Our research efforts will center on the possible cellular mechanisms of hepatocyte transformation focusing on the hypothesis that the insulin receptor substrate-1 (IRS-1) pathway activation in HCC provides the stimulus for constitutive hepatocyte proliferation; it is in this milieu that other secondary events, i.e., cellular injury, cirrhosis and/or genetic mutation(s), may occur leading/contributing to subsequent tumor development. Prior studies demonstrate overexpression of IRS-1, or activation of this insulin/IGF-1 stimulated growth pathway(s) in 95 percent of human HCC tumors (compared to adjacent uninvolved normal liver) representing the most common cellular event described thus far. In vivo and in vitro studies of downstream genes upregulated by IRS-1 overexpression have begun to be defined, and we postulate that human aspartyl (asparaginyl) beta hydroxylase (HAAH) may be important in tumor pathogenesis with expression and function apparently linked to the IRS-1 signal transduction cascade. It is known that IRS-1 overexpression produces hepatocyte proliferation, malignant transformation of NIH 3T3 cells, and inhibits TGF-beta1 induced programmed cell death of hepatocytes. If HAAH is a downstream effector gene in this pathway, it will be important to: 1] establish a connection between IRS-1 and HAAH gene upregulation. In this regard HAAH expression levels on malignant phenotype generation will be studied. To determine possible molecular mechanisms FOCUS HCC, NIH 3T3 and 32D cells will be stably transfected with dominant-negative IRS-1 cDNAs mutated in the plextrin and phosphotryosine (PTB) domains, and Grb2, Syp and P13K binding motifs located in the C-terminus of the protein. 2] Evaluate the biologic consequences of increased HAAH expression with respect to acquisition of cellular resistance to apoptosis, alteration of phenotype leading to increased invasive growth, and enhanced cellular proliferation. 3] Finally, we will examine the hypothesis that initiation of a second cellular event(s) leads to HCC development in vivo. A unique transgenic mouse model will be used for IRS-1 expression which produces a nontransforming, but continuous, hepatocyte proliferative stimulus. Secondary events will be introduced through breeding of the IRS-1 transgenic mouse with p53 knockout mice, Hbx transgenic mice, and initiation of acute/chronic cellular injury and cirrhosis by intraperitoneal injection of thioacetamide. The IRS-1 transgenic model may provide an ideal cellular milieu in which the influence of secondary genetic events completing the in vivo hepatocytic transformation process may be studied.
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