Berberine downregulates MDM2 by interaction with DAXX in cancer cells

描述（由申请人提供）：多功能癌蛋白MDM 2在癌症的发生、进展和耐药性的发展中起关键作用。我们研究了儿科癌症患者，包括急性淋巴细胞白血病（ALL）和神经母细胞瘤（NB）患者，发现预后不良的患者通常具有组成性高水平表达MDM 2的癌细胞。MDM 2的一个重要功能是由于与DAXX和HAUSP的相互作用，肿瘤抑制因子p53的泛素化（作为E3连接酶）和降解。有趣的是，这些相互作用的破坏可以导致MDM 2的自我泛素化和p53激活。因此，我们相信通过破坏MDM 2-DAXX-HAUSP相互作用来靶向MDM 2是开发用于MDM 2过表达癌症患者的新疗法的创新方法。小檗碱（BBR）是一种来源于传统中草药的异喹啉生物碱，具有抗肿瘤增殖和促凋亡作用。我们的初步数据支持BBR以前未被认识的作用机制：它通过抑制DAXX，破坏MDM 2-DAXX-HAUSP相互作用来下调MDM 2。与诱导先前p53激活和随后MDM 2上调的常规化疗药物阿霉素（Dox）相反，我们发现BBR强烈诱导MDM 2的持续下调，随后是p53的稳态激活，导致MDM 2过表达癌细胞（包括Dox耐药的癌细胞）的有效凋亡。该提议旨在验证我们的中心假设，即通过DAXX抑制调节MDM 2是BBR在发挥其抗癌作用方面不同于Dox的主要机制。该项目的长期目标是：表征BBR在下调MDM 2以克服化疗耐药性方面的关键作用，并确定BBR作为难治性癌症患者治疗试剂的潜力。本研究的具体目的是：1）充分阐明BBR下调MDM 2的细胞/分子机制; 2）确定MDM 2的下调是BBR发挥其抗癌作用的主要机制; 3）评估BBR逆转化疗耐药性的能力及其作为难治性癌症患者治疗的潜力。这些特定目标的成功完成将在儿科癌症患者中进行这种天然产物的未来临床试验之前产生关于BBR作用机制的知识，将BBR作为克服化学耐药性的非常有用的新药候选物引入，并且还将为靶向DAXX-MDM 2途径的其他治疗方法的合理设计提供基本框架。

DESCRIPTION (provided by applicant): The multifunctional oncoprotein MDM2 plays critical roles in cancer initiation, progression and the development of resistance to therapy. We studied pediatric cancer patients, including those with acute lymphoblastic leukemia (ALL) and neuroblastoma (NB), finding that patients with a poor prognosis commonly had cancer cells that expressed constitutively high levels of MDM2. An important function of MDM2 is the ubiquitination (as an E3 ligase) and degradation of the tumor suppressor p53, due to interactions with DAXX and HAUSP. Interestingly, disruption of these interactions can result in self-ubiquitination of MDM2 and p53 activation. Thus, we believe targeting MDM2 by disrupting MDM2-DAXX-HAUSP interactions is an innovative approach to develop new therapeutics for MDM2-overexpressing cancer patients. Berberine (BBR), an isoquinoline alkaloid derived from a traditional Chinese medicinal herb, has been shown to have anti-proliferative and pro-apoptotic effects on human cancer. Our preliminary data supports a previously unrecognized mechanism of action for BBR: It downregulates MDM2 by inhibiting DAXX, disrupting MDM2-DAXX-HAUSP interactions. In contrast to the conventional chemotherapeutic drug doxorubicin (Dox) that induces prior p53 activation and a subsequent upregulation of MDM2, we discovered that BBR strongly induced persistent downregulation of MDM2, followed by a steady-state activation of p53, resulting in potent apoptosis of MDM2-overexpressing cancer cells, including those that are Dox-resistant. This proposal is designed to test our central hypothesis that regulation of MDM2 by DAXX inhibition is the major mechanism by which BBR differs from Dox in exerting its anti-cancer effect. The project's long term objectives are: characterization of the critical role BBR plays in the downregulation of MDM2 to overcome chemoresistance and determination of BBR's potential as a therapeutic reagent for refractory cancer patients. The specific aims of this study are: 1) To fully elucidate the cellular/molecular mechanisms by which BBR downregulates MDM2; 2) To establish that downregulation of MDM2 is the primary mechanism by which BBR exerts its anticancer effects; and 3) To assess the ability of BBR to reverse chemoresistance and its potential as a treatment for refractory cancer patients. Successful completion of these specific aims will generate knowledge with respect to the mechanisms of action for BBR prior to future clinical trials of this natural product in pediatric cancer patients, should introduce BBR as a highly useful new drug candidate for overcoming chemoresistance, and also will provide a basic framework for the rational design of other therapeutic approaches targeting the DAXX-MDM2 pathway.