Juan Ramón Tejedor, Alfonso Peñarroya, Javier Gancedo-Verdejo, Pablo Santamarina-Ojeda, Raúl F Pérez, Sara López-Tamargo, Ana Díez-Borge, Juan J Alba-Linares, Nerea González-Del-Rey, Rocío G Urdinguio, Cristina Mangas, Annalisa Roberti, Virginia López, Teresa Morales-Ruiz, Rafael R Ariza, Teresa Roldán-Arjona, Mónica Meijón, Luis Valledor, María Jesús Cañal, Daniel Fernández-Martínez, María Fernández-Hevia, Paula Jiménez-Fonseca, Luis J García-Flórez, Agustín F Fernández, Mario F Fraga
{"title":"CRISPR/dCAS9介导的DNA去甲基化筛选鉴定癌症的功能性表观遗传决定簇。","authors":"Juan Ramón Tejedor, Alfonso Peñarroya, Javier Gancedo-Verdejo, Pablo Santamarina-Ojeda, Raúl F Pérez, Sara López-Tamargo, Ana Díez-Borge, Juan J Alba-Linares, Nerea González-Del-Rey, Rocío G Urdinguio, Cristina Mangas, Annalisa Roberti, Virginia López, Teresa Morales-Ruiz, Rafael R Ariza, Teresa Roldán-Arjona, Mónica Meijón, Luis Valledor, María Jesús Cañal, Daniel Fernández-Martínez, María Fernández-Hevia, Paula Jiménez-Fonseca, Luis J García-Flórez, Agustín F Fernández, Mario F Fraga","doi":"10.1186/s13148-023-01546-1","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.</p><p><strong>Results: </strong>In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53<sup>-/-</sup> cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.</p><p><strong>Conclusions: </strong>These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.</p>","PeriodicalId":48652,"journal":{"name":"Clinical Epigenetics","volume":null,"pages":null},"PeriodicalIF":5.7000,"publicationDate":"2023-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10464368/pdf/","citationCount":"0","resultStr":"{\"title\":\"CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.\",\"authors\":\"Juan Ramón Tejedor, Alfonso Peñarroya, Javier Gancedo-Verdejo, Pablo Santamarina-Ojeda, Raúl F Pérez, Sara López-Tamargo, Ana Díez-Borge, Juan J Alba-Linares, Nerea González-Del-Rey, Rocío G Urdinguio, Cristina Mangas, Annalisa Roberti, Virginia López, Teresa Morales-Ruiz, Rafael R Ariza, Teresa Roldán-Arjona, Mónica Meijón, Luis Valledor, María Jesús Cañal, Daniel Fernández-Martínez, María Fernández-Hevia, Paula Jiménez-Fonseca, Luis J García-Flórez, Agustín F Fernández, Mario F Fraga\",\"doi\":\"10.1186/s13148-023-01546-1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). 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CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.
Background: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.
Results: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.
Conclusions: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.
Clinical EpigeneticsBiochemistry, Genetics and Molecular Biology-Developmental Biology
CiteScore
8.90
自引率
5.30%
发文量
150
审稿时长
12 weeks
期刊介绍:
Clinical Epigenetics, the official journal of the Clinical Epigenetics Society, is an open access, peer-reviewed journal that encompasses all aspects of epigenetic principles and mechanisms in relation to human disease, diagnosis and therapy. Clinical trials and research in disease model organisms are particularly welcome.