RESUMO
How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.
Assuntos
Apresentação de Antígeno/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/imunologia , Oncogenes , RNA Longo não Codificante/genética , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Adenoma/genética , Adenoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
Assuntos
Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Tonsila Palatina/imunologia , Animais , Antígenos CD34/metabolismo , Antígeno CD56/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Ativação Linfocitária , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
The 2i-media, composed of two small molecule inhibitors (PD0325901 and CHIR99021) against MEK and GSK3-kinases, respectively, is known to establish naïve ground state pluripotency in mouse embryonic stem cells (mESCs). These inhibitors block MEK-mediated differentiation, while driving ß-catenin dependent de-repression of pluripotency promoting targets. However, accumulating evidence suggest that ß-catenin's association with activating TCFs (TCF7 and TCF7L2) can induce expression of several lineage-specific prodifferentiation genes. We posited that CHIR-induced upregulation of ß-catenin levels could therefore compromise the stability of the naïve state in long-term cultures. Here, we investigated whether replacing CHIR with iCRT3, a small molecule that abrogates ß-catenin-TCF interaction, can still retain ground state pluripotency in mESCs. Our data suggests that iCRT3 + PD mediated coinhibition of MEK and ß-catenin/TCF-dependent transcriptional activity over multiple passages significantly reduces expression of differentiation markers, as compared to 2i. Furthermore, the ability to efficiently contribute toward chimera generation and germline transmission suggests that the inhibition of ß-catenin's TCF-dependent transcriptional activity, independent of its protein expression level, retains the naïve ground state pluripotency in mESCs. Additionally, growth medium containing iCRT3 + PD can provide an alternative to 2i as a stable culture method. Stem Cells 2017;35:1924-1933.
Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Benzamidas/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Oxazóis/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/ß-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of ß-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that ß-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of ß-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/ß-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that ß-catenin, but not its transcriptional activity, is central to pluripotency acting through a ß-catenin/Oct4 complex.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/fisiologia , beta Catenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Fator 3 de Transcrição de Octâmero/fisiologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in the fly Drosophila melanogaster is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, we performed genome-wide transcriptional profiling of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, we identified 23 genes that have sex-differential transcript levels at all three time points, of which 13 encode transcription factors, a significant enrichment. We focus here on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. We show that, in female genital discs, Dsx activates lz and represses Dr and AP-2. We further show that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes we observe upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. Our time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors.
Assuntos
Padronização Corporal/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Genitália/embriologia , Caracteres Sexuais , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Genitália/metabolismo , Masculino , Organogênese/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan(-/-) cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas do Olho/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Proteínas do Domínio Armadillo/antagonistas & inibidores , Linhagem Celular , Drosophila/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Olho/metabolismo , Proteínas do Olho/genética , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Ligação Proteica , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Fatores de Transcrição/antagonistas & inibidoresRESUMO
BACKGROUND: Exercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy. METHODS: Here, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography-mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof's effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models. RESULTS: We demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity. CONCLUSIONS: Our study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.
Assuntos
Diferenciação Celular/genética , Mutação com Ganho de Função , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Obesidade/terapia , RNA Longo não Codificante/genética , Animais , Biomarcadores , Proteínas de Transporte , Células Cultivadas , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Distrofina/genética , Distrofina/metabolismo , Imunofluorescência/métodos , Terapia Genética , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Obesidade/diagnóstico , Obesidade/etiologia , Obesidade/metabolismo , Fosforilação , Ligação ProteicaRESUMO
The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.
Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias/genética , RNA Longo não Codificante/genética , Acetilgalactosamina , Animais , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Biopterinas/uso terapêutico , Dieta , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Conformação de Ácido Nucleico , Fenilalanina/administração & dosagem , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/metabolismo , Ligação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/uso terapêuticoRESUMO
Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Humanos , Células Matadoras Naturais , Receptores KIR , Análise de Sequência de RNARESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
An improved understanding of the biochemical alterations that accompany tumor progression and metastasis is necessary to inform the next generation of diagnostic tools and targeted therapies. Metabolic reprogramming is known to occur during the epithelial-mesenchymal transition (EMT), a process that promotes metastasis. Here, we identify metabolic enzymes involved in extracellular matrix remodeling that are upregulated during EMT and are highly expressed in patients with aggressive mesenchymal-like breast cancer. Activation of EMT significantly increases production of hyaluronic acid, which is enabled by the reprogramming of glucose metabolism. Using genetic and pharmacological approaches, we show that depletion of the hyaluronic acid precursor UDP-glucuronic acid is sufficient to inhibit several mesenchymal-like properties including cellular invasion and colony formation in vitro, as well as tumor growth and metastasis in vivo. We found that depletion of UDP-glucuronic acid altered the expression of PPAR-gamma target genes and increased PPAR-gamma DNA-binding activity. Taken together, our findings indicate that the disruption of EMT-induced metabolic reprogramming affects hyaluronic acid production, as well as associated extracellular matrix remodeling and represents pharmacologically actionable target for the inhibition of aggressive mesenchymal-like breast cancer progression.
Assuntos
Neoplasias da Mama/patologia , Ácido Hialurônico/biossíntese , Uridina Difosfato Glucose Desidrogenase/metabolismo , Animais , Mama/patologia , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide , Progressão da Doença , Transição Epitelial-Mesenquimal , Matriz Extracelular/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , PPAR gama/metabolismo , RNA-Seq , Análise Serial de Tecidos , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Ácido Glucurônico/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chemotherapy resistant prostate cancer is a major clinical problem. When the prostate cancer has become androgen deprivation resistant, one of the few treatment regimens left is chemotherapy. There is a strong connection between a cancer's stem cell like characteristics and drug resistance. By performing RNA-seq we observed several factors associated with stem cells being strongly up-regulated by the estrogen receptor ß variants, ß2 and ß5. In addition, most of these factors were also up-regulated by hypoxia. One mechanism of chemotherapy resistance was expression of the hypoxia-regulated, drug transporter genes, where especially ABCG2 and MDR1 were shown to be expressed in recurrent prostate cancer and to cause chemotherapy resistance by efficiently transporting drugs like docetaxel out of the cells. Another mechanism was expression of the hypoxia-regulated Notch3 gene, which causes chemotherapy resistance in urothelial carcinoma, although the mechanism is unknown. It is well known that hypoxic signaling is involved in increasing chemotherapy resistance. Regulation of the hypoxic factors, HIF-1α and HIF-2α is very complex and extends far beyond hypoxia itself. We have recently shown that two of the estrogen receptor ß variants, estrogen receptor ß2 and ß5, bind to and stabilize both HIF-1α and HIF-2α proteins leading to expression of HIF target genes. This study suggests that increased expression of the estrogen receptor ß variants, ß2 and ß5, could be involved in development of a cancer's stem cell characteristics and chemotherapy resistance, indicating that targeting these factors could prevent or reverse chemotherapy resistance and cancer stem cell expansion.
RESUMO
Long noncoding RNAs (lncRNAs) are commonly dysregulated in tumors, but only a handful are known to play pathophysiological roles in cancer. We inferred lncRNAs that dysregulate cancer pathways, oncogenes, and tumor suppressors (cancer genes) by modeling their effects on the activity of transcription factors, RNA-binding proteins, and microRNAs in 5,185 TCGA tumors and 1,019 ENCODE assays. Our predictions included hundreds of candidate onco- and tumor-suppressor lncRNAs (cancer lncRNAs) whose somatic alterations account for the dysregulation of dozens of cancer genes and pathways in each of 14 tumor contexts. To demonstrate proof of concept, we showed that perturbations targeting OIP5-AS1 (an inferred tumor suppressor) and TUG1 and WT1-AS (inferred onco-lncRNAs) dysregulated cancer genes and altered proliferation of breast and gynecologic cancer cells. Our analysis indicates that, although most lncRNAs are dysregulated in a tumor-specific manner, some, including OIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergistically dysregulate cancer pathways in multiple tumor contexts.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA Longo não Codificante/genética , Linhagem Celular , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Genes Supressores de Tumor , Humanos , OncogenesRESUMO
Dysregulation of the Wnt pathway leading to accumulation of ß-catenin (CTNNB1) is a hallmark of colorectal cancer (CRC). Nuclear CTNNB1 acts as a transcriptional coactivator with TCF/LEF transcription factors, promoting expression of a broad set of target genes, some of which promote tumor growth. However, it remains poorly understood how CTNNB1 interacts with different transcription factors in different contexts to promote different outcomes. While some CTNNB1 target genes are oncogenic, others regulate differentiation. Here, we found that TCF7L1, a Wnt pathway repressor, buffers CTNNB1/TCF target gene expression to promote CRC growth. Loss of TCF7L1 impaired growth and colony formation of HCT116 CRC cells and reduced tumor growth in a mouse xenograft model. We identified a group of CTNNB1/TCF target genes that are activated in the absence of TCF7L1, including EPHB3, a marker of Paneth cell differentiation that has also been implicated as a tumor suppressor in CRC. Knockdown of EPHB3 partially restores growth and normal cell cycle progression of TCF7L1-Null cells. These findings suggest that while CTNNB1 accumulation is critical for CRC progression, activation of specific Wnt target genes in certain contexts may in fact inhibit tumor growth.
Assuntos
Neoplasias Colorretais/metabolismo , Receptor EphB3/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Neoplasias Colorretais/patologia , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Xenoenxertos , Humanos , Camundongos Nus , Receptor EphB3/genética , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/ß-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how ß-catenin's function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of ß-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that ß-catenin's function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein-protein interactions drive stem cell fate.
Assuntos
Diferenciação Celular , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células-Tronco Embrionárias Murinas/fisiologia , beta Catenina/metabolismo , Animais , Autorrenovação Celular , Células Cultivadas , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Camundongos , Oxazóis/farmacologia , Transcrição Gênica , beta Catenina/antagonistas & inibidoresRESUMO
MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of â-catenin (ß-cat), miR-1 and miR-613 act upstream of ß-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive ß-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.