Abstract 1733: Fibulin 1 is a novel tumor suppressor gene detected in hepatocellular carcinoma using a double combination array analysis
Authors: Shuji Nomoto, Mitsuro Kanda, Yukiyasu Okamura, Masamichi Hayashi, Tsutomu Fujii, Hiroyuki Sugimoto, Shin Takeda, Akimasa Nakao
Journal: Cancer Research (2010)

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide, but its underlying molecular mechanisms contributing to hepatocarcinogenesisare still not clear. Microarray techniques have offered considerable advances, and the expression status of various genes in malignant tumors has been investigated coinstantaneously through the use of microarray. Furthermore, the advent of single nucleotide polymorphism (SNP) arrays has enabled us to detect a large number of DNA polymorphic loci simultaneously and to analyze copy number changes. In this study, we devised a double combination array method, conducting both expression array and SNP array using the same surgical specimen of a patient with HCC, and reported a new candidate for tumor suppressor gene in HCC and the gene silencing mechanism. Materials &amp; Methods: A 68-year-old woman with chronic hepatitis type C was diagnosed with HCC and underwent hepatectomy. The tumor specimen and corresponding normal tissues were used for SNP array, Affymetrix GeneChip Mapping 500K array, and expression profile, Affymetrix HGU133A and B. We consequently analyzed the identified gene, using quantitative RT-PCR, methylation and un-methylation specific PCR in five HCC cell lines, in addition to immunohistochemical staining with FBLN1 antibody in 48 primary HCC tissues and corresponding non-cancerous tissues collected at Nagoya University Hospital. We then compared these results with clinico-pathological data. Results: According to the results of expression array, FBLN1 gene showed decreased expression in tumor tissue. The copy number of chromosome 22q13, where FBLN1 exists, did not show chromosomal deletion. We found many CpG dinucreotides in the promoter region of the FBLN1 gene and confirmed promoter methylation by sequence analysis. Re-activation of the FBLN1 expression was seen by 5’-aza-dC treatment using HCC cell lines. Moreover, we found 24 of 48 (50%) HCC samples showed promoter hypermethylation. In quantitative real-time PCR, mRNA expression of FBLN1 was significantly decreased in HCC samples compared with that in corresponding non-cancerous liver tissues (P=0.0139). Downregulation in HCC tissues was also confirmed by immunohistochemical staining at the protein level. Finally, we evaluated the methylation status and the clinico-pathological findings. Although the difference was not significant, the methylated cases were corelated with poorer prognosis than the methylation negative cases. Promoter hypermethylation of FBLN1 in HCCs was significantly associated with advanced stage, multiple tumors and tumor size≥3cm (P=0.0022, P=0.0346 and P=0.0088, respectively). Conclusion: Double array analysis revealed a novel tumor suppressor gene, FBLN1, for HCC. Promoter hypermethylation was associated with down-regulation of FBLN1 expression and was correlated with advanced stage of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1733.