Hepatoselüler malignansi fenotipinin heterojenliği

Hepatocellular carcinoma (HCC) is one of the most wide-spread carcinomasthroughout the world - responsible for more than 600,000 deaths annually - and isstrongly associated with several etiological factors; including aflatoxin B1, alcohol, andHepatitis virus B and C. In HCC, many genes undergo somatic aberrations with atendency to cluster at genes involved in cell cycle regulation, in the p53 and canonicalWnt signaling pathways, and in the TGF-β/IGF axis. Almost a third of HCCs displaymutations affecting canonical Wnt signalling. However, the role of canonical Wntsignaling aberrations in HCC is not known in detail, since transgenic mice which expressmutant β-catenin (an integral component of canonical Wnt signaling) do not develop livertumours.To study the heterogeneity of hepatocellular malignancies, we have concentratedon canonical Wnt signaling in HCC cell lines. We have found that canonical Wntsignaling was active in 80% of well-differentiated, and 14% of poorly-differentiated celllines respectively. Furthermore, ectopic expression of a mutant β-catenin resulted instrong canonical Wnt activity in well-differentiated, but not in poorly-differentiated HCCcells. Our findings suggested that heterogeneity in HCC exists even in the same pathwayas exemplified by differential canonical Wnt signaling activity in well- and poorly-differentiated HCC cell lines. During this study, we produced monoclonal antibodiesagainst β-catenin to distinguish between the pools of nuclear/cytoplasmic and membrane-associated β-catenin in cells, since it is believed that the nuclear β-catenin pool is morepotent in tumorigenesis. Monoclonal antibody (MAb), 4C9 recognised β-catenin out ofadherens junctions, while another MAb, 9E10 recognised all β-catenin forms eventhough their epitopes were adjacent.iDifferential Wnt signaling activity in HCC cell lines prompted us to investigatethe interactions between β-catenin and other molecules, which have important functionsin hepatocytes and may affect β-catenin/TCF transcriptional activity. C/EBPα is a potentinhibitor of cell proliferation in HCC cell lines and is involved in liver-specific geneexpression, and some somatic alterations of it have been observed in AML and HCC. Weinvestigated the effect of C/EBPα on β-catenin signalling. We have found that C/EBPαinhibits mutant β-catenin-TCF transcriptional activity, and physically interacts with β-catenin in HCC cells.While we were analyzing some stably mock-transfected Huh7 clones to use ascontrols, we observed heterogeneity in their proliferation rates. Further analysis of theseclones revealed that some clones ceased to proliferate when passaged extensively. One ofthese clones (C3) was not tumorigenic in immunodeficient mice. Based on theseobservations, we hypothesized that some cancer cells could produce senescencentprogeny in cell culture. Indeed, we showed that breast- and liver-cancer-derived cellsdisplay senescencent phenotypes at variable ratios. By using our experimental system, wealso showed that replicative senescence program may work independently of functionalp53 and p16 pathways, and the SIP1 gene is partially responsible for replicative senescentphenotypes in our Huh7-derived senescent clone C3. Overexpression of mouse SIP1 inp53- and Rb-deficient Hep3B cells induced partial senescent phenotypes at earlypassages. However, stable Hep3B cells repressed mouse SIP1 expression by an unknownmechanism and escaped senescent arrest in late passages.Our results suggest that Wnt pathway may have a dual role in hepatocellularmalignancy, as it is active/easily inducible in well-differentiated HCC cells andinactive/repressed in poorly-differentiated ones. The further study of β-catenin in tumorsamples by using our monoclonal antibodies may reveal new aspects in β-cateninsignaling. However, the mechanism of these phenomena and the inhibition of β-catenin-TCF signaling by C/EBPα require more study to reach a more comprehensiveconclusion. The study of reprogramming of replicative senescence in HCC-derived cellsindicated that senescence program may work independent of p53 and p16 pathways andheterogeneity of hepatocellular malignancy exists even within the established HCCderived cell lines.ii