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ABSTRACT: Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.
Journal of Biological Chemistry 04/2012; 287(24):20037-46. · 4.77 Impact Factor
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ABSTRACT: Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer-initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse-type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5-8% of the human diffuse-type gastric carcinoma cells, OCUM-2MLN and HSC-39; were more tumourigenic than ALDH1- cells; and were able to self-renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet-derived family, member 4) as one of the genes up-regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony-forming ability of OCUM-2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF-β signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse-type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF-β signalling. These findings illustrate that, in diffuse-type gastric carcinoma, REG4 is up-regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF-β down-regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse-type gastric carcinoma. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
The Journal of Pathology 03/2012; 228(3):391-404. · 6.32 Impact Factor
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ABSTRACT: A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma.
American Journal Of Pathology 12/2011; 179(6):2920-30. · 4.89 Impact Factor
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ABSTRACT: Transforming growth factor-β (TGF-β) elicits both tumor-suppressive and tumor-promoting functions during cancer progression. Here, we describe the tumor-promoting functions of TGF-β and how these functions play a role in cancer progression. Normal epithelial cells undergo epithelial-mesenchymal transition (EMT) through the action of TGF-β, while treatment with TGF-β and fibroblast growth factor (FGF)-2 results in transdifferentiation into activated fibroblastic cells that are highly migratory, thereby facilitating cancer invasion and metastasis. TGF-β also induces EMT in tumor cells, which can be regulated by oncogenic and anti-oncogenic signals. In addition to EMT promotion, invasion and metastasis of cancer are facilitated by TGF-β through other mechanisms, such as regulation of cell survival, angiogenesis, and vascular integrity, and interaction with the tumor microenvironment. TGF-β also plays a critical role in regulating the cancer-initiating properties of certain types of cells, including glioma-initiating cells. These findings thus may be useful for establishing treatment strategies for advanced cancer by inhibiting TGF-β signaling.
Upsala journal of medical sciences 11/2011; 117(2):143-52. · 0.73 Impact Factor
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Akira Watanabe,
Hideki Ogiwara, Shogo Ehata,
Akitake Mukasa,
Shumpei Ishikawa,
Daichi Maeda,
Keisuke Ueki,
Yasushi Ino,
Tomoki Todo,
Yasuhiro Yamada,
Masashi Fukayama,
Nobuhito Saito,
Kohei Miyazono,
Hiroyuki Aburatani
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ABSTRACT: Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells.
Proceedings of the National Academy of Sciences 07/2011; 108(30):12384-9. · 9.68 Impact Factor
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ABSTRACT: The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-β induced EMT and that long-term exposure to TGF-β elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-β induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-β-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-β and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-β and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.
The EMBO Journal 02/2011; 30(4):783-95. · 9.20 Impact Factor
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ABSTRACT: The epithelial–mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-β induced EMT and that long-term exposure to TGF-β elicited the epithelial–myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-β induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-β-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-β and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-β and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.
The EMBO Journal 01/2011; 30(4):783-795. · 9.20 Impact Factor
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ABSTRACT: Cancer cells undergo multi-step processes in obtaining the ability to metastasize, and are constantly exposed to signals that induce apoptosis. Acquisition of anti-apoptotic properties by cancer cells is important for metastasis, and recent studies suggest that transforming growth factor (TGF)-β promotes the survival of certain types of cancer cells. Here, we found that in highly metastatic breast cancer cells, JygMC(A), JygMC(B) and 4T1, TGF-β ligands were produced in autocrine fashion. Pharmacological inhibition of endogenous TGF-β signalling by a TGF-β type I receptor kinase inhibitor in serum-free conditions increased the expression of BH3-only protein, Bim (also known as Bcl2-like 11) in JygMC(A) and JygMC(B) cells, and caused apoptotic cell death. We also found that induction of Bim by TGF-β was not observed in Foxc1 knocked-down cancer cells. These findings suggest that TGF-β plays a crucial role in the regulation of survival of certain types of cancer cells through the TGF-β-Foxc1-Bim pathway, and that specific inhibitors of TGF-β signalling might be useful as apoptosis inducers in breast cancer cells.
Journal of biochemistry 09/2010; 149(1):55-65. · 1.95 Impact Factor
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ABSTRACT: Transforming growth factor-beta (TGF-beta) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13) as a downstream target of TGF-beta signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-beta type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-beta induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-beta type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-beta type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-beta-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-beta-mediated cell survival and metastasis of cancer.
Cancer Research 11/2007; 67(20):9694-703. · 7.86 Impact Factor
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Shogo Ehata,
Aki Hanyu,
Makoto Fujime,
Yoko Katsuno,
Erina Fukunaga,
Kouichiro Goto,
Yuichi Ishikawa,
Kimie Nomura,
Hiroshi Yokoo,
Toshiyuki Shimizu,
Etsuro Ogata,
Kohei Miyazono,
Kiyoshi Shimizu,
Takeshi Imamura
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ABSTRACT: Transforming growth factor (TGF)-beta signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for treatment of patients with such cancers. In this study, we investigated the effects of a novel TGF-beta type I receptor (TbetaR-I) kinase inhibitor, Ki26894, on bone metastasis of a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Ki26894 blocked TGF-beta signaling in MDA-231-D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF-beta-responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-beta in vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and interleukin-11 (IL-11) mRNA of MDA-231-D cells, which were stimulated by TGF-beta. X-ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA-231-D cells into the left ventricle of BALB/cnu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA-231-D cells compared to vehicle-treated mice. These findings suggest that TbetaR-I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers.
Cancer Science 02/2007; 98(1):127-33. · 3.33 Impact Factor
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ABSTRACT: c-Ski is a proto-oncogene product that induces morphologic transformation, anchorage independence, and myogenic differentiation when it is over-expressed in mesenchymal cells. c-Ski also inhibits signaling of transforming growth factor-beta (TGF-beta) superfamily members through interaction with Smad proteins. Although c-Ski is predominantly localized in the nucleus, aberrant cytoplasmic localization of it has also been reported in some tumor tissues and cell lines. In the present study, we identified the nuclear localization signal (NLS) in c-Ski. By introducing a mutation to abolish NLS activity, we examined the function of cytoplasmic c-Ski. Although cytoplasmic c-Ski suppressed TGF-beta superfamily-induced Smad signaling through sequestration of activated Smad complex to the cytoplasm, it failed to exhibit some of the activities that require nuclear localization of c-Ski, including suppression of basal transcription of the Smad7 gene. These findings indicate that subcellular localization of c-Ski affects its biologic activities. We also found that c-Ski accumulated in the cytoplasm when proteasome activity was inhibited. Mapping of the regions required for cytoplasmic accumulation by proteasome inhibitors suggests that subcellular localization of c-Ski may be regulated by proteasome-sensitive processes through amino acid residues 94-210 and 491-548.
Genes to Cells 12/2006; 11(11):1267-80. · 2.68 Impact Factor
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Ken Shirakawa,
Shingo Maeda,
Tomomi Gotoh,
Makoto Hayashi,
Kenichi Shinomiya, Shogo Ehata,
Riko Nishimura,
Masataka Mori,
Kikuo Onozaki,
Hidetoshi Hayashi,
Satoshi Uematsu,
Shizuo Akira,
Etsuro Ogata,
Kohei Miyazono,
Takeshi Imamura
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ABSTRACT: Differentiation of committed osteoblasts is controlled by complex activities involving signal transduction and gene expression, and Runx2 and Osterix function as master regulators for this process. Recently, CCAAT/enhancer-binding proteins (C/EBPs) have been reported to regulate osteogenesis in addition to adipogenesis. However, the roles of C/EBP transcription factors in the control of osteoblast differentiation have yet to be fully elucidated. Here we show that C/EBP homologous protein (CHOP; also known as C/EBPzeta) is expressed in bone as well as in mesenchymal progenitors and primary osteoblasts. Overexpression of CHOP reduces alkaline phosphatase activity in primary osteoblasts and suppresses the formation of calcified bone nodules. CHOP-deficient osteoblasts differentiate more strongly than their wild-type counterparts, suggesting that endogenous CHOP plays an important role in the inhibition of osteoblast differentiation. Furthermore, endogenous CHOP induces differentiation of calvarial osteoblasts upon bone morphogenetic protein (BMP) treatment. CHOP forms heterodimers with C/EBPbeta and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBPbeta, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBPbeta and prevents osteoblast differentiation but promotes BMP signaling in a cell-type-dependent manner. Thus, endogenous CHOP may have dual roles in regulating osteoblast differentiation and bone formation.
Molecular and Cellular Biology 09/2006; 26(16):6105-16. · 5.53 Impact Factor
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Haruhito Azuma, Shogo Ehata,
Hideyo Miyazaki,
Tetsuro Watabe,
Osamu Maruyama,
Takeshi Imamura,
Takeshi Sakamoto,
Satoshi Kiyama,
Yuko Kiyama,
Takanobu Ubai,
Teruo Inamoto,
Shiro Takahara,
Yuko Itoh,
Yoshinori Otsuki,
Yoji Katsuoka,
Kohei Miyazono,
Shigeo Horie
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ABSTRACT: Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. We used adenovirus-mediated gene transfer of these natural inhibitors in a mouse model of breast cancer to examine the roles of TGF-beta superfamily signaling in tumor growth and metastasis.
We systemically administered, by intravenous injection, adenoviruses (AdCMV) containing the mouse cDNAs for Smad7, Smad6, c-Ski, the c-Ski mutant c-Ski (ARPG), or LacZ (control) to nude mice (>19 mice/group) bearing tumors derived from mouse mammary carcinoma JygMC(A) cells, which spontaneously metastasize to lung and liver, and examined their effects on survival and metastasis. High-throughput western blotting analysis was used to examine the expression levels for 47 signal transduction proteins in JygMC(A) cells and primary tumors. We also investigated the proliferation, migration, and invasion of JygMC(A) cells that stably overexpressed Smad6 or Smad7. Nonparametric comparisons were done by Kruskal-Wallis H statistic and Wilcoxon's rank sum tests. Parametric comparisons were done by one-way analysis of variance or two-sided unpaired Student's t tests. All statistical tests were two-sided.
Control mice bearing tumors derived from JygMC(A) cells showed many metastases to the lung and liver; all animals died by 50 days after cell inoculation. By contrast, mice treated with AdCMV-Smad7 or AdCMV-c-Ski demonstrated a dramatic decrease in metastasis and statistically significantly longer survival than control mice (Smad7 versus LacZ: medium survival = 55 days versus 41 days, difference = 14 days [95% confidence interval {CI} = 6 days to 22 days], P < .001), whereas mice treated with AdCMV-Smad6 or AdCMV-c-Ski (ARPG) did not. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and tight junctions, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells.
Smad7 inhibits metastasis, possibly by regulating cell-cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers.
CancerSpectrum Knowledge Environment 12/2005; 97(23):1734-46. · 14.07 Impact Factor
