Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells

Abstract Background Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. Methods We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9‐Tet1CD‐based CDO1‐targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. Results CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p < .05). The CRISPR/dCas9‐based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non‐invasive biomarker for early diagnosis of BC. Conclusions CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

[1]  Ana-Teresa Maia,et al.  THOR is a targetable epigenetic biomarker with clinical implications in breast cancer , 2022, Clinical epigenetics.

[2]  J. Shao,et al.  Targeted demethylation at ZNF154 promotor upregulates ZNF154 expression and inhibits the proliferation and migration of Esophageal Squamous Carcinoma cells , 2022, Oncogene.

[3]  Yunli Zhou,et al.  Soluble POSTN is a novel biomarker complementing CA153 and CEA for breast cancer diagnosis and metastasis prediction , 2022, BMC cancer.

[4]  J. Kocher,et al.  DNA methylation signature predicts cancer response to demethylation agents from profiling diverse cancer cell lines , 2022, Cancer communications.

[5]  D. Segal,et al.  Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer , 2022, British Journal of Cancer.

[6]  Yancheng Xu,et al.  Sigma-1 receptor overexpression promotes proliferation and ameliorates cell apoptosis in β-cells , 2022, Molecular medicine reports.

[7]  S. R. Hanumanth,et al.  In silico identification of single nucleotide variations at CpG sites regulating CpG island existence and size , 2022, Scientific Reports.

[8]  Shaw-Hwa Hwang,et al.  A High-Performance Deep Neural Network Model for BI-RADS Classification of Screening Mammography , 2022, Sensors.

[9]  Yihan Wang,et al.  MicroRNA expression is deregulated by aberrant methylation in B-cell acute lymphoblastic leukemia mouse model , 2022, Molecular Biology Reports.

[10]  Yunfeng Zhou,et al.  A Novel Gene Prognostic Signature Based on Differential DNA Methylation in Breast Cancer , 2021, Frontiers in Genetics.

[11]  Ran Ma,et al.  Aberrant promoter hypermethylation inhibits RGMA expression and contributes to tumor progression in breast cancer , 2021, Oncogene.

[12]  Jing Yang,et al.  USP25 Regulates the Proliferation and Apoptosis of Ovarian Granulosa Cells in Polycystic Ovary Syndrome by Modulating the PI3K/AKT Pathway via Deubiquitinating PTEN , 2021, Frontiers in Cell and Developmental Biology.

[13]  Chungyeul Kim,et al.  RNA-sequencing identification and validation of genes differentially expressed in high-risk adenoma, advanced colorectal cancer, and normal controls , 2021, Functional & Integrative Genomics.

[14]  T. C. Evans,et al.  Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA , 2021, Genome research.

[15]  J. Sandoval,et al.  Cancer Epigenetic Biomarkers in Liquid Biopsy for High Incidence Malignancies , 2021, Cancers.

[16]  J. Ferlay,et al.  Cancer statistics for the year 2020: An overview , 2021, International journal of cancer.

[17]  Jincai Lu,et al.  Cimicifuga dahurica extract inhibits the proliferation, migration and invasion of breast cancer cells MDA-MB-231 and MCF-7 in vitro and in vivo. , 2021, Journal of ethnopharmacology.

[18]  B. Stillman,et al.  Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication. , 2021, Molecular cell.

[19]  R. Xu,et al.  Liquid Biopsy of Methylation Biomarkers in Cell-Free DNA. , 2021, Trends in molecular medicine.

[20]  Lei Cao,et al.  B7-H3 promotes the cell cycle-mediated chemoresistance of colorectal cancer cells by regulating CDC25A , 2020, Journal of Cancer.

[21]  C. Stirzaker,et al.  The DNA methylation landscape in cancer , 2019, Essays in biochemistry.

[22]  Christopher D. Heinen,et al.  Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways , 2019, Nature Communications.

[23]  S. Cai,et al.  In vivo metabolism of 8,2'-diprenylquercetin 3-methyl ether and the distribution of its metabolites in rats by HPLC-ESI-IT-TOF-MSn. , 2019, Fitoterapia.

[24]  Lei Zhang,et al.  The interplay of circulating tumor DNA and chromatin modification, therapeutic resistance, and metastasis , 2019, Molecular Cancer.

[25]  M. Conrad,et al.  Role of GPX4 in ferroptosis and its pharmacological implication. , 2019, Free radical biology & medicine.

[26]  Xiang-Nan Li,et al.  RNA sequencing reveals the expression profiles of circRNA and indicates that circDDX17 acts as a tumor suppressor in colorectal cancer , 2018, Journal of Experimental & Clinical Cancer Research.

[27]  Xiang-Nan Li,et al.  RNA sequencing reveals the expression profiles of circRNA and indicates that circDDX17 acts as a tumor suppressor in colorectal cancer , 2018, Journal of experimental & clinical cancer research : CR.

[28]  S. Gurunathan,et al.  Cytotoxic Potential and Molecular Pathway Analysis of Silver Nanoparticles in Human Colon Cancer Cells HCT116 , 2018, International journal of molecular sciences.

[29]  C. Sutter,et al.  Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients , 2018, International journal of cancer.

[30]  K. Yamashita,et al.  Prognostic significance of promoter DNA hypermethylation of the cysteine dioxygenase 1 (CDO1) gene in primary gallbladder cancer and gallbladder disease , 2017, PloS one.

[31]  J. Yu,et al.  Cysteine Dioxygenase 1 Mediates Erastin-Induced Ferroptosis in Human Gastric Cancer Cells1 , 2017, Neoplasia.

[32]  O. Elemento,et al.  TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells , 2017, Nature Genetics.

[33]  H. Pass,et al.  A Panel of Novel Detection and Prognostic Methylated DNA Markers in Primary Non–Small Cell Lung Cancer and Serum DNA , 2017, Clinical Cancer Research.

[34]  Y. Tao,et al.  EGLN1/c-Myc Induced Lymphoid-Specific Helicase Inhibits Ferroptosis through Lipid Metabolic Gene Expression Changes , 2017, Theranostics.

[35]  Luca Pinello,et al.  Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments , 2017, Nature Protocols.

[36]  Johannes Stein,et al.  CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients , 2016, Epigenetics.

[37]  M. Pellegrini,et al.  Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells. , 2016, Cell reports.

[38]  Á. Carracedo,et al.  Development of a methylation marker set for forensic age estimation using analysis of public methylation data and the Agena Bioscience EpiTYPER system. , 2016, Forensic science international. Genetics.

[39]  Howard Cedar,et al.  DNA Methylation in Cancer and Aging. , 2016, Cancer research.

[40]  Lei Zhang,et al.  A CRISPR-based approach for targeted DNA demethylation , 2016, Cell Discovery.

[41]  Rui Henrique,et al.  Diagnostic and prognostic epigenetic biomarkers in cancer. , 2015, Epigenomics.

[42]  P. A. van den Brandt,et al.  Promoter Methylation of CDO1 Identifies Clear-Cell Renal Cell Cancer Patients with Poor Survival Outcome , 2015, Clinical Cancer Research.

[43]  P. Altevogt,et al.  L1CAM is expressed in triple-negative breast cancers and is inversely correlated with Androgen receptor , 2014, BMC Cancer.

[44]  D. Sidransky,et al.  Detection of Methylated CDO1 in Plasma of Colorectal Cancer; A PCR Study , 2014, PloS one.

[45]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

[46]  Ludmila V. Danilova,et al.  Frequent Inactivation of Cysteine Dioxygenase Type 1 Contributes to Survival of Breast Cancer Cells and Resistance to Anthracyclines , 2013, Clinical Cancer Research.

[47]  P. Defossez,et al.  On how mammalian transcription factors recognize methylated DNA , 2013, Epigenetics.

[48]  J. Pennings,et al.  A Bead-Based Multiplexed Immunoassay to Evaluate Breast Cancer Biomarkers for Early Detection in Pre-Diagnostic Serum , 2012, International journal of molecular sciences.

[49]  K. Hunt,et al.  Local-regional control according to surrogate markers of breast cancer subtypes and response to neoadjuvant chemotherapy in breast cancer patients undergoing breast conserving therapy , 2012, Breast Cancer Research.

[50]  K. Ramos,et al.  Epigenetic Therapies for Cancer , 2011 .

[51]  G. Ming,et al.  Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain , 2011, Cell.

[52]  D. Lambrechts,et al.  A new approach to imprinting mutation detection in GNAS by Sequenom EpiTYPER system. , 2010, Clinica chimica acta; international journal of clinical chemistry.

[53]  Yi Zhang,et al.  Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification , 2010, Nature.

[54]  J. Foekens,et al.  RESEARCH ARTICLE Open Access Research article CDO1 Promoter Methylation is a Biomarker for Outcome Prediction of Anthracycline Treated, Estrogen Receptor-Positive, Lymph Node-Positive Breast Cancer Patients , 2022 .

[55]  Mitsuyoshi Nakao,et al.  Sequence-specific recognition of methylated DNA by human zinc-finger proteins , 2010, Nucleic acids research.

[56]  David R. Liu,et al.  The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing , 2010, PloS one.

[57]  David R. Liu,et al.  Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1 , 2009 .

[58]  D. Richardson,et al.  Growth arrest and DNA damage-45 alpha (GADD45alpha). , 2009, The international journal of biochemistry & cell biology.

[59]  J. Dominy,et al.  Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels , 2009, Amino Acids.

[60]  F. Lyko,et al.  Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine , 2008, International journal of cancer.

[61]  M. Maroney,et al.  Cysteine dioxygenase: structure and mechanism. , 2007, Chemical communications.

[62]  J. Moskovitz,et al.  Ablation of the mammalian methionine sulfoxide reductase A affects the expression level of cysteine deoxygenase. , 2007, Biochemical and biophysical research communications.

[63]  Shuji Ogino,et al.  Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. , 2006, The Journal of molecular diagnostics : JMD.

[64]  P. Laird,et al.  Sex differential in methylation patterns of selected genes in Singapore Chinese , 2005, Human Genetics.

[65]  L. Busino,et al.  Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis , 2004, Oncogene.

[66]  Long-Cheng Li,et al.  MethPrimer: designing primers for methylation PCRs , 2002, Bioinform..

[67]  A. Fornace,et al.  Genomic instability, centrosome amplification, cell cycle checkpoints and Gadd45a , 2002, Oncogene.

[68]  M. Esteller.  CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future , 2002, Oncogene.

[69]  Albert B. Reynolds,et al.  The p120ctn-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides , 2002, Nucleic Acids Res..

[70]  A. Bird,et al.  The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor. , 2001, Genes & development.

[71]  P. Laird,et al.  MethyLight: a high-throughput assay to measure DNA methylation. , 2000, Nucleic acids research.

[72]  Sung-Hoon Kim,et al.  CNOT2 promotes proliferation and angiogenesis via VEGF signaling in MDA-MB-231 breast cancer cells. , 2018, Cancer letters.

[73]  E. Novellino,et al.  CDC25 phosphatase inhibitors: an update. , 2012, Mini reviews in medicinal chemistry.