BETA-CATENIN AS A THERAPEUTIC TARGET IN HEPATOCELLULAR CANCER

描述(申请人提供)：连环蛋白的异常激活发生在包括肝癌在内的许多癌症中。尽管-连环蛋白在肝细胞癌中的激活被证明是多因素的，但所有的事件都集中在-连环蛋白上，使其成为肝细胞癌有吸引力的治疗靶点。我们已经在转基因小鼠的肝脏、肝脏再生、肝脏发育和肝细胞培养中展示了β-连环蛋白的促增殖作用。为了更有效地阐明其在肝脏生物学中的作用，我们使用cre-lox系统产生了条件性连环蛋白基因敲除小鼠。将β-连环蛋白克隆小鼠(EX2-6)分别与白蛋白-Cre或甲胎蛋白-白蛋白-Cre小鼠杂交，分别产生条件缺失小鼠：CTNNB1 loxP/loxP：Alb-Cre或CTNNB1 loxP/loxP：AFP-Alb-Cre。这两只小鼠都是正常出生的，在出生2周时，β-连环蛋白约有95%的丢失，在前者的正常寿命中持续存在，而在后者，在4-5个月时出现100%的-连环蛋白丢失，并因肝脏大小和功能减小而死亡。此外，我们最近还鉴定了白蛋白启动子/增强子作用下正常连环蛋白过表达的转基因(TG)小鼠。这些小鼠有较高的基础肝细胞增殖，随之而来的是肝肿大。最近，我们培育出了过度表达稳定形式连环蛋白(Ser45突变)的转基因小鼠。这些小鼠的特征是，表现出比正常的-连环蛋白TG小鼠更强健的表型。这些模型为我们提供了一个独特的机会来明确连环蛋白在肝细胞癌的诱导和进展中的作用。我们建议使用DEN/苯巴比妥模型来研究在不存在或存在稳定的-连环蛋白的情况下肝癌的发生。此外，我们建议在基因敲除和转基因小鼠中研究卵圆细胞激活，这是肝脏中的一种癌前事件，以确定？-catenin在这一事件中的作用。最后，我们将探讨治疗性抑制β-连环蛋白在转基因模型中的作用。我们已经确定了R-依托度酸(NSAID Etolodolac的对映体，缺乏COX-2抑制)对肝癌细胞中β-catenin的抑制作用。该药正在进行难治性慢性淋巴细胞性白血病的二期临床试验。我们建议在我们的转基因小鼠和肿瘤异种移植模型中检测R-依托度酸在一系列体外和体内研究中的效果，以确定其作为抗连环蛋白在肝癌治疗或化学预防中的作用。因此，这一建议将为启动针对肝细胞癌中β-连环蛋白的临床研究奠定基础。

DESCRIPTION (provided by applicant): Aberrant activation of ¿-Catenin occurs in many cancers including HCC. Although the activation of ¿-catenin in HCC has been shown to be multifactorial, all events converge at ¿-catenin, making it an attractive therapeutic target in HCC. We have shown pro-proliferative effect of ¿-catenin in liver in transgenic mice, liver regeneration, liver development & in hepatocyte cultures. To more efficiently elucidate its role in hepatic biology, we have generated conditional ¿-catenin knockout mice using the cre- lox system. ¿-Catenin floxed mice (Ex2-6) were bred to Albumin-Cre or a-fetoprotein-albumin-Cre mice to generate ¿-catenin conditional null mice: Ctnnb1 loxP/loxp:Alb-Cre OR Ctnnb1 loxP/loxp:aFP-Alb-Cre respectively. Both these mice are born normally & show about 95% loss of ¿-catenin by 2 weeks that persists throughout their normal life span in the former, while 100% loss of ¿-catenin occurs in the latter by 4-5 months and succumb due to diminished liver size & function. In addition, we have recently characterized the normal ¿-catenin over-expressing transgenic (TG) mice under albumin promoter/enhancer. These mice have a higher basal hepatocyte proliferation, with ensuing hepatomegaly. More recently we have generated TG mice that over expresses stable form of ¿-catenin (Ser45 mutated). These mice are being characterized, and demonstrate a more robust phenotype than normal ¿-catenin TG mice. These models give us a unique opportunity to conclusively address the role of ¿-catenin in HCC induction and progression. We propose to employ the DEN/phenobarbital model to investigate hepatocarcinogenesis in absence of ¿-catenin or presence of stable ¿-catenin. In addition, we propose to investigate oval cell activation, a preneoplastic event in liver, in knockout and transgenic mice to assertively address role of ¿-catenin in this event. Lastly, we would explore the role of therapeutic inhibition of ¿-catenin in transgenic models. We have identified role of R-Etodolac (enantiomer of NSAID Etolodolac, lacking cox-2 inhibition) in inhibiting ¿-catenin in hepatoma cells. This drug is in phase-ll clinical trials in refractory CLL. We propose to examine the effect of R-Etodolac in our transgenic mice and in tumor xenograft models in a series of both in vitro and in vivo studies to ascertain its role as an anti- ¿-catenin for treatment or chemoprophylaxis in HCC. Thus this proposal will lay the ground work for initiating clinical studies directed against ¿-catenin in HCC.