CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

IF 5.7 2区 医学 Q1 Medicine Clinical Epigenetics Pub Date : 2023-08-24 DOI:10.1186/s13148-023-01546-1
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
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Abstract

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.

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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。
背景:在癌症恶性转化过程中,肿瘤抑制基因的启动子高甲基化是常见的。然而,这种表观遗传机制在癌症中是否起作用,或者仅仅是致癌过程的结果,还有待阐明。结果:在这项工作中,我们采用了一种综合的多组学方法来识别人类CRC样本和一组8个结肠癌癌症细胞系中DNA甲基化与基因表达之间具有强相关性的候选基因。作为概念验证,我们将最近的CRISPR-Cas9表观基因组编辑工具(dCas9-TET1,dCas9-TET-IM)与定制的阵列gRNA文库相结合,以调节先前与CRC中的强表观遗传抑制相关的56个启动子的DNA甲基化状态,并通过高含量细胞增殖筛查监测这种DNA甲基化缺失的潜在功能后果。总体而言,这些DNA甲基化区域中的大多数的表观遗传调节对基因表达的重新激活和癌症细胞的生存能力有轻微影响。有趣的是,我们发现肿瘤背景下RSPO2的表观遗传再激活与p53-/-neneneba癌症细胞系中细胞增殖的显著损伤相关,并且对人类样本的进一步验证表明,RSPO2表观遗传沉默是腺瘤-癌序列中的中晚期事件。结论:这些结果突出了DNA甲基化作为CRC驱动机制的潜在作用,并为基于某些肿瘤抑制基因的表观遗传学再激活识别新的治疗窗口铺平了道路。
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来源期刊
Clinical Epigenetics
Clinical Epigenetics Biochemistry, 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.
期刊最新文献
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