Automatic Detection of the Circulating Cell-Free Methylated DNA Pattern of GCM2, ITPRIPL1 and CCDC181 for Detection of Early Breast Cancer and Surgical Treatment Response

Wang, Sheng Chao; Liao, Li Min; Ansar, Muhamad; Lin, Shih Yun; Hsu, Wei‐Wen; Su, Chih-Ming; Chung, Yu Mei; Liu, Cai Cing; Hung, Chin Sheng; Lin, Ruo Kai

doi:10.3390/cancers13061375

Cited by 14 publications

(15 citation statements)

References 96 publications

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“…Most importantly, all six DMCs of ENPP2 located at TSS in the promoter or at the 1st exon showed increased methylation across different cancer types, including HCC, melanoma, CRC, LC and PC. These results corroborate and expand recent observations showing a hypermethylated ENPP2 promoter in primary tumors of LC and squamous cell carcinoma patients [ 45 ] and in breast cancer [ 13 , 22 , 46 ].…”

Section: Discussionsupporting

confidence: 92%

ENPP2 Methylation in Health and Cancer

Παναγοπούλου

Fanidis

Aidinis

et al. 2021

IJMS

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Autotaxin (ATX) encoded by Ectonucleotide Pyrophosphatase/Phosphodiesterase 2 (ENPP2) is a key enzyme in Lysophosphatidic Acid (LPA) synthesis implicated in cancer. Although its aberrant expression has been reported, ENPP2 methylation profiles in health and malignancy are not described. We examined in silico the methylation of ENPP2 analyzing publicly available methylome datasets, to identify Differentially Methylated CpGs (DMCs) which were then correlated with expression at gene and isoform levels. Significance indication was set to be FDR corrected p-value < 0.05. Healthy tissues presented methylation in all gene body CGs and lower levels in Promoter Associated (PA) regions, whereas in the majority of the tumors examined (HCC, melanoma, CRC, LC and PC) the methylation pattern was reversed. DMCs identified in the promoter were located in sites recognized by multiple transcription factors, suggesting involvement in gene expression. Alterations in methylation were correlated to an aggressive phenotype in cancer cell lines. In prostate and lung adenocarcinomas, increased methylation of PA CGs was correlated to decreased ENPP2 mRNA expression and to poor prognosis parameters. Collectively, our results corroborate that methylation is an active level of ATX expression regulation in cancer. Our study provides an extended description of the methylation status of ENPP2 in health and cancer and points out specific DMCs of value as prognostic biomarkers.

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Section: Discussionsupporting

confidence: 92%

ENPP2 Methylation in Health and Cancer

Παναγοπούλου

Fanidis

Aidinis

et al. 2021

IJMS

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“…Analysis showed that ENPP2 hypermethylation was detected more often in ccfDNA of BrCa patients than in healthy individuals in a statistically significant manner. In a previous study addressing ENPP2 methylation in ccfDNA from 22 healthy and 45 Taiwanese BrCa patients, no significant differences were found [ 28 ], although ENPP2 methylation showed a twofold increase in BrCa in relation to adjacent normal tissue. Methodological differences or even population genetic variations might explain these different findings.…”

Section: Discussionmentioning

confidence: 93%

ENPP2 Promoter Methylation Correlates with Decreased Gene Expression in Breast Cancer: Implementation as a Liquid Biopsy Biomarker

Παναγοπούλου

Drosouni

Fanidis

et al. 2022

IJMS

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Autotaxin (ATX), encoded by the ctonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) gene, is a key enzyme in lysophosphatidic acid (LPA) synthesis. We have recently described ENPP2 methylation profiles in health and multiple malignancies and demonstrated correlation to its aberrant expression. Here we focus on breast cancer (BrCa), analyzing in silico publicly available BrCa methylome datasets, to identify differentially methylated CpGs (DMCs) and correlate them with expression. Numerous DMCs were identified between BrCa and healthy breast tissues in the gene body and promoter-associated regions (PA). PA DMCs were upregulated in BrCa tissues in relation to normal, in metastatic BrCa in relation to primary, and in stage I BrCa in relation to normal, and this was correlated to decreased mRNA expression. The first exon DMC was also investigated in circulating cell free DNA (ccfDNA) isolated by BrCa patients; methylation was increased in BrCa in relation to ccfDNA from healthy individuals, confirming in silico results. It also differed between patient groups and was correlated to the presence of multiple metastatic sites. Our data indicate that promoter methylation of ENPP2 arrests its transcription in BrCa and introduce first exon methylation as a putative biomarker for diagnosis and monitoring which can be assessed in liquid biopsy.

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“…Circulating cell-free DNA was extracted from plasma (3.5 mL) that had been isolated from 10 mL of peripheral blood within 2 h of collection. Circulating cell-free DNA (ccfDNA) was extracted from 15 of the plasma samples using the MagMAX Cell-Free DNA Isolation Kit (Thermo Fisher Scientific, Austin, TX, USA) according to the manufacturer's recommended protocol (Yan et al 2018;Hung et al 2018;Wang et al 2021). CcfDNA was extracted from 46 of the plasma samples using the iCatcher Circulating cfDNA 1000 kit (CatchGene, New Taipei City, Taiwan) according to the manufacturer's recommended protocol.…”

Section: Genomic Dna Circulating Cell-free Dna and Rna Extractionmentioning

confidence: 99%

Hypermethylation of TMEM240 predicts poor hormone therapy response and disease progression in breast cancer

Lin

et al. 2022

Mol Med

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Background Approximately 25% of patients with early-stage breast cancer experience cancer progression throughout the disease course. Alterations in TMEM240 in breast cancer were identified and investigated to monitor treatment response and disease progression. Methods Circulating methylated TMEM240 in the plasma of breast cancer patients was used to monitor treatment response and disease progression. The Cancer Genome Atlas (TCGA) data in Western countries and Illumina methylation arrays in Taiwanese breast cancer patients were used to identify novel hypermethylated CpG sites and genes related to poor hormone therapy response. Quantitative methylation-specific PCR (QMSP), real-time reverse transcription PCR, and immunohistochemical analyses were performed to measure DNA methylation and mRNA and protein expression levels in 394 samples from Taiwanese and Korean breast cancer patients. TMEM240 gene manipulation, viability, migration assays, RNA-seq, and MetaCore were performed to determine its biological functions and relationship to hormone drug treatment response in breast cancer cells. Results Aberrant methylated TMEM240 was identified in breast cancer patients with poor hormone therapy response using genome-wide methylation analysis in the Taiwan and TCGA breast cancer cohorts. A cell model showed that TMEM240, which is localized to the cell membrane and cytoplasm, represses breast cancer cell proliferation and migration and regulates the expression levels of enzymes involved in estrone and estradiol metabolism. TMEM240 protein expression was observed in normal breast tissues but was not detected in 88.2% (67/76) of breast tumors and in 90.0% (9/10) of metastatic tumors from breast cancer patients. QMSP revealed that in 54.5% (55/101) of Taiwanese breast cancer patients, the methylation level of TMEM240 was at least twofold higher in tumor tissues than in matched normal breast tissues. Patients with hypermethylation of TMEM240 had poor 10-year overall survival (p = 0.003) and poor treatment response, especially hormone therapy response (p < 0.001). Circulating methylated TMEM240 dramatically and gradually decreased and then diminished in patients without disease progression, whereas it returned and its levels in plasma rose again in patients with disease progression. Prediction of disease progression based on circulating methylated TMEM240 was found to have 87.5% sensitivity, 93.1% specificity, and 90.2% accuracy. Conclusions Hypermethylation of TMEM240 is a potential biomarker for treatment response and disease progression monitoring in breast cancer.

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Automatic Detection of the Circulating Cell-Free Methylated DNA Pattern of GCM2, ITPRIPL1 and CCDC181 for Detection of Early Breast Cancer and Surgical Treatment Response

Cited by 14 publications

References 96 publications

ENPP2 Methylation in Health and Cancer

ENPP2 Methylation in Health and Cancer

ENPP2 Promoter Methylation Correlates with Decreased Gene Expression in Breast Cancer: Implementation as a Liquid Biopsy Biomarker

Hypermethylation of TMEM240 predicts poor hormone therapy response and disease progression in breast cancer

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