RESUMO
Latent transforming growth factor ß-binding protein 4 (LTBP4) is an extracellular matrix molecule that is a member of important connective tissue networks and is needed for the correct folding and the secretion of TGF-ß1. LTBP4 is downregulated in carcinomas of various tissues. Here we show that LTBP4 is also downregulated in adenocarcinomas and squamous cell carcinomas of the esophagus in vitro and in vivo. Re-expression of LTBP4 in esophageal cancer cell lines reduced cell migration ability, whereas cell viability and cell proliferation remained unchanged. Hypermethylation of the promoter regions of the two main human LTBP4 transcriptional forms, LTBP4L and LTBP4S, was found to be involved in LTBP4 silencing. Detailed investigations of the methylation patterns of the promoter regions of LTBP4L and LTBP4S identified GATA1, SP1, E2F4 and SMAD3 as potential transcription factors involved in LTBP4 expression. In in vitro transcription factor activity studies we discovered E2F4 as novel powerful regulator for LTBP4S expression.
Assuntos
Metilação de DNA , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a TGF-beta Latente/genética , Regiões Promotoras Genéticas , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Sítios de Ligação , Carcinoma/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Decitabina , Progressão da Doença , Regulação para Baixo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Humanos , Estadiamento de Neoplasias , Ligação Proteica , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
BACKGROUND: Transforming growth factor beta (TGF-ß) is able to inhibit the proliferation of epithelial cells and is involved in the carcinogenesis of mammary tumors. Three latent transforming growth factor-ß binding proteins (LTBPs) are known to modulate TGF-ß functions. METHODS: The current study analyses the expression profiles of LTBP4, its isoforms LTBP1 and LTBP3, and TGF-ß1, TGF-ß2, TGF-ß3, and SMAD2, SMAD3 and SMAD4 in human and murine (WAP-TNP8) DCIS compared to invasive mammary tumors. Additionally mammary malignant (MCF7, Hs578T, MDA-MB361) and non malignant cell lines (Hs578BsT) were analysed. Microarray, q-PCR, immunoblot, immunohistochemistry and immunofluorescence were used. RESULTS: In comparison to non-malignant tissues (n = 5), LTBP4 was downregulated in all human and mouse DCIS (n = 9) and invasive mammary adenocarcinomas (n = 5) that were investigated. We also found decreased expression of bone morphogenic protein 4 (BMP4) and increased expression of its inhibitor gremlin (GREM1). Treatment of the mammary tumor cell line (Hs578T) with recombinant TGF-ß1 rescued BMP4 and GREM1 expression. CONCLUSION: We conclude that the lack of LTBP4-mediated targeting in malignant mammary tumor tissues may lead to a possible modification of TGF-ß1 and BMP bioavailability and function.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Regulação para Baixo/genética , Proteínas de Ligação a TGF-beta Latente/genética , Neoplasias Mamárias Animais/genética , Animais , Proteína Morfogenética Óssea 4/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Citocinas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Proteínas de Neoplasias/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismoRESUMO
BACKGROUND: The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma. Since DCIS is a curable disease, it would be highly desirable to identify molecular markers that allow early detection. Mice transgenic for the WAP-SV40 early genome region were used as a model for DCIS development. Gene expression profiling was carried out on DCIS-bearing mice and control animals. Additionally, a set of human DCIS and invasive mammary tumors were analyzed in a similar fashion. Enhanced expression of these marker genes in human and murine samples was validated by quantitative RT-PCR. Besides, marker gene expression was also validated by immunohistochemistry of human samples. Furthermore in silico analyses using an online microarray database were performed. RESULTS: In DCIS-mice seven genes were identified that were significantly up-regulated in DCIS: DEPDC1, NUSAP1, EXO1, RRM2, FOXM1, MUC1 and SPP1. A similar up-regulation of homologues of the murine genes was observed in human DCIS samples. Enhanced expression of these genes in DCIS and IDC (invasive ductal carcinoma) was validated by quantitative RT-PCR and immunohistochemistry. CONCLUSIONS: By comparing murine markers for the ductal carcinoma in situ (DCIS) of the mammary gland with genes up-regulated in human DCIS-samples we were able to identify a set of genes which might allow early detection of DCIS and invasive carcinomas in the future. The similarities between gene expression in DCIS and invasive carcinomas in our data suggest that the early detection and treatment of DCIS is of utmost relevance for the survival of patients who are at high risk of developing breast carcinomas.
