Heterogeneity of hepatocellular malignant phenotype
Author(s)
Advisor
Öztürk, MehmetDate
2006Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
Hepatocellular carcinoma (HCC) is one of the most wide-spread carcinomas
throughout the world - responsible for more than 600,000 deaths annually - and is
strongly associated with several etiological factors; including aflatoxin B1, alcohol, and
Hepatitis virus B and C. In HCC, many genes undergo somatic aberrations with a
tendency to cluster at genes involved in cell cycle regulation, in the p53 and canonical
Wnt signaling pathways, and in the TGF-β/IGF axis. Almost a third of HCCs display
mutations affecting canonical Wnt signalling. However, the role of canonical Wnt
signaling aberrations in HCC is not known in detail, since transgenic mice which express
mutant β-catenin (an integral component of canonical Wnt signaling) do not develop liver
tumours.
To study the heterogeneity of hepatocellular malignancies, we have concentrated
on canonical Wnt signaling in HCC cell lines. We have found that canonical Wnt
signaling was active in 80% of well-differentiated, and 14% of poorly-differentiated cell
lines respectively. Furthermore, ectopic expression of a mutant β-catenin resulted in
strong canonical Wnt activity in well-differentiated, but not in poorly-differentiated HCC
cells. Our findings suggested that heterogeneity in HCC exists even in the same pathway
as exemplified by differential canonical Wnt signaling activity in well- and poorlydifferentiated
HCC cell lines. During this study, we produced monoclonal antibodies
against β-catenin to distinguish between the pools of nuclear/cytoplasmic and membraneassociated
β-catenin in cells, since it is believed that the nuclear β-catenin pool is more
potent in tumorigenesis. Monoclonal antibody (MAb), 4C9 recognised β-catenin out of
adherens junctions, while another MAb, 9E10 recognised all β-catenin forms even
though their epitopes were adjacent.
Differential Wnt signaling activity in HCC cell lines prompted us to investigate
the interactions between β-catenin and other molecules, which have important functions
in hepatocytes and may affect β-catenin/TCF transcriptional activity. C/EBPα is a potent
inhibitor of cell proliferation in HCC cell lines and is involved in liver-specific gene
expression, and some somatic alterations of it have been observed in AML and HCC. We
investigated the effect of C/EBPα on β-catenin signalling. We have found that C/EBPα
inhibits 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 as
controls, we observed heterogeneity in their proliferation rates. Further analysis of these
clones revealed that some clones ceased to proliferate when passaged extensively. One of
these clones (C3) was not tumorigenic in immunodeficient mice. Based on these
observations, we hypothesized that some cancer cells could produce senescencent
progeny in cell culture. Indeed, we showed that breast- and liver-cancer-derived cells
display senescencent phenotypes at variable ratios. By using our experimental system, we
also showed that replicative senescence program may work independently of functional
p53 and p16 pathways, and the SIP1 gene is partially responsible for replicative senescent
phenotypes in our Huh7-derived senescent clone C3. Overexpression of mouse SIP1 in
p53- and Rb-deficient Hep3B cells induced partial senescent phenotypes at early
passages. However, stable Hep3B cells repressed mouse SIP1 expression by an unknown
mechanism and escaped senescent arrest in late passages.
Our results suggest that Wnt pathway may have a dual role in hepatocellular
malignancy, as it is active/easily inducible in well-differentiated HCC cells and
inactive/repressed in poorly-differentiated ones. The further study of β-catenin in tumor
samples by using our monoclonal antibodies may reveal new aspects in β-catenin
signaling. However, the mechanism of these phenomena and the inhibition of β-cateninTCF
signaling by C/EBPα require more study to reach a more comprehensive
conclusion. The study of reprogramming of replicative senescence in HCC-derived cells
indicated that senescence program may work independent of p53 and p16 pathways and
heterogeneity of hepatocellular malignancy exists even within the established HCC
derived cell lines.