Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma

Kate Revill, Tim Wang, Anja Lachenmayer, Kensuke Kojima, Andrew Harrington, Jinyu Li, Yujin Hoshida, Josep M. Llovet, Scott Powers

Research output: Contribution to journalArticle

103 Citations (Scopus)

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 HumanBeadchip27K 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.

Original languageEnglish (US)
Pages (from-to)1424-1435.e25
JournalGastroenterology
Volume145
Issue number6
DOIs
StatePublished - Dec 2013
Externally publishedYes

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Tumor Suppressor Genes
Epigenomics
Sphingomyelin Phosphodiesterase
Methylation
Hepatocellular Carcinoma
Genome
Intermediate Filaments
DNA Methylation
Gene Expression
Cell Line
Peptides
Recurrence
Gene Expression Regulation
Subcutaneous Injections
Microarray Analysis
Liver Neoplasms
Small Interfering RNA
Cell Movement
Transfection
Neoplasms

Keywords

  • 5-aza-2-deoxycitidine
  • NEFH
  • nSMase2
  • Sphingomyelin Phosphodiesterase

ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

Cite this

Revill, K., Wang, T., Lachenmayer, A., Kojima, K., Harrington, A., Li, J., ... Powers, S. (2013). Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma. Gastroenterology, 145(6), 1424-1435.e25. https://doi.org/10.1053/j.gastro.2013.08.055

Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma. / Revill, Kate; Wang, Tim; Lachenmayer, Anja; Kojima, Kensuke; Harrington, Andrew; Li, Jinyu; Hoshida, Yujin; Llovet, Josep M.; Powers, Scott.

In: Gastroenterology, Vol. 145, No. 6, 12.2013, p. 1424-1435.e25.

Research output: Contribution to journalArticle

Revill, K, Wang, T, Lachenmayer, A, Kojima, K, Harrington, A, Li, J, Hoshida, Y, Llovet, JM & Powers, S 2013, 'Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma', Gastroenterology, vol. 145, no. 6, pp. 1424-1435.e25. https://doi.org/10.1053/j.gastro.2013.08.055
Revill, Kate ; Wang, Tim ; Lachenmayer, Anja ; Kojima, Kensuke ; Harrington, Andrew ; Li, Jinyu ; Hoshida, Yujin ; Llovet, Josep M. ; Powers, Scott. / Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma. In: Gastroenterology. 2013 ; Vol. 145, No. 6. pp. 1424-1435.e25.
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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 HumanBeadchip27K 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.",
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AU - Hoshida, Yujin

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N2 - 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 HumanBeadchip27K 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.

AB - 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 HumanBeadchip27K 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.

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