Kazunobu Isogaya

Arkadia Induces Degradation of SnoN and c-Ski to Enhance Transforming Growth Factor-β Signaling

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators that target either signaling receptors or activated Smad complexes. Among the negative regulators, Smad7 antagonizes TGF-β signaling mainly through targeting the signaling receptors, whereas SnoN and c-Ski repress signaling at the transcriptional level through inactivation of Smad complexes. We previously found that Arkadia is a positive regulator of TGF-β signaling that induces ubiquitin-dependent degradation of Smad7 through its C-terminal RING domain. We report here that Arkadia induces degradation of SnoN and c-Ski in addition to Smad7. Arkadia interacts with SnoN and c-Ski in their free forms as well as in the forms bound to Smad proteins, and constitutively down-regulates levels of their expression. Arkadia thus appears to effectively enhance TGF-β signaling through simultaneous down-regulation of two distinct types of negative regulators, Smad7 and SnoN/c-Ski, and may play an important role in determining the intensity of TGF-β family signaling in target cells.

An Id‐like molecule, HHM, is a synexpression group‐restricted regulator of TGF‐β signalling

Transforming growth factor (TGF)‐β induces various cellular responses principally through Smad‐dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad‐cofactor complexes are denoted a synexpression group. We found that an Id‐like helix‐loop‐helix protein, human homologue of Maid (HHM), is a synexpression group‐restricted regulator of TGF‐β signalling. HHM suppressed TGF‐β‐induced growth inhibition and cell migration but not epithelial—mesenchymal transition. In addition, HHM inhibited TGF‐β‐induced expression of plasminogen activator inhibitor‐type 1 (PAI‐1), PDGF‐B, and p21WAF, but not Snail. We identified a basic‐helix‐loop‐helix protein, Olig1, as one of the Smad‐binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF‐β stimulation, and was involved in transcriptional activation of PAI‐1 and PDGF‐B. HHM, but not Id proteins, inhibited TGF‐β signalling‐dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF‐β target genes including the Olig1‐Smad synexpression group. HHM is the first example of a cellular response‐selective regulator of TGF‐β signalling with clearly determined mechanisms.

A Smad3 and TTF-1/NKX2-1 complex regulates Smad4-independent gene expression

Thyroid transcription factor-1 (TTF-1, also known as NKX2-1) is a tissue-specific transcription factor in lung epithelial cells. Although TTF-1 inhibits the epithelial-to-mesenchymal transition induced by transforming growth factor-β (TGF-β) in lung adenocarcinoma cells, the mechanism through which TTF-1 inhibits the functions of TGF-β is unknown. Here we show that TTF-1 disrupts the nuclear Smad3-Smad4 complex without affecting the nuclear localization of phospho-Smad3. Genome-wide analysis by chromatin immunoprecipitation followed by sequencing revealed that TTF-1 colocalizes with Smad3 on chromatin and alters Smad3-binding patterns throughout the genome, while TTF-1 generally inhibits Smad4 binding to chromatin. Moreover, Smad3 binds to chromatin together with TTF-1, but not with Smad4, at some Smad3-binding regions when TGF-β signaling is absent, and knockdown of Smad4 expression does not attenuate Smad3 binding in these regions. Thus, TTF-1 may compete with Smad4 for interaction with Smad3, and in the presence of TTF-1, Smad3 regulates the transcription of certain genes independently of Smad4. These findings provide a new model of regulation of TGF-β-Smad signaling by TTF-1.

RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor growth in lung adenocarcinoma.

RNA-binding proteins provide a new layer of posttranscriptional regulation of RNA during cancer progression. We identified RNA-binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-β in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition. TGF-β repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin 3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-β. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor-suppressive roles for RBM47 through the inhibition of Nrf2 activity.RNA-binding proteins provide a new layer of posttranscriptional regulation of RNA during cancer progression. We identified RNA-binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-β in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition. TGF-β repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin 3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-β. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor-suppressive roles for RBM47 through the inhibition of Nrf2 activity.

Oligodendrocyte Transcription Factor 1 (Olig1) Is a Smad Cofactor Involved in Cell Motility Induced by Transforming Growth Factor-β

Background: To date, the Smad cofactor involved in cell motility induced by transforming growth factor-β (TGF-β) has not been identified. Results: Knockdown of oligodendrocyte transcription factor-1 (Olig1), as well as inhibition of the Olig1-Smad interaction, resulted in attenuation of TGF-β-induced cell motility. Conclusion: Olig1 is involved in TGF-β-induced cell motility. Significance: This study enhances understanding of the regulation of TGF-β-induced cell motility. Transforming growth factor (TGF)-β plays crucial roles in embryonic development and adult tissue homeostasis by eliciting various cellular responses in target cells. TGF-β signaling is principally mediated through receptor-activated Smad proteins, which regulate expression of target genes in cooperation with other DNA-binding transcription factors (Smad cofactors). In this study, we found that the basic helix-loop-helix transcription factor Olig1 is a Smad cofactor involved in TGF-β-induced cell motility. Knockdown of Olig1 attenuated TGF-β-induced cell motility in chamber migration and wound healing assays. In contrast, Olig1 knockdown had no effect on bone morphogenetic protein-induced cell motility, TGF-β-induced cytostasis, or epithelial-mesenchymal transition. Furthermore, we observed that cooperation of Smad2/3 with Olig1 is regulated by a peptidyl-prolyl cis/trans-isomerase, Pin1. TGF-β-induced cell motility, induction of Olig1-regulated genes, and physical interaction between Smad2/3 and Olig1 were all inhibited after knockdown of Pin1, indicating a novel mode of regulation of Smad signaling. We also found that Olig1 interacts with the L3 loop of Smad3. Using a synthetic peptide corresponding to the L3 loop of Smad3, we succeeded in selectively inhibiting TGF-β-induced cell motility. These findings may lead to a new strategy for selective regulation of TGF-β-induced cellular responses.

Identification of a phosphorylation site in c-Ski as serine 515

c-Ski has been known to be phosphorylated at serine residue(s), which results in slower migration of c-Ski in SDS-polyacrylamide gel electrophoresis. The position(s) of phosphorylation, however, has not been determined. In the present study, we identified a phosphorylation site of c-Ski which affects its electrophoretic motility as serine 515 using MALDI-TOF mass spectrometry. A phosphorylation-resistant mutant, c-Ski S515A, did not exhibit a phosphatase-sensitive band shift. In addition, we confirmed that endogenous c-Ski is phosphorylated at serine 515, using a specific antibody. The phosphorylation status of c-Ski, however, does not appear to affect its stability or effects on TGF-β signalling. Identification of the phosphorylation site of c-Ski would allow us further examination of physiological significance of c-Ski phosphorylation.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix‐loop‐helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant‐negative HLH (dnHLH) proteins lack DNA‐binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho‐physiological processes. We report the first structure of a free‐standing human dnHLH protein, HHM (Human homologue of murine maternal Id‐like molecule). HHM adopts a V‐shaped conformation, with N‐terminal and C‐terminal five‐helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N‐ and C‐terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V‐shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V‐shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V‐shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

TUFT1 interacts with RABGAP1 and regulates mTORC1 signaling

The mammalian target of rapamycin (mTOR) pathway is commonly activated in human cancers. The activity of mTOR complex 1 (mTORC1) signaling is supported by the intracellular positioning of cellular compartments and vesicle trafficking, regulated by Rab GTPases. Here we showed that tuftelin 1 (TUFT1) was involved in the activation of mTORC1 through modulating the Rab GTPase-regulated process. TUFT1 promoted tumor growth and metastasis. Consistently, the expression of TUFT1 correlated with poor prognosis in lung, breast and gastric cancers. Mechanistically, TUFT1 physically interacted with RABGAP1, thereby modulating intracellular lysosomal positioning and vesicular trafficking, and promoted mTORC1 signaling. In addition, expression of TUFT1 predicted sensitivity to perifosine, an alkylphospholipid that alters the composition of lipid rafts. Perifosine treatment altered the positioning and trafficking of cellular compartments to inhibit mTORC1. Our observations indicate that TUFT1 is a key regulator of the mTORC1 pathway and suggest that it is a promising therapeutic target or a biomarker for tumor progression.

Dynamics of chromatin accessibility during TGF- β- induced EMT of Ras-transformed mammary gland epithelial cells

Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor (TGF)-β and facilitates tumor progression. We here performed global mapping of accessible chromatin in the mouse mammary gland epithelial EpH4 cell line and its Ras-transformed derivative (EpRas) using formaldehyde-assisted isolation of regulatory element (FAIRE)-sequencing. TGF-β and Ras altered chromatin accessibility either cooperatively or independently, and AP1, ETS, and RUNX binding motifs were enriched in the accessible chromatin regions of EpH4 and EpRas cells. Etv4, an ETS family oncogenic transcription factor, was strongly expressed and bound to more than one-third of the accessible chromatin regions in EpRas cells treated with TGF-β. While knockdown of Etv4 and another ETS family member Etv5 showed limited effects on the decrease in the E-cadherin abundance and stress fiber formation by TGF-β, gene ontology analysis showed that genes encoding extracellular proteins were most strongly down-regulated by Etv4 and Etv5 siRNAs. Accordingly, TGF-β-induced expression of Mmp13 and cell invasiveness were suppressed by Etv4 and Etv5 siRNAs, which were accompanied by the reduced chromatin accessibility at an enhancer region of Mmp13 gene. These findings suggest a mechanism of transcriptional regulation during Ras- and TGF-β-induced EMT that involves alterations of accessible chromatin, which are partly regulated by Etv4 and Etv5.

Identification of a novel fusion gene HMGA2-EGFR in glioblastoma: HMGA2-EGFR in glioblastoma progression

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA‐seq data from TGS‐01 human glioma‐initiating cells, and identified a novel fusion gene (HMGA2‐EGFR), encoding a protein comprising the N‐terminal region of the high‐mobility group AT‐hook protein 2 (HMGA2) fused to the C‐terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor‐forming capacity in cell culture and in vivo. Mechanistically, HMGA2‐EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in‐frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2‐EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS‐01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in‐frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma.