Shogo Ehata

Bone morphogenetic protein signaling enhances invasion and bone metastasis of breast cancer cells through Smad pathway.

Transforming growth factor (TGF)-β is known to promote tumor invasion and metastasis. Although bone morphogenetic proteins (BMPs), members of the TGF-β family, are expressed in a variety of human carcinoma cell lines, their roles in tumor progression have not been fully clarified. In this study, we sought to determine the roles of BMPs in the progression of breast cancer bone metastasis using human breast cancer samples and a mouse xenograft model. Immunohistochemical analysis of samples from breast cancer patients as well as a mouse xenograft model of MDA-231-D, highly metastatic human breast cancer cells, revealed phospho-Smad2 and phospho-Smad1/5/8 staining in the nuclei of cancer cells in primary tumor and/or bone metastasis. Using a functional in vivo bioluminescence imaging system, we showed that TGF-β- and BMP-induced transcriptional pathways are active in bone metastatic lesions in vivo. In addition, both TGF-β3 and BMP-2 promoted the motility and invasiveness of the MDA-231-D cells in vitro. Moreover, expression of dominant-negative receptors for TGF-β and/or BMPs in the MDA-231-D cells inhibited invasiveness in vitro and bone metastasis in the xenograft model. These results suggest that BMPs as well as TGF-β promote invasion and bone metastasis of breast cancer.

Ki26894, a novel transforming growth factor‐β type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line

Transforming growth factor (TGF)‐β signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF‐β 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‐β type I receptor (TβR‐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‐β signaling in MDA‐231‐D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF‐β‐responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA‐231‐D cells induced by TGF‐βin 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‐β. 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/c nu/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 TβR‐I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers. (Cancer Sci 2007; 98: 127–133)

TGF-β regulates isoform switching of FGF receptors and epithelial-mesenchymal transition.

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.

Transforming Growth Factor-β Promotes Survival of Mammary Carcinoma Cells through Induction of Antiapoptotic Transcription Factor DEC1

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.

CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP) Regulates Osteoblast Differentiation

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/EBPζ) 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/EBPβ and inhibits the DNA-binding activity as well as Runx2-binding activity of C/EBPβ, leading to inhibition of osteocalcin gene transcription. These findings indicate that CHOP acts as a dominant-negative inhibitor of C/EBPβ 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.

Transforming growth factor-β decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-β (TGF-β) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-β repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-β, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-β negatively contributes to maintain the CICs within the cancer.

Tumor-promoting functions of transforming growth factor-β in progression of cancer

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.

Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-β.

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

Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim

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.

Nuclear and cytoplasmic c-Ski differently modulate cellular functions

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‐β (TGF‐β) 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‐β 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.