RESUMEN
The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.
Asunto(s)
Proliferación Celular , Reprogramación Celular , Colitis Ulcerosa/metabolismo , Neoplasias Asociadas a Colitis/metabolismo , Enfermedad de Crohn/metabolismo , Epigénesis Genética , Activación de Linfocitos , Linfocitos T Reguladores/metabolismo , Vía de Señalización Wnt , Animales , Estudios de Casos y Controles , Células Cultivadas , Colitis Ulcerosa/genética , Colitis Ulcerosa/inmunología , Neoplasias Asociadas a Colitis/genética , Neoplasias Asociadas a Colitis/inmunología , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Fenotipo , Factor 1 de Transcripción de Linfocitos T , Linfocitos T Reguladores/inmunologíaRESUMEN
Thymocyte development requires a complex orchestration of multiple transcription factors. Ablating either TCF-1 or HEB in CD4+CD8+ thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and fewer late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that TCF-1 and HEB share ~7,000 DNA-binding sites genome wide and promote chromatin accessibility. The binding of both TCF-1 and HEB was required at these shared sites for epigenetic and transcriptional gene regulation. Binding of TCF-1 and HEB to their conserved motifs in the enhancer regions of genes associated with T cell differentiation promoted their expression. Binding to sites lacking conserved motifs in the promoter regions of cell-cycle-associated genes limited proliferation. TCF-1 displaced nucleosomes, allowing for chromatin accessibility. Importantly, TCF-1 inhibited Notch signaling and consequently protected HEB from Notch-mediated proteasomal degradation. Thus, TCF-1 shifts nucleosomes and safeguards HEB, thereby enabling their cooperation in establishing the epigenetic and transcription profiles of CD4+CD8+ thymocytes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Regulación de la Expresión Génica/inmunología , Factor Nuclear 1-alfa del Hepatocito/inmunología , Linfopoyesis/inmunología , Timocitos/inmunología , Animales , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
UNLABELLED: Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and increased growth in the mouse intestine. However, Ptk6-null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial-mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. IMPLICATIONS: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563-73. ©2016 AACR.
Asunto(s)
Neoplasias del Colon/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Células HCT116 , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinasas/genética , Transducción de SeñalRESUMEN
Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that has distinct functions in normal epithelia and cancer. It is expressed primarily in nondividing epithelial cells in the normal intestine, where it promotes differentiation. However, after DNA damage, PTK6 is induced in proliferating progenitor cells, where it contributes to apoptosis. We examined links between PTK6 and the tumor suppressor p53 in the isogenic p53(+/+) and p53(-/-) HCT116 colon tumor cell lines. We found that p53 promotes expression of PTK6 in HCT116 cells, and short hairpin RNA-mediated knockdown of PTK6 leads to reduced induction of the cyclin-dependent kinase inhibitor p21. Knockdown of PTK6 enhances apoptosis in HCT116 cells with wild-type p53, following treatment of cells with γ-radiation, doxorubicin, or 5-fluorouracil. No differences in the activation of AKT, ERK1/2, or ERK5, known PTK6-regulated prosurvival signaling proteins, were detected. However, activity of STAT3, a PTK6 substrate, was impaired in cells with knockdown of PTK6 following DNA damage. In contrast to its role in the normal epithelium following DNA damage, PTK6 promotes survival of cancer cells with wild-type p53 by promoting p21 expression and STAT3 activation. Targeting PTK6 in combination with use of chemotherapeutic drugs or radiation may enhance death of colon tumor cells with wild-type p53.
Asunto(s)
Apoptosis , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Daño del ADN , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Rayos gamma , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Ratones , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Previous studies indicate that green tea extract may inhibit breast cancer progression by blocking angiogenesis, although the molecular mechanisms are not well defined. We demonstrate that administration of Polyphenon E (Poly E), a standardized green tea extract, inhibited MDA-MB231 breast cancer and human dermal microvascular endothelial (HMVEC) cell migration and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9). In addition, Poly E inhibited VEGF-induced neovascularization in vivo. We also demonstrate that Poly E blocked signal transducers and activators of transcription (STAT) signaling by suppressing interferon-gamma (IFN-gamma)-induced gene transcription via IFN-gamma-activating sequence (GAS) elements and downstream STAT3 activation by inhibiting STAT1 and STAT3 dimerization in MDA-MB231 cells. Transient expression of constitutively active STAT3 significantly reduced the inhibitory effect of Poly E on cell migration and VEGF and MMP9 expression. Taken together, these observations indicate that green tea extract inhibits angiogenesis partly through the disruption of STAT3-mediated transcription of genes, including VEGF.
Asunto(s)
Neoplasias de la Mama/metabolismo , Catequina/análogos & derivados , Catequina/farmacología , Neovascularización Patológica/metabolismo , Factor de Transcripción STAT3/efectos de los fármacos , Té/química , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunoprecipitación , Interferón gamma/efectos de los fármacos , Interferón gamma/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Neovascularización Patológica/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismo , Transfección , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
Previous studies show inhibitory effects of green tea in chemically induced mammary tumors or human tumor explants, but not in spontaneous tumor models that are more representative of human breast cancer. The C3(1)/SV40 mouse model is particularly suited for breast cancer prevention studies because it produces spontaneous ductal adenocarcinomas and a predictable time course for mammary tumorigenesis through a multistage progression similar to that occurring in humans. We therefore used this model to test the chemoprotective effects of green tea. Administration of 0.5% Polyphenon E (Poly E) (a standardized preparation of green tea extract) in drinking water delayed tumor onset and suppressed tumor growth by 40%, compared to tap water-fed animals, with no adverse side effects. Histological analysis of mammary glands showed that green tea slowed the progression of ductal lesions to advanced mammary intraepithelial neoplasias and suppressed tumor invasiveness. Green tea inhibited the proliferation of ductal epithelial cells and tumors and, overall, disrupted post-pubertal ductal growth. Immunohistochemical analyses also demonstrated that green tea inhibited angiogenesis through a decrease in both ductal epithelial and stromal VEGF expression and a decrease in intratumoral microvascular density. Our data strongly support the potential use of green tea as a breast cancer chemopreventive agent.