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Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation

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...

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Autores principales: Montenegro, María F., Sáez-Ayala, Magali, Piñero-Madrona, Antonio, Cabezas-Herrera, Juan, Rodríguez-López, José Neptuno
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522638/
https://www.ncbi.nlm.nih.gov/pubmed/23251702
http://dx.doi.org/10.1371/journal.pone.0052231
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author Montenegro, María F.
Sáez-Ayala, Magali
Piñero-Madrona, Antonio
Cabezas-Herrera, Juan
Rodríguez-López, José Neptuno
author_facet Montenegro, María F.
Sáez-Ayala, Magali
Piñero-Madrona, Antonio
Cabezas-Herrera, Juan
Rodríguez-López, José Neptuno
author_sort Montenegro, María F.
collection PubMed
description 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/SIGNIFICANCE: 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.
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spelling pubmed-35226382012-12-18 Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation Montenegro, María F. Sáez-Ayala, Magali Piñero-Madrona, Antonio Cabezas-Herrera, Juan Rodríguez-López, José Neptuno PLoS One Research Article 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/SIGNIFICANCE: 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. Public Library of Science 2012-12-14 /pmc/articles/PMC3522638/ /pubmed/23251702 http://dx.doi.org/10.1371/journal.pone.0052231 Text en © 2012 Montenegro et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Montenegro, María F.
Sáez-Ayala, Magali
Piñero-Madrona, Antonio
Cabezas-Herrera, Juan
Rodríguez-López, José Neptuno
Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation
title Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation
title_full Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation
title_fullStr Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation
title_full_unstemmed Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation
title_short Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation
title_sort reactivation of the tumour suppressor rassf1a in breast cancer by simultaneous targeting of dna and e2f1 methylation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522638/
https://www.ncbi.nlm.nih.gov/pubmed/23251702
http://dx.doi.org/10.1371/journal.pone.0052231
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