RESUMO
Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-ß regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-ß. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-ß. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.
Assuntos
DNA , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Fator de Crescimento Transformador beta/metabolismo , Células Hep G2 , DNA/metabolismo , Ligação Proteica , Proteína Smad3/metabolismo , Proteína Smad2/metabolismo , Células A549 , Células HaCaT , Proteínas Smad/metabolismoRESUMO
Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Carcinoma , Infecções por Vírus Epstein-Barr , Neoplasias Gástricas , Humanos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Genoma Viral , Carcinoma/genética , RNA Viral/genética , Microambiente TumoralRESUMO
Transforming growth factor-ß (TGF-ß) signaling promotes cancer progression. In particular, the epithelial-mesenchymal transition (EMT) induced by TGF-ß is considered crucial to the malignant phenotype of cancer cells. Here, we report that the EMT-associated cellular responses induced by TGF-ß 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 ß4 region in the Smad3 MH1 domain is essential for TGF-ß-induced cell motility, but is not essential for other EMT-associated responses including epithelial marker downregulation. TGF-ß was previously reported to enhance cell motility by activating Rac1 via phosphoinositide 3-kinase. Intriguingly, TGF-ß-dependent signaling mediated by Smad3's ß4 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-ß type I receptor contribute cooperatively to sustained Rac1 activation, thereby leading to enhanced cell motility.
Assuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Proteínas Ativadoras de GTPase/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Células A549 , Proteínas Ativadoras de GTPase/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteína Smad3/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
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)-ß 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-ß (TGF-ß) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-ß signaling by RLR ligands with respect to IFN-ß-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-ß, which inhibits the growth of the surrounding cancer cells. In addition, IFN-ß-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-ß 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-ß that suppresses IFN-ß expression is suggested when RLR-mediated cancer treatment is used in TNBC.
Assuntos
Interferon beta/metabolismo , Poli I-C/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/metabolismo , Poli I-C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-ß (TGF-ß) 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-ß comprises three isoforms, TGF-ß1, -ß2, and -ß3, and transduces intracellular signals via TGF-ß type I receptor (TßRI) and TGF-ß type II receptor (TßRII). 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 TßRII (TßRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-ß ligands, but several lines of evidence indicate that TßRII-Fc more effectively traps TGF-ß1 and TGF-ß3 than TGF-ß2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-ß receptor containing both TßRI and TßRII (TßRI-TßRII-Fc) and found that TßRI-TßRII-Fc trapped all TGF-ß isoforms, leading to inhibition of both the TGF-ß signal and TGF-ß-induced EMT of oral cancer cells, whereas TßRII-Fc failed to trap TGF-ß2. Furthermore, we found that TßRI-TßRII-Fc suppresses tumor growth and angiogenesis more effectively than TßRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-ß expression. These results suggest that TßRI-TßRII-Fc may be a promising tool for targeting all TGF-ß isoforms in the TME.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Receptores Fc/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Carcinoma de Células Escamosas/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Receptores Fc/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Microambiente TumoralRESUMO
Smad proteins are transcriptional regulators activated by TGF-ß. 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-ß. 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.
Assuntos
Proteína Smad3/química , Proteína Smad3/metabolismo , Proteína Smad4/química , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Ligação Proteica , Elementos de Resposta , Proteína Smad2/química , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad4/genética , Ativação TranscricionalRESUMO
It is well established that transforming growth factor-ß (TGFß) 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 TGFß 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. TGFß-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 TGFß-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGFß-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 TGFß-induced cellular phenotypes of epithelial cells.
Assuntos
Neoplasias da Mama/genética , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Embrião não Mamífero , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Invasividade Neoplásica , Ligação Proteica , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Peixe-ZebraRESUMO
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.
Assuntos
Ativinas/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Receptores de Estrogênio/metabolismo , Proteína Smad2/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Citostáticos/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Smad2/genéticaRESUMO
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.
Assuntos
Receptores ErbB/genética , Glioblastoma/genética , Proteína HMGA2/genética , Proteínas de Fusão Oncogênica/genética , Idoso , Animais , Linhagem Celular , Linhagem Celular Tumoral , Éxons/genética , Feminino , Amplificação de Genes/genética , Deleção de Genes , Glioma/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação/genética , Transdução de Sinais/genéticaRESUMO
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-ß (TGF-ß) signaling and regulates TGF-ß-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 ß1 as a binding partner of NORAD and found that knockdown of NORAD partially inhibits the physical interaction of importin ß1 with Smad3, inhibiting the nuclear accumulation of Smad complexes in response to TGF-ß. Our findings may provide a new mechanism underlying the function of NORAD in cancer cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Pulmonares/patologia , RNA Longo não Codificante/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células A549 , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Fenótipo , Transdução de SinaisRESUMO
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.
Assuntos
Fatores de Transcrição/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
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)-ß family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-ß and bone morphogenetic proteins. Treatment with TGF-ß attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-ß regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-ß negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.
Assuntos
Aldeído Desidrogenase/biossíntese , Regulação Neoplásica da Expressão Gênica/fisiologia , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Proteína Smad4/metabolismo , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Xenoenxertos , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno , Retinal Desidrogenase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transforming growth factor-ß (TGF-ß) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-ß 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-ß did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-ß 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-ß-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-ß suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-ß 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.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Fator de Transcrição MafK/genética , Fator de Crescimento Transformador beta/farmacologia , Antioxidantes/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células HEK293 , Heme Oxigenase-1/metabolismo , Humanos , Hidroquinonas/farmacologia , Immunoblotting , Fator de Transcrição MafK/metabolismo , Microscopia de Fluorescência , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMO
Transforming growth factor (TGF)-ß exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-ß 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-ß. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-ß 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-ß. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-ß 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-ß of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-ß-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-ß functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-ß signaling.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Linfotoxina-alfa/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Smad7/metabolismo , Animais , Sequência de Bases , Northern Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/genéticaRESUMO
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.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Motivos de Nucleotídeos , Elementos Reguladores de Transcrição , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Elementos Facilitadores Genéticos , Genoma Humano , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/citologia , Receptores Notch/metabolismo , Análise de Sequência de DNA , Proteínas Serrate-JaggedRESUMO
Specific regulation of target genes by transforming growth factor-ß (TGF-ß) 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-ß. 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-ß 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-ß 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-ß and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-ß-Smad signaling pathway.
Assuntos
Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Motivos de Aminoácidos , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Humanos , Especificidade de Órgãos/fisiologia , Ligação Proteica , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Transforming growth factor-ß (TGF-ß) 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-ß signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-ß signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-ß signaling, and knockdown of endogenous RB1CC1 attenuated TGF-ß-induced expression of target genes as well as TGF-ß-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-ß signaling by restricting substrate specificity of Arkadia.
Assuntos
Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Apoptose/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Neoplasias Renais/patologiaRESUMO
Smad proteins transduce signals downstream of transforming growth factor-ß (TGF-ß) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-ß and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-ß induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-ß was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-ß. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-ß-induced MAB21L4 up-regulates the gene expression induced by TGF-ß, possibly through the inhibition of c-Ski via physical interaction in the cytosol.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Queratinócitos/metabolismo , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
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-ß 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.