Elsevier

Journal of Hepatology

Volume 49, Issue 5, November 2008, Pages 746-757
Journal of Hepatology

Gain of GRHL2 is associated with early recurrence of hepatocellular carcinoma

https://doi.org/10.1016/j.jhep.2008.06.019Get rights and content

Background/Aims

The aim of this study is to identify genomic changes that might be implicated in hepatocellular carcinoma (HCC) progression, and evaluate the associations with clinico-pathological features.

Methods

The genomic DNA of 17 hepatoma cell lines was analyzed using Affymetrix GeneChip Human Mapping 50 K high-density oligonucleotide arrays. We selected representative genes from recurrent amplified regions and measured the copy number of these genes in 70 HCC clinical samples.

Results

We found 10 recurrent high-grade gain regions spanning less than 3 Mb in at least two hepatoma cell lines, and selected 10 representative genes. The copy number was almost normal in non-cancerous tissue and frequently amplified in Edmondson grade II or III HCC compared to Edmondson grade I HCC. Gain of TAX1BP1 in 7p15.2-1 was associated with larger tumor size and positivity of HCV antibody, and gain of CCND1 in 11q13.2-3 was associated with larger tumor size by multivariate analysis. Furthermore, a gain of GRHL2 in 8q22.3 was associated with early recurrence of HCC, controlling for clinical parameters. Decreased GRHL2 expression by RNA interference inhibits the growth of hepatoma cells, suggesting its association with cell proliferation.

Conclusions

A gain of GRHL2 might be a predictive marker for HCC recurrence.

Introduction

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide, and its incidence continues to increase in the United States and Europe due to the spread of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection [1]. The treatment of HCC is limited because of underlying cirrhosis and a high rate of recurrence; the cumulative 5-year survival rate is 53.4% with hepatic resection, 42.0% with local ablation therapy, and 22.6% with transcatheter arterial embolization in Japan [2]. Radio-frequency ablation therapy has been recently introduced and has improved the survival rate. Nevertheless, even in small HCC, the 4-year survival rate is only 74% [3]. The recurrence and prognosis of HCC is highly dependent on tumor extension and liver function. Prognostic scoring systems, such as CLIP score [4], BCLC score [5], JIS score [6] and Tokyo score [7] have been widely used to evaluate the prognosis of HCC patients. However, each system cannot always be sufficient for prediction of recurrence and prognosis, therefore identification of molecular markers that complement clinical information for recurrence could help attain a better follow-up of patients.

The genesis and progress of HCC are considered to be due to the accumulation of genetic alterations, including point mutations in oncogenes or tumor suppressor genes (TSGs). At the same time, copy number aberration or loss of heterozygosity (LOH) in the chromosome can lead to the activation of oncogenes and inactivation of TSGs, resulting in carcinogenesis [8], [9]. The regions of copy number gain and loss are supposed to contain putative oncogenes or TSGs respectively, and some reports revealed that they are associated with clinico-pathological features or recurrence of HCC [10], [11]. However, other reports failed to show any correlation between them [12], [13], [14], and predictive potential of allelic imbalance for clinical parameters is still uncertain.

Recently, single nucleotide polymorphism (SNP) arrays have been applied to detect SNPs as well as DNA copy number changes at the genome level [15], [16]. SNP arrays allow the genome-wide measurement of gene copy number changes and LOH simultaneously with high accuracy for several types of cancers, including HCC [17]. In this report, 36 HCCs were analyzed using Affymetrix GeneChip Human Mapping 10 K high-density oligonucleotide arrays. However, association between allelic imbalance and clinico-pathological parameters was not shown.

To find the molecular markers that associate with clinical parameters, we analyzed the genome of 17 hepatoma cell lines using Affymetrix GeneChip Human Mapping 50 K high-density oligonucleotide arrays, and evaluated the association between allelic imbalance and clinical parameters. In this study, we focused on the regions that contain putative oncogenes and analyzed a copy number of representative 10 genes (derived from 10 recurrent amplified regions of high-grade gain spanning less than 3 Mb in at least two cell lines) in HCC clinical samples. An analysis of the gain of genes and clinico-pathological features revealed that the gain of some chromosomal regions is associated with certain clinical characters such as tumor size and HCV infection. Moreover, the gain of GRHL2 in 8q22.3 was associated with recurrence-free survival, suggesting that gain of GRHL2 might be a novel genetic marker for predicting HCC recurrence.

Section snippets

Cell lines

We used 17 cell lines derived from human hepatomas; PLC/PRF/5, HuH-1, HuH-6, HuH-7, HepG2, Hep3B, HLE, HLF, SK-Hep1, JHH-1, JHH-4, JHH-7, SNU-398, SNU-449, SSP-25, HT17 and RBE (Table 1). HuH-1, HuH-7, HLE, HLF, JHH-1, JHH-4, and JHH-7 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). HT17 was obtained from the Cell Resource Center for Biomedical Research (Sendai, Japan). PLC/PRF/5, HuH-6, HepG2, SSP-25, and RBE were obtained from RIKEN Bioresource Center

Overview of copy number alterations in hepatoma cell lines

We analyzed 17 liver cancer cell lines, 13 hepatocellular carcinomas, 3 cholangiocellular carcinomas, and 1 hepatoblastoma (Table 1) using Genechip and CNAG. The absolute copy number was measured and estimated with FACS. The DNA of 17 human hepatoma cell lines was hybridized to SNP arrays containing over 50,000 mapped SNP loci. CNAG, a copy number detection algorithm, allowed the assessment of copy number and the identification of genomic gains and deletions by HMM.

Fig. 1 shows an overview of

Discussion

In this study, we surveyed 17 hepatoma cell lines using Affymetrix GeneChip Human Mapping 50 K arrays and a data analysis tool, CNAG, for determining chromosomal imbalance. Furthermore, we assessed the association between a gain of 10 genes and clinico-pathological features, and showed the possibility that a gain of GRHL2 is a novel genetic marker to predict HCC recurrence.

The genomes of hepatoma cell lines were broadly amplified due to polyploidity. However, the tendency for a gain and loss of

Acknowledgments

We thank Ms. Mitsuko Tsubouchi and Ms. Yasuko Ogino for technical assistance. This study was supported in part by Health and Labor Sciences Research Grants for Research on Hepatitis from the Ministry of Health, Labour and Welfare, Japan, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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    The authors declare that they do not have anything to disclose regarding funding from industries or conflict of interest with respect to this manuscript.

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