Role of HSP90 Family Chaperone Proteins in Cellular Signal Transduction

HSP90 家族伴侣蛋白在细胞信号转导中的作用

• 批准号: 9154266
• 负责人: Leonard Neckers
• 金额: $ 92.95万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9154266
• 关键词: Address; Affect; Androgen Receptor; Antibodies; Area; Biological Assay; Biological Factors; Breast Cancer Cell; C-terminal; Catalytic Domain; Cell Proliferation; Cell membrane; Cell physiology; Cell surface; Cells; Citric Acid Cycle; Client; Clinical; Complex; Data; Dependence; Detection; Development; Dose; Evaluation; Family; Fibroblasts; Foundations; Generations; Glucocorticoids; HSF1; Heat-Shock Proteins 90; Human; Lead; Ligands; Malignant Neoplasms; Malignant Pheochromocytoma; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Mammary Neoplasms; Mediating; Metabolic; Metastatic Pheochromocytoma; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Target; Mus; Mutation; N-terminal; Neoplasm Metastasis; Neurosecretory Systems; Normal Cell; Normal tissue morphology; Null Lymphocytes; Oncogenic; Optics; Oxidative Phosphorylation; PC12 Cells; PIK3CA gene; Patients; Phenotype; Pheochromocytoma; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Property; Proteins; Radiolabeled; Reactive Oxygen Species; Regulation; Renal carcinoma; Reporting; Research; Resistance; Respiration; Role; SRC gene; Serum; Signal Pathway; Signal Transduction; Therapeutic; Time; Transmembrane Transport; Tumor Necrosis Factor Receptor; Tumor Suppressor Proteins; Tyrosine Kinase Inhibitor; Urologic Diseases; aerobic glycolysis; base; cancer cell; cancer site; cell motility; cell transformation; cytotoxicity; drug development; fatty acid oxidation; glucose metabolism; improved; inhibitor/antagonist; insight; kinase inhibitor; malignant breast neoplasm; member; migration; mutant; neoplastic cell; non-invasive imaging; novel; oncology; overexpression; phosphoinositide-dependent kinase 1; preference; prevent; radiotracer; research clinical testing; response; success; tacrolimus binding protein 4; targeted treatment; therapeutic target; therapy resistant; tumor; tumor growth; ubiquitin ligase

Hsp90 is a cellular chaperone that stabilizes several signal transduction networks important to cancer cells. In FY14, our research accomplishments include further development and evaluation of a novel lead compound that interferes with Hsp90/FKBP52-dependent chaperoning of the androgen receptor. This compound acts via a mechanism that is unique compared to other androgen receptor antagonists. Also, we found that the HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitor crizotinib in both crizotinib-sensitive and -resistant MET-driven tumor models. The clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. MET is an HSP90-dependent kinase, and in this report we show that HSP90 preferentially interacts with and stabilizes activated MET, regardless of whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET-TKI show a preference for the inactive form of the kinase, and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET-TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in both TKI-sensitive and -resistant MET-driven tumor models. These data suggest that inclusion of an HSP90 inhibitor can partially restore TKI sensitivity to previously resistant MET mutants, and they provide the foundation for clinical evaluation of this therapeutic combination in patients with MET-driven cancers. In addition, we showed that the Hsp90-related and mitochondrially localized molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis. TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor. We collaborated on a study targeting heat shock protein 90 for the treatment of malignant pheochromocytoma. Metastatic pheochromocytoma represents one of the major clinical challenges in the field of neuroendocrine oncology. Recent molecular characterization of pheochromocytoma suggests new treatment options with targeted therapies. In this study, we investigated Hsp90 as a potential therapeutic target for advanced pheochromocytoma. Both the first generation, natural product Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), and the second-generation synthetic Hsp90 inhibitor STA-9090 (ganetespib) demonstrated potent inhibition of proliferation and migration of pheochromocytoma cell lines and induced degradation of key Hsp90 clients. Furthermore, ganetespib induced dose-dependent cytotoxicity in primary pheochromocytoma cells. Using metastatic models of pheochromocytoma, we demonstrated the efficacy of 17-AAG and ganetespib in reducing metastatic burden and increasing survival. These findings suggest that targeting Hsp90 may benefit patients with advanced pheochromocytoma. We collaborating on a study showing that optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells. These tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein occurs at the plasma membrane. In mice, we observed exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors that target ectopic Hsp90 can be used to detect breast cancer malignancies through noninvasive imaging. Finally, we identified coordinated regulation of serum- and glucocorticoid-inducible kinase 3 (SGK3) by a C-terminal hydrophobic motif and Hsp90-Cdc37 chaperone complex. SGK3 mediates a variety of cellular processes including membrane transport, cell proliferation, and survival, and it has been implicated in Akt-independent signaling downstream of oncogenic PIK3CA mutations (activating mutations in the catalytic subunit of PI3K) in human cancers. However, the regulation of SGK3 is poorly understood. Here we report that SGK3 stability and kinase activation are regulated by the Hsp90-Cdc37 chaperone complex. Hsp90-Cdc37 associates with the kinase domain of SGK3 and acts in concert with a C-terminal hydrophobic motif of SGK3 to prevent Hsp70 association and ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein)-mediated degradation. Phosphorylation of hydrophobic motif triggers release of Cdc37 and concomitant association of 3-phosphoinositide dependent kinase 1 (PDK1) to activate SGK3. Our study provides new insights into regulation of SGK3 stability and activation and the rationale for application of Hsp90 inhibitors in treating SGK3-dependent cancers.

Hsp90是一种细胞伴侣，它稳定了对癌细胞重要的几个信号转导网络。在2014财年，我们的研究成果包括进一步开发和评估一种干扰Hsp90/ fkbp52依赖性雄激素受体伴侣的新型先导化合物。与其他雄激素受体拮抗剂相比，这种化合物通过一种独特的机制起作用。此外，我们发现HSP90抑制剂ganetespib在克里唑替尼敏感和耐药MET驱动的肿瘤模型中与MET激酶抑制剂克里唑替尼协同作用。MET导向的酪氨酸激酶抑制剂（TKI）的临床成功一直受到限制，部分原因是MET激酶结构域的突变导致了治疗耐药性。规避这一问题仍然是改善接受met靶向治疗的患者持久反应的关键挑战。MET是一种依赖于HSP90的激酶，在本报告中，我们发现HSP90优先与活化的MET相互作用并使其稳定，而不管活化是依赖于配体还是激酶结构域突变的结果。相反，许多MET- tki表现出对激酶的非活性形式的偏好，并且MET的激活突变可以赋予抗性。将HSP90抑制剂ganetespib与MET- tki克里唑替尼联合使用，在tki敏感和耐药的MET驱动肿瘤模型中实现了MET、其下游信号通路和肿瘤生长的协同抑制。这些数据表明，纳入HSP90抑制剂可以部分恢复TKI对先前耐药MET突变体的敏感性，并为MET驱动型癌症患者的这种治疗组合的临床评估提供了基础。此外，我们发现hsp90相关的线粒体定位分子伴侣TRAP1调节线粒体呼吸和有氧糖酵解之间的代谢转换。TRAP1 （TNF受体相关蛋白）是HSP90伴侣蛋白家族的一员，主要存在于线粒体中。TRAP1被广泛认为是一种抗癌分子靶点。然而，目前的抑制剂无法区分HSP90和TRAP1，这使得它们作为TRAP1特异性功能探针的实用性受到质疑。与正常组织相比，一些癌症表达的TRAP1较少，这表明TRAP1在正常细胞和转化细胞的线粒体中的功能比以前所认识的要复杂得多。我们使用TRAP1缺失细胞和短暂的TRAP1沉默/过表达来证明，在永生化小鼠成纤维细胞和人类肿瘤细胞中，TRAP1调节氧化磷酸化和有氧糖酵解之间的代谢转换。trap1缺乏促进线粒体呼吸和脂肪酸氧化的增加，以及三羧酸循环中间体、ATP和活性氧的细胞积累。同时，葡萄糖代谢受到抑制。trap1缺陷细胞也表现出显著增强的侵袭性。TRAP1与线粒体c-Src的相互作用和调控为这些表型提供了机制基础。综上所述，在几种癌症中，TRAP1的表达与肿瘤分级呈负相关，这些数据表明，在某些情况下，这种线粒体分子伴侣可能具有肿瘤抑制作用。我们合作进行了一项针对热休克蛋白90治疗恶性嗜铬细胞瘤的研究。转移性嗜铬细胞瘤是神经内分泌肿瘤学领域的主要临床挑战之一。最近嗜铬细胞瘤的分子特征提出了靶向治疗的新治疗选择。在这项研究中，我们研究了Hsp90作为晚期嗜铬细胞瘤的潜在治疗靶点。第一代天然产物Hsp90抑制剂17-烯丙基氨基-17-demethoxygeldanamycin （17-AAG, tanespimycin）和第二代合成Hsp90抑制剂sta9090 （ganetespib）均显示出对嗜铬细胞瘤细胞系增殖和迁移的有效抑制，并诱导关键Hsp90客户的降解。此外，ganetespib在原发性嗜铬细胞瘤细胞中诱导剂量依赖性细胞毒性。通过嗜铬细胞瘤转移模型，我们证明了17-AAG和ganetespib在减少转移负担和提高生存率方面的疗效。这些发现表明，靶向Hsp90可能对晚期嗜铬细胞瘤患者有益。我们合作的一项研究表明，光学和放射性碘系固Hsp90抑制剂揭示了恶性乳腺肿瘤细胞中异位Hsp90的选择性内在化。这些栓系抑制剂选择性地识别表达异位Hsp90的细胞并被内化。内化过程被Hsp90抗体阻断，表明该蛋白的活性循环发生在质膜上。在小鼠中，我们观察到在乳腺肿瘤中以非常敏感的水平积累了氟系缚型。基于细胞的放射标记检测显示，在表达异位Hsp90的细胞中可以检测到皮摩尔。我们的研究结果表明，靶向异位Hsp90的氟系或放射性标记抑制剂可用于通过无创成像检测乳腺癌恶性肿瘤。最后，我们确定了c端疏水基序和Hsp90-Cdc37伴侣复合物对血清和糖皮质激素诱导的激酶3 （SGK3）的协调调节。SGK3介导多种细胞过程，包括膜转运、细胞增殖和存活，并且在人类癌症中与致癌PIK3CA突变（PI3K催化亚基的激活突变）下游的akt不依赖信号传导有关。然而，我们对SGK3的调控知之甚少。在这里，我们报道了SGK3的稳定性和激酶激活是由Hsp90-Cdc37伴侣复合物调节的。Hsp90-Cdc37与SGK3的激酶结构域结合，并与SGK3的C端疏水基序协同作用，阻止Hsp70结合和泛素连接酶CHIP （hsc70相互作用蛋白的C端）介导的降解。疏水基序的磷酸化触发Cdc37的释放，并伴随3-磷酸肌肽依赖性激酶1 （PDK1）的结合，从而激活SGK3。我们的研究为SGK3的稳定性和激活调控以及应用Hsp90抑制剂治疗SGK3依赖性癌症提供了新的见解。