Cloning of suppressor gene of metastasis using subtraction hybridization method

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A gene (HFREP-1) that is overexpressed in hepatocellular carcinomas (HCCs) has been identified using a subtractive and differential cDNA cloning approach. We isolated the gene from a lambdagt10 cDNA library that was constructed from the poly(A)+-enriched RNA of a HCC specimen. The expression patterns observed in HCCs from 20 patients were classified into three types ; HFREP-1 was overexpressed in HCCs compared with non-tumorous regions (NTRs) in 55%, expressed weakly in both regions (15%) and strongly in both (30%). The largest cDNA insert contained 1231 base pairs coding 312 amino acids. The deduced protein sequence contained a hydrophobic leader peptide with a site for cleavage. The putative protein sequence showed strong homology with fibrinogen beta- and gamma- subunites and other fibrinogen-related proteins (FRPs), including four cysteine conserved residues. However, the HFREP-1 protein lacked a platelet-binding site, cross-linking region and thrombin-sensitive site, which are crucial for fibrin clot formation. The gene expression was studied in various organs in the rat and was specific to the liver, but little or none was detected in the fetal rat liver. Of the human carcinoma cell lines investigated, HFREP-1 was detected only in the HCC cell lines. We suggest that the HFREP-1 gene is a new member of the family of fibrinogens and has different features from other known FRPs and its expression is regulated oncodevelopmentally in hepatocytes.