title: Reactivation of the tumour suppressor RASSF1A in breast cancer by simultaneous targeting of DNA and E2F1 methylation.
abstract: BACKGROUND
  Tumour suppressor genes are often transcriptionally silenced by promoter hypermethylation, and recent research has implicated alterations in chromatin structure as the mechanistic basis for this repression. In addition to DNA methylation, other epigenetic post-translational modifications that modulate the stability and binding of specific transcription factors to gene promoters have emerged as important mechanisms for controlling gene expression. The aim of this study was to analyse the implications of these mechanisms and their molecular connections in the reactivation of RASSF1A in breast cancer.
  
  
  METHODS
  Compounds that modulate the intracellular concentration of adenosine, such as dipyridamole (DIPY), greatly increase the antiproliferative effects of 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin (TMCG), a synthetic antifolate derived from the structure of tea catechins. Quantitative real-time PCR arrays and MALDI-TOF mass spectrometry indicated that this combination (TMCG/DIPY) induced apoptosis in breast cancer cells by modulating the methylation levels of DNA and proteins (such as E2F1), respectively. Chromatin immunoprecipitation (ChIP) assays were employed to confirm that this combination induced chromatin remodelling of the RASSF1A promoter and increased the occupancy of E2F1 at the promoter of this tumour suppressor gene.
  
  
  RESULTS
  The TMCG/DIPY combination acted as an epigenetic treatment that reactivated RASSF1A expression and induced apoptosis in breast cancer cells. In addition to modulating DNA methylation and chromatin remodelling, this combination also induced demethylation of the E2F1 transcription factor. The ChIP assay showed enhancement of E2F1 occupancy at the unmethylated RASSF1A promoter after TMCG/DIPY treatment. Interestingly, inhibition of E2F1 demethylation using an irreversible inhibitor of lysine-specific demethylase 1 reduced both TMCG/DIPY-mediated RASSF1A expression and apoptosis in MDA-MB-231 cells, suggesting that DNA and protein demethylation may act together to control these molecular and cellular processes.
  
  
  CONCLUSIONS
  This study demonstrates that simultaneous targeting of DNA and E2F1 methylation is an effective epigenetic treatment that reactivates RASSF1A expression and induces apoptosis in breast cancer cells.
authors.affiliation: Department of Biochemistry and Molecular Biology A, School of Biology, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Espinardo, Murcia, Spain.
authors.forename: María F
authors.initials: MF
authors.lastname: Montenegro
authors.forename: Magali
authors.initials: M
authors.lastname: Sáez-Ayala
authors.forename: Antonio
authors.initials: A
authors.lastname: Piñero-Madrona
authors.forename: Juan
authors.initials: J
authors.lastname: Cabezas-Herrera
authors.forename: José Neptuno
authors.initials: JN
authors.lastname: Rodríguez-López
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002843:Chromatin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000235:genetics*; D004176:Dipyridamole / Q000494:pharmacology; D004357:Drug Synergism; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C559677:3-O-(3,4,5-trimethoxybenzoyl)catechin; D002843:Chromatin; D004273:DNA, Neoplasm; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D004176:Dipyridamole; D002392:Catechin