Genome-Wide Methylation Analysis and Epigenetic Unmasking Identify Tumor Suppressor Genes in Hepatocellular Carcinoma

Abstract

BACKGROUND & AIMS Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. METHODS We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina Human Beadchip 27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). RESULTS Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6–6.5 in multivariate analysis). CONCLUSIONS Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.

Genome-wide analysis of DNA methylation in hepatoblastoma tissues

“…For instance, Revill et al revealed sphingomyelin phosphodiesterase 3 as a potent tumor suppressor gene, which is hypermethylated in primary hepatocellular carcinoma (13). The present study aimed to determine whether changes in methylation status could affect HB by analyzing HB tissue samples and paired distant noncancerous tissues to profile differentially methylated genes in the disease.…”

Epigenetic silencing of neurofilament genes promotes an aggressive phenotype in breast cancer

“…14,15 Neurofilaments have been implicated in the pathology of neurological diseases, [16][17][18] but recent data also point to a role in the pathology of cancer. [19][20][21] For example, NEFL and NEFM are both located within the 8p21 chromosome region and LOH of this region has been described in several cancers including breast cancer. [22][23][24] Furthermore, aberrant expression and methylation of neurofilament genes were recently detected in esophageal squamous cell, renal cell and hepatocellular carcinoma as well as in Ewing sarcoma.…”

“…[22][23][24] Furthermore, aberrant expression and methylation of neurofilament genes were recently detected in esophageal squamous cell, renal cell and hepatocellular carcinoma as well as in Ewing sarcoma. 19,21,25,26 In the present study we used a comprehensive approach to study the epigenetic inactivation of neurofilament genes in breast cancer and the functional significance of these events.…”

Molecular alterations in the carcinogenesis and progression of hepatocellular carcinoma: Tumor factors and background liver factors

“…Wong et al (81) detected the downregulation of miRNA-223 expression levels in HCC tissues and investigated stathmin 1 (STMN1) as a downstream target. Methylation array analysis has been used to detect cancer-specific epigenetic alterations in HCC (82)(83)(84)(85)(86)(87)(88)(89)(90)(91). Shen et al (82) analyzed tumor and adjacent non-tumor tissue samples from 62 Taiwanese patients with HCC, using Illumina ® methylation arrays, and identified several genes that were hyper-or hypo-methylated in tumor tissues, as compared with adjacent non-tumor tissues (82).…”

“…The tumor-suppressor gene candidates were efficiently identified by detecting downregulation and methylation in tumor tissues, and without chromosomal deletion of the loci of the targeted genes. Revill et al (90) performed a genome-wide methylation array analysis of HCC samples and a microarray analysis of gene re-expression in HCC cell lines, and then combined the data to locate an epigenetically-silenced tumor-suppressor gene. These studies demonstrate that the majority of hepatocarcinogenesis studies focus on the changes in tumor tissues and the comparison of HCC cell lines or tumor tissues with adjacent normal liver tissues.…”