Genetic analysis of Smad4 gene in TGF-Beta signalling pathway in human liver cancer

HCC is a multistep genetic disease in which many genomic changes occur as a result of uncontrolled proliferation of hepatocytes. Molecular events leading to HCC is still unclear. Until now, neither an oncogene nor a tumor suppressor gene has been shown to be prefentially altered in HCC. Genetic alterations other than p53, pl6, BRCA2 (Breast Carcinoma Associated Protein), M6P/IGFIIR (lyiannose 6 Phosphate/ Insulin Like Growth Factor II Receptor), Rb (Retinoblastoma), PRLTS (Platelet Derived Growth Factor Receptor-|3-Like Tumor Suppressor Gene), and Tg737 (Candidate polycyctic kidney disease gene) genes remain unknown. TGF-P is a strong inhibitor of hepatocyte proliferation. In HCC and cirrhosis increased levels of TGF-P is observed, so this shows that the presence of high levels of TGF-3 does not avoid hepatocyte proliferation. Thus, there may be a disruption in the signalling pathway of TGF-p. The common mediator Smad4 gene, which is among the genes located in TGF-P signalling pathway, is found to be mutated in many cancer types. We decided to do the mutational analysis of Smad4 gene, which is located in the signalling pathway of the hepatocyte antiproliferative factor, TGF-p. Exons 8, 9, 10, and 11 which are in MH2 region, and exon 2 which is in MHl region of Smad4 is mutationally analysed by SSCP for 35 HCC cases. In the 35 HCC tumors, 5 alterations were observed (14%), 3 of them being in exon 8, one of them being in exon 9a, and the last one being in exon 10 of Smad4 gene. In the samples we tested, no big deletions were observed, but the alterations observed are probably single base changes. Also HCC cell lines namely, HepG2, Hep3B, Huh-7, FOCUS, Mahlavu, and PLC/PRF/5 were checked for their mutations and cell lines other then PLC/PRF/5 were analysed for their mRNA transcription. There were no big deletions or alterations in Nand C- terminals of the cell lines and we have shown mRNA transcription for all cell lines except Hep3B in which PCR has revealed very weak amplification. Our results suggest that Smad4 might be involved in at least a part of primary HCC tumor development.