Our research program continues to focus on the molecular mechanisms of hepatocarcinogenesis with particular? emphasis on characterization of signal transduction pathways involved in cell proliferation, migration, and invasion.? During the last four years, we have made several significant observations: 1) the insulin receptor substrate-1 (IRS-1)? signal transduction cascade plays a major role in hepatocyte proliferation. It is overexpressed in human HCC tumors? and produces hepatic growth in an IRS-1 transgenic mouse model. The liver eventually stops growing due to activation? of pro-apoptotic pathways since there is striking upregulation of Fas receptor (FasR). 2) We have recently identified a downstream effector gene that is regulated through the IRS-I growth factor cascade and important in cell migration such as aspartyl-asparaginyl-13-hydroxylase (AAH); further characterization is a major goal of this research. 3) Because GSK-3-13 is a major component in the IRS-1 cascade that sends survival signals to hepatocytes, we investigated? potential cross-talk with the Wnt/13-catenin pathway. It was found that this pathway is activated very early in the transformation process both in murine and human HCC. More important, constitutive pathway activation is achieved through over-expression of Frizzled 7 (FZDT) receptors. Further characterization of this pathway including the identification of the Wnt ligands will be a major focus of our research for the next grant period. Finally, we are interested in determining what factors are necessary and sufficient to transform hepatocytes in vivo. In this context, we have made a striking preliminary observation that activation of the IRS-1 signal transduction cascade in conjunction with expression of the hepatitis Bx protein (HBx) in a double transgenic animal model is sufficient to induce the formation of dysplastic foci followed by the development of HCC. This exciting observation has led us to propose experiments to explore the cellular mechanisms involved. We plan to do the following: ? Specific Aim #1 Determine the cellular mechanisms involved in the transformation of hepatocytes in the IRS-1/HBx double transgenic mouse model. ? Specific Aim #2 Further characterize a critical downstream effector gene (AAH) upregulated through activation of the IRS-1 signal transduction pathway and important in cell migration and invasion.
Specific Aim #3 Examine the expression and activation of FZD7 and other components of the Wntl_-catenin pathway in early dysplastic foci and HCC tumors compared to adjacent normal liver following laser microdissection. These experiments will further define? the molecular mechanisms involved in the generation of this devastating disease.? ? ? ? SPECIFIC AIMS? Our research program continues to focus on the molecular mechanisms of hepatocarcinogenesis with particular? emphasis on characterization of signal transduction pathways involved in cell proliferation, migration, and invasion as? shown by the cartoon depicted in Fig. 1. During the last four years, we have made several significant observations: 1) the? IRS-1 signal transduction cascade plays a major role in hepatocyte proliferation. It is overexpressed in human HCC tumors and produces hepatic growth in a transgenic mouse model. The liver eventually stops growing due to activation of proapoptotic pathways since there is striking upregulation of Fas receptor (FasR). 2) We have identified a downstream effector gene that is regulated through the IRS- 1 growth factor cascade and important in cell migration such as aspartyl-asparaginyl- 13-hydroxylase (AAH); further characterization is a major goal of this research. 3) Because GSK-3-_ is a major component in the IRS-1 cascade that sends survival signals to hepatocytes, we investigated potential cross-talk with the Wnt/13-catenin pathway. It was found that this pathway is activated very early in the transformation process both in murine and human? HCC. More important, constitutive pathway activation is achieved through over-expression of Frizzled 7 (FZD7) receptors.? Further characterization of this pathway including the identification of the Wnt ligands will be a major focus of our research for the next grant period. Finally, we are interested in determining what factors are necessary and sufficient to transform hepatocytes in vivo. In this context, we have made a striking preliminary observation that activation of the IRS-1 signal transduction cascade in conjunction with expression of the hepatitis Bx protein (HBx) in a double transgenic animal model is sufficient to induce the formation of dysplastic foci followed by the development of HCC. This exciting observation has led us to propose experiments to explore the cellular mechanisms involved.? ? We plan to do the following:? ? Specific Aim #1. Determine the cellular mechanisms involved in the transformation of hepatocytes in the IRS-1/HBx? double transgenic mouse model.? a. Determine the influence of HBx expression on various components of the IRS-1 growth factor signaling cascade? and the downstream activation of Ras, Raf and MAPK kinases.? b. Evaluate if HBx expression alters the high level of Fas receptor expression, and/or induces the loss of pro-apoptotic? mechanisms observed in the single IRS-1 transgenic mice.? ? Specific Aim #2 - Further characterize a critical downstream effector gene (AAH) upregulated through activation of the IRS-1 signal transduction pathway and important in cell migration and invasion.? a. Evaluate the role of AAH protein phosphorylation on cellular expression levels and stability? b. Characterize the pattern of AAH translocation within the cell in relationship to phosphorylation status.? c. Determine if translocation to the cell surface is related to enhanced cell migration and invasion.? ? Specific Aim #3 - Examine if the expression and activation of FZD7 and other components of the Wnt/fl-catenin pathway in? early dysplastic foci and HCC tumors compared to adjacent normal liver following laser microdissection.? a.Employ proteomic approaches including SELDI mass spectrometry in HCC cell lines where Wnt signaling has been? shown to be up- or down-regulated to identify Wnt ligand(s).? b. Analyze expression of the twenty known Wnt ligand genes by real-time PCR from HCC cell lines and tumor tissue? samples.? c. Examine downstream proteins in the Wnt/fl-catenin signaling pathway by inhibiting the actions of Wnt ligands using either specific synthetic peptides or antibodies against these proteins.? d.Study activation of known downstream effector genes of the Wnt/fl-catenin pathway such as WISP, cyclin-D, and c-myc to further confirm the importance of this pathway in the pathogenesis of HCC.? ? These experiments will further define the molecular mechanisms involved in the generation of this devastating disease.?
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