THE ROLE OF hCdc4 IN HEPATOCELLULAR CARCINOMA

描述（由申请人提供）：本研究的总体目标是研究hCdc4作为肿瘤抑制基因在肝细胞癌（HCC）中的潜在作用。我们最近的研究表明，在70%的HCC中过表达的癌基因cyclin E在增殖和存活中发挥了重要作用，可能成为HCC的一个有希望的治疗靶点。我们还发现，hCdc4（一种最近在乳腺癌中发现的肿瘤抑制候选因子）的下调可以显著触发细胞周期蛋白E蛋白在HCC细胞中的积累。我们推测hCdc4缺乏是导致细胞周期蛋白E在HCC中过表达的关键因素之一。为了验证这一假设，将分析HCC标本在DNA、RNA和蛋白质水平上的hCdc4改变。具体而言，将在德克萨斯州休斯顿地区收集100例HCC样本及其相应的非癌性肝组织。来自HCC及其匹配的非癌性肝组织的RT-PCR产物将通过直接测序用于筛选hCdc4突变。为了确定转录或转录后水平是否发生改变，hCdc4基因完整的HCC标本将通过Northern blot和Western blot评估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过表达相关，以及hCdc4是否可以作为HCC患者的预后因素。除了对患者样本进行研究外，还将使用转基因小鼠模型评估hCdc4缺乏对HCC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干扰方法产生抑制hCdc4表达的转基因小鼠。我们已经证明，在小鼠细胞系中稳定表达Cdc4的小干扰RNA导致Cdc4表达降低和细胞周期蛋白E蛋白水平升高。我们将测试类似的效果是否可以在转基因小鼠中扩展和复制。我们将对这两种方法产生的转基因系进行hCdc4和cyclin E表达的系统分析。组织病理学研究也将在转基因中进行，以确定hcc相关表型。

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.