The overall goal of this study is to address the potential role of hCdc4 as a tumor suppressor gene in hepatocellular carcinoma (HCC). Our recent studies indicated that cyclin E, an oncogene overexpressed in 70% of HCC, played a substantial role on proliferation and survival and could serve as a promising therapeutic target for HCC. We also showed that downregulation of hCdc4, a recently-identified tumor suppressor candidate in breast cancer, could markedly trigger cyclin E protein accumulation in HCC cells. We hypothesize that deficiency of hCdc4 is one of the key factors causing cyclin E overexpression in HCC. To test this hypothesis, HCC specimen will be analyzed for hCdc4 alteration at DNA, RNA, and protein levels. Specifically, 100 HCC samples and their corresponding noncarcerous liver tissues will be collected at Houston area in Texas. RT-PCR product from HCC and their matched noncarcerous liver tissues will be applied to screen for hCdc4 mutation by direct sequencing. To determine if any alteration occurs in transcriptional or post-transcriptional level, the expression level of hCdc4 will be assessed by Northern blot and Western blot from HCC specimen with intact hCdc4 gene. We will also determine if hCdc4 status is correlated to cyclin E overexpression and if hCdc4 can serve as a prognostic factor for HCC patients. In addition to studies in patient samples, the effects caused from hCdc4 deficiencies on HCC will be evaluated using a transgenic mouse model. We will generate transgenic mice expressing a dominant negative hCdc4 mutant driven by a liver specific promoter. Alternatively, we will generate transgenic mice with hCdc4 expression suppressed by RNA interference approach. We have demonstrated that stable expression of a small interfering RNA against Cdc4 in a mouse cell line led to decrease of Cdc4 expression and elevation of cyclin E protein level. We will test whether the similar effects can be extended and reproduced in the transgenic mice. The transgenic lines generated by these two approaches will be systematically analyzed for both hCdc4 and cyclin E expression. Histopathological studies will also be performed in the transgenics to determine HCC-related phenotypes.
