HSP90 Family Chaperones in Cellular Signal Transduction

HSP90和GRP94是分别存在于细胞质和内质网的同源细胞伴侣。几年前，我们发现包括herbimycin A和geldanamycin （GA）在内的苯醌类抗生素的成员与HSP90和GRP94结合，并破坏了这些蛋白质所属的某些多分子复合物。我们利用HSP90和GRP94活性的药理学破坏来研究这些伴侣蛋白在细胞信号转导中的功能。多种信号转导蛋白与这些charperones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导致信号蛋白的明显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。我们进行了新的观察，发现HSP90与胞质激酶RIP相关，而胞质激酶RIP是导致NFkB激活的TNF信号通路的关键成分。我们已经确定，格尔达霉素破坏RIP稳定性可以阻止TNF诱导NFkB，但不能阻止TNF向Jnk信号传导，从而使细胞对TNF的凋亡特性敏感。我们还观察到另一种与细胞存活相关的激酶Akt对格尔达霉素敏感。格尔达霉素可阻断除TNF以外的多种刺激对NFkB的诱导，包括化疗药物和IL-1。它这样做的能力可能与它对Akt的不稳定有关。苯醌类ansamyins（格尔达霉素）是唯一能够特异性干扰HSP90/GRP94功能的药物。最近，我们发现了一种新的能够与HSP90结合的天然产物。根霉素和反霉素都在氨基末端的核苷酸口袋中与热休克蛋白90结合。最近，我们发现了第三类天然产物，新生物素，它也与HSP90结合，尽管其亲和力低于苯醌类抗霉素或根瘤素。尽管如此，新生物素对“客户蛋白”的生物学作用似乎与反霉素和根瘤素相同。令人惊讶的是，新生物素似乎与HSP90上的羧基末端区域相互作用，这实际上是一个以前未被识别的第二核苷酸结合位点。初步的动物试验显示，每天两次服用新生物素超过一个月后没有毒性。该方案在erbb2驱动的乳腺癌转基因小鼠模型中显示出显著的抗肿瘤活性。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。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。由于erbb是跨膜蛋白，它们可能同时与Hsp90和Grp94接触，其中一个或两个伴侣蛋白可能对ErbB2的格尔达霉素敏感性负责。我们目前的数据表明，ErbB2的激酶结构域介导其对格尔达霉素的反应，并且Hsp90在成熟蛋白中与该结构域结合。相比之下，成熟的ErbB1对格尔达霉素的敏感性远低于ErbB2，不与Hsp90相关。格尔达那霉素诱导的新生ErbB2的不稳定性也由其激酶结构域介导，我们几乎没有证据支持Grp94在ErbB2成熟中的作用。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（内质网中的应激反应）的正常功能联系起来。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于氧和vhl的调节HIF稳定性的途径，这是一种可以使用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。

HSP90 and GRP94 are homologous cellular chaperones found in cytosol and endoplasmic reticulum, respectively. Several years ago, we discovered that members of the benzoquinone ansamycin class of antibiotics, including herbimycin A and geldanamycin (GA) bound to HSP90 and GRP94 and disrupted certain multi-molecular complexes of which these proteins were a part. We have utilized pharmacologic disruption of HSP90 and GRP94 activity to study the function of these chaperones in cellular signal transduction. Multiple signal transduction proteins interact with these charperones, 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 signalling protein. The instability is due to stimulation of targeted degradation of the signalling protein by the 26S proteasome proteolytic complex following chaperone dissociation. We made the novel observation that HSP90 associates with the cytosolic kinase RIP, a key component of the TNF signalling pathway which leads to NFkB activation. We have determined that disruption of RIP stability by geldanamycin prevents NFkB induction by TNF, but not TNF signalling to Jnk, thus sensitizing cells to the apoptotic properties of TNF. We have additionally observed that another kinase associated with cell survival, Akt, is sensitive to geldanamycin. Geldanamycin blocks NFkB induction by a wide variety of stimuli other than TNF, including chemotherapeutic drugs and IL-1. Its ability to do this may relate to its destabilization of Akt. Benzoquinone ansamycins (geldanamycin) had been the only agents capable of specifically interfering in HSP90/GRP94 function. Recently, we identified radicicol as representing a novel class of natural product capable of binding to HSP90. Both radicicol and the ansamycins bind to HSP90 at an amino terminal nucleotide pocket. Most recently, we have identified a third class of natural product, novobiocin, which also binds to HSP90, although at a lower affinity than either benzoquinone ansamycins or radicicol. Nonetheless, novobiocin appears to cause the same biologic effects on "client proteins" as ansamycins and radicicol. Surprisingly, novobiocin appears to interact with a carboxyl terminal region on HSP90, which is in fact a previously unrecognized second nucleotide binding site. Preliminary animal testing has revealed no toxicity after twice daily administration of novobiocin for more than one month. This regimen demonstrates significant anti-tumor activity in a transgenic murine model of erbB2-driven breast cancer. 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.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. Since the ErbBs are transmembrane proteins, they are likely to come in contact with both Hsp90 and Grp94, and one or both chaperones may be responsible for ErbB2's geldanamycin sensitivity. Our current data demonstrate that the kinase domain of ErbB2 mediates its geldanamycin responsiveness and that Hsp90 binds to this domain in the mature protein. In contrast, mature ErbB1, much less sensitive to geldanamycin than ErbB2, does not associate with Hsp90. Geldanamycin-induced instability of nascent ErbB2 is also mediated by its kinase domain, and we can find little evidence to support a role for Grp94 in ErbB2 maturation.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 stress response as it occurs in the endoplasmic reticulum. 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 - 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 of an 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 invastion and metastasis.