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: 8554037
• 负责人: Leonard Neckers
• 金额: $ 69.4万
• 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/8554037
• 关键词: 26S proteasome; Address; Affect; Affinity; Androgen Receptor; Ansamycin Antineoplastic Antibiotic; Antibiotics; Apoptosis; Area; Benzoquinones; Binding; Biological Models; Bladder; Breast; C-terminal; Cancer Model; Cancer cell line; Cell Line; Cell surface; Cells; Characteristics; Charge; Clear Cell; Client; Clinic; Clinical Trials; Collaborations; Complex; Data; Dependence; Deposition; Development; Disseminated Malignant Neoplasm; Dissociation; Dose; Drug Combinations; E-Cadherin; Endoplasmic Reticulum; ErbB Receptor Family Protein; Family; Family member; Fostering; Geldanamycin; Growth; HIF1A gene; Heat-Shock Proteins 90; Herbimycin; Hydrophobicity; Hypoxia; Imatinib; In Vitro; Information Technology; Kansas; Lead; Link; MMP2 gene; Malignant neoplasm of cervix uteri; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Mast-Cell Leukemia; Mediating; Melanoma Cell; Membrane; Metalloproteases; Metastatic Fibrosarcoma; Modeling; Molecular Chaperones; Molecular Conformation; Mutate; N-terminal; National Institute of Allergy and Infectious Disease; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Normal Cell; Novobiocin; Oxygen; Papillary; Pathway interactions; Phase; Phase II Clinical Trials; Phosphotransferases; Post-Translational Protein Processing; Property; Prostate; Proteasome Inhibition; Proteasome Inhibitor; Protein Tyrosine Kinase; Proteins; Radiation-Sensitizing Agents; Recruitment Activity; Renal Cell Carcinoma; Renal carcinoma; Research; Research Personnel; Resistance; Rifabutin; Role; Series; Signal Transduction; Signaling Protein; Site; Specificity; Stimulus; Surface; Testing; Toxic effect; Tyrosine Phosphorylation; Ubiquitination; United States National Institutes of Health; Urologic Diseases; Urologic Oncology; base; beta catenin; cancer cell; cancer site; cell motility; collaborative trial; drug development; improved; in vivo; inhibitor/antagonist; mastocytosis; meetings; melanoma; member; multicatalytic endopeptidase complex; mutant; neoplastic cell; novel; phase 2 study; prevent; protein complex; response; src-Family Kinases; tacrolimus binding protein 4; transcription factor; tumor; ubiquitin-protein ligase

HSP90 is a cellular chaperone that stabilizes several signal transduction networks important to cancer cells. Several years ago, we discovered that members of the benzoquinone ansamycin class of antibiotics, including herbimycin A and geldanamycin (GA) bound to HSP90 and disrupted its function. Multiple signal transduction proteins interact with these chaperones, including the kinases src, erbB2, c-raf-1, Akt, Kit, Met, Bcr-Abl, the transcription factor HIF-1alpha, and mutated (but not wild type) p53. A general consequence of pharmacologic disruption of the chaperone/signal protein complex is the resultant marked instability and incorrect subcellular localization of the signaling protein. The instability is due to stimulation of targeted degradation of the signaling protein by the 26S proteasome proteolytic complex following chaperone dissociation. We have observed that geldanamycin reverses beta-catenin tyrosine phosphorylation in melanoma cells, probably due to the rapid loss of erbB2 from these cells. In untreated cells, erbB2 and beta-catenin can be readily co-precipitated. Loss of beta-catenin tyrosine phosphorylation leads to an increased association with E-cadherin and decreased cell motility in vitro. This is the first indication that modulation of the tyrosine phosphorylation status of beta catenin in melanoma cells is associated with decreased motility. The fact that beta-catenin tyrosine phosphorylation seems to be mediated, in 3/3 melanoma cell lines examined, by erbB2 - a geldanamycin-sensitive tyrosine kinase - suggests that geldanamycin treatment may be anti-metastatic. This hypothesis is currently being tested in an in vivo metastasis model.In collaboration with Brian Blagg of the Univ. of Kansas, we have identified a series of novobiocin derivatives that demonstrate improved binding affinity and anti-Hsp90 activity compared to the parental compound, and we have demonstrated the ability of several of these derivatives to deplete Hsp90 client proteins in tumor cells.The ErbB family of receptor tyrosine kinases contains four members. We have found that ErbB2, the only ligandless member of the family, is one of the most sensitive geldanamycin substrates. In contrast, mature ErbB1 is much less sensitive to geldanamycin than ErbB2. However, ErbB1 mutants found in NSCLC are highly dependent on Hsp90 and sensitive to its pharmacologic inhibition.In collaboration with Yong Lee of the Center for Information Technology, NIH, we have identified a short loop in the kinase domain of ErbB2 whose hydrophobicity and charge determine Hsp90 binding. We have shown that Hsp90 client kinases and non-client kinases differ in the charge distribution and hydrophobicity of this small region of the kinase domain.We recently identified the novel E3 ubiquitin ligase Chip as being recruited to ErbB2-associated chaperone complexes in the presence of geldanamycin. Chip mediates geldanamycin induced ErbB2 ubiquitination, which is necessary for, and precedes, its degradation by the proteasome. We have recently identified the site within the kinase domain of ErbB2 at which Hsp90 binds and we have proposed a model to explain remodeling of ErbB2-associated chaperone complexes in the presence of geldanamycin and other Hsp90 inhibitors.We have demonstrated that combination of low doses of geldanamycin and a proteasome inhibitor currently in clinical trial increases the toxicity toward tumor cells compared to that observed with each agent alone. We showed that this property was unique to tumor cells, in that the combination was not toxic to non-transformed cells at the concentrations tested. Further, we proposed a model to explain these results that invokes proteasome overload and deposition in the cell of insoluble proteins, leading to initiation of apoptosis. These data have led to initiation of a phase I combination clinical trial of an Hsp90 inhibitor and a proteasome inhibitor, in collaboration with investigators at the Mayo Clinic.We demonstrated that the trans-membrane endoplasmic reticulum signaling proteins, IRE1 and PERK, are Hsp90 client proteins and are thus sensitive to Hsp90 inhibitors. These data were the first to link Hsp90 to proper function of the unfolded protein response. The data further underline the crucial role of Hsp90 in allowing cells to survive stressful stimuli.We recently demonstrated that HIF-1alpha, a transcription factor whose expression is upregulated by hypoxia and loss of VHL, is an Hsp90 client protein and is sensitive to Hsp90 inhibition. These data were the first to identify an oxygen- and VHL-independent pathway regulating HIF stability and they identify a pathway that is amenable to pharmacologic manipulation using Hsp90 inhibitors. These data have led to initiation, in the Urologic Oncology Branch, of a phase II clinical trial of benzoquinone ansamycins in clear cell kidney cancer (lacking VHL and expressing HIF-1alpha at constitutively high levels).We identified kinase-mutated KIT protein to be sensitive to Hsp90 inhibition. Kinase-mutated KIT is resistant to imatinib and other KIT inhibitors currently in the clinic. Kinase-mutated KIT is characteristic of mastocytosis and mast cell leukemia. Based on our findings, a phase II study ofan Hsp90 inhibitor to treat mastocytosis has been initiated as an NCI/NIAID/Mayo Clinic collaborative trial.We demonstrated that MET and mutated MET protein, characteristic of papillary renal cell cancer, is sensitive to Hsp90 inhibition. Based on these findings, a phase II clinical trial of an Hsp90 inhibitor in papillary renal cell cancer is being initiated in the Urologic Oncology Branch.We identified Hsp90 on the surface of metastatic fibrosarcoma cells and demonstrated that specific inhibition of surface Hsp90 (using cell impermeable geldanamycin derivatives) significantly inhibits cell invasiveness. Further, we showed that inhibition of surface of Hsp90 prevented the maturation of MMP2, a metalloproteinase that has been implicated in fostering invasion and metastasis. Thus, we are now examining the model that surface Hsp90 may be a specific target in metastatic cancer cells. We have gone on to show, in a bladder cancer model system, that cell-impermeable Hsp90 inhibitors have dramatic inhibitory effects on cell migration and invasiveness, while having minimal growth inhibitory activity. We have extended these studies to include bladder, breast, prostate, and melanoma cancer cell lines with similar results.During the past year we have also shown that the combination of Hsp90 inhibition with proteasome inhibition is especially toxic to highly secretory tumor cells (while sparing normal cells). We have provided evidence that this drug combination disrupts the endoplasmic reticulum and therefore hypothesize that the endoplasmic reticulum is a viable target in cancer cells.We have also shown that Hsp90 inhibition is a potent radiosensitizer, in a cervical cancer model, both in vitro and in vivo.We have provided evidence that geldanamycin cannot bind to Hsp90 without first being isomerized by the chaperone. This may provide binding specificity for activated forms of Hsp90, the state of the chaperone which is characteristic of cancer cells, but not normal cells. We speculate, therefore, that making use of this property may permit us to synthesize more potent, tumor Hsp90-prefering Hsp90 inhibitors.We have initial evidence that not all Hsp90 inhibitors recognize/trap the same conformation of the chaperone.Finally, we have identified a novel lead compound that interferes with Hsp90/FKBP52-dependent chaperoning of the androgen receptor. This compound acts in a mechanism that is unique compared to other androgen receptor antagonists.

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。几年前，我们发现苯醌类抗生素ansamycin的成员，包括herbimycin A和geldanamycin （GA）结合到HSP90并破坏其功能。多种信号转导蛋白与这些伴侣蛋白相互作用，包括激酶src、erbB2、c-raf-1、Akt、Kit、Met、Bcr-Abl、转录因子hif -1 α和突变（但不是野生型）p53。伴侣/信号蛋白复合物的药理学破坏的一般后果是信号蛋白的显著不稳定和不正确的亚细胞定位。这种不稳定性是由于在伴侣解离后，26S蛋白酶体蛋白水解复合物刺激了信号蛋白的靶向降解。我们观察到格尔达霉素在黑色素瘤细胞中逆转β -连环蛋白酪氨酸磷酸化，可能是由于这些细胞中erbB2的快速丢失。在未经处理的细胞中，erbB2和β -连环蛋白可以很容易地共沉淀。在体外，β -连环蛋白酪氨酸磷酸化的缺失导致与e -钙粘蛋白的关联增加和细胞运动性降低。这是第一个表明黑色素瘤细胞中β -连环蛋白酪氨酸磷酸化状态的调节与运动能力下降有关的迹象。事实上，在3/3的黑色素瘤细胞系中，β -连环蛋白酪氨酸磷酸化似乎是由erbB2介导的，erbB2是一种格尔达霉素敏感的酪氨酸激酶，这表明格尔达霉素治疗可能具有抗转移性。这一假设目前正在体内转移模型中进行验证。与堪萨斯大学的Brian Blagg合作，我们鉴定了一系列新生物素衍生物，与亲本化合物相比，它们具有更好的结合亲和力和抗Hsp90活性，并且我们已经证明了这些衍生物中有几种能够消耗肿瘤细胞中的Hsp90客户蛋白。ErbB受体酪氨酸激酶家族包含四个成员。我们已经发现ErbB2，家族中唯一的无配体成员，是最敏感的格尔达霉素底物之一。相比之下，成熟的ErbB1对格尔达霉素的敏感性远低于ErbB2。然而，在NSCLC中发现的ErbB1突变体高度依赖于Hsp90，并且对其药理学抑制敏感。与NIH信息技术中心的Yong Lee合作，我们在ErbB2的激酶结构域中发现了一个短环，其疏水性和电荷决定了Hsp90的结合。我们已经证明，Hsp90客户端激酶和非客户端激酶在这个激酶结构域的小区域的电荷分布和疏水性方面有所不同。我们最近发现新的E3泛素连接酶Chip在格尔达霉素存在下被募集到erbb2相关的伴侣复合物中。芯片介导格尔德霉素诱导的ErbB2泛素化，这是蛋白酶体降解ErbB2的必要条件和前提。我们最近确定了ErbB2激酶结构域内Hsp90结合的位点，并提出了一个模型来解释在格尔达霉素和其他Hsp90抑制剂存在下ErbB2相关伴侣复合物的重塑。我们已经证明，与单独使用每种药物相比，目前临床试验中低剂量格尔达霉素和蛋白酶体抑制剂的联合使用增加了对肿瘤细胞的毒性。我们发现这种特性是肿瘤细胞所特有的，因为在测试的浓度下，这种组合对未转化的细胞没有毒性。此外，我们提出了一个模型来解释这些结果，该模型调用蛋白酶体过载和细胞中不溶性蛋白的沉积，导致细胞凋亡的启动。这些数据促使与梅奥诊所的研究人员合作，启动了一项Hsp90抑制剂和蛋白酶体抑制剂的I期联合临床试验。我们证明了跨膜内质网信号蛋白IRE1和PERK是Hsp90的客户蛋白，因此对Hsp90抑制剂敏感。这些数据首次将Hsp90与未折叠蛋白反应的适当功能联系起来。这些数据进一步强调了Hsp90在使细胞在应激刺激中存活的关键作用。我们最近证明hif -1 α是一种Hsp90客户蛋白，对Hsp90抑制敏感。hif -1 α是一种转录因子，其表达会因缺氧和VHL缺失而上调。这些数据首次确定了一种不依赖氧和vhl的途径调节HIF的稳定性，并确定了一种可以使用Hsp90抑制剂进行药理学操作的途径。这些数据促使泌尿肿瘤科启动了一项苯醌类ansamyins治疗透明细胞肾癌（缺乏VHL且hif -1 α表达水平高）的II期临床试验。我们发现激酶突变的KIT蛋白对Hsp90抑制敏感。目前临床上，激酶突变的KIT对伊马替尼和其他KIT抑制剂具有耐药性。激酶突变的KIT是肥大细胞增多症和肥大细胞白血病的特征。基于我们的发现，一种Hsp90抑制剂治疗肥大细胞增多症的II期研究已经作为NCI/NIAID/Mayo Clinic的合作试验启动。我们证明了MET和突变的MET蛋白，乳头状肾细胞癌的特征，对Hsp90抑制敏感。基于这些发现，一种Hsp90抑制剂治疗乳头状肾细胞癌的II期临床试验正在泌尿肿瘤科启动。我们在转移性纤维肉瘤细胞表面发现了Hsp90，并证明对表面Hsp90的特异性抑制（使用细胞不渗透的格尔达霉素衍生物）显著抑制了细胞的侵袭性。此外，我们发现抑制Hsp90表面可以阻止MMP2的成熟，MMP2是一种金属蛋白酶，与促进侵袭和转移有关。因此，我们现在正在研究表面Hsp90可能是转移性癌细胞的特定靶点的模型。在膀胱癌模型系统中，我们已经证明，不渗透细胞的Hsp90抑制剂对细胞迁移和侵袭具有显著的抑制作用，同时具有最小的生长抑制活性。我们已经将这些研究扩展到膀胱癌、乳腺癌、前列腺癌和黑色素瘤细胞系，结果相似。在过去的一年中，我们还表明，Hsp90抑制与蛋白酶体抑制的结合对高分泌性肿瘤细胞具有特别的毒性（同时保留正常细胞）。我们提供的证据表明，这种药物组合破坏内质网，因此假设内质网是癌细胞的可行靶点。我们还表明，在体外和体内的宫颈癌模型中，抑制Hsp90是一种有效的放射增敏剂。我们已经提供的证据表明，格尔达霉素不能与Hsp90结合，除非先被伴侣分子异构化。这可能为活化形式的Hsp90提供结合特异性，这是癌细胞的特征，而不是正常细胞的伴侣状态。因此，我们推测，利用这一特性可能使我们能够合成更有效的肿瘤Hsp90偏好抑制剂。我们有初步证据表明，并非所有的Hsp90抑制剂都能识别/捕获伴侣蛋白的相同构象。最后，我们发现了一种新的先导化合物，它可以干扰Hsp90/ fkbp52依赖性雄激素受体的陪伴作用。与其他雄激素受体拮抗剂相比，这种化合物的作用机制是独特的。