Analysis of TGF-β/BMP signaling during bone metastasis and development of cancer therapy

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Members of the transforming growth factor-b (TGF-β) superfamily, including TGF-β and bone morphogenetic proteins (BMPs), are multifunctional cytokines that regulate a wide range of cellular responses, including cell proliferation, differentiation, adhesion, migration, and apoptosis. TGF-β signaling facilitates tumor growth and metastasis in advanced cancer. The TGF-β signaling pathway has correspondingly become an attractive target for drug development in the field of oncology.We investigated the roles of TGF-β and BMP signaling during bone metastasis. We first established a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Next, we examined the effects of a novel TGF-β type I receptor (TBR-I) kinase inhibitor, Ki26894, on bone metastasis of MDA-231-D cells. 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, Ki … More 26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-β 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-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.In addition, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stral3) as a downstream target of TGF-β signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC (A) and 4T1, the TBR-β 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-β induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TβR-I kinase inhibitors as well as by expression of dominant-negative TGF-β 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-β-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-β-mediated cell survival and metastasis of cancer. Less