THE ROLE OF hCdc4 IN HEPATOCELLULAR CARCINOMA

描述(由申请人提供)：本研究的总体目标是探讨hCDC4作为肿瘤抑制基因在肝细胞癌中的潜在作用。我们最近的研究表明，细胞周期蛋白E是一种在70%的肝细胞癌中过度表达的癌基因，它在肝细胞癌的增殖和生存中起着重要作用，有望成为治疗肝细胞癌的靶点。我们还发现，最近发现的乳腺癌候选抑癌基因hCDC4的下调可以显著地触发肝癌细胞中细胞周期蛋白E的积聚。我们推测hCDC4的缺失是导致肝细胞癌细胞周期蛋白E过表达的关键因素之一。为了验证这一假设，我们将在DNA、RNA和蛋白质水平上分析肝细胞癌标本中hCDC4的变化。具体地说，将在德克萨斯州休斯顿地区收集100份肝癌样本及其相应的非癌肝组织。来自肝细胞癌及其配对的非癌肝组织的RT-PCR产物将被用于通过直接测序来筛查hCDC4突变。为了确定hCDC4基因在转录或转录后水平是否发生改变，将通过Northern印迹和Western印迹检测hCDC4基因完整的肝细胞癌标本中hCDC4的表达水平。我们还将确定hCDC4状态是否与细胞周期蛋白E过度表达相关，以及hCDC4是否可以作为肝细胞癌患者的预后因素。除了对患者样本的研究外，还将使用转基因小鼠模型来评估hCdc4缺陷对肝癌的影响。我们将产生转基因小鼠，表达由肝脏特异性启动子驱动的显性阴性hCdc4突变体。或者，我们将通过RNA干扰的方法获得hCDC4表达被抑制的转基因小鼠。我们已经证明了针对CDC4的小干扰RNA在小鼠细胞系中的稳定表达导致了CDC4表达的降低和细胞周期蛋白E水平的升高。我们将测试类似的效应是否可以在转基因小鼠身上延伸和重现。通过这两种方法产生的转基因株系将被系统地分析hCDC4和细胞周期蛋白E的表达。组织病理学研究也将在转基因中进行，以确定与肝癌相关的表型。

DESCRIPTION (provided by applicant): 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.