Revill, Kate, Wang, Tim, Lachenmayer, Anja, Kojima, Kensuke, Harrington, Andrew, Li, Jinyu, Hoshida, Yujin, Llovet, Josep M., Powers, R. Scott (September 2013) Genome-wide Methylation Analysis and Epigenetic Unmasking Identify Tumor Suppressor Genes in Hepatocellular Carcinoma. Gastroenterology, 145 (6). pp. 1424-1435. ISSN 0016-5085
Abstract
AbstractBackground & 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 following epigenetic unmasking. Methods We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines following 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 following epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, SMPD3 and 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, NEFH P=.022), and increased their ability to form tumors following subcutaneous injection or orthotopic transplant into mice, confirming their role as tumor suppressor genes in HCC cells. 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.
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