Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths, with an estimated 250,000 new cases annually worldwide. HCC frequently spreads to distant organs, including the portal vein, lymph nodes, and lungs, resulting in the demise of the patient. Therefore, understanding the molecular mechanisms underlying metastatic spread in this disease is of great importance. We recently generated a mouse model for HCC in which lung metastases occur in mice with liver-specific deletion of the p53 and Ink4a/Arftumor suppressor loci, but not in mice with liver-specific deletion of p53 alone. This finding implies a role for the Ink4a/Arflocus in regulating HCC metastasis. We have isolated two cell lines - HCC-I, derived from a tumor with deletion of the p53 and Ink4a/Arf oc ', and HCC-NI, derived from a tumor with deletion of p53 - and find that these cell lines recapitulate, in cell culture assays, the metastatic phenotypes of the tumors from which they are derived. Gene expression profiling identified several genes that are differentially expressed between the two cell lines, including Twist and Pdgfr-fi, which have been previously implicated in tumor cell invasion and metastasis. Given these findings, we propose to: 1) Explore whether the induction of metastasis is dependent on the loss of either the p16 or p19 tumor suppressor proteins (encoded by the Ink4a/Arf locus), or both. Further, through the expression either wild- type or mutant proteins, we will determine whether the inhibition of metastasis by p16 is dependent on its interaction with Cdk4; and whether the ability of p19 to block this process is dependent on its interactions with Mdm2 and CtBP. 2) Determine whether the induction of Twist is required for tumor cell migration, invasion and metastasis. In addition, using gene expression microarrays and chromatin immunoprecipitation (ChIP) experiments, we will identify potential Twist transcriptional targets involved in this process. Finally, through the analysis of reporter constructs regulated by the Twist gene regulatory elements, and ChIP experiments, we will explore potential mechanisms regulating Twist expression in HCC. 3) Ascertain whether activated PDGF signaling is involved in HCC metastasis. Further, using PDGFR receptor mutants defective in signaling through specific downstream effector molecules, we will identify the PDGF-regulated signaling pathways that are critical mediators of the effects of PDGF in HCC tumor cell migration, invasion, and metastasis. Our studies will therefore increase our knowledge of the molecular mechanisms underlying metastasis of HCC, and may potentially lead to the development of effective targeted interventions, thereby reducing the public health impact of this disease.
