BETA-CATENIN AS A THERAPEUTIC TARGET IN HEPATOCELLULAR CANCER

描述（由申请人提供）：在包括HCC在内的许多癌症中，均发生<$-连环蛋白的异常激活。虽然激活了在HCC中的β-连环蛋白已被证明是多因素的，所有事件都集中在β-连环蛋白，使其成为HCC中有吸引力的治疗靶点。我们已经在转基因小鼠的肝脏、肝脏再生、肝脏发育和肝细胞培养物中显示了<$-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敲除小鼠。将<$-连环蛋白floxed小鼠（Ex 2 -6）与白蛋白-Cre或甲胎蛋白-白蛋白-Cre小鼠交配以产生<$-连环蛋白条件性无效小鼠：分别为Ctnnb 1 loxP/loxp：Alb-Cre或Ctnnb 1 loxP/loxp：aFP-Alb-Cre。这两种小鼠都是正常出生的，并且在2周内表现出约95%的<$-连环蛋白损失，并且在前者的整个正常寿命中持续存在，而在后者中，<$-连环蛋白的100%损失发生在4-5个月内，并且由于肝脏大小和功能减少而死亡。此外，我们最近的特点是正常的β-连环蛋白过表达转基因（TG）小鼠白蛋白启动子/增强子。这些小鼠具有较高的基础肝细胞增殖，伴随着肝肿大。最近，我们已经产生了过度表达稳定形式的β-连环蛋白（Ser 45突变）的TG小鼠。这些小鼠正在被表征，并且表现出比正常的β-连环蛋白TG小鼠更稳健的表型。这些模型为我们提供了一个独特的机会，最终解决的作用，连环蛋白在肝癌的诱导和发展。我们建议采用DEN/苯巴比妥模型来研究在不存在<$-连环蛋白或存在稳定<$-连环蛋白的情况下的肝癌发生。此外，我们建议调查卵圆细胞激活，在肝脏中的癌前事件，在敲除和转基因小鼠中，以肯定地解决的作用，连环蛋白在这一事件中。最后，我们将探索在转基因模型中治疗性抑制连环蛋白的作用。我们已经确定了R-依托度酸（NSAID依托度酸的对映体，缺乏考克斯-2抑制作用）在抑制肝癌细胞中的连环蛋白中的作用。这种药物在难治性CLL的II期临床试验中。我们建议在一系列体外和体内研究中检查R-依托度酸在我们的转基因小鼠和肿瘤异种移植模型中的作用，以确定其作为抗-连环蛋白用于治疗或化学预防HCC的作用。因此，该建议将为启动针对HCC中的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.