記述言語：英語   掲載種別：研究論文（学術雑誌）  

Autosomal dominant "sterile alpha motif domain containing 9 (Samd9) and Samd9L (Samd9/9L) syndromes" are a large subgroup of currently established inherited bone marrow failure syndromes that include MIRAGE, ataxia pancytopenia, and familial monosomy 7 syndromes. Samd9/9L genes are located in tandem on chromosome 7 and have been known to be the genes responsible for myeloid malignancies associated with monosomy 7. Additionally, as interferon-inducible genes, Samd9/9L are crucial for protection against viruses. Samd9/9L syndromes are caused by gain-of-function mutations and develop into infantile myelodysplastic syndromes associated with monosomy 7 (MDS/-7) at extraordinarily high frequencies. We generated mice expressing Samd9LD764N, which mimic the MIRAGE syndrome presenting with growth retardation, a short life, bone marrow failure, and multi-organ degeneration. In hematopoietic cells, Samd9LD764N downregulates the endocytosis of transferrin and c-Kit resulting in a rare cause of anemia and a low bone marrow reconstitutive potential that ultimately causes MDS/-7. By contrast, in non-hematopoietic cells we tested, Samd9LD764N upregulated the endocytosis of EGFR by Ship2 phosphatase translocation to the cytomembrane and activated lysosomes, resulting in the reduced expression of surface receptors and signaling. Thus Samd9/9L is a downstream regulator of interferon that controls receptor metabolism, with constitutive activation leading to multi-organ dysfunction.

DOI： 10.1172/JCI140147

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Uncontrolled self-renewal of hematopoietic progenitors induces leukemia. To self-renew, leukemia cells must continuously activate genes that were previously active in their mother cells. Here, we describe the circuitry of a transactivation system responsible for oncogenic self-renewal. MLL recruits RNA polymerase II (RNAP2) to unmethylated CpG-rich promoters by its CXXC domain and activates transcription by transcriptional regulators, including the AF4 family/ENL family/P-TEFb complex, DOT1L, and p300/CBP histone acetyl transferases. MOZ also targets a broad range of CpG-rich promoters through association with RNAP2 and MLL. Leukemic fusion proteins such as MOZ-TIF2 and MLL-AFX constitutively activate CpG-rich promoters by aberrantly recruiting p300/CBP. Pharmacological inhibition of MLL or DOT1L induces differentiation of MOZ-TIF2-transformed cells. These results reveal that activation of unmethylated CpG-rich promoters mediated by MLL is the central mechanism of oncogenic self-renewal in MOZ-rearranged leukemia and indicate that the molecularly targeted therapies intended for MLL-rearranged leukemia can be applied for MOZ-rearranged leukemia.

DOI： 10.1016/j.celrep.2020.108200

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIO-PROTOCOL  

Numerous oncogenes have been identified to cause leukemia. For example, chromosomal translocation generates various fusion genes of the mixed-lineage leukemia (MLL) gene and a partner gene in leukemia, whose gene products transform primary myeloid progenitors into an immortalized state. To characterize the transforming ability of leukemic oncogenes, researchers in the field have developed an ex vivo murine myeloid transformation assay using retroviral gene transduction and its protocol has been improved over the past 10 years. Here, we provide the detailed procedure for this assay.

DOI： 10.21769/BioProtoc.2626

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIO-PROTOCOL  

Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models using retroviral gene transduction, xenograft, or Cre-mediated chromosomal translocation have been developed over the past twenty years. Particularly, a retroviral gene transduction-mediated murine leukemia model has been frequently used in the leukemia research field. Here, we describe the detailed protocol for this model.

DOI： 10.21769/BioProtoc.2627

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4-ENL-P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. However, the functional interrelationship among those 3 complexes in leukemic transformation remains largely elusive. Here, we have shown that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to fully transform hematopoietic progenitors. These results reveal a cooperative transcriptional activation mechanism of AEP and DOT1L and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion-AF4 complex and DOT1L for more effective treatment of MLL-rearranged leukemia.

DOI： 10.1172/JCI91406

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Gene rearrangement of the mixed lineage leukemia (MLL) gene causes leukemia by inducing the constitutive expression of a gene subset normally expressed only in the immature haematopoietic progenitor cells. MLL gene rearrangements often generate fusion products of MLL and a component of the AF4 family/ENL family/P-TEFb (AEP) complex. MLL-AEP fusion proteins have the potential of constitutively recruiting the P-TEFb elongation complex. Thus, it is hypothesized that relieving the promoter proximal pausing of RNA polymerase II is the rate-limiting step of MLL fusion-dependent transcription. AEP also has the potential to recruit the mediator complex via MED26. We recently showed that AEP activates transcription initiation by facilitating TBP loading to the TATA element through the SL1 complex. In the present study, we show that the key activity responsible for the oncogenic property of MLL-AEP fusion proteins is the TBP loading activity, and not the mediator recruitment or transcriptional elongation activities. Thus, we propose that TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription.

DOI： 10.1080/15384101.2016.1222337

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

WNT signaling is well known to play an important role in the regulation of development, cell proliferation and cell differentiation in a wide variety of normal and cancerous tissues. Despite the wealth of knowledge concerning when and where various WNT genes are expressed and downstream events under their control, there is surprisingly little published evidence of how they are regulated. We have recently reported that aberrant WNT7A is observed in serous ovarian carcinomas, and WNT7A is the sole ligand accelerating ovarian tumor progression through CTNNB1 (β-catenin)/TCF signaling in the absence of CTNNB1 mutations. In the present study, we report that WNT7A is a direct target of miR-15b in ovarian cancer. We showed that a luciferase reporter containing the putative binding site of miR-15b in the WNT7A 3'-UTR was significantly repressed by miR-15b. Mutation of the putative binding site of miR-15b in the WNT7A 3'-UTR restored luciferase activity. Furthermore, miR-15b was able to repress increased levels of TOPFLASH activity by WNT7A, but not those induced by S33Y. Additionally, miR-15b dose-dependently decreased WNT7A expression. When we evaluated the prognostic impact of WNT7A and miR-15b expression using TCGA datasets, a significant inverse correlation in which high-expression of WNT7A and low-expression of miR-15b was associated with reduced survival rates of ovarian cancer patients. Treatment with decitabine dose-dependently increased miR-15b expression, and silencing of DNMT1 significantly increased miR-15b expression. These results suggest that WNT7A is post-transcriptionally regulated by miR-15b, which could be down-regulated by promoter hypermethylation, potentially via DNMT1, in ovarian cancer.

DOI： 10.1371/journal.pone.0156109

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Gene rearrangements generate MLL fusion genes, which can lead to aggressive leukemia. In most cases, MLL fuses with a gene encoding a component of the AEP (AF4 family/ENL family/P-TEFb) coactivator complex. MLL-AEP fusion proteins constitutively activate their target genes to immortalize haematopoietic progenitors. Here we show that AEP and MLL-AEP fusion proteins activate transcription through selectivity factor 1 (SL1), a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). The pSER domain of AF4 family proteins associates with SL1 on chromatin and loads TATA-binding protein (TBP) onto the promoter to initiate RNA polymerase II (RNAP2)-dependent transcription. These results reveal a previously unknown transcription initiation mechanism involving AEP and a role for SL1 as a TBP-loading factor in RNAP2-dependent gene activation.

DOI： 10.1038/ncomms9869

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously characterized the link between WNT7A and the progression of ovarian cancer. Other groups have identified FGF1 as a relevant risk factor in ovarian cancer. Here, we show a linkage between these two signaling pathways that may be exploited to improve treatment and prognosis of patients with ovarian cancer. High expression of WNT7A and FGF1 are correlated in ovarian carcinomas and poor overall patient survival. A chromatin immunoprecipitation assay demonstrated that WNT7A/β-catenin signaling directly regulates FGF1 expression via TCF binding elements in the FGF1-1C promoter locus. In vitro gene manipulation studies revealed that FGF1 is sufficient to drive the tumor-promoting effects of WNT7A. In vivo xenograft studies confirmed that the stable overexpression of WNT7A or FGF1 induced a significant increase in tumor incidence, whereas FGF1 knockdown in WNT7A overexpressing cells caused a significant reduction in tumor size. Niclosamide most efficiently abrogated WNT7A/β-catenin signaling in our model, inhibited β-catenin transcriptional activity and cell viability, and increased cell death. Furthermore, niclosamide decreased cell migration following an increase in E-cadherin subsequent to decreased levels of SLUG. The effects of niclosamide on cell functions were more potent in WNT7A-overexpressing cells. Oral niclosamide inhibited tumor growth and progression in an intraperitoneal xenograft mouse model representative of human ovarian cancer. Collectively, these results indicate that FGF1 is a direct downstream target of WNT7A/β-catenin signaling and this pathway has potential as a therapeutic target in ovarian cancer. Moreover, niclosamide is a promising inhibitor of this pathway and may have clinical relevance.

DOI： 10.1038/onc.2014.277

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mixed-lineage leukemia (MLL) maintains the expression of cellular memory genes during development, while leukemic MLL fusion proteins aberrantly maintain expression of hematopoietic stem cell program genes such as HOXA9 to cause leukemia. However, the molecular mechanism of gene activation is unclear. Here we show that only two functional modules are necessary and sufficient for target recognition: those that bind to non-methylated CpGs and di-/tri-methylated histone H3 lysine 36 (H3K36me2/3). An artificial protein composed of the two targeting modules and an interaction domain for AF4-family coactivators can functionally substitute for MLL fusion proteins. Because H3K36me2/3 markers are indicative of active transcription, MLL fusion proteins target previously active CpG-rich genes and activate transcription by recruiting coactivators thereto. Our results indicate that such chromatin context-dependent gene activation is the fundamental mechanism by which MLL fusion proteins maintain the expression of the cellular memory/hematopoietic stem cell program genes.

DOI： 10.1093/nar/gkt1394

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL-1β which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.

DOI： 10.1002/emmm.201201623

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Despite significant improvement in survival rates of patients with breast cancer, prognosis of metastatic disease is still dismal. Cancer stem-like cells (CSC) are considered to play a role in metastatic progression of breast cancer; however, the exact pathologic role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24(-)/CD44(+)/ESA(+)) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ-specific manner. The results of our microRNA (miRNA) profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this miRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain but not bone metastasis-free survival of patients with breast cancer, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or upregulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer.

DOI： 10.1158/0008-5472.CAN-12-2037

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary, or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally or subcutaneously with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the intraperitoneal group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histologic examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared with the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving subcutaneous injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Cotransfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by cotransfection with SFRP2 or dominant negative TCF4. Overexpression of WNT7A stimulated matrix metalloproteinase 7 (MMP7) promoter, and mutation of TCF-binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that reexpression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway.

DOI： 10.1158/1541-7786.MCR-11-0177

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Wnt signalling has pivotal roles in tumour progression and metastasis; however, the exact molecular mechanism of Wnt signalling in the metastatic process is as yet poorly defined. Here we demonstrate that the tumour metastasis suppressor gene, NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signalling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumour cells. Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumour cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a novel negative master regulator of Wnt signalling during the metastatic progression, which opens an opportunity to define a potential therapeutic target for metastatic disease.

DOI： 10.1002/emmm.201100190

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

DOI： 10.1158/0008-5472.CAN-11-1678

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Metastatic disease is the major cause of cancer deaths, and recurrent tumors at distant organs are a critical issue. However, how metastatic tumor cells become dormant and how and why tumors recur in target organs are not well understood. In this study, we demonstrate that BMP7 (bone morphogenetic protein 7) secreted from bone stromal cells induces senescence in prostate cancer stem-like cells (CSCs) by activating p38 mitogen-activated protein kinase and increasing expression of the cell cycle inhibitor, p21, and the metastasis suppressor gene, NDRG1 (N-myc downstream-regulated gene 1). This effect of BMP7 depended on BMPR2 (BMP receptor 2), and BMPR2 expression inversely correlated with recurrence and bone metastasis in prostate cancer patients. Importantly, this BMP7-induced senescence in CSCs was reversible upon withdrawal of BMP7. Furthermore, treatment of mice with BMP7 significantly suppressed the growth of CSCs in bone, whereas the withdrawal of BMP7 restarted growth of these cells. These results suggest that the BMP7-BMPR2-p38-NDRG1 axis plays a critical role in dormancy and recurrence of prostate CSCs in bone and suggest a potential therapeutic utility of BMP7 for recurrent metastatic disease.

DOI： 10.1084/jem.20110840

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24(-)/CD44(+)/ESA(+)) that were isolated from both ER+ and ER- breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.

DOI： 10.1007/s10549-010-1300-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In this study, we aimed to define the mechanism of Notch-ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch ligands in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly upregulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a γ-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting Akt phosphorylation. Importantly, Jagged2 was also found to be upregulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma has a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer.

DOI： 10.1038/onc.2011.122

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously isolated cacalol as a free radical-scavenging compound from Cacalia delphiniifolia which is a traditional Asian herbal plant and is believed to have medicinal effects on cancer. In this report, we demonstrated that cacalol has strong anti-proliferation effect on breast cancer cells and induces apoptosis by activating a pro-apoptotic pathway. We also found that a combination of cacalol and other chemotherapeutic drugs (Taxol and cyclophosphamide) synergistically induced apoptosis and partially overcame chemo-resistance. To further gain a mechanistic insight, we tested a potential inhibitory effect of cacalol on fatty acid synthase gene (FAS) in breast cancer cells, and found that cacalol significantly modulated the expression of the FAS gene, which resulted in apoptosis through activation of DAPK2 and caspase 3. We have also shown that cacalol significantly suppressed the Akt-sterol regulatory element-binding proteins (SREBP) signaling pathway and concomitant transcriptional activation of FAS. In a xenograft model of nude mouse, when cacalol was administered intraperitoneally, tumor growth was significantly suppressed. Importantly, oral administration of cacalol before implanting tumors showed significant preventive effect on tumor growth in the same animal model. Furthermore, the treatment of mice with a combination of low dose of Taxol and cacalol significantly suppressed the tumor growth. Taken together, our results indicate that cacalol induces apoptosis in breast cancer cells and impairs mammary tumor growth in vivo by blocking the expression of the FAS gene through modulation of Akt-SREBP pathway, suggesting that cacalol has potential utility as a chemopreventive and chemotherapeutic agent for breast cancer.

DOI： 10.1007/s10549-010-1076-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFκB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade.

DOI： 10.1074/jbc.M111.232637

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Re-programming of metabolic pathways is a hallmark of physiological changes in cancer cells. The expression of certain genes that directly control the rate of key metabolic pathways including glycolysis, lipogenesis and nucleotide synthesis are drastically altered at different stages of tumor progression. These alterations are generally considered as an adaptation of tumor cells; however, they also contribute to the progression of tumor cells to become more aggressive phenotypes. This review summarizes the recent information about the mechanistic link of these genes to oncogenesis and their potential utility as diagnostic markers as well as for therapeutic targets. We particularly focus on three groups of genes; GLUT1, G6PD, TKTL1 and PGI/AMF in glycolytic pathway, ACLY, ACC1 and FAS in lipogenesis and RRM2, p53R2 and TYMS for nucleotide synthesis. All these genes are highly up-regulated in a variety of tumor cells in cancer patients, and they play active roles in tumor progression rather than expressing merely as a consequence of phenotypic change of the cancer cells. Molecular dissection of their orchestrated networks and understanding the exact mechanism of their expression will provide a window of opportunity to target these genes for specific cancer therapy. We also reviewed existing database of gene microarray to validate the utility of these genes for cancer diagnosis.

DOI： 10.1016/j.bbcan.2010.01.005

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

RhoC is a member of the Ras-homologous family of genes which have been implicated in tumorigenesis and tumor progression. However, the exact role of RhoC is controversial and is yet to be clarified. We have examined the effect of RhoC on prostate tumor cells and found that RhoC had no effect on cell proliferation in vitro or on tumor growth in mice. However, RhoC significantly enhanced the metastatic ability of the tumor cells in these animals, suggesting that RhoC affects only the metastasis but not the growth of prostate tumor cells. The results of our immunohistochemical analyses on tumor specimens from 63 patients with prostate cancer indicate that RhoC expression had no significant correlation with Gleason grade. However, the expression of RhoC showed significant positive correlation with both lymph node and distant metastasis, and it was inversely correlated with patient survival. We also found that RhoC significantly augmented the invasion and motility of prostate tumor cells by activating matrix metalloproteinases 2 and 9 (MMP2 and MMP9) in vitro. The results of our antibody array analysis for signal molecules revealed that RhoC significantly activated kinases including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt, and Pyk2. Inhibition of Pyk2 kinase blocked the RhoC-dependent activation of FAK, MAPK, and Akt, followed by the suppression of MMP2 and MMP9. Inhibitors of both MAPK and Akt also significantly blocked the activities of these MMPs. Therefore, our results indicate that RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt followed by the up-regulation of MMP2 and MMP9, which results in the stimulation of invasiveness of tumor cells.

DOI： 10.1158/0008-5472.CAN-07-6700

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.

DOI： 10.1074/jbc.M710057200

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PESTICIDE SCI SOC JAPAN  

The actions of neonicotinoid insecticides on nicotinic acetylcholine receptors (nAChRs) in the terminal abdominal ganglion neurons of the American cockroach were investigated using whole-cell patch-clamp electrophysiology. Neonicotinoids possessing a nitromethylene group showed higher agonist affinity than the corresponding nitroimine analogues, whereas compounds with an acyclic guanidine moiety showed greater agonist efficacy than the corresponding cyclic compounds. Imidacloprid showed the lowest agonist efficacy of all neonicotinoids and low concentrations of imidacloprid attenuated acetylcholine-induced currents. However, such blocking actions were minimal with other neonicotinoids. The diverse actions of neonicotinoids on nAChRs, combined with target accessibility based on hydrophobicity, appears to account for their insecticidal potency on cockroaches measured in the presence of metabolic inhibitors. (c) Pesticide Science Society of Japan.

DOI： 10.1584/jpestics.31.35

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

4'-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of gamma-aminobutyric acid (GABA)-gated Cl- channels (GABARs) of insects and mammals, and gamma-hexachlorocyclohexane (gamma-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and gamma-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and gamma-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and gamma-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and gamma-HCH on the GABA- and glutamate-induced responses were compared by determining IC50 values under steady state condition. The IC50 values for the actions of EBOB on GABAR and GluCls differed less than those of gamma-HCH.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Atherosclerosis is a vascular disorder involving inflammation, a narrowed vascular lumen in the entire tunica intima, and reduced elasticity of the arterial wall. It has been found that Hsp60 from Chlamydia pneumoniae, an obligate bacterial pathogen associated with atheroma lesions, mimics human Hsp60, thereby causing attacks by immune cells on stressed endothelial cells expressing endogenous Hsp60 on their surface. Furthermore, Hsp60 from C. pneumoniae has been shown to promote the growth of vascular smooth muscle cells (VSMCs). To explore probes that can be used for studying signal transduction elicited by the chlamydial Hsp60, we have tested several natural products for their inhibitory actions on the Hsp60-induced proliferation of rat arterial smooth muscle cells. Sesamol, vanillyl alcohol, and trans-ferulic acid exhibited moderate inhibitory actions on the Hsp60-induced cell proliferation; zerumbone, humulene, and caryophylene effectively inhibited it at low concentrations with IC(50) values of 529, 122, and 110 nM, respectively. The results indicated that the 11-membered alicyclic ring is favorable for interactions with receptors involved in the Hsp60-induced VSMC proliferation.

PubMed

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

1. Neonicotinoid insecticides are agonists of insect nicotinic acetylcholine receptors (AChRs) and show selective toxicity for insects over vertebrates. To elucidate the molecular basis of the selectivity, amino acid residues influencing neonicotinoid sensitivity were investigated by site-directed mutagenesis of the chicken alpha7 nicotinic AChR subunit, based on the crystal structure of an ACh binding protein (AChBP). 2. In the ligand binding site of AChBP, Q55 in loop D is close to Y164 in loop F that corresponds to G189 of the alpha7 nicotinic receptor. Since Q55 of AChBP is preserved as Q79 in the alpha7 nicotinic receptor and the G189D and G189E mutations have been found to reduce the neonicotinoid sensitivity, we investigated effects of Q79E, Q79K and Q79R mutations on the neonicotinoid sensitivity of the alpha7 receptor expressed in Xenopus laevis oocytes to evaluate contributions of the glutamine residue to nicotinic AChR-neonicotinoid interactions. 3. The Q79E mutation markedly reduced neonicotinoid sensitivity of the alpha7 nicotinic AChR whereas the Q79K and Q79R mutations increased sensitivity, suggesting electronic interactions of the neonicotinoids with the added residues. 4. By contrast, the Q79E mutation scarcely influenced responses of the alpha7 nicotinic receptor to ACh, (-)-nicotine and desnitro-imidacloprid (DN-IMI), an imidacloprid derivative lacking the nitro group, whereas the Q79K and Q79R mutations reduced the sensitivity to these ligands. The results indicate that the glutamine residue of the alpha7 nicotinic receptor is likely to be located close to the nitro group of the insecticides in the nicotinic receptor-insecticide complex.

PubMed

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2011-3409

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2011-1028

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2011-979

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