17-AAG, an Hsp90 inhibitor, ameliorates polyglutamine-mediated motor neuron degeneration

Heat-shock protein 90 (Hsp90) functions as part of a multichaperone complex that folds, activates and assembles its client proteins. Androgen receptor (AR), a pathogenic gene product in spinal and bulbar muscular atrophy (SBMA), is one of the Hsp90 client proteins. We examined the therapeutic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a potent Hsp90 inhibitor, and its ability to degrade polyglutamine-expanded mutant AR. Administration of 17-AAG markedly ameliorated motor impairments in the SBMA transgenic mouse model without detectable toxicity, by reducing amounts of monomeric and aggregated mutant AR. The mutant AR showed a higher affinity for Hsp90-p23 and preferentially formed an Hsp90 chaperone complex as compared to wild-type AR; mutant AR was preferentially degraded in the presence of 17-AAG in both cells and transgenic mice as compared to wild-type AR. 17-AAG also mildly induced Hsp70 and Hsp40. 17-AAG would thus provide a new therapeutic approach to SBMA and probably to other related neurodegenerative diseases.     

Dihydroquinone ansamycins: toward resolving the conflict between low in vitro affinity and high cellular potency of geldanamycin derivatives

Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.     

Protection of murine neural progenitor cells by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin in the low nanomolar concentration range

Stem cell-based approaches provide hope as a potential therapy for neurodegenerative diseases and stroke. One of the major scientific hurdles for stem cell therapy is the poor survival rate of the newly formed or transplanted neural stem cells. In this study, we found that low-dose treatment with the Heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heavily investigated anti-cancer drug, prevented neural progenitor cells from either naturally-occurring or stress-induced apoptosis, although it induced apoptosis at higher doses. This stress adaptation effect mediated by low-dose 17-AAG is accompanied by activation of multiple cell survival pathways, including the stress response pathway (induction of Hsp70), the MAPK pathway, and the PI3K/Akt pathway. When administered in vivo, 17-AAG led to Akt and glycogen synthase kinase 3β phosphorylation, and more 5-bromo-2'-deoxyuridine positive cells in the mouse brain. These findings could have profound implications in stem cell therapy for neurodegenerative diseases and stroke.     

Protein quality control of DYRK family protein kinases by the Hsp90-Cdc37 molecular chaperone

The DYRK (Dual-specificity tYrosine-phosphorylation Regulated protein Kinase) family consists of five related protein kinases (DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4). DYRKs show homology to Drosophila Minibrain, and DYRK1A in human chromosome 21 is responsible for various neuronal disorders including human Down syndrome. Here we report identification of cellular proteins that associate with specific members of DYRKs. Cellular proteins with molecular masses of 90, 70, and 50-kDa associated with DYRK1B and DYRK4. These proteins were identified as molecular chaperones Hsp90, Hsp70, and Cdc37, respectively. Microscopic analysis of GFP-DYRKs showed that DYRK1A and DYRK1B were nuclear, while DYRK2, DYRK3, and DYRK4 were mostly cytoplasmic in COS7 cells. Overexpression of DYRK1B induced nuclear re-localization of these chaperones with DYRK1B. Treatment of cells with specific Hsp90 inhibitors, geldanamycin and 17-AAG, abolished the association of Hsp90 and Cdc37 with DYRK1B and DYRK4, but not of Hsp70. Inhibition of Hsp90 chaperone activity affected intracellular dynamics of DYRK1B and DYRK4. DYRK1B and DYRK4 underwent rapid formation of cytoplasmic punctate dots after the geldanamycin treatment, suggesting that the chaperone function of Hsp90 is required for prevention of protein aggregation of the target kinases. Prolonged inhibition of Hsp90 by geldanamycin, 17-AAG, or ganetespib, decreased cellular levels of DYRK1B and DYRK4. Finally, DYRK1B and DYRK4 were ubiquitinated in cells, and ubiquitinated DYRK1B and DYRK4 further increased by Hsp90 inhibition with geldanamycin. Taken together, these results indicate that Hsp90 and Cdc37 discriminate specific members of the DYRK kinase family and play an important role in quality control of these client kinases in cells.     

SNX-25a,a novel Hsp90 inhibitor,inhibited human cancer growth more potently than 17-AAG

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (<1 μM). It showed low cytotoxicity in L-02, HDF and MRC5 normal human cells. Compared with 17-AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail.     

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol–based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922–sensitive cell lines lacked NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.     

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

PIWI-interacting RNAs (piRNAs) defend the genome against transposon activity in animal gonads. The Hsp90 chaperone machinery has been implicated in the piRNA pathway, but its exact role remains obscure. Here, we examined the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on the piRNA pathway. In the silkworm ovary-derived BmN4 cells, 17-AAG treatment reduced the level of piRNAs and PIWI proteins. In vitro, the 5′-nucleotide preference upon precursor piRNA loading was compromised by 17-AAG, whereas 3′-end trimming and 2′-O-methylation were unaffected. Our data highlight a role of Hsp90 in accurate loading of precursor piRNAs into PIWI proteins.     

Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors

Hepatocyte growth factor/scatter factor (HGF/SF) receptor c-Met is implicated in growth, invasion and metastasis of many tumors. Tumor cells harboring MET gene amplification are initially sensitive to c-Met tyrosine kinase inhibitors (TKI), but escape from long-term treatment has not been investigated. C-Met is a client of heat shock protein 90 (Hsp90) and is destabilized by Hsp90 inhibitors, suggesting that these drugs may inhibit tumors driven by MET amplification, although tumor escape under these conditions also has not been explored. Here, we evaluated the initial inhibitory effects of, and the likelihood of escape from, the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and the c-Met TKI SU11274, using two cell lines harboring MET gene amplification. 17-AAG inhibited cell growth in both cell lines and induced substantial apoptosis, whereas SU11274 was only growth inhibitory in one cell line. In both cell lines, c-Met-dependent Akt, Erk and/or STAT3 signaling, as well as activation of the EGFR family, resumed shortly after treatment with c-Met TKI despite sustained c-Met inhibition. PKC δ upregulation may participate in reactivation of c-Met downstream signaling in both cell lines. In contrast to c-Met TKI, 17-AAG destabilized c-Met protein and durably blocked reactivation of downstream signaling pathways and EGFR family members. Our data demonstrate that downstream signaling in tumor cells over-expressing c-Met is not stably suppressed by c-Met TKI, even though c-Met remains fully inhibited. In contrast, Hsp90 inhibitors provide long-lasting suppression of c-Met-dependent signaling, and these drugs should be further evaluated in tumors driven by MET gene amplification.     

Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells

Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1^Thr163 phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells.     

Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors

The development of therapeutic inhibitors of key signaling pathways has been hampered by the inability to assess the effect of a drug on its target in the patient. 17-allylaminogeldanamycin (17-AAG) is the first Hsp90 inhibitor to be tested in a clinical trial. It causes the degradation of HER2 and other Hsp90 targets, and has antitumor activity in preclinical models. We have developed a method for imaging the inhibition of Hsp90 by 17-AAG. We labeled an F(ab')2 fragment of the anti-HER2 antibody Herceptin with 68Ga, a positron emitter, which allows the sequential positron-emission tomographic imaging of HER2 expression. We have used this method to quantify as a function of time the loss and recovery of HER2 induced by 17-AAG in animal tumors. This approach allows noninvasive imaging of the pharmacodynamics of a targeted drug and will facilitate the rational design of combination therapy based on target inhibition.     

Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.     

ErbB2 degradation mediated by the co-chaperone protein CHIP

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.     

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K_d values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 × 10^− 4 min^− 1 μM^− 1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.     

Lamin-A interacting protein Hsp90 is required for DNA damage repair and chemoresistance of ovarian cancer cells

Ovarian cancer is the most malignant gynecologic cancer. Previous studies found that lamin-A was associated with DNA damage repair proteins but the underlying mechanism remains unclear. We speculate that this may be related to its interacting proteins, such as Hsp90. The aim of this study is to investigate the effects of Hsp90 on DNA damage repair and chemoresistance of ovarian cancer cells. In our research, co-immunoprecipitation (co-IP) and mass spectrometry (MS) were used to identify proteins interacting with lamin-A and the interaction domain. Next, the relationship between lamin-A and Hsp90 was explored by Western blotting (WB) and immunofluorescence staining. Then, effect of Hsp90 inhibition on DNA damage repair was assessed through detecting Rad50 and Ku80 by WB. Furthermore, to test the roles of 17-AAG on cell chemosensitivity, CCK-8 and colony formation assay were carried out. Meanwhile, IC50 of cells were calculated, followed by immunofluorescence to detect DNA damage. At last, the mouse xenograft model was used in determining the capacity of 17-AAG and DDP to suppress tumor growth and metastatic potential. The results showed that lamin-A could interact with Hsp90 via the domain of lamin-A^1-430. Besides, the distribution of Hsp90 could be affected by lamin-A. After lamin-A knockdown, Hsp90 decreased in the cytoplasm and increased in the nucleus, suggesting that the interaction between lamin-A and Hsp90 may be related to the nucleocytoplasmic transport of Hsp90. Moreover, inhibition of Hsp90 led to an obvious decrease in the expression of DSBs (DNA double-strand break) repair proteins, as well as cell proliferation ability upon DDP treatment and IC50 of DDP, causing more serious DNA damage. In addition, the combination of 17-AAG and DDP restrained the growth of ovarian cancer efficiently in vivo and prolonged the survival time of tumor-bearing mice.Subject terms: Cancer therapy, Gynaecological cancer, Gynaecological cancer     

Cooperative enhancement of radiosensitivity after combined treatment of 17-(allylamino)-17-demethoxygeldanamycin and celecoxib in human lung and colon cancer cell lines

We investigated whether the combined treatment of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat-shock protein 90 (hsp90), and celecoxib, an inhibitor of cyclooxygenase-2, can cooperatively enhance the radiosensitivity of various human cancer cells. Combined treatment with 17-AAG and celecoxib, at clinically relevant concentrations, cooperatively induced radiosensitization in all tested cancer cells, but not in normal cells. Cooperative radiosensitization by the drug combination was also shown in a human tumor xenograft system. We found that ataxia-telangiectasia and rad3-related (ATR) and ataxia-telangiectasia mutated (ATM) are novel client proteins of hsp90. Combined treatment with 17-AAG and celecoxib cooperatively induced downregulation of ATR and ATM. In conclusion, combined treatment with 17-AAG and celecoxib at clinically relevant concentrations may significantly enhance the therapeutic efficacy of ionizing radiation.     

Utility of 17-(allylamino)-17-demethoxygeldanamycin treatment for skeletal muscle injury

Repeated eccentric contractions can injure skeletal muscle and result in functional deficits that take several weeks to fully recover. The 70-kDa heat shock protein (Hsp70) is a stress-inducible molecular chaperone that maintains protein quality and plays an integral role in the muscle’s repair processes following injury. Here, we attempted to hasten this recovery by pharmacologically inducing Hsp70 expression in mouse skeletal muscle with 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) (40 mg/kg) both prior to and throughout the first 7 days after an injurious bout of 150 maximal eccentric contractions. Hsp70 content in the injured skeletal muscle was strongly induced following the eccentric contractions and remained elevated over the next 7 days as the muscle underwent repair. Treatment with 17-AAG increased Hsp70 content ～fivefold; however, this was significantly less than that induced by the injury. Moreover, 17-AAG treatment did not recover the decrements to in vivo isometric torque production following the bout of eccentric contractions. Together, these findings demonstrate that although Hsp70 content was induced in the uninjured skeletal muscle, treatment of 17-AAG (40 mg/kg) was not a preventive measure to either reduce the severity of skeletal muscle damage or enhance functional recovery following a bout of maximal eccentric contractions.     

Therapeutic and diagnostic implications of Hsp90 activation

The molecular chaperone heat-shock protein 90 (Hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. Hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is presently in clinical trials. Recent work has identified the role of Hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by Hsp90 inhibitors is the result of an activated, high-affinity conformation of Hsp90 in tumors. This review discusses these recent advances in the understanding of tumor Hsp90 for the treatment and diagnosis of cancer. In addition, the role of Hsp90 in non-oncological diseases will also be discussed.     

Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90beta,a property that can be exploited in screens for new Hsp90 chaperone inhibitors

Heat shock protein 90 (Hsp90) is essential for activation of many of the most important regulatory proteins of eukaryotic cells. It is an extremely conserved protein, such that heterologous expressions of either human Hsp90beta or Caenorhabditis elegans Hsp90 will provide the essential Hsp90 function in yeast. The ability of these metazoan Hsp90s to provide this Hsp90 function to yeast cells requires Sti, a Hsp90 system cochaperone. Yeast that is expressing human Hsp90beta in place of the normal native yeast Hsp90 is selectively hypersensitised to Hsp90 inhibitor drugs. Hsp90 drugs are promising anticancer agents, their administration simultaneously destabilizing a number of the proteins critical to multistep carcinogenesis. Though one of these drugs (17-allylaminogeldanamycin, 17-AAG) is now progressing to Phase 2 clinical trials, there is a pressing need to identify selective Hsp90 inhibitors that are more soluble than 17-AAG. High-throughput screening for chemical agents that exert greater inhibitory effects against yeast expressing the human Hsp90beta relative to yeast expressing its native Hsp90 should therefore facilitate the search for new Hsp90 inhibitors.