RESUMO
A pharmacological-based global screen for epigenetically silenced tumor suppressor genes was performed in MCF-7 and MDA-MB-231 breast cancer cells. Eighty-one genes in MCF-7 cells and 131 in MDA-MB-231 cells were identified, that had low basal expression and were significantly upregulated following treatment. Eighteen genes were studied for methylation and/or expression in breast cancer; PTCH, the receptor for the hedgehog (Hh) pathway and a known tumor suppressor gene, was selected for further analysis. Methylation of the PTCH promoter was found in MCF-7 cells and in breast cancer samples, and correlated with low PTCH expression. Immunohistochemical analysis of breast tissue arrays revealed high expression of PTCH in normal breast compared to ductal carcinomas in situ (DCIS) and invasive ductal carcinomas; furthermore, association was found between PTCH expression and favorable prognostic factors. PTCH is an inhibitor of the Hh pathway, and its silencing activates the pathway and promotes growth. Indeed, high activity of the Hh pathway was identified in MCF-7 cells and overexpression of PTCH inhibited the pathway. Moreover, treatment with cyclopamine, an inhibitor of the pathway, reduced cell growth and slowed the cell cycle in these cells. Thus, unmasking of epigenetic silencing in breast cancer enabled us to discover a large number of candidate tumor suppressor genes. Further analysis suggested a role of one of these genes, PTCH, in breast cancer tumorigenesis.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/genética , Sequência de Bases , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Feminino , Perfilação da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Patched , Receptor Patched-1 , Células Tumorais CultivadasRESUMO
Prostaglandin E(2) plays a growth-stimulatory role in breast cancer, and the rate-limiting enzyme in its synthesis, cyclooxygenase-2, is often overexpressed in these cancers. Little is known about the role of the key prostaglandin catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in breast cancer pathogenesis. Using a pharmacologically based screen for epigenetically silenced genes, we found low levels of 15-PGDH in MDA-MB-231 cells [estrogen receptor (ER) negative] but high levels in MCF-7 cells (ER positive) and observed its up-regulation following demethylation treatment. Further analysis revealed methylation of the 15-PGDH promoter in one breast cancer cell line and 30% of primary tumors. Analysis of 15-PGDH expression revealed low levels in 40% of primary breast tumors and identified a correlation between 15-PGDH and ER expression. Transfection assays showed that transient up-regulation of 15-PGDH levels in MDA-MB-231 cells resulted in a decreased clonal growth, and stable up-regulation significantly decreased the ability of these cells to form tumors in athymic mice. In contrast, transient silencing of 15-PGDH in MCF-7 cells resulted in their enhanced proliferation, and a stable silencing in these cells enhanced cell cycle entry in vitro and tumorigenicity in vivo. Forced expression of 15-PGDH inhibited the ER pathway and silencing of 15-PGDH up-regulated expression of aromatase. In addition, 15-PGDH levels were down-regulated by estrogen but up-regulated by the tumor suppressor gene CAAT/enhancer binding protein alpha. Our results indicate for the first time that 15-PGDH may be a novel tumor suppressor gene in breast cancer, and suggest that this enzyme can modulate the ER pathway.
