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 in 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 rare 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.

1- Analysis of the viral regulatory protein HBx (Christine Neuveut, Yuanfei WU, 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 HBx subcellular localization and its role in viral replication and oncogenesis. Our recent data indicate that HBx is acetylated in vivo, and we are investigating the effects of this modification on known HBx functions in transcriptional transactivation and apoptosis. A second theme is the search for cellular genes that are regulated by HBx in primary human hepatocytes and in mouse liver. 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, Charlotte Labalette, Christine Neuveut, Claire-Angélique Renard, Carolina Armengol, Stefano Cairo)

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.

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. 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.

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, and we are currently studying the role of these genes in liver carcinogenesis. 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. The aim of our project is to generate new molecular markers for tumor diagnosis and prognosis, and novel approaches for liver cancer therapy.

3- Murine models of liver tumorigenesis (Claire-Angélique Renard, Charlotte Labalette, Yu Wei)

Our lab has established WHV/myc transgenic mice, in which liver-specific expression of the c-myc or N-myc oncogene driven by hepatitis virus regulatory elements leads to the development of primary liver tumors in all animals within 12 months in average. These models are used in different collaborative projects to analyze the oncogenic steps in liver oncogenesis. A major project is to analyze gene expression profiles and genetic alterations of liver tumors induced by Myc in the context of the CIT program of the Ligue Nationale contre le Cancer. In a collaborative project recently initiated within Pasteur Institute (PTR 136), the WHV/myc transgenic mice will be used to monitor potential anti-tumor effects of proapoptotic biopeptides.

The importance of the coactivator FHL2 in tumorigenesis will be evaluated in murine models. FHL2-null mice have been crossed with different oncomice that develop liver or colon tumors (collaboration: Ju Chen, UCSD, and Christine Perret, ICGM, Paris). Transgenic mice expressing FHL2 in liver and intestine under control of the ApoA-IV promoter-enhancer have been established.

Photos :

Photo 1 : The HBx transactivator is a multifunctional protein. It activates the Src and Ras signaling pathways, stimulates the activity of several transcription factors (NF-kB, NF-AT, AP1, CREB), and interacts with various factors of the general transcription machinary (TBP, TFIIB, TFIIH, RPB5), as well as with the DNA repair protein UV-DDB.

Photo 2 : Transient overexpression of ß-catenin leads to the co-localization of FHL2 and p300 in nuclear structures (HeLa cells, confocal microscopy).

Keywords: cancer, virology, hepatitis, transcription regulation, acetylation, transgenic mice

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