Aki Hanyu

Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-β

Transforming growth factor (TGF)‐β signaling facilitates tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF‐β signaling may thus be a novel strategy for the treatment of patients with such cancer. In this study, we synthesized and characterized a small molecule inhibitor, A‐83‐01, which is structurally similar to previously reported ALK‐5 inhibitors developed by Sawyer et al. (2003) and blocks signaling of type I serine/threonine kinase receptors for cytokines of the TGF‐β superfamily (known as activin receptor‐like kinases; ALKs). Using a TGF‐β‐responsive reporter construct in mammalian cells, we found that A‐83‐01 inhibited the transcriptional activity induced by TGF‐β type I receptor ALK‐5 and that by activin type IB receptor ALK‐4 and nodal type I receptor ALK‐7, the kinase domains of which are structurally highly related to those of ALK‐5. A‐83‐01 was found to be more potent in the inhibition of ALK5 than a previously described ALK‐5 inhibitor, SB‐431542, and also to prevent phosphorylation of Smad2/3 and the growth inhibition induced by TGF‐β. In contrast, A‐83‐01 had little or no effect on bone morphogenetic protein type I receptors, p38 mitogen‐activated protein kinase, or extracellular regulated kinase. Consistent with these findings, A‐83‐01 inhibited the epithelial‐to‐mesenchymal transition induced by TGF‐β, suggesting that A‐83–01 and related molecules may be useful for preventing the progression of advanced cancers. (Cancer Sci 2005; 96: 791–800)

Arkadia amplifies TGF-β superfamily signalling through degradation of Smad7

Arkadia was originally identified as a protein that enhances signalling activity of Nodal and induces mammalian nodes during early embryogenesis; however, the mechanisms by which Arkadia affects transforming growth factor‐β (TGF‐β) superfamily signalling have not been determined. Here we show that Arkadia is widely expressed in mammalian tissues, and that it enhances both TGF‐β and bone morphogenetic protein (BMP) signalling. Arkadia physically interacts with inhibitory Smad, Smad7, and induces its poly‐ubiquitination and degradation. In contrast to Smurf1, which interacts with TGF‐β receptor complexes through Smad7 and degrades them, Arkadia fails to associate with TGF‐β receptors. In contrast to Smad7, expression of Arkadia is down‐regulated by TGF‐β. Silencing of the Arkadia gene resulted in repression of transcriptional activities induced by TGF‐β and BMP, and accumulation of the Smad7 protein. Arkadia may thus play an important role as an amplifier of TGF‐β superfamily signalling under both physiological and pathological conditions.

The N domain of Smad7 is essential for specific inhibition of transforming growth factor-β signaling

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-β (TGF-β) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-β and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-β signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-β and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-β signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-β type I receptor (TβR-I) more efficiently, and were more potent in repressing TGF-β signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-β receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-β signaling.

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)

Estrogen Inhibits Transforming Growth Factor β Signaling by Promoting Smad2/3 Degradation

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERα and ERβ, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-β acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERα and TGF-β/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERα inhibits TGF-β signaling by decreasing Smad protein levels. ERα-mediated reductions in Smad levels did not require the DNA binding ability of ERα, implying that ERα opposes the effects of TGF-β via a novel non-genomic mechanism. Our analysis revealed that ERα formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERα.

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.

Smurf2 Induces Ubiquitin-dependent Degradation of Smurf1 to Prevent Migration of Breast Cancer Cells

Ubiquitin-dependent protein degradation is involved in various biological processes, and accumulating evidence suggests that E3 ubiquitin ligases play important roles in cancer development. Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf2 are E3 ubiquitin ligases, which suppress transforming growth factor-β (TGF-β) family signaling through degradation of Smads and receptors for TGF-β and bone morphogenetic proteins. In addition, Smurf1 has been reported to promote RhoA ubiquitination and degradation and regulate cell motility, suggesting the involvement of Smurf1 in cancer progression. However, the regulation and biological function of Smurf1 and Smurf2 in cancer development remain to be elucidated. In the present study, we show the post-translational regulation of Smurf1 by Smurf2 and the functional differences between Smurf1 and Smurf2 in the progression of breast cancer cells. Smurf2 interacted with Smurf1 and induced its ubiquitination and degradation, whereas Smurf1 failed to induce degradation of Smurf2. Knockdown of Smurf2 in human breast cancer MDA-MB-231 cells resulted in increases in the levels of Smurf1 protein, and enhancement of cell migration in vitro and bone metastasis in vivo. Of note, knockdown of Smurf1, but not of Smurf2, enhanced TGF-β signaling in MDA-MB-231 cells, suggesting that increased an protein level of Smurf1 offsets the effect of Smurf2 knockdown on TGF-β signaling. These results indicate that two related E3 ubiquitin ligases, Smurf1 and Smurf2, act in the same direction in TGF-β family signaling but play opposite roles in cell migration.

SKI and MEL1 Cooperate to Inhibit Transforming Growth Factor-β Signal in Gastric Cancer Cells

Chromosomal amplification occurs frequently in solid tumors and is associated with poor prognosis. Several reports demonstrated the cooperative effects of oncogenic factors in the same amplicon during cancer development. However, the functional correlation between the factors remains unclear. Transforming growth factor (TGF)-β signaling plays important roles in cytostasis and normal epithelium differentiation, and alterations in TGF-β signaling have been identified in many malignancies. Here, we demonstrated that transcriptional co-repressors of TGF-β signaling, SKI and MDS1/EVI1-like gene 1 (MEL1), were aberrantly expressed in MKN28 gastric cancer cells by chromosomal co-amplification of 1p36.32. SKI and MEL1 knockdown synergistically restored TGF-β responsiveness in MKN28 cells and reduced tumor growth in vivo. MEL1 interacted with SKI and inhibited TGF-β signaling by stabilizing the inactive Smad3-SKI complex on the promoter of TGF-β target genes. These findings reveal a novel mechanism where distinct transcriptional co-repressors are co-amplified and functionally interact, and provide molecular targets for gastric cancer treatment.