ErbB2 Downregulation Through HSP90 Inhibition

ErbB 2（Her 2/Neu）过表达在20-30%的乳腺癌患者以及其他癌症中发现，并且表示预后不良。人源化抗ErbB 2抗体的成功进一步验证了ErbB 2作为治疗靶标。安莎霉素HSP 90抑制剂，如17 AAG，作为新型抗癌剂处于I/II期临床研究中，因为它们靶向ErbB 2和其他信号传导蛋白以进行泛素依赖性降解。基于我们和其他人最近的研究，我们假设CHIP（HSP 70-Interacting Protein的C-末端）和另外的迄今未知的泛素连接酶通过蛋白酶体或通过溶酶体降解介导17 AAG诱导的ErbB 2下调。在这里，我们提出了全面的策略来测试这些假设。 我们将进行细胞生物学，分子和生物化学研究，以区分是否热休克蛋白90受体驱动的ErbB 2下调是通过蛋白酶体，溶酶体或两者的降解介导。我们将研究CHIP和几个通过生物信息学鉴定的候选蛋白在17 AAG诱导的ErbB 2泛素化和降解，ErbB 2过表达乳腺癌细胞系的增殖和存活以及它们在裸鼠体内生长中的作用，使用过表达，显性负突变表达和RNAi敲低策略。如果其他未知的泛素连接酶似乎可能介导17 AAG诱导的ErbB 2降解，我们将采用蛋白质组学和酵母双杂交方法来鉴定这些蛋白质，并将其功能表征为CHIP和其他候选物。通过这种综合性的方法，我们希望阐明热休克蛋白90抑制剂下调ErbB 2的生物学基础及其抗癌活性。这些研究的成功将为ErbB 2驱动的癌症以及由EGFRvIII引起的癌症开辟新的治疗途径，EGFRvIII是一种与ErbB 2具有生物学相似性的ErbB 1变体。 与公共卫生的相关性：该提案将研究干扰致癌蛋白Her 2/Neu的新方法，该蛋白在20-30%预后最差的乳腺癌中高度增加。拟议的研究可以为针对乳腺癌和其他癌症的更新，更有效的靶向治疗提供基础。

DESCRIPTION (provided by applicant): Technical Description: ErbB2 (Her2/Neu) overexpression is found in 20-30% of breast cancer patients as well as other cancers, and signifies poor prognosis. Success with humanized anti-ErbB2 antibodies has further validated ErbB2 as a therapeutic target. Ansamycin HSP90 inhibitors, such as 17AAG, are in phase l/ll clinical studies as novel anticancer agents as they target ErbB2 and other signaling proteins for ubiquitin-dependent degradation. Based on recent studies by us and others, we hypothesize that CHIP (C-terminus of HSP70-lnteracting Protein) and additional hitherto unknown ubiquitin ligases mediate 17AAG-induced ErbB2 downregulation either through proteasomal or through lysosomal degradation. Here, we propose comprehensive strategies to test these hypotheses. We will carry out cell biological, molecular and biochemical studies to distinguish whether HSP90 inhibitor-driven ErbB2 downregulation is mediated through degradation in the proteasome, lysosome or both. We will examine the role of CHIP and several candidate proteins identified through bioinformatics in 17AAG-induced ErbB2 ubiquitinylation and degradation, proliferation and survival of ErbB2 overexpressing breast cancer cell lines and in their in vivo growth in nude mice, using overexpression, dominant-negative mutant expression and RNAi knockdown strategies. If additional unknown ubiquitin ligases appear likely to mediate 17AAG-induced ErbB2 degradation, we will employ proteomics and yeast two-hybrid approaches to identify these proteins and characterize them functionally as for CHIP and other candidates. Through this comprehensive approach, we hope to elucidate the biological basis of ErbB2 downregulation by HSP90 inhibitory drugs and their anticancer activity. Success of these studies will open new therapeutic avenues for ErbB2-driven cancers as well those caused by EGFRvlll, an ErbB1 variant with biological similarities with ErbB2. Relevance to Public Health: This proposal will investigate new means of interfering with a cancer-causing protein Her2/Neu that is highly increased in 20-30% breast cancers with worst prognosis. The proposed studies could provide a basis for newer, more effective targeted therapies against breast and other cancers.