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Nat Commun ; 12(1): 4897, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385432


Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.

Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Elementos Reguladores de Transcrição/genética , Sítios de Ligação/genética , Western Blotting , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos
Cancer Res ; 81(8): 2116-2127, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33574090


Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in ß-catenin target genes, suggesting that YAP is required for nuclear ß-catenin activity. Mechanistically, nuclear YAP interacted with ß-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, ß-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/ß-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. SIGNIFICANCE: These findings show that YAP cooperates with ß-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer. GRAPHICAL ABSTRACT:

Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinogênese , Linhagem Celular Tumoral , Transdiferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Proteínas Musculares/metabolismo , Células-Tronco Neoplásicas/patologia , Proteômica , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/prevenção & controle , Neoplasias de Mama Triplo Negativas/terapia , Regulação para Cima , Proteínas Wnt/metabolismo , beta Catenina/genética
Stem Cell Res ; 33: 120-124, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30343101


Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times. Both new cell lines offer the opportunity to study basic stem cell biology and model human disease. They can be applied as reference cell lines for creating isogenic clones bearing disease mutations on a well-characterized genomic background, as both cell lines have demonstrated excellent differentiation capacity in multiple labs. Resource table.

Feto/fisiopatologia , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular , Feminino , Fibroblastos/citologia , Humanos
Cancer Immun ; 13: 12, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23882157


During cancer progression, malignant cells may evade immunosurveillance. However, evidence for immunological escape in humans is scarce. We report here the clinical course of a melanoma patient whose initial tumor was positive for the antigens NY-ESO-1, MAGE-C1, and Melan-A. Upon immunization with a recombinant vaccinia/fowlpox NY-ESO-1 construct, the patient experienced a mixed clinical response and spreading of the NY-ESO-1 epitopes in the CD4+ T cell compartment. After NY-ESO-1 protein + CpG immunization, the patient's anti-NY-ESO-1 IgG response increased. Over the following years, progressing lesions were resected and found to be NY-ESO-1-negative while being positive for MAGE-C1, Melan-A, and MHC-I. The fatal, inoperable brain metastasis was analyzed after his death and also proved to be NY-ESO-1-negative, while being positive for MAGE-C1 and Melan-A, as well as MHC-I. We propose that cancer control and cancer escape in this patient were governed by NY-ESO-1-specific immunological pressure. Our findings provide evidence for the existence of immunoediting and immunoescape in this cancer patient.

Antígenos de Neoplasias/imunologia , Melanoma/imunologia , Proteínas de Membrana/imunologia , Humanos , Imuno-Histoquímica , Masculino , Melanoma/patologia , Melanoma/secundário , Pessoa de Meia-Idade
Cancer Immun ; 13: 3, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23390374


We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.

Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Membrana/imunologia , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Clonagem Molecular , Apresentação Cruzada/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Mapeamento de Epitopos , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/patologia , Resultado do Tratamento
J Immunol ; 189(2): 558-66, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22685313


Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity.

Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/radioterapia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/radioterapia , Animais , Apresentação do Antígeno/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos da radiação , Carcinoma Pulmonar de Lewis/patologia , Diferenciação Celular/efeitos da radiação , Células Dendríticas/metabolismo , Células Dendríticas/efeitos da radiação , Relação Dose-Resposta à Radiação , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos