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. 2019 May 6;14(5):e0216374.
doi: 10.1371/journal.pone.0216374. eCollection 2019.

LINE-1 hypomethylation in human hepatocellular carcinomas correlates with shorter overall survival and CIMP phenotype

Sumadi Lukman Anwar  1   2 , Britta Hasemeier  2 , Elisa Schipper  2 , Arndt Vogel  3 , Hans Kreipe  2 , Ulrich Lehmann  2
Affiliations

LINE-1 hypomethylation in human hepatocellular carcinomas correlates with shorter overall survival and CIMP phenotype

Sumadi Lukman Anwar et al. PLoS One. .

Abstract

Reactivation of interspersed repetitive sequences due to loss of methylation is associated with genomic instability, one of the hallmarks of cancer cells. LINE-1 hypomethylation is a surrogate marker for global methylation loss and is potentially a new diagnostic and prognostic biomarker in tumors. However, the correlation of LINE-1 hypomethylation with clinicopathological parameters and the CpG island methylator phenotype (CIMP) in patients with liver tumors is not yet well defined, particularly in Caucasians who show quite low rates of HCV/HBV infection and a higher incidence of liver steatosis. Therefore, quantitative DNA methylation analysis of LINE-1, RASSF1A, and CCND2 using pyrosequencing was performed in human hepatocellular carcinomas (HCC, n = 40), hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5), and corresponding peritumoral liver tissues as well as healthy liver tissues (n = 5) from Caucasian patients. Methylation results were correlated with histopathological findings and clinical data. We found loss of LINE-1 DNA methylation only in HCC. It correlated significantly with poor survival (log rank test, p = 0.007). An inverse correlation was found for LINE-1 and RASSF1A DNA methylation levels (r2 = -0.47, p = 0.002). LINE-1 hypomethylation correlated with concurrent RASSF1/CCND2 hypermethylation (Fisher's exact test, p = 0.02). Both LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation were not found in benign hepatocellular tumors (HCA and FNH). Our results show that LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation are epigenetic aberrations specific for the process of malignant liver transformation. In addition, LINE-1 hypomethylation might serve as a future predictive biomarker to identify HCC patients with unfavorable overall survival.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. LINE-1 DNA methylation levels in HCC cell lines and hepatocyte cell lines.
Seven HCC lines showed significant lower DNA methylation levels than hepatocytes lines (mean methylation37.52 ± 3.12 vs. 50.73 ± 0.02 respectively, p = 0.005). For DNA methylation levels of individual CpG sites see S2 Table.
Fig 2
Fig 2. LINE-1 DNA methylation in HCC, adjacent peritumoral tissues, and healthy liver.
Frequent loss of LINE-1 DNA methylation was observed in HCC primary tissues. Means of DNA methylation were 46.5, 56.1, and 57.1 in HCC, peritumoral, and healthy liver tissues respectively. LINE-1 DNA methylation levels were significantly lower in the HCC primary tissues compared to the adjacent peritumoral tissues and significant difference was not observed between peritumoral tissues and healthy liver tissues. For DNA methylation levels of individual CpG sites see S3 Table.
Fig 3
Fig 3. RASSF1A and CCND2 DNA methylation levels in HCC, adjacent peritumoral tissues, and healthy liver.
DNA methylation levels were significantly higher in HCC compared to pertitumoral tissues both for RASSF1A (mean methylation levels were 39.8 and 16.2, p<0.0001, respectively) and CCND2 (mean methylation levels were 20.2 and 9.4, p<0.0001, respectively). For DNA methylation levels of individual CpG sites see S4 Table.
Fig 4
Fig 4. LINE-1, RASSF1A, and CCND2 DNA methylation in benign liver tumors (HCA and FNH) and the adjacent peritumoral tissues.
DNA methylation levels at the LINE-1, RASSF1A, and CCND2 loci were not significantly different in benign liver tumors and the peritumoral tissues. For DNA methylation levels of individual CpG sites see S5 Table.
Fig 5
Fig 5
A) Correlation of LINE-1 hypomethylation and poor survival in HCC. In compared to without methylation changes, LINE-1 hypomethylation was significantly correlated with shorter HCC survival (median survival 41 and 490 weeks respectively, log rank Mantel-Cox test p = 0.007), B) significant inverse correlation between LINE-1 and RASSF1 DNA methylation levels in HCC (Spearman r2 = -0.47, p = 0.002). C) Concordant hypermethylation of RASSF1A and CCND2 was associated with LINE-1 hypomethylation (Fisher exact test p = 0.02) but was not correlated with CTNNB1 positive mutation. Black box represents hypermethylation, white box represents hypomethylation, blue box represents CTNNB1 wild type, and red box represent CTNNB1 mutation.
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Grants and funding

This study was supported by a research grant to UL and HK from the Deutsche Forschungsgemeinschaft (DFG, http://www.dfg.de/) SFB-TRR77 “Liver cancer” (Project B1). SLA received a PhD fellowship from Molecular Medicine program of the Hannover Biomedical Research School (HBRS, https://www.mh-hannover.de/hbrs.html), Hannover Medical School, Germany. The funding bodies did not have any role in the study design, data evaluation, and preparation of the manuscript.