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1.
J Cell Mol Med ; 24(1): 418-430, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730267

RESUMO

Numerous genetic and epigenetic alterations cause functional changes in cell biology underlying cancer. These hallmark functional changes constitute potentially tissue-independent anticancer therapeutic targets. We hypothesized that RNA-Seq identifies gene expression changes that underly those hallmarks, and thereby defines relevant therapeutic targets. To test this hypothesis, we analysed the publicly available TCGA-TARGET-GTEx gene expression data set from the University of California Santa CruzToil recompute project using WGCNA to delineate co-correlated 'modules' from tumour gene expression profiles and functional enrichment of these modules to hierarchically cluster tumours. This stratified tumours according to T cell activation, NK-cell activation, complement cascade, ATM, Rb, angiogenic, MAPK, ECM receptor and histone modification signalling. These correspond to the cancer hallmarks of avoiding immune destruction, tumour-promoting inflammation, evading growth suppressors, inducing angiogenesis, sustained proliferative signalling, activating invasion and metastasis, and genome instability and mutation. This approach did not detect pathways corresponding to the cancer enabling replicative immortality, resisting cell death or deregulating cellular energetics hallmarks. We conclude that RNA-Seq stratifies tumours along some, but not all, hallmarks of cancer and, therefore, could be used in conjunction with other analyses collectively to inform precision therapy.


Assuntos
Neoplasias/genética , RNA-Seq , Regulação Neoplásica da Expressão Gênica , Humanos , Especificidade de Órgãos/genética , Transdução de Sinais/genética
2.
Mol Cell Proteomics ; 11(8): 303-16, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22493178

RESUMO

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.


Assuntos
Separação Celular/métodos , Glicoproteínas/análise , Imunofenotipagem/métodos , Proteínas de Membrana/análise , Células-Tronco Pluripotentes/metabolismo , Proteômica/métodos , Animais , Células Cultivadas , Receptor gp130 de Citocina/análise , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Espectrometria de Massas , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Microscopia Confocal , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Teratoma/metabolismo , Teratoma/patologia
3.
JCO Precis Oncol ; 7: e2300189, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37883719

RESUMO

Germline pathogenic loss-of-function (pLOF) variants in DICER1 are associated with a predisposition for a variety of solid neoplasms, including pleuropulmonary blastoma and Sertoli-Leydig cell tumor (SLCT). The most common DICER1 pLOF variants include small insertions or deletions leading to frameshifts, and base substitutions leading to nonsense codons or altered splice sites. Larger deletions and pathogenic missense variants occur less frequently. Identifying these variants can trigger surveillance algorithms with potential for early detection of DICER1-related cancers and cascade testing of family members. However, some patients with DICER1-associated tumors have no pLOF variants detected by germline or tumor testing. Here, we present two patients with SLCT whose tumor sequencing showed only a somatic missense DICER1 RNase IIIb variant. Conventional exon-directed germline sequencing revealed no pLOF variants. Using a custom capture panel, we discovered novel intronic variants, ENST00000343455.7: c.1752+213A>G and c.1509+16A>G, that appear to interfere with normal splicing. We suggest that when no DICER1 pLOF variants or large deletions are discovered in exonic regions despite strong clinical suspicion, intron sequencing and splicing analysis should be performed.


Assuntos
Neoplasias Ovarianas , Tumor de Células de Sertoli-Leydig , Masculino , Feminino , Humanos , Tumor de Células de Sertoli-Leydig/genética , Tumor de Células de Sertoli-Leydig/patologia , Neoplasias Ovarianas/genética , Íntrons/genética , Mutação em Linhagem Germinativa/genética , Mutação , Ribonuclease III/genética , RNA Helicases DEAD-box/genética
5.
Stem Cells Transl Med ; 6(4): 1096-1108, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28186685

RESUMO

Due to their capacity to self-renew, proliferate and generate multi-lineage cells, adult-derived stem cells offer great potential for use in regenerative therapies to stop and/or reverse degenerative diseases such as diabetes, heart failure, Alzheimer's disease and others. However, these subsets of cells can be isolated from different niches, each with differing potential for therapeutic applications. The stromal vascular fraction (SVF), a stem cell enriched and adipose-derived cell population, has garnered interest as a therapeutic in regenerative medicine due to its ability to secrete paracrine factors that accelerate endogenous repair, ease of accessibility and lack of identified major adverse effects. Thus, one can easily understand the rush to employ adipose-derived SVF to treat human disease. Perhaps faster than any other cell preparation, SVF is making its way to clinics worldwide, while critical preclinical research needed to establish SVF safety, efficacy and optimal, standardized clinical procedures are underway. Here, we will provide an overview of the current knowledge driving this phenomenon, its regulatory issues and existing studies, and propose potential unmapped applications. Stem Cells Translational Medicine 2017;6:1096-1108.


Assuntos
Tecido Adiposo/citologia , Adipócitos/citologia , Animais , Humanos , Células-Tronco Mesenquimais/citologia , Medicina Regenerativa/métodos , Transplante de Células-Tronco , Células-Tronco/citologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-27087855

RESUMO

BACKGROUND: The cohesin complex consists of multiple core subunits that play critical roles in mitosis and transcriptional regulation. The cohesin-associated protein Wapal plays a central role in off-loading cohesin to facilitate sister chromatid separation, but its role in regulating mammalian gene expression is not understood. We used embryonic stem cells as a model, given that the well-defined transcriptional regulatory circuits were established through master transcription factors and epigenetic pathways that regulate their ability to maintain a pluripotent state. RESULTS: RNAi-mediated depletion of Wapal causes a loss of pluripotency, phenocopying loss of core cohesin subunits. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), we determine that Wapal occupies genomic sites distal to genes in combination with CTCF and core cohesin subunits such as Rad21. Interestingly, genomic sites occupied by Wapal appear enriched for cohesin, implying that Wapal does not off-load cohesin at regions it occupies. Wapal depletion induces derepression of Polycomb group (PcG) target genes without altering total levels of Polycomb-mediated histone modifications, implying that PcG enzymatic activity is preserved. By integrating ChIP-seq and gene expression changes data, we identify that Wapal binding is enriched at the promoters of PcG-silenced genes and is required for proper Polycomb repressive complex 2 (PRC2) recruitment. Lastly, we demonstrate that Wapal is required for the interaction of a distal cis-regulatory element (CRE) with the c-Fos promoter. CONCLUSIONS: Collectively, this work indicates that Wapal plays a critical role in silencing of PcG target genes through the interaction of distal CREs with promoters.

7.
Exp Hematol ; 43(1): 53-64.e1-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25246269

RESUMO

Sal-like protein 4 (SALL4) is a transcription factor that exists in two splice isoforms, SALL4a and SALL4b, and regulates transcription in embryonic stem cells, hematopoiesis, and acute myeloid leukemia. Constitutive overexpression of SALL4 in mice induces acute myeloid leukemia. Interestingly, a potential benefit of using SALL4 to facilitate ex vivo hematopoietic stem cell expansion has been proposed. However, distinct roles for how SALL4 contributes to normal versus malignant processes remain undefined. Here we show that SALL4b is the predominant isoform in murine hematopoietic stem cells and progenitors. Overexpression of either SALL4 isoform in hematopoietic stem cells or progenitors impairs hematopoietic colony formation and expansion in vitro. Lineage-negative bone marrow overexpressing SALL4b fails to engraft and reconstitute hematopoiesis when transplanted. We found that both SALL4a and SALL4b overexpression impair hematopoiesis, in part through dose-dependent repression of BMI1. Additionally, we have identified the following potential novel SALL4 target genes in hematopoiesis: ARID5B (SALL4a and SALL4b), EZH2, and KLF2 (SALL4a). Lastly, we found that SALL4 expression is variable in acute myeloid leukemia, ranging from no expression to levels comparable to embryonic stem cells. These results show that SALL4 isoforms contribute to only a subset of acute myeloid leukemia and that overexpression of SALL4 isoforms impairs hematopoiesis through repression of BMI1. Together these data demonstrate the sensitivity of hematopoiesis to appropriately balanced SALL4 expression, highlighting the importance of regulating this dynamic in potential therapeutic applications such as ex vivo stem cell expansion.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/fisiologia , Animais , Células Sanguíneas/metabolismo , Medula Óssea/metabolismo , Divisão Celular , Transformação Celular Neoplásica/genética , Criança , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Regulação da Expressão Gênica , Sobrevivência de Enxerto , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 1/biossíntese , Complexo Repressor Polycomb 1/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Quimera por Radiação , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
8.
Epigenetics ; 8(12): 1303-20, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24135681

RESUMO

Enhancers are cis-acting elements capable of regulating transcription in a distance and orientation-independent manner. A subset of enhancers are occupied by RNA polymerase II (RNAP II) and transcribed to produce long non-coding RNAs termed eRNAs. We thoroughly investigated the association between eRNA productivity and various chromatin marks and transcriptional regulators in mouse embryonic stem cells (ESCs) through an integrative approach. We found that eRNA-producing enhancers exhibited elevated levels of the active mark H3K27Ac, decreased DNA methylation, and enrichment for the DNA hydroxylase Tet1. Many eRNA-producing enhancers have recently been characterized as "super-enhancers," suggesting an important role in the maintenance of pluripotency. Using experimental methods, we focally investigated a well-characterized enhancer linked to the Nanog locus and confirmed its exclusive eRNA productivity in ESCs. We further demonstrate that the binding of Sall4 and Tet family proteins were required for eRNA productivity at this locus. Collectively, we demonstrate that Tet1 binding and DNA hypomethylation are hallmarks of eRNA production.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Células NIH 3T3 , Proteína Homeobox Nanog , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , RNA/genética , Elementos Reguladores de Transcrição
9.
Pediatr Dev Pathol ; 16(2): 67-73, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23286282

RESUMO

Posttransplant lymphoproliferative disorders (PTLD) involving the central nervous system (CNS) in children are uncommon and can prove diagnostically challenging. The clinical and imaging characteristics of CNS PTLD can overlap with those of infection, hemorrhage, and primary CNS tumors. Some cases of CNS PTLD remain clinically unsuspected and are diagnosed postmortem. We report 6 instances of CNS PTLD in children, 2 of which were limited to the CNS and were unsuspected before autopsy. In our autopsy series, PTLD was found outside the CNS in 4 out of 6 cases. Since CNS PTLD has a poor prognosis and the presentation can be subtle, unsuspected, and high grade, it is important to maintain a high index of suspicion and to perform imaging and brain biopsy whenever clinically appropriate. In the presence of leptomeningeal involvement, the diagnosis could be made by cerebral spinal fluid examination.


Assuntos
Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/patologia , Transtornos Linfoproliferativos/etiologia , Transtornos Linfoproliferativos/patologia , Transplante de Órgãos/efeitos adversos , Adolescente , Autopsia , Criança , Humanos , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia
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