RESUMO
RNA and protein analysis revealed the consistent upregulation of the neural transcription factors ZIC1 and ZIC4 in desmoid tumors and other fibroproliferative disorders. The 5' flanking region of the ZIC1 promoter was unmethylated in desmoid tumor fibroblasts, while a hypermethylated ZIC1 promoter was found in human and mouse cell lines not expressing the gene. In addition, expressing cells showed a H3K4me2 at the ZIC1 promoter, whereas non-expressing cells showed higher levels of H3K9me2 in the same region. To our knowledge, this is the first report describing ZIC1 expression in mesenchymal proliferations and a role for DNA methylation in the control of ZIC1 expression.
Assuntos
Metilação de DNA , Fibromatose Agressiva/genética , Regulação da Expressão Gênica , Histonas/metabolismo , Fatores de Transcrição/genética , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Proliferação de Células , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Fibromatose Agressiva/patologia , Humanos , Mesoderma/química , Mesoderma/metabolismo , Mesoderma/patologia , Metilação , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Regiões Promotoras Genéticas , Fatores de Transcrição/análiseRESUMO
Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which beta-catenin-mediated TCF-3-dependent transcription is activated. To provide more insight into the pathophysiology of these tumors, expression profiles were generated using oligonucleotide arrays (Affymetrix). In total, 69 differentially expressed genes were identified in desmoids compared to normal fibroblasts (fascia) from the same patients. The differential expression of a selection of genes was confirmed using RT-PCR and Northern blotting. We further evaluated the insulin-like growth factor-binding protein 6 (IGFBP-6), a gene that was consistently downregulated in all desmoids tested. Promotor studies and electromobility shift assays revealed two functional beta-catenin/TCF-responsive elements in the human IGFBP-6 promoter. These findings suggest that IGFBP-6 is directly downregulated by the beta-catenin/TCF complex in desmoid tumors, and imply a role for the IGF axis in the proliferation of desmoid tumors.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Regulação para Baixo/fisiologia , Fibroma/genética , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Transativadores/fisiologia , Animais , Sequência de Bases , Northern Blotting , Células CHO , Cricetinae , Primers do DNA , Fibroma/patologia , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , beta CateninaRESUMO
Here for the first time we showed, despite the oncogenic mutations in beta-Catenin, that TGF-beta is a modulator of beta-Catenin levels in tumoral fibroblasts as well as non-tumoral fibroblasts. The results show that the TGF-beta pathway is active in desmoids cells and in in situ tumors. A dose dependent increase in beta-Catenin protein levels was observed after TGF-beta treatment in combination with an increased repression of GSK-3beta both in normal and tumoral fibroblasts. TGF-beta stimulation also led to an altered -- up to 5 fold -- transcriptional activity of beta-Catenin responsive promoters, such as IGFBP6 as well as increase of TOPflash activity. TGF-beta stimulation increased cell proliferation and BrdU incorporation 2.5 times. Taken together, we propose that TGF-beta is a modulator of beta-Catenin levels in tumoral fibroblasts and non-tumoral fibroblasts, despite the oncogenic mutations already present in this gene in tumoral fibroblasts of desmoid tumors. This modulation of beta-Catenin levels by TGF-beta may be involved in determining the tumoral phenotype of the cells.
Assuntos
Fibromatose Agressiva/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibromatose Agressiva/genética , Fibromatose Agressiva/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Fosforilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors in which beta-catenin/TCF3 mediated Wnt signaling is activated. More than 80% of desmoid tumors contain activating mutations in beta-catenin. It has been shown that the Wnt signaling pathway interacts with Wilms' tumor gene 1 (WT1) in normal kidney development and plays a role in the genesis of some Wilms' tumors. About 15% of Wilms' tumors contain WT1 mutations and of these, about 50% contain beta-catenin mutations. This overlap in mutation pattern of WT1 and beta-catenin in Wilms' tumor suggests that these 2 genes may collaborate in the genesis of a subset of Wilms' tumors. To investigate whether this hypothesis could be extended to other Wnt-dependent tumor types, we searched for WT1 mutations and studied WT1 expression in beta-catenin mutant desmoid tumors. We investigated the expression of WT1 mRNA and protein in desmoid tumors. Medium to high abundant levels of WT1 mRNA were detected by TaqMan quantitative PCR in all tested desmoid cells, whereas adjacent normal fibroblasts showed less expression of WT1. Western blot analysis and immunohistochemistry confirmed this overexpression at the protein level. A mutational screen of the WT1 zinc-finger region by sequence analysis did not identify any mutations. Finally, we investigated a possible role of beta-catenin on WT1 regulation and vice versa. Overexpression of different beta-catenin mutants in the HEK293T cell line did not modulate WT1 promoter activity and WT1 did not affect beta-catenin /TCF transcriptional activity in this cell line. These results show that the wild-type WT1 gene is strongly overexpressed in beta-catenin mutant desmoid tumors and may play a role in tumorigenesis of desmoid tumors, similar to what has been suggested in some epithelial malignancies.