Berberine downregulates MDM2 by interaction with DAXX in cancer cells

描述(由申请人提供)：多功能癌蛋白MDM2在癌症的发生、发展和对治疗的耐药性的形成中发挥关键作用。我们研究了儿童癌症患者，包括那些患有急性淋巴细胞白血病(ALL)和神经母细胞瘤(NB)的患者，发现预后较差的患者通常具有结构性高表达MDM2的癌细胞。MDM2的一个重要功能是泛素化(作为E3连接酶)和降解肿瘤抑制因子p53，这是由于与DAXX和Hausp的相互作用。有趣的是，这些相互作用的破坏可以导致MDM2和P53激活的自我泛素化。因此，我们认为，通过干扰MDM2-DAXX-Hausp相互作用来靶向MDM2是开发MDM2过表达癌症患者新疗法的一种创新方法。黄连素(berberine，BBR)是一种从中草药中提取的异喹啉生物碱，已被证明对人类癌症具有抗增殖和促凋亡作用。我们的初步数据支持BBR以前未知的作用机制：它通过抑制DAXX下调MDM2，破坏MDM2-DAXX-Hausp相互作用。与传统化疗药物阿霉素(Dox)诱导先前的p53激活和随后的MDM2上调不同，我们发现BBR强烈地诱导MDM2的持续下调，随后P53的稳定激活，导致MDM2过表达的癌细胞，包括那些对Dox耐药的癌细胞的有效凋亡。这一建议旨在验证我们的中心假设，即DAXX抑制对MDM2的调节是BBR发挥抗癌作用与Dox不同的主要机制。该项目的长期目标是：表征BBR在下调MDM2以克服化疗耐药性方面所起的关键作用，并确定BBR作为难治性癌症患者治疗试剂的潜力。这项研究的具体目的是：1)全面阐明BBR下调MDM2的细胞/分子机制；2)确定下调MDM2是BBR发挥抗癌作用的主要机制；3)评估BBR逆转化疗耐药的能力及其作为治疗难治性癌症患者的潜力。这些特定目标的成功完成将在这种天然产品未来在儿科癌症患者中进行临床试验之前产生关于BBR作用机制的知识，应该会引入BBR作为克服化疗耐药的高度有用的新药候选药物，还将为合理设计针对DAXX-MDM2途径的其他治疗方法提供基本框架。

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.