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hCdc 4作为肝细胞癌（HCC）抑癌基因的潜在作用。我们最近的研究表明，细胞周期蛋白E（cyclin E）是一种在70%的肝癌中过表达的癌基因，它对肝癌的增殖和生存起着重要的作用，可能成为肝癌治疗的一个有希望的靶点。我们还发现，下调hCdc 4，一个最近确定的乳腺癌肿瘤抑制候选人，可以显着触发细胞周期蛋白E蛋白在肝癌细胞中的积累。我们推测hCdc 4的缺陷是导致细胞周期蛋白E过度表达的关键因素之一。为了检验这一假设，将在DNA、RNA和蛋白质水平上分析HCC标本的hCdc 4改变。具体而言，将在德克萨斯州休斯顿地区收集100份HCC样本及其相应的非癌肝组织。采用RT-PCR方法检测肝癌组织及癌旁肝组织中hCdc 4基因的突变。为了确定是否在转录或转录后水平发生任何改变，将通过北方印迹和Western印迹从具有完整hCdc 4基因的HCC标本中评估hCdc 4的表达水平。我们还将确定hCdc 4状态是否与细胞周期蛋白E过表达相关，以及hCdc 4是否可以作为HCC患者的预后因素。除了在患者样本中进行的研究外，还将使用转基因小鼠模型评估hCdc 4缺陷对HCC的影响。我们将产生转基因小鼠表达显性负性hCdc 4突变驱动的肝脏特异性启动子。或者，我们将产生通过RNA干扰方法抑制hCdc 4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针对Cdc 4的小干扰RNA导致Cdc 4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。通过这两种方法产生的转基因株系将被系统地分析hCdc 4和细胞周期蛋白E的表达。还将在转基因动物中进行组织学研究，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.