Abstract 323: Use of triple combination array analysis to identify STEAP family member 4 (STEAP4), a novel tumor suppressor gene in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide, but the molecular mechanisms underlying hepatocarcinogenesis are not clear. Array technology has made it possible to identify novel genes with alterations in liver cancer. In this study, we performed triple combination array analysis, expression profiling, karyotyping analysis using SNP array, and array-based DNA methylation profiling in the same HCC sample and attempted to find a novel tumor suppressor gene as a prognostic marker. Materials & Methods: A 68-year-old woman with chronic hepatitis C was diagnosed with HCC. The tumor and corresponding normal tissues were used for extraction of DNA and RNA. SNP chip array experiments were performed according to the standard protocol of Affymetrix GeneChip Mapping 500K arrays for DNA. mRNA expression profiles were determined using Affimetrix HGU133A and B Gene-Chips. Bisulphite-converted genomic DNA was analyzed using Illumina's Infinium Human Methylation27 Beadchip Kit. We consequently analyzed the identified gene using quantitative RT-PCR, methylation and un-methylation-specific PCR in nine HCC cell lines and in 48 primary HCC tissues and corresponding non-cancerous tissues collected at Nagoya University Hospital, and then compared these results with clinico-pathological data. Results: The STEAP4 gene, is located on 7q21.12, and expression was decreased at a Log2 ratio of -4.2 to -2.0 in cancerous tissue. There were two copies of 7q21.12, indicating no loss of heterozygosity (LOH) in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.870 compared to 0.234 in non-cancerous tissue, indicating high methylation in tumor tissue in the Methylation array. Re-activation of STEAP4 expression was seen after 5-aza-dC treatment in HCC cell lines. Moreover, we found that 32 of 48 (67%) HCC samples showed promoter hypermethylation. In 32 methylated cases, the expression values of STEAP4 gene in tumor tissues by real-time RT-PCR were tended to decrease compared to corresponding normal samples. In addition, methylated cases were significantly correlated with positive results for Hepatitis B and α-feto protein and had a tendency for poor prognosis (p=0.098). Conclusion: The STEAP4 gene is a tumor suppressor gene in HCC and might be a marker of poor prognosis. The triple combination array was a useful technique to identify the novel methylated tumor suppressor gene in HCC. Citation Format: {Authors}. {Abstract title} [ab