RESUMO
Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.
Assuntos
Cognição/fisiologia , Suturas Cranianas/fisiopatologia , Craniossinostoses/fisiopatologia , Regeneração/fisiologia , Crânio/fisiopatologia , Animais , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Craniossinostoses/genética , Dura-Máter/patologia , Dura-Máter/fisiopatologia , Gelatina/farmacologia , Perfilação da Expressão Gênica , Força da Mão , Pressão Intracraniana/efeitos dos fármacos , Pressão Intracraniana/fisiologia , Locomoção/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Metacrilatos/farmacologia , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Crânio/patologia , Proteína 1 Relacionada a Twist/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Padronização Corporal/efeitos da radiação , Proteínas de Drosophila/genética , Embrião não Mamífero , Luz , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteólise/efeitos da radiação , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.
Assuntos
Carcinogênese , Desenvolvimento Muscular , Proteína MyoD/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Células Cultivadas , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Transição Epitelial-Mesenquimal , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Sequências Hélice-Alça-Hélice , Humanos , Proteína MyoD/química , Mioblastos/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/química , Proteína 1 Relacionada a Twist/químicaRESUMO
Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis have overlooked transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual regulatory element-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. The glucocorticoid receptor activates transcription by inducing RNA polymerase pause release, whereas SP and AP-1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome Twist2 -/- patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.
Assuntos
Adipócitos , Redes Reguladoras de Genes , Proteína 1 Relacionada a Twist , Animais , Camundongos , Linhagem Celular , Adipócitos/citologia , Adipócitos/metabolismo , Fatores de Transcrição/metabolismo , Adipogenia , Transcrição Gênica , Elementos Reguladores de Transcrição , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Tumor metastasis is the major cause of breast cancer morbidity and mortality. It has been reported that the F-box protein FBXO3 functions as an E3 ubiquitin ligase in regulating various biological processes, including host autoimmune, antiviral innate immunity, and inflammatory response. However, the role of FBXO3 in tumor metastasis remains elusive. We have previously shown that ΔNp63α is a common inhibitory target in oncogene-induced cell motility and tumor metastasis. In this study, we show that FBXO3 plays a vital role in PI3K-mediated breast cancer metastasis independent of its E3 ligase activity and ΔNp63α in breast cancer cells and in mouse. FBXO3 can bind to and stabilize USP4, leading to Twist1 protein stabilization and increased breast cancer cell migration and tumor metastasis. Mechanistically, FBXO3 disrupts the interaction between USP4 and aspartyl aminopeptidase (DNPEP), thereby protecting USP4 from DNPEP-mediated degradation. Furthermore, p110αH1047R facilitates the phosphorylation and stabilization of FBXO3 in an ERK1-dependent manner. Knockdown of either FBXO3 or USP4 leads to significant inhibition of PI3K-induced breast cancer metastasis. Clinically, elevated expression of p110α/FBXO3/USP4/Twist1 is associated with poor overall survival (OS) and recurrence-free survival (RFS) of breast cancer patients. Taken together, this study reveals that the FBXO3-USP4-Twist1 axis is pivotal in PI3K-mediated breast tumor metastasis and that FBXO3/USP4 may be potential therapeutic targets for breast cancer treatment.
Assuntos
Neoplasias da Mama , Melanoma , Neoplasias Cutâneas , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Fosfatidilinositol 3-Quinases/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Global energy balance in mammals is controlled by the actions of circulating hormones that coordinate fuel production and utilization in metabolically active tissues. Bone-derived osteocalcin, in its undercarboxylated, hormonal form, regulates fat deposition and is a potent insulin secretagogue. Here, we show that insulin receptor (IR) signaling in osteoblasts controls osteoblast development and osteocalcin expression by suppressing the Runx2 inhibitor Twist2. Mice lacking IR in osteoblasts have low circulating undercarboxylated osteocalcin and reduced bone acquisition due to decreased bone formation and deficient numbers of osteoblasts. With age, these mice develop marked peripheral adiposity and hyperglycemia accompanied by severe glucose intolerance and insulin resistance. The metabolic abnormalities in these mice are improved by infusion of undercarboxylated osteocalcin. These results indicate the existence of a bone-pancreas endocrine loop through which insulin signaling in the osteoblast ensures osteoblast differentiation and stimulates osteocalcin production, which in turn regulates insulin sensitivity and pancreatic insulin secretion.
Assuntos
Osteoblastos/metabolismo , Osteogênese , Receptor de Insulina/metabolismo , Adiposidade , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Resistência à Insulina , Masculino , Camundongos , Osteoblastos/citologia , Osteocalcina/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.
Assuntos
Núcleo Celular/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Protozoários/metabolismo , RNA Interferente Pequeno/metabolismo , Tetrahymena thermophila/metabolismo , Sequência de Aminoácidos , Conjugação Genética , Citoplasma/metabolismo , Proteínas de Protozoários/química , Tetrahymena thermophila/citologia , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Genome wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) associated with the risk of common disorders. However, since the large majority of these risk SNPs reside outside gene-coding regions, GWAS generally provide no information about causal mechanisms regarding the specific gene(s) that are affected or the tissue(s) in which these candidate gene(s) exert their effect. The 'gold standard' method for understanding causal genes and their mechanisms of action are laborious basic science studies often involving sophisticated knockin or knockout mouse lines, however, these types of studies are impractical as a high-throughput means to understand the many risk variants that cause complex diseases like coronary artery disease (CAD). As a solution, we developed a streamlined, data-driven informatics pipeline to gain mechanistic insights on complex genetic loci. The pipeline begins by understanding the SNPs in a given locus in terms of their relative location and linkage disequilibrium relationships, and then identifies nearby expression quantitative trait loci (eQTLs) to determine their relative independence and the likely tissues that mediate their disease-causal effects. The pipeline then seeks to understand associations with other disease-relevant genes, disease sub-phenotypes, potential causality (Mendelian randomization), and the regulatory and functional involvement of these genes in gene regulatory co-expression networks (GRNs). Here, we applied this pipeline to understand a cluster of SNPs associated with CAD within and immediately adjacent to the gene encoding HDAC9. Our pipeline demonstrated, and validated, that this locus is causal for CAD by modulation of TWIST1 expression levels in the arterial wall, and by also governing a GRN related to metabolic function in skeletal muscle. Our results reconciled numerous prior studies, and also provided clear evidence that this locus does not govern HDAC9 expression, structure or function. This pipeline should be considered as a powerful and efficient way to understand GWAS risk loci in a manner that better reflects the highly complex nature of genetic risk associated with common disorders.
Assuntos
Doença da Artéria Coronariana , Estudo de Associação Genômica Ampla , Proteína 1 Relacionada a Twist/metabolismo , Animais , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Histona Desacetilases/metabolismo , Desequilíbrio de Ligação , Camundongos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Proteínas Repressoras/metabolismoRESUMO
Synovial chondromatosis (SC) is a disorder of the synovium characterized by the formation of osteochondral nodules within the synovium. This study aimed to identify the abnormally differentiated progenitor cells and possible pathogenic signaling pathways. Loose bodies and synovium were obtained from patients with SC during knee arthroplasty. Single-cell RNA sequencing was used to identify cell subsets and their gene signatures in SC synovium. Cells derived from osteoarthritis (OA) synovium were used as controls. Multi-differentiation and colony-forming assays were used to identify progenitor cells. The roles of transcription factors and signaling pathways were investigated through computational analysis and experimental verification. We identified an increased proportion of CD34+ sublining fibroblasts in SC synovium. CD34+CD31- cells and CD34-CD31- cells were sorted from SC synovium. Compared with CD34- cells, CD34+ cells had larger alkaline phosphatase (ALP)-stained area and calcified area after osteogenic induction. In addition, CD34+ cells exhibited a stronger tube formation ability than CD34- cells. Our bioinformatic analysis suggested the expression of TWIST1, a negative regulator of osteogenesis, in CD34- sublining fibroblasts and was regulated by the TGF-ß signaling pathway. The experiment showed that CD34+ cells acquired the TWIST1 expression during culture and the combination of TGF-ß1 and harmine, an inhibitor of Twist1, could further stimulate the osteogenesis of CD34+ cells. Overall, CD34+ synovial fibroblasts in SC synovium have multiple differentiation potentials, especially osteogenic differentiation potential, and might be responsible for the pathogenesis of SC.
Assuntos
Antígenos CD34 , Condromatose Sinovial , Fibroblastos , Osteogênese , Membrana Sinovial , Humanos , Antígenos CD34/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Condromatose Sinovial/patologia , Condromatose Sinovial/metabolismo , Membrana Sinovial/patologia , Membrana Sinovial/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Diferenciação Celular , Idoso , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteínas NuclearesRESUMO
Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high-confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1-dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1-regulated genes identified herein may be prioritized for future mechanistic and functional studies.
Assuntos
Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/metabolismo , Biomarcadores/metabolismo , Matriz Extracelular/metabolismo , Ligação Proteica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development and progression of many cancers. Partial EMT (pEMT) could represent a critical step in tumor migration and dissemination. Sarcomatoid renal cell carcinoma (sRCC) is an aggressive form of renal cell carcinoma (RCC) composed of a carcinomatous (sRCC-Ca) and sarcomatous (sRCC-Sa) component. The role of (p)EMT in the progression of RCC to sRCC remains unclear. The aim of this study was to investigate the involvement of (p)EMT in RCC and sRCC. Tissue samples from 10 patients with clear cell RCC (ccRCC) and 10 patients with sRCC were selected. The expression of main EMT markers (miR-200 family, miR-205, SNAI1/2, TWIST1/2, ZEB1/2, CDH1/2, VIM) was analyzed by qPCR in ccRCC, sRCC-Ca, and sRCC-Sa and compared to non-neoplastic tissue and between both groups. Expression of E-cadherin, N-cadherin, vimentin and ZEB2 was analyzed using immunohistochemistry. miR-200c was downregulated in sRCC-Ca compared to ccRCC, while miR-200a was downregulated in sRCC-Sa compared to ccRCC. CDH1 was downregulated in sRCC-Sa when compared to any other group. ZEB2 was downregulated in ccRCC and sRCC compared to corresponding non-neoplastic kidney. A positive correlation was observed between CDH1 expression and miR-200a/b/c. Our results suggest that full EMT is not present in sRCC. Instead, discreet molecular differences exist between ccRCC, sRCC-Ca, and sRCC-Sa, possibly representing distinct intermediary states undergoing pEMT.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Renais , Transição Epitelial-Mesenquimal , Neoplasias Renais , MicroRNAs , Vimentina , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Humanos , Transição Epitelial-Mesenquimal/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , MicroRNAs/genética , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Vimentina/metabolismo , Vimentina/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Idoso , Caderinas/genética , Caderinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Antígenos CD/genética , Antígenos CD/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/metabolismo , Adulto , Proteínas NuclearesRESUMO
BACKGROUND: Although Huaier granules can be used as prospective anti-cholangiocarcinoma drugs, the mechanism of action of Huaier granules in cholangiocarcinoma is not clear. The anti-cholangiocarcinoma effect of Huaier granules was validated in cell line research. In vitro experiments were conducted to investigate the signalling pathways affected by Huaier in CCA cells. METHODS AND RESULTS: Real-time quantitative PCR (RTâqPCR) and Western blot analysis were performed to analyse gene expression in CCA cells. MTT assays, scratch tests, and Transwell assays were used to explore the effects on the proliferation and metastasis of CCA cells. Chromatin immunoprecipitation assays were performed to reveal the potential underlying mechanisms involved. Twist1 was upregulated in human CCA tissues. In addition, its expression levels were negatively related to FBP1 expression levels. Mechanistically, Twist1 can bind to the region of the FBP1 promoter to reduce its expression. Huaier plays an indispensable role in suppressing Twist1 expression to inhibit the Twist1/FBP1/Wnt/ß-catenin axis. Then, we verified the effect of Huaier in vitro. CONCLUSIONS: These findings suggested that Huaier granules were capable of inhibiting CCA development through regulating the Twist1/FBP1/Wnt/ß-catenin signalling axis and provided a novel orientation for the development of novel anti-CCA drugs.
Assuntos
Neoplasias dos Ductos Biliares , Proliferação de Células , Colangiocarcinoma , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares , Proteína 1 Relacionada a Twist , Via de Sinalização Wnt , beta Catenina , Humanos , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Colangiocarcinoma/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Linhagem Celular Tumoral , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , beta Catenina/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genéticaRESUMO
The transcriptional coactivator PGC-1alpha promotes mitochondrial biogenesis and thermogenic programs in brown adipose tissue. Pan et al. (2009) identify the transcription factor twist-1 as a negative feedback regulator of PGC-1alpha.
Assuntos
Tecido Adiposo Marrom/metabolismo , Metabolismo Energético , Proteína 1 Relacionada a Twist/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , PPAR gama/metabolismo , Transativadores/metabolismoRESUMO
Brown fat is specialized for energy expenditure, a process that is principally controlled by the transcriptional coactivator PGC-1alpha. Here, we describe a molecular network important for PGC-1alpha function and brown fat metabolism. We find that twist-1 is selectively expressed in adipose tissue, interacts with PGC-1alpha, and is recruited to the promoters of PGC-1alpha's target genes to suppress mitochondrial metabolism and uncoupling. In vivo, transgenic mice expressing twist-1 in the adipose tissue are prone to high-fat-diet-induced obesity, whereas twist-1 heterozygous knockout mice are obesity resistant. These phenotypes are attributed to their altered mitochondrial metabolism in the brown fat. Interestingly, the nuclear receptor PPARdelta not only mediates the actions of PGC-1alpha but also regulates twist-1 expression, suggesting a negative-feedback regulatory mechanism. These findings reveal an unexpected physiological role for twist-1 in the maintenance of energy homeostasis and have important implications for understanding metabolic control and metabolic diseases.
Assuntos
Tecido Adiposo Marrom/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/genética , Proteína 1 Relacionada a Twist/metabolismo , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Animais , Metabolismo Energético , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Obesidade/metabolismo , PPAR delta/genética , PPAR delta/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
BACKGROUND: Diagnosis of early mycosis fungoides (eMF) is challenging and often delayed as many of its clinical and histopathologic features may mimic various benign inflammatory dermatoses (BIDs). The products of the thymocyte selection-associated high mobility group box (TOX), twist family BHLH transcription factor 1 (TWIST1), signal transducer and activator of transcription 4 (STAT4), and special AT-rich sequence-binding protein 1 (SATB1) genes function as transcription factors and are involved in the pathogenesis of MF. OBJECTIVES: We aim to determine the diagnostic value of TOX, TWIST1, STAT4, and SATB1 protein expressions in eMF. METHODS: This non-randomized, controlled, prospective analytic study was conducted by performing immunohistochemistry staining with TOX, TWIST1, STAT4, and SATB1 polyclonal antibodies in lesional skin biopsies of eMF and BID patients. Nuclear staining of lymphocytes was compared between eMF and BIDs, and the capacity of these antibodies to predict eMF was determined. RESULTS: Immunostainings with anti-TWIST1 showed an increase in protein expression (p = 0.003) and showed a decrease with anti-SATB1 antibodies in eMF compared to BIDs (p = 0.005) while anti-TOX and anti-STAT4 antibodies did not exhibit significant differences (p = 0.384; p = 0.150). Receiver operating characteristic analysis showed that immunohistochemical evaluations of TWIST1 and SATB1 protein expressions can differentiate eMF (area under the curve [AUC]: 0.728, 95% confidence interval [CI]: 0.605-0.851, p = 0.002; AUC: 0.686, 95% CI: 0.565-0.807, p = 0.013). CONCLUSIONS: TWIST1 and SATB1 are potential diagnostic markers for the histologic diagnosis of eMF.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Micose Fungoide , Neoplasias Cutâneas , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Micose Fungoide/patologia , Proteínas Nucleares/metabolismo , Estudos Prospectivos , Neoplasias Cutâneas/patologia , Fator de Transcrição STAT4/metabolismo , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Twist has been shown to cause treatment failure, cancer progression, and cancer-related death. However, strategies that directly target Twist are not yet conceivable. Here we reveal that K63-linked ubiquitination is a crucial regulatory mechanism for Twist activation. Through an E3 ligase screen and biochemical studies, we unexpectedly identified that RNF8 functions as a direct Twist activator by triggering K63-linked ubiquitination of Twist. RNF8-promoted Twist ubiquitination is required for Twist localization to the nucleus for subsequent EMT and CSC functions, thereby conferring chemoresistance. Our histological analyses showed that RNF8 expression is upregulated and correlated with disease progression, EMT features, and poor patient survival in breast cancer. Moreover, RNF8 regulates cancer cell migration and invasion and cancer metastasis, recapitulating the effect of Twist. Together, our findings reveal a previously unrecognized tumor-promoting function of RNF8 and provide evidence that targeting RNF8 is an appealing strategy to tackle tumor aggressiveness and treatment resistance.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Lisina/metabolismo , Células MCF-7 , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Proteína 1 Relacionada a Twist/metabolismo , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Gliomas are one of the most common and lethal brain tumors among adults. One process that contributes to glioma progression and recurrence is the epithelial to mesenchymal transition (EMT). EMT is regulated by a set of defined transcription factors which tightly regulate this process, among them is the basic helix-loop-helix family member, TWIST1. Here we show that TWIST1 is methylated on lysine-33 at chromatin by SETD6, a methyltransferase with expression levels correlating with poor survival in glioma patients. RNA-seq analysis in U251 glioma cells suggested that both SETD6 and TWIST1 regulate cell adhesion and migration processes. We further show that TWIST1 methylation attenuates the expression of the long-non-coding RNA, LINC-PINT, thereby promoting EMT in glioma. Mechanistically, TWIST1 methylation represses the transcription of LINC-PINT by increasing the occupancy of EZH2 and the catalysis of the repressive H3K27me3 mark at the LINC-PINT locus. Under un-methylated conditions, TWIST1 dissociates from the LINC-PINT locus, allowing the expression of LINC-PINT which leads to increased cell adhesion and decreased cell migration. Together, our findings unravel a new mechanistic dimension for selective expression of LINC-PINT mediated by TWIST1 methylation.
Assuntos
Glioma , Proteínas Metiltransferases , RNA Longo não Codificante , Proteína 1 Relacionada a Twist , Humanos , Transição Epitelial-Mesenquimal , Proteínas Nucleares/genética , Proteínas Metiltransferases/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Glioma/metabolismo , Glioma/patologia , RNA Longo não Codificante/metabolismo , Linhagem Celular TumoralRESUMO
A prominent function of TGIF1 is suppression of transforming growth factor beta (TGF-ß) signaling, whose inactivation is deemed instrumental to the progression of pancreatic ductal adenocarcinoma (PDAC), as exemplified by the frequent loss of the tumor suppressor gene SMAD4 in this malignancy. Surprisingly, we found that genetic inactivation of Tgif1 in the context of oncogenic Kras, KrasG12D , culminated in the development of highly aggressive and metastatic PDAC despite de-repressing TGF-ß signaling. Mechanistic experiments show that TGIF1 associates with Twist1 and inhibits Twist1 expression and activity, and this function is suppressed in the vast majority of human PDACs by KrasG12D /MAPK-mediated TGIF1 phosphorylation. Ablating Twist1 in KrasG12D ;Tgif1KO mice completely blunted PDAC formation, providing the proof-of-principle that TGIF1 restrains KrasG12D -driven PDAC through its ability to antagonize Twist1. Collectively, these findings pinpoint TGIF1 as a potential tumor suppressor in PDAC and further suggest that sustained activation of TGF-ß signaling might act to accelerate PDAC progression rather than to suppress its initiation.
Assuntos
Carcinoma Ductal Pancreático/patologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteína 1 Relacionada a Twist/genéticaRESUMO
BACKGROUND: In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. METHODS: We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. RESULTS: TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. CONCLUSIONS: Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.
Assuntos
Fibrose Pulmonar Idiopática , Miofibroblastos , Humanos , Camundongos , Animais , Miofibroblastos/metabolismo , Cromatina , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Fibroblastos/metabolismo , Colágeno/genética , Colágeno/metabolismo , Fibrose , Bleomicina , Fatores de Transcrição/genética , RNA/metabolismo , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Cutaneous squamous cell carcinoma (cSCC) represents an important clinical problem requiring novel approaches for both prevention and treatment. The transcription factor, Twist-related protein 1 (Twist1), has been identified as having a key mechanistic role in the development and progression of cSCC. Studies in relevant mouse models of cSCC have shown that Twist1 regulates epithelial-mesenchymal transition (EMT) and stemness driving progression and metastasis of cSCC. In addition, further research has shown that Twist1 regulates the balance between keratinocyte proliferation and differentiation and therefore impacts earlier stages of cSCC development. Through use of keratinocyte specific Twist1 knockout models, a role for this gene in keratinocyte stem cell homeostasis has been revealed. As a transcription factor, Twist1 regulates a large number of genes both in a positive, as well as a negative manner across several interdependent pathways. Studies in keratinocyte specific knockout models have shown that Twist1 upregulates the expression of genes involved in proliferation, stemness, and EMT while downregulating the expression of genes associated with differentiation. Furthermore, a number of compounds, including naturally occurring compounds, have been identified that target Twist1 and can block its effects in cancer cells and in keratinocytes in vivo. Collectively, the current understanding of Twist1 function in cSCC development and progression suggests that it represents a potential target for prevention and treatment of cSCC.