activity towards hydroxylation of HIF-1 alpha and co-localizes with Siah2, suggesting that Siah2 limits the availability of the more active form of PHD3. These findings provide new insight into the mechanism underlying the regulation of PHD3 availability and activity in hypoxia by the E3 ligase Siah2.

DOI： 10.1042/BJ20061135

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1 alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.

DOI： 10.1074/jbc.M606568200

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Hypoxia-inducible factor-1alpha (HIF1alpha) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. Here, we demonstrate that the abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2, under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression was completely inhibited in Siah1a/2 null cells, yet could be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia displayed an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia.

DOI： 10.1016/j.cell.2004.06.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

DOI： 10.1128/MCB.23.15.5331-5345.2003

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

A novel cDNA EZI isolated as an oncostatin M-inducible gene encoded a protein containing 12 C2H2-type zinc fingers. EZI was found to transactivate the promoters that are also responsive to STAT3 and activated the acute phase response element (APRE) synergistically with STAT3. Co-immunoprecipitation demonstrated the association of EZI with STAT3, which was mediated by the N-terminal region (1-183) of EZI. The EZI mutant lacking this region showed reduced transcriptional activity, indicating that EZI and STAT3 function cooperatively through physical interaction. While EZI predominantly localized in the nucleus and enhanced the nuclear localization of STAT3, the EZI mutant lacking 11 zinc finger motifs failed to translocate into the nucleus and also inhibited nuclear localization of STAT3 as well as STAT3-mediated transactivation. These results indicate that EZI is a novel nuclear zinc finger protein that augments STAT3 activity by keeping it in the nucleus.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Oncostatin M (OSM) is a member of the IL-6 family cytokines that use gp130 as a common signal transducer and exhibits both growth stimulatory as well as growth inhibitory activity depending on the cells. To analyze the mechanism of OSM function, we isolated immediate early responsive genes upon OSM stimulation. Here we describe the novel OSM-inducible gene OIG37 that is related to MyD118 and GADD45, The MyD118 gene has been described as an immediate early gene induced by IL-6 in M1 monocytic cells, and GADD45 was identified as a gene induced by UV or gamma-ray irradiation. Both are considered to function in growth arrest and/or DNA repair. Although the expression of OIG37, MyD118, and GADD45 was rather ubiquitous, it was differentially regulated. As the gp130 mutant defective for activating the STAT3 pathway showed the reduced induction of OIG37 by cytokine stimulation and expression of dominant negative STATE inhibited the induction of OIG37 by OSM, STAT3 is involved in OIG37 induction by IL-6 family cytokines, To examine the function of OIG37, we expressed it in NIH3T3 and IL-3-dependent BaF3 cells and found that OIG37 suppressed cell growth without any evidence of apoptosis. Whereas both MyD118 and OIG37 suppressed cell growth in both cell lines, suppression by OIG37 was more efficient than by MyD118. Immunoprecipitation experiments indicated that OIG37 associates with p21, a cyclin-dependent kinase inhibitor, and proliferating cell nuclear antigen.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：OXFORD UNIV PRESS  

Cells induce the hypoxia responses to adapt to the environment when organisms are exposed to a low oxygen environment. The hypoxia response leads to the activation of multiple cellular signalling pathways involved in regulation of respiration, metabolism, cell survival and so forth. Hypoxia-Inducible-Factor (HIF) pathway plays a central role during the hypoxia response as its expression and activity are regulated in an oxygen-dependent manner and it also regulates the expression of multiple hypoxia responsive genes. The expression of HIF is regulated by proline hydroxylation, which is mediated by HIF prolyl-hydroxylase named PHD. The hydroxylated HIF-alpha subunit is degraded via the ubiquitin-proteasome pathway. The PHD activity needs to be strictly regulated to ensure the stabilization of HIF under hypoxic conditions, because PHD leads to HIF degradation. This review describes the regulatory mechanism of HIF stability and activity under normoxia and hypoxic conditions. Furthermore, the role of the HIF-independent pathways during the hypoxia response, which is as important as the HIF pathway, will also be described.

DOI： 10.1093/jb/mvp167

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Prolyl-hydroxylation of HIF-1alpha is a prerequisite for pVHL binding to HIF-1alpha, which results in degradation of HIF-1alpha by the ubiquitin-proteasome pathway. Hydroxylation of HIF-1alpha is mediated by the family of prolyl-hydroxylase proteins (PHD). In hypoxia, HIF-1alpha is stabilized as a result of inhibition of HIF-1alpha hydroxylation, which in part is achieved by decreased activity of PHD enzymes at very low oxygen concentrations. We recently demonstrated that in hypoxia the stability of 2 of 3 PHDs (1 and 3) is regulated by the E3 ligases Siah1/2. Consequently, in hypoxia Siah determines the availability of PHD1/3, which otherwise modify HIF-1alpha to enable its association-dependent degradation by pVHL. These findings define a newly discovered layer in the regulation of HIF-1alpha in hypoxia. The roles of Siah activities in hypoxia responses are discussed.

DOI： 10.4161/cc.3.11.1207

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC HEMATOLOGY  

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Venue：神戸  

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Venue：Barga, Italy  

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Venue：San Diego, USA  

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Venue：Breckenridge, USA  

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Venue：京都  

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Venue：福岡  

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Venue：福岡  

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Venue：Banff, Canada  

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