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Transforming growth factor β (TGF-β) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-β also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-β confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-β, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-β generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-β-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis., Competing Interests: Declaration of interests The authors do have a patent related to this work (patent application number [Japan Patent Office]: P2021-214914)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-β that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-β (TGF-β) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-β signaling by RLR ligands with respect to IFN-β-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-β, which inhibits the growth of the surrounding cancer cells. In addition, IFN-β-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-β expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-β that suppresses IFN-β expression is suggested when RLR-mediated cancer treatment is used in TNBC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor β (TGF-β) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-β family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo . Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Development of innovative therapeutic modalities would address an unmet clinical need in the treatment of triple negative breast cancer (TNBC). Activation of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) such as melanoma differentiation-associated gene 5 (MDA5) and RIG-I in cancer cells is suggested to suppress tumor progression by inducing cell death. Transfection of polyI:C, a conventionally used synthetic double-stranded RNA (dsRNA) analogue that activates RLRs, has been evaluated in clinical trials. However, detailed mechanisms of tumor suppression by RLRs, especially interactions with other signaling pathways, remain elusive. Here, we showed that transfection of polyI:C suppressed transforming growth factor-β (TGF-β) signaling in a MDA5- and RIG-I-dependent manner. We found that suppression of TGF-β signaling by polyI:C promoted cancer cell death, which was attenuated by forced expression of constitutively active Smad3. More detailed analysis suggested that cell death by polyI:C transfection exhibited characteristics of pyroptosis, which is distinct from apoptosis. Therapeutic efficacy of polyI:C transfection was also demonstrated using a mouse model. These results indicated that intratumor administration of polyI:C and related dsRNA analogues may be promising treatments for TNBC through inhibition of the anti-pyroptotic function of TGF-β., (© 2020 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Transforming growth factor-β (TGF-β) signaling promotes cancer progression. In particular, the epithelial-mesenchymal transition (EMT) induced by TGF-β is considered crucial to the malignant phenotype of cancer cells. Here, we report that the EMT-associated cellular responses induced by TGF-β are mediated by distinct signaling pathways that diverge at Smad3. By expressing chimeric Smad1/Smad3 proteins in SMAD3 knockout A549 cells, we found that the β4 region in the Smad3 MH1 domain is essential for TGF-β-induced cell motility, but is not essential for other EMT-associated responses including epithelial marker downregulation. TGF-β was previously reported to enhance cell motility by activating Rac1 via phosphoinositide 3-kinase. Intriguingly, TGF-β-dependent signaling mediated by Smad3's β4 region causes the downregulation of multiple mRNAs that encode GTPase activating proteins that target Rac1 (ARHGAPs), thereby attenuating Rac1 inactivation. Therefore, two independent pathways downstream of TGF-β type I receptor contribute cooperatively to sustained Rac1 activation, thereby leading to enhanced cell motility., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

BMP signaling has been found to have tumor-promoting as well as tumor-suppressing effects in different types of tumors. In this study, we investigated the effects of BMP signaling and of BMP inhibitors on ovarian cancer (OC) cells in vitro and in vivo. High expression of BMP receptor 2 (BMPR2) correlated with poor overall survival of OC patients in the TCGA dataset. Both BMP2 and BMPR2 enhanced OC cell proliferation, whereas BMP receptor kinase inhibitors inhibited OC cell growth in cell culture as well as in a mouse model. BMP2 also augmented sphere formation, migration, and invasion of OC cells, and induced EMT. High BMP2 expression was observed after chemotherapy of OC patients in the GSE109934 dataset. In accordance, carboplatin, used for the treatment of OC patients, increased BMP2 secretion from OC cells, and induced EMT partially via activation of BMP signaling. Our data suggest that BMP signaling has tumor-promoting effects in OC, and that BMP inhibitors might be useful therapeutic agents for OC patients. Considering that carboplatin treatment augmented BMP2 secretion, the possibility to use a combination of BMP inhibitors and carboplatin in the treatment of OC patients, would be worth exploring.

Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-β (TGF-β) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-β comprises three isoforms, TGF-β1, -β2, and -β3, and transduces intracellular signals via TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TβRII (TβRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-β ligands, but several lines of evidence indicate that TβRII-Fc more effectively traps TGF-β1 and TGF-β3 than TGF-β2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-β receptor containing both TβRI and TβRII (TβRI-TβRII-Fc) and found that TβRI-TβRII-Fc trapped all TGF-β isoforms, leading to inhibition of both the TGF-β signal and TGF-β-induced EMT of oral cancer cells, whereas TβRII-Fc failed to trap TGF-β2. Furthermore, we found that TβRI-TβRII-Fc suppresses tumor growth and angiogenesis more effectively than TβRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-β expression. These results suggest that TβRI-TβRII-Fc may be a promising tool for targeting all TGF-β isoforms in the TME., Competing Interests: Conflict of interest—Y. A. is an employee of Nippon Kayaku, Co., Ltd., (© 2020 Takahashi et al.)

Adult bone structural integrity is maintained by remodeling via the coupling of osteoclastic bone resorption and osteoblastic bone formation. Osteocytes or osteoblasts express receptor activator of nuclear factor κ-B ligand (Rankl) or osteoprotegerin (Opg) to promote or inhibit osteoclastogenesis, respectively. Bone morphogenetic protein (BMP) is a potent bone inducer, but its major role in adult bone is to induce osteocytes to upregulate sclerostin (Sost) and increase the Rankl/Opg expression ratio, resulting in promotion of osteoclastogenesis. However, the precise effect of BMP-target gene(s) in osteoblasts on the Rankl/Opg expression ratio remains unclear. In the present study, we identified atonal homolog 8 ( Atoh8 ), which is directly upregulated by the BMP-Smad1 axis in osteoblasts. In vivo, Atoh8 was detected in osteoblasts but not osteocytes in adult mice. Although global Atoh8-knockout mice showed only a mild phenotype in the neonate skeleton, the bone volume was decreased and osteoclasts were increased in the adult phase. Atoh8-null marrow stroma cells were more potent than wild-type cells in inducing osteoclastogenesis in marrow cells. Atoh8 loss in osteoblasts increased Runx2 expression and the Rankl/Opg expression ratio, while Runx2 knockdown normalized the Rankl/Opg expression ratio. Moreover, Atoh8 formed a protein complex with Runx2 to inhibit Runx2 transcriptional activity and decrease the Rankl/Opg expression ratio. These results suggest that bone remodeling is regulated elaborately by BMP signaling; while BMP primarily promotes bone resorption, it simultaneously induces Atoh8 to inhibit Runx2 and reduce the Rankl/Opg expression ratio in osteoblasts, suppressing osteoclastogenesis and preventing excessive BMP-mediated bone resorption., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Thyroid transcription factor-1 (TTF-1, encoded by the NKX2-1 gene) is highly expressed in small-cell lung carcinoma (SCLC) and lung adenocarcinoma (LADC), but how its functional roles differ between SCLC and LADC remains to be elucidated. Here, we compared the genome-wide distributions of TTF-1 binding regions and the transcriptional programs regulated by TTF-1 between NCI-H209 (H209), a human SCLC cell line, and NCI-H441 (H441), a human LADC cell line, using chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq). TTF-1 binding regions in H209 and H441 cells differed by 75.0% and E-box motifs were highly enriched exclusively in the TTF-1 binding regions of H209 cells. Transcriptome profiling revealed that TTF-1 is involved in neuroendocrine differentiation in H209 cells. We report that TTF-1 and achaete-scute homolog 1 (ASCL1, also known as ASH1, an E-box binding basic helix-loop-helix transcription factor, and a lineage-survival oncogene of SCLC) are coexpressed and bound to adjacent sites on target genes expressed in SCLC, and cooperatively regulate transcription. Furthermore, TTF-1 regulated expression of the Bcl-2 gene family and showed antiapoptotic function in SCLC. Our findings suggest that TTF-1 promotes SCLC growth and contributes to neuroendocrine and antiapoptotic gene expression by partly coordinating with ASCL1., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Smad proteins are transcriptional regulators activated by TGF-β. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis -acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-β. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo ., (© 2019 Itoh et al.)

Cyclin-dependent kinase (CDK) 4 and CDK6 inhibitors are effective therapeutic options for hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. Although CDK4/6 inhibitors mainly target the cyclin D-CDK4/6-retinoblastoma tumor suppressor protein (RB) axis, little is known about the clinical impact of inhibiting phosphorylation of other CDK4/6 target proteins. Here, we focused on other CDK4/6 targets, SMAD proteins. We showed that a CDK4/6 inhibitor palbociclib and activin-SMAD2 signaling cooperatively inhibited cell cycle progression of a luminal-type breast cancer cell line T47D. Palbociclib enhanced SMAD2 binding to the genome by inhibiting CDK4/6-mediated linker phosphorylation of the SMAD2 protein. We also showed that cyclin G2 plays essential roles in SMAD2-dependent cytostatic response. Moreover, comparison of the SMAD2 ChIP-seq data of T47D cells with those of Hs578T (triple-negative breast cancer cells) indicated that palbociclib augmented different SMAD2-mediated functions based on cell type, and enhanced SMAD2 binding to the target regions on the genome without affecting its binding pattern. In summary, palbociclib enhances the cytostatic effects of the activin-SMAD2 signaling pathway, whereas it possibly strengthens the tumor-promoting aspect in aggressive breast cancer., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Long noncoding RNAs are involved in a variety of cellular functions. In particular, an increasing number of studies have revealed the functions of long noncoding RNA in various cancers; however, their precise roles and mechanisms of action remain to be elucidated. NORAD, a cytoplasmic long noncoding RNA, is upregulated by irradiation and functions as a potential oncogenic factor by binding and inhibiting Pumilio proteins (PUM1/PUM2). Here, we show that NORAD upregulates transforming growth factor-β (TGF-β) signaling and regulates TGF-β-induced epithelial-to-mesenchymal transition (EMT)-like phenotype, which is a critical step in the progression of lung adenocarcinoma, A549 cells. However, PUM1 does not appear to be involved in this process. We thus focused on importin β1 as a binding partner of NORAD and found that knockdown of NORAD partially inhibits the physical interaction of importin β1 with Smad3, inhibiting the nuclear accumulation of Smad complexes in response to TGF-β. Our findings may provide a new mechanism underlying the function of NORAD in cancer cells., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Tumor microenvironment plays a pivotal role in cancer progression; however, little is known regarding how differences in the microenvironment affect characteristics of cancer cells. Here, we investigated the effects of tumor microenvironment on cancer cells by using mouse tumor models. After three cycles of inoculation and extraction of human pancreatic cancer cells, including SUIT-2 and Panc-1 cells, from tumors, distinct cancer cell lines were established: 3P cells from the pancreas obtained using the orthotopic tumor model and 3sc cells from subcutaneous tissue obtained using the subcutaneous tumor model. On re-inoculation of these cells, the 3sc cells and, more prominently, the 3P cells, exhibited higher tumorigenic activity than the parental cells. The 3P cells specifically exhibited low E-cadherin expression and high invasiveness, suggesting that they were endowed with the highest malignant characteristics. RNA-sequence analysis demonstrated that distinct signaling pathways were activated in each cell line and that the 3P cells acquired a cancer stem cell-like phenotype. Among cancer stem cell-related genes, those specifically expressed in the 3P cells, including NES, may be potential new targets for cancer therapy. The mechanisms underlying the development of highly malignant cancer cell lines were investigated. Individual cell clones within the parental cells varied in tumor-forming ability, indicating the presence of cellular heterogeneity. Moreover, the tumor-forming ability and the gene expression profile of each cell clone were altered after serial orthotopic inoculations. The present study thus suggests that both selection and education processes by tumor microenvironment are involved in the development of highly malignant cancer cells.

It is well established that transforming growth factor-β (TGFβ) switches its function from being a tumor suppressor to a tumor promoter during the course of tumorigenesis, which involves both cell-intrinsic and environment-mediated mechanisms. We are interested in breast cancer cells, in which SMAD mutations are rare and interactions between SMAD and other transcription factors define pro-oncogenic events. Here, we have performed chromatin immunoprecipitation (ChIP)-sequencing analyses which indicate that the genome-wide landscape of SMAD2/3 binding is altered after prolonged TGFβ stimulation. De novo motif analyses of the SMAD2/3 binding regions predict enrichment of binding motifs for activator protein (AP)1 in addition to SMAD motifs. TGFβ-induced expression of the AP1 component JUNB was required for expression of many late invasion-mediating genes, creating a feed-forward regulatory network. Moreover, we found that several components in the WNT pathway were enriched among the late TGFβ-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGFβ-induced breast cancer cell invasion, while inhibition of the WNT pathway reduced this process. Our study thereby helps to explain how accumulation of pro-oncogenic stimuli switches and stabilizes TGFβ-induced cellular phenotypes of epithelial cells.

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., Competing Interests: N.K., K.I., D.K., and K.M. have submitted a patent related to this work to the Japan Patent Office under application no. 2015-089220. The other authors declare that they have no competing interests.

Zinc finger E-box binding protein 1 (ZEB1) and ZEB2 induce epithelial-mesenchymal transition (EMT) and enhance cancer progression. However, the global view of transcriptional regulation by ZEB1 and ZEB2 is yet to be elucidated. Here, we identified a ZEB1-regulated inflammatory phenotype in breast cancer cells using chromatin immunoprecipitation sequencing and RNA sequencing, followed by gene set enrichment analysis (GSEA) of ZEB1-bound genes. Knockdown of ZEB1 and/or ZEB2 resulted in the downregulation of genes encoding inflammatory cytokines related to poor prognosis in patients with cancer, including IL6 and IL8, therefore suggesting that ZEB1 and ZEB2 have similar functions in terms of the regulation of production of inflammatory cytokines. Antibody array and ELISA experiments confirmed that ZEB1 controlled the production of the IL-6 and IL-8 proteins. The secretory proteins regulated by ZEB1 enhanced breast cancer cell proliferation and tumor growth. ZEB1 expression in breast cancer cells also affected the growth of fibroblasts in cell culture, and the accumulation of myeloid-derived suppressor cells in tumors in vivo. These findings provide insight into the role of ZEB1 in the progression of cancer, mediated by inflammatory cytokines, along with the initiation of EMT., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

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.

ELF4 (also known as MEF) is a member of the ETS family of transcription factors. An oncogenic role for ELF4 has been demonstrated in hematopoietic malignancies, but its function in epithelial tumors remains unclear. Here, we show that ELF4 can function as a tumor suppressor and is somatically inactivated in a wide range of human tumors. We identified a missense mutation affecting the transactivation potential of ELF4 in oral squamous cell carcinoma cells. Restoration of the transactivation activity through introduction of wild-type ELF4 significantly inhibited cell proliferation in vitro and tumor xenograft growth. Furthermore, we found that ELF1 and ELF2, closely related transcription factors to ELF4, also exerted antiproliferative effects in multiple cancer cell lines. Mutations in ELF1 and ELF2, as in ELF4, were widespread across human cancers, but were almost all mutually exclusive. Moreover, chromatin immunoprecipitation coupled with high-throughput sequencing revealed ELF4-binding sites in genomic regions adjacent to genes related to cell-cycle regulation and apoptosis. Finally, we provide mechanistic evidence that the antiproliferative effects of ELF4 were mediated through the induction of HRK, an activator of apoptosis, and DLX3, an inhibitor of cell growth. Collectively, our findings reveal a novel subtype of human cancer characterized by inactivating mutations in the ELF subfamily of proteins, and warrant further investigation of the specific settings where ELF restoration may be therapeutically beneficial. Cancer Res; 76(7); 1814-24. ©2016 AACR., (©2016 American Association for Cancer Research.)

Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor-β (TGF-β) induces apoptosis in many types of cancer cells and acts as a tumor suppressor. We performed a functional analysis of TGF-β signaling to identify a molecular mechanism that regulated survival in small cell lung cancer cells. Here, we found low expression of TGF-β type II receptor (TβRII) in most small cell lung cancer cells and tissues compared to normal lung epithelial cells and normal lung tissues, respectively. When wild-type TβRII was overexpressed in small cell lung cancer cells, TGF-β suppressed cell growth in vitro and tumor formation in vivo through induction of apoptosis. Components of polycomb repressive complex 2, including enhancer of zeste 2 (EZH2), were highly expressed in small cell lung cancer cells; this led to epigenetic silencing of TβRII expression and suppression of TGF-β-mediated apoptosis. Achaete-scute family bHLH transcription factor 1 (ASCL1; also known as ASH1), a Smad-dependent target of TGF-β, was found to induce survival in small cell lung cancer cells. Thus, EZH2 promoted small cell lung cancer progression by suppressing the TGF-β-Smad-ASCL1 pathway.

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor (TGF)-β exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-β is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-β. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-β in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-β. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-β type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-β of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-β-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-β functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-β signaling., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

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.

Transforming growth factor-β (TGF-β) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-β suppresses the transcriptional activation of the heme oxygenase-1 (HO-1) gene, which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor Nrf2. TGF-β did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-β induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-β-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-β suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-β is activated after tissue injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.

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.

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.

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) and pulmonary arterial smooth muscle cells (PASMCs) are implicated in human genetic disorders. Here, we generated genome-wide maps of Smad1/5 binding sites in ECs and PASMCs. Smad1/5 preferentially bound to the region outside the promoter of known genes, and the binding was associated with target gene upregulation. Cell-selective Smad1/5 binding patterns appear to be determined mostly by cell-specific differences in baseline chromatin accessibility patterns. We identified, for the first time, a Smad1/5 binding motif in mammals, and termed GC-rich Smad binding element (GC-SBE). Several sequences in the identified GC-SBE motif had relatively weak affinity for Smad binding, and were enriched in cell type-specific Smad1/5 binding regions. We also found that both GC-SBE and the canonical SBE affect binding affinity for the Smad complex. Furthermore, we characterized EC-specific Smad1/5 target genes and found that several Notch signaling pathway-related genes were induced by BMP in ECs. Among them, a Notch ligand, JAG1 was regulated directly by Smad1/5, transactivating Notch signaling in the neighboring cells. These results provide insights into the molecular mechanism of BMP signaling and the pathogenesis of vascular lesions of certain genetic disorders, including hereditary hemorrhagic telangiectasia.

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.

Specific regulation of target genes by transforming growth factor-β (TGF-β) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In this study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and we compared them with those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-β. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells but not in HaCaT cells, and the HNF4α-binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. Chromatin immunoprecipitation sequencing analysis of HNF4α binding regions under TGF-β stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4α bindings. MIXL1 was identified as a new combinatorial target of HNF4α and Smad2/3, and both the HNF4α protein and its binding motif were required for the induction of MIXL1 by TGF-β in HepG2 cells. These findings generalize the importance of binding of HNF4α on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-β and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-β-Smad signaling pathway.

Smad4, the common partner Smad, is a key molecule in transforming growth factor-beta (TGF-beta) family signaling. Loss of Smad4 expression is found in several types of cancer, including pancreatic cancer and colon cancer, and is related to carcinogenesis. Here we identified Smad4 binding sites in the promoter regions of over 25 500 known genes by chromatin immunoprecipitation on a microarray (ChIP-chip) in HaCaT human keratinocytes. We identified 925 significant Smad4 binding sites. Approximately half of the identified sites overlapped the binding regions of Smad2 and Smad3 (Smad2/3, receptor-regulated Smads in TGF-beta signaling), while the rest of the regions appeared dominantly occupied by Smad4 even when a different identification threshold for Smad2/3 binding regions was used. Distribution analysis showed that Smad4 was found in the regions relatively distant from the transcription start sites, while Smad2/3 binding regions were more often present near the transcription start sites. Motif analysis also revealed that activator protein 1 (AP-1) sites were especially enriched in the sites common to Smad2/3 and Smad4 binding regions. In contrast, GC-rich motifs were enriched in Smad4-dominant binding regions. We further determined putative target genes of Smad4 whose expression was regulated by TGF-beta. Our findings revealed some general characteristics of Smad4 binding regions, and provide resources for examining the role of Smad4 in epithelial cells and cancer pathogenesis.