Unit: Oncogenesis and Molecular Virology - Inserm U579
Director: Marie Annick BUENDIA
Our Unit is investigating the molecular mechanisms of liver carcinogenesis in humans and in murine models. Our projects aim at better understanding the role of hepatitis B virus, notably the regulatory protein HBx, and at defining the factors implicated in liver cell transformation upon abnormal reactivation of the WNT/ß-catenin signaling pathway in hepatocellular carcinoma and hepatoblastoma.
Hepatocellular carcinoma (HCC) is the predominant type of primary liver cancer, affecting epithelial cells of the hepatic parenchyma, the hepatocytes. This tumor ranks among the commonest human cancers, and because it is usually fatal within a few months of diagnosis, it represents a leading cause of cancer death worldwide. HCC is also one of the human neoplasms epidemiologically related to viral infections : over 70% of HCC cases are associated with chronic infection with hepatitis B (HBV) or hepatitis C virus (HCV). In particular, HBV infection remains a major risk factor, despite the availability of an efficient and safe vaccine. However, the molecular basis of liver carcinogenesis remains incompletely understood. Several oncogenic pathways have been implicated, including abnormal reactivation of WNT/ß-catenin signaling. This pathway plays a predominant role in the development of hepatoblastoma, a rare embryonic liver tumor originating from immature liver progenitor cells.
Current projects of our research Unit aim at defining the mechanisms that regulate the functional activity of two proteins involved in liver oncogenesis : the transcriptional transactivator HBx of hepatitis B virus, and the oncoprotein ß-catenin, which is frequently mutated in human cancers. Our major objectives are to identify the cellular partners and the genes that are activated by these proteins, and to better understand their role in human and murine hepatocarcinogenesis. A new project has been recently initiated to analyze gene expression profiles and genetic as well as epigenetic alterations in hepatoblastoma.
1- Analysis of the viral regulatory protein HBx
(Christine Neuveut, Delphine Cougot)
The X (or HBx) protein of hepatitis B virus is a transcriptional transactivator endowed with weak oncogenic properties, involved in cell cycle control and apoptosis. Our previous studies have implicated HBx as a co-factor in liver carcinogenesis. To better understand the regulation of HBx transcriptional activity, we are currently analyzing the interactions between HBx histone acetyltransferases and deacetylases, and their impact on viral replication and oncogenesis. Our recent data indicate that HBx interacts directly with CBP/p300 in vivo and in vitro. Using deletion mutants, the interaction domains have been mapped on both partners. This interaction plays a crucial role in HBx activity on CREB-dependent transcription, probably by enhancing the formation and stabilization of the CREB/CBP complex on target DNA. Moreover, we have evidenced a direct interaction between HBx and HDAC1 . Current studies are aimed at confirming and extending these findings to other experimental systems and better understanding the role of HATs and HDACs in the context of HBV replication in HepG2 cells. By its activities in the viral life cycle and in liver tumorigenesis, HBx represents a major therapeutic target for combating HBV-induced carcinogenesis.
2- Oncogenic activation of ß-catenin
(Yu Wei, Claire-Angélique Renard, Charlotte Labalette, Christine Neuveut, Yann Nouet)
We have previously demonstrated that the Wnt pathway is frequently activated by stabilizing mutations of ß-catenin in HCC and in hepatoblastoma. Our objectives are to characterize the network of factors involved in liver carcinogenesis induced by oncogenic ß-catenin, including interacting partners and downstream Wnt target genes in the liver context. Our recent work has shed new light on the interaction of ß-catenin with the histone acetyltransferase CBP/p300, showing that acetylation of ß-catenin residue K345 by CBP/p300 reinforces ß-catenin binding affinity for Tcf4, leading to increased transcriptional activity of the ß-catenin/Tcf4 complex (Lévy et al, MCB, 2004). We have recently identified a new ß-catenin binding protein : the LIM protein FHL2 (Four and a Half LIM-only protein-2). LIM domains are made of double zinc fingers rich in cysteines and histidines, and LIM proteins are viewed as adaptors capable of mediating protein interactions. We have shown that FHL2 behaves as a coactivator of the nuclear ß-catenin/Tcf complex. Moreover, FHL2 physically interacts with CBP/p300, and these two coactivators form a tripartite complex with ß-catenin. FHL2 stimulates p300-mediated acetylation of ß-catenin, leading to enhanced transcriptional activation of Wnt-responsive genes (Labalette et al, MCB, 2004). We are currently attempting to delineate the contribution of FHL2 in ß-catenin transforming properties, using primary fibroblasts from FHL2-null mice or inhibition of FHL2 expression by small interfering RNAs. New murine models of liver and intestine tumorigenesis have been generated in collaboration with C. Perret (Inserm U567, Institut Cochin, Paris) and J. Chen (UCSD).
The second aspect of our project is to identify downstream targets of activated ß-catenin using microarray analysis in different hepatic contexts : primary human hepatocytes, murine hepatocellular carcinoma, and human hepatoblastoma. Our recent work has allowed us to characterize a number of ß-catenin target genes in the liver context, including IL-8, HIP/PAP and TBX3, and we are currently studying the role of these genes in liver carcinogenesis.
3- Genetic and epigenetic studies of hepatoblastoma
(Carolina Armengol, Claire-Angélique Renard, Stefano Cairo)
Our group is the main investigator for studies of hepatoblastoma in a national program of tumor expression profiling (CIT project of the Ligue Nationale contre le Cancer), in collaboration with different French hospitals. Hepatoblastoma is a rare pediatric liver tumor and etiological factors are mostly unknown. Analysis of gene expression profiles in a series of hepatoblastomas is associated with studies of genetic alterations by CGH array, as well as epigenetic alterations such as genome-wide promoter methylation and expression of microRNAs. Data are then correlated with a number of clinico-pathological parameters. Les données sont ensuite confrontées à divers paramètres clinico-pathologiques. Nos objectifs sont de mieux comprendre les bases moléculaires du développement de cette tumeur, et d'identifier des marqueurs moléculaires pour le diagnostic ainsi que de nouvelles pistes thérapeutiques. Le développement d'un modèle murin d'hépatoblastome est en cours d'étude. The aims of our project are to better understand the molecular pathogenesis of hepatoblastoma, and to generate new molecular markers for tumor diagnosis and prognosis, and novel approaches for therapy.
Figure 1 : Activation of CREB-dependent transcription by the transcriptional activator HBx. Phosphorylation of CREB by PKA induces the recrutement of the CBP/p300 histone acetyltransferases. Interaction of HBx with CBP/p300 might stimulate the formation and the stabilization of the CREB/CBP complex on cognate DNA.
Figure 2 : Expression of ß-catenin in normal liver (a) and in two hepatoblastomas of fetal (b) and embryonic (c) types by immunohistochemistry. ß-catenin is localized at cellular membrane in normal hepatocytes and mutated ß-catenin accumulates in the cytoplasm (b) and in the nucleus (c) of tumor cells.
Keywords: cancer, virology, hepatitis, transcription regulation, acetylation, transgenic mice