Inhibition Of Hsp90 Function Research Articles

Hsp90-Mediated Multi-Drug Resistance in DNA Polymerase-Defective Strains of Candida albicans.

The incidence of infections caused by Candida species, specifically by drug-resistant isolates, is a major health concern as they can disseminate to and colonize most vital organs, enhancing morbidity and mortality. Several molecular mechanisms have been reported to be involved in drug resistance. These are mostly drug- and isolate-specific. Here, we characterized three different genetically modified strains of C. albicans that were multi-drug-resistant (MDR) and deciphered a uniform mechanism responsible for resistance. DNA polymerase epsilon (Polε) is a leading strand-specific polymerase consisting of four subunits, namely, Pol2, Dpb2, Dpb3, and Dpb4. The deletion of one or both of the Dpb3 and Dpb4 subunits in C. albicans rendered multi-drug resistance. A detailed characterization of these strains revealed that acquired mutagenesis, drug efflux pumps, and other known mechanisms did not play a significant role because the complemented strain showed drug sensitivity. More importantly, the function of heat shock protein 90 (Hsp90) in these knockout strains is critical for reducing susceptibility to several antifungal drugs. Cell wall deformity and composition in these strains can add to such a phenotype. The inhibition of Hsp90 function by geldanamycin and tricostatin A sensitized the MDR strains to antifungals. Considering our earlier research and this report, we suggest that replication stress induces Hsp90 expression and activity in order to orchestrate a cellular stress response circuit and thus develop fungal drug resistance. Thus, Hsp90 is an important drug target for use in combinatorial therapy.

Spotlight on 17‐AAG as an Hsp90 inhibitor for molecular targeted cancer treatment

Hsp90 is a ubiquitous chaperone with important roles in the organization and maturation of client proteins that are involved in the progression and survival of cancer cells. Multiple oncogenic pathways can be affected by inhibition of Hsp90 function through degradation of its client proteins. That makes Hsp90 a therapeutic target for cancer treatment. 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is a potent Hsp90 inhibitor that binds to Hsp90 and inhibits its chaperoning function, which results in the degradation of Hsp90's client proteins. There have been several preclinical studies of 17-AAG as a single agent or in combination with other anticancer agents for a wide range of human cancers. Data from various phases of clinical trials show that 17-AAG can be given safely at biologically active dosages with mild toxicity. Even though 17-AAG has suitable pharmacological potency, its low water solubility and high hepatotoxicity could significantly restrict its clinical use. Nanomaterials-based drug delivery carriers may overcome these drawbacks. In this paper, we review preclinical and clinical research on 17-AAG as a single agent and in combination with other anticancer agents. In addition, we highlight the potential of using nanocarriers and nanocombination therapy to improve therapeutic effects of 17-AAG.

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer.

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.

Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells

Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells.

Abstract 1683: Autophagy inhibition for cancer therapy.

Abstract In tumor but not normal cells, the stress-inducible heat shock protein 70 (HSP70) is abundantly expressed and becomes incorporated into lysosome membranes, where it serves to stabilize and protect lysosome function. Inhibition of HSP70 thus results in defective lysosome function, along with impaired autophagy. We previously reported that the HSP70 inhibitor phenylethynesulfonamide (PES) is a potent and effective autophagy inhibitor (1). PES shows marked cytotoxicity to tumor cells, but minimal effects in normal, non-transformed cells. We previously reported that PES binds to both HSP70 and to the constitutively-expressed family member HSC70; these are critical co-chaperones for HSP90, and we previously reported that treatment of tumor cells with PES leads to decreased function of HSP90 client proteins like HER2, AKT and CDK4 (2). We have recently performed a preliminary structure-activity relationship for PES. These studies revealed a novel PES analogue that we call PES-Cl, which shows 10-fold decreased IC50 for tumor cells, minimal cytotoxicity to normal cells, and a greatly enhanced ability to inhibit autophagy. In vitro we show that PES-Cl is cytotoxic to melanoma cells, including those with both intrinsic and acquired resistance to BRAF inhibitors, but shows minimal cytotoxicity to primary melanocytes. In a pre-clinical model for B-cell lymphoma (Eu-myc transgenic mouse), we show that PES-Cl demonstrates significant ability to extend the life of mice (p=0.000006), with no evidence for liver pathology or toxicity. We report that PES binds to the substrate-binding domain of HSP70, and requires the C-terminal helical ‘lid’ of this protein (amino acids 573-616) in order to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in HSP70, and we identify point mutants that fail to interact with this compound. Our cell cycle analyses of PES-Cl-treated cells show that this compound induces G2/M arrest. Interestingly, we also show that this HSP70 inhibitor impairs the activity of the Anaphase Promoting Complex/Cyclosome (APC/C) in cell-free extracts. PES-Cl is thus a promising new HSP70 inhibitor that binds to the C-terminal ‘lid’ of HSP70, and multiple mechanisms of action: inhibition of autophagy, inhibition of HSP90 function, and inhibition of the Anaphase Promoting Complex/Cyclosome (APC/C). 1. Leu et al, Molecular Cell, 6(1):15-27, 2009 2. Leu et al, Molecular Cancer Research, 9(7):936-47, 2011 Citation Format: Gregor Balaburski, Julia I-Ju Leu, Neil Beeharry, Seth Hayik, Mark D. Andrake, Gao Zhang, Meenhard Herlyn, Jessie Villanueva, Roland L. Dunbrack, Tim Yen, Donna L. George, Maureen E. Murphy. Autophagy inhibition for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2013-1683

Abstract 2771: HSP90 inhibitor NVP-AUY922 exhibits the potent antiproliferative effect in gastrointestinal cancer cells with a lack of PTEN expression

Abstract Background: The heat shock protein 90 (HSP90), a molecular chaperone, has provoked great interest as a promising molecular target for cancer treatment, since it is involved in regulating the conformation, stability and functions of key oncogenic proteins. At present, a variety of chemical compounds targeting HSP90 have been developed and have shown convincing anti-neoplastic activity in various preclinical tumor models. Several inhibitors of HSP90 have been reported exhibiting more antitumor efficacy in PTEN-null tumor xenograft. The aim of our study was to evaluate the antitumor effects of a novel HSP90 inhibitor, NVP-AUY922, in human malignant cells, especially in PTEN-null cancer cells. Materials and Methods: Human esophageal cancer (TE-1, TE-4, TE-8, TE-10), gastric cancer (NUGC-3, NUGC-3/IL), pancreatic cancer (PANC-1, MIAPACA-2, Bxpc3) and mutant c-kit-driven lymphocytes (Ba/F3-KE11, Ba/F3-K642, Ba/F3-820Mz), mimicking gastrointestinal stromal tumor cells, were examined. WST assay and/or trypan blue exclusion assay were conducted to assess the anti-proliferation effects of NVP-AUY922 in those cells mentioned above. The activity of cell growth-related molecules such as AKT, ERK, and PTEN were investigated by western blot analysis. Genetic modification using siRNA and expression vector were applied for silencing and overexpression of PTEN, respectively, in cancer cells. Results: Cell viability assays showed that NVP-AUY922 potently inhibits the proliferation of human cancer cell lines, regardless of 5-FU resistance or c-Kit mutation, by which the sensitivity to imatinib can be affected. PTEN-null TE-4 cells exhibited more sensitivity to NVP-AUY922. Interestingly, The sensitivity to NVP-AUY922 was increased by silencing PTEN expression in intact PTEN-expressing TE-10 cells and decreased by exogenous transduction of PTEN in TE-4 cells. Furthermore, NVP-AUY922 significantly inhibited the activity of AKT and ERK in TE-4 cells, but not in TE-10 cells. Conclusion: Inhibition of Hsp90 function by NVP-AUY922 exhibited strong cytotoxicity against most of the cancer cells, including some anticancer drug-resistant cells, revealing its potential for a novel therapeutic alternative to cancer treatment. In addition, this study suggests that the expression status of PTEN determines the sensitivity to HSP90 inhibitor for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2771. doi:1538-7445.AM2012-2771

Fibroblast Growth Factor Receptor 3 (FGFR3) Is a Strong Heat Shock Protein 90 (Hsp90) Client: IMPLICATIONS FOR THERAPEUTIC MANIPULATION

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.

Activation of heat shock factor 1 plays a role in pyrrolidine dithiocarbamate-mediated expression of the co-chaperone BAG3

Adaptive responses to physical and inflammatory stressors are mediated by transcription factors and molecular chaperones. The transcription factor heat shock factor 1 (HSF1) has been implicated in extending lifespan in part by increasing expression of heat shock response genes. Pyrrolidine dithiocarbamate (PDTC) is a small thiol compound that exerts in vivo and in vitro anti-inflammatory properties through mechanisms that remain unclear. Here we report that PDTC induced the release of monomeric HSF1 from the molecular chaperone heat shock protein 90 (Hsp90), with concomitant increase in HSF1 trimer formation, translocation to the nucleus, and binding to promoter of target genes in human HepG2 cells. siRNA-mediated silencing of HSF1 blocked BAG3 gene expression by PDTC. The protein levels of the co-chaperone BAG3 and its interaction partner Hsp72 were stimulated by PDTC in a dose-dependent fashion, peaking at 6 h. Inhibition of Hsp90 function by geldanamycin derivatives and novobiocin elicited a pattern of HSF1 activation and BAG3 expression that was similar to PDTC. Chromatin immunoprecipitation studies showed that PDTC and the inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin enhanced the binding of HSF1 to the promoter of several target genes, including BAG3, HSPA1A, HSPA1B, FKBP4, STIP1 and UBB. Cell treatment with PDTC increased significantly the level of Hsp90α thiol oxidation, a posttranslational modification known to inhibit its chaperone function. These results unravel a previously unrecognized mechanism by which PDTC and related compounds could confer cellular protection against inflammation through HSF1-induced expression of heat shock response genes.

Abstract 3793: Celastrol inhibits Hsp90 and triggers ER stress-induced cell death in non-small cell lung cancer cells

Abstract Celastrol, a plant-derived triterpenoid, is a known inhibitor of Hsp90 and NF-kB activation pathways and has recently been suggested to be of therapeutic importance in various cancers. However, the molecular mechanisms of celastrol-mediated effects in lung cancer are not known. Inhibition of Hsp90, commonly linked with the suppression of multiple cell-survival pathways, is also associated with induction of maladaptive endoplasmic reticulum (ER)-stress response. Resistance to ER-stress response is essential for the growth and survival of cancer cells. In response to cellular stress from cytotoxic agents an initial protective ER-stress response is initiated within the cell with the goal of rescuing the cell; however, sustained ER stress overwhelms the cellular-protective mechanisms, triggering cell death through specific programmed cell death pathways. The role of ER-stress outcomes in mediating celastrol-induced cell death is not known. In this study, we have examined the response of two NSCLC cell lines to celastrol and explore the molecular mechanisms to determine the proteins governing the cellular responses. We observed that celastrol inhibited the proliferation of A549 and H1299 non-small lung carcinoma cells in a time- and dose-dependent manner as indexed by MTT assay. Evaluation of downstream targets of celastrol-induced effects in H1299 cells by immunoblotting revealed an increase in Hsp70 and Hsp90 protein expression indicating cellular stress, a hallmark response of inhibiton of Hsp90 function. Disruption of Hsp90 function has also been shown to inhibit TNFα-mediated NF-kB activation as was also observed in our studies where a 4 h celastrol pre-treatment of H1299 cells completely abrogated TNFα-induced NF-kB activation. Celastrol treatment also up regulated the expression of ER-stress chaperones Grp78, and pPERK. These changes in ER-stress mediators were paralleled by an increase in apoptotic response as evidenced by increased annexin-V/PI staining evaluated by FACS and immunoblotting which showed a time- and dose-dependent upregulation of the ER stress specific pro-apoptotic transcription factor, GADD153/CHOP, alteration of Bax/Bcl2 ratio and enhanced cleavage of PARP (a downstream effector of caspase-3 activation). Interestingly, however, celastrol treatment also induced the activation of pro-survival Erk1/2 and Akt pathways’, suggesting that celastrol concomitantly activates pro-survival and pro-apoptotic mechanisms, with the pro-apoptotic pathways taking precedence resulting in cell death. Together, our data demonstrate the chemotherapeutic potential of celastrol in lung cancer via the inhibition of Hsp90 function and induction of chronic ER-stress-mediated cell death. (Supported from KLCRP grant and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3793.

Targeting Heat Shock Protein 90 Overrides the Resistance of Lung Cancer Cells by Blocking Radiation-Induced Stabilization of Hypoxia-Inducible Factor-1α

Hypoxia-inducible factor-1 (HIF-1) has been suggested to play a major role in tumor radioresistance. However, the mechanisms through which irradiation regulates HIF-1alpha expression remain unclear. The purpose of this study was to investigate the mechanisms that mediate HIF-1 activation and thus radioresistance. Here, we show that irradiation induces survival and angiogenic activity in a subset of radioresistant lung cancer cell lines by elevating HIF-1alpha protein expression. Radiation induced HIF-1alpha protein expression mainly through two distinct pathways, including an increase in de novo protein synthesis via activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and stabilization of HIF-1alpha protein via augmenting the interaction between heat shock protein 90 (Hsp90) and HIF-1alpha protein. Whereas the PI3K/Akt/mTOR pathway was activated by irradiation in all the lung cancer cells examined, the Hsp90-HIF-1alpha interaction was enhanced in the resistant cells only. Inhibition of Hsp90 function by 17-allylamino-17-demethoxygeldanamycin or deguelin, a novel natural inhibitor of Hsp90, suppressed increases in HIF-1alpha/Hsp90 interaction and HIF-1alpha expression in radioresistant cells. Furthermore, combined treatment of radiation with deguelin significantly decreased the survival and angiogenic potential of radioresistant lung cancer cells in vitro. We finally determined in vivo that systemic administration of deguelin resulted in profound inhibition of tumor growth and angiogenesis when combined with radiation. These results provide a strong rationale to target Hsp90 as a means to block radiation-induced HIF-1alpha and thus to circumvent radioresistance in lung cancer cells.

Co-Treatment with JAK2 Inhibitor TG101209 and Panobinostat or hsp90 Inhibitor AUY922 Attenuates Mutant JAK2-V617F Levels and Activity in Human Myeloproliferative Disorder Cells

The mutant JAK2-V617F is present and constitutively active in myeloproferative disorders (MPD), including polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis (MF). JAK2-V617F activates the signal transduction through the STAT, RAS/MAPK and PI3K/AKT pathways, conferring proliferative and survival advantage in the MPD hematopoietic progenitor cells (HPCs). We have previously reported that the panhistone deacetylase (HDAC) inhibitors e.g., panobinostat (Novartis Pharmaceuticals Inc), induces growth arrest and apoptosis of neoplastic HPCs. Panobinostat (PS), by inhibiting HDAC6, induces hyperacetylation of heat shock protein (hsp) 90, which attenuates the ATP binding and chaperone function of hsp90. This is associated with polyubiquitylation and degradation of hsp90 client protein signaling kinases, e.g, FLT-3, Bcr-Abl, AKT and c-RAF. Additionally, the mutant versions of signaling oncoprotein kinases, e.g., FLT-3, Bcr-Abl, B-RAF and c-KIT, may be even more dependent on hsp90 chaperone function for maintaining their proper folding and active conformation. This makes the mutant oncoprotein kinase even more susceptible to misfolding and proteasomal degradation upon inhibition of hsp90 function. In the present studies we determined the effects of PS or the non-geldanamycin analogue hsp90 inhibitor (AUY922, Novartis) and/or the JAK-2 inhibitor TG101209 (TG) (TargeGen, San Diego, CA) in the cultured (human erythroleukemia HEL and mouse BaF3 cells expressing JAK2-V617F) and primary human MPD cells (from two patients with PV and MF. Treatment with PS (10 to 30 nM) dose-dependently increased % of cells in G1 and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of HEL and BaF3/JAK2-V617F cells. This was associated with attenuation in the levels of JAK2-V617F, p-JAK2, p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310), GATA1 and Bcl-xL in HEL cells. PS induced lysine acetylation of hsp90 but not of JAK2. Similar effects were observed, following treatment with AUY922 (AUY). Both PS and AUY induced significantly less apoptosis of BaF3/hEpoR versus BaF3/JAK2-V617F cells. PS and AUY also reduced the chaperone association of JAK2-V617F with hsp90. Treatment with TG (0.2 to 2.0 uM) also dose-dependently attenuated the levels of p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310) and Bcl-xL, which was associated with apoptosis of HEL and BaF3/JAK2-V617F cells. Co-treatment with PS and TG induced more apoptosis of the cultured and primary MPD cells than treatment with either agent alone. AUY also increased apoptosis of HEL cells induced by TG. Co-treatment with PS and AUY synergistically induced apoptosis of HEL cells (combination indices < 1.0). Co-treatment with 20 nM of PS or 15 nM of AUY augmented TG mediated attenuation of p-STAT3, p-STAT5 and Bcl-xL levels in HEL and BaF3/JAK2-V617F cells. These findings demonstrate for the first time that pan-HDAC inhibitor PS and hsp90 inhibitor AUY attenuate the levels and signaling of JAK2-V617F and enhance the anti-JAK2-V617F activity of TG in human MPD cells. Our findings also support the rationale to determine the in vivo activity of PS or AUY in combination with JAK2 inhibitors against human MPD.

Recent Advances in Hsp90 Inhibitors as Antitumor Agents

One promising therapeutic strategy for treating cancer is to specifically target signal transduction pathways that have a key role in oncogenic transformation and malignant progression. Hsp90 is an emerging therapeutic target of interest for the treatment of cancer. It is responsible for modulating cellular response to stress by maintaining the function of numerous signalling proteins - known as 'client proteins' - that are associated with cancer cell survival and proliferation. Many cancers result from specific mutations in, or aberrant expression of, these client proteins. Small molecule Hsp90 inhibitors bind to the ATP binding pocket, inhibit chaperone function and could potentially result in cytostasis or cell death. Consequently, many client proteins are targeted for degradation via the ubiquitin-proteasome pathway including receptor and non receptor kinases (Erb-B2, epidermal growth factor receptor, and Src family kinases), serine/threonine kinases (c-Raf-1 and Cdk4), steroid hormone receptors (androgen and estrogen), and apoptosis regulators such as mutant p53. Inhibition of Hsp90 function has also proven effective in killing cancer cells that have developed resistance to targeted therapies such as kinase inhibitors. This review is intended to update recent developments in new Hsp90 inhibitors as antitumors agents, the design, biological evaluation and their clinical trials studies.

P.2.e.010 Efficacy of amisulpride in bipolar mania

Adaptive responses to physical and inflammatory stressors are mediated by transcription factors and molecular chaperones. The transcription factor heat shock factor 1 (HSF1) has been implicated in extending lifespan in part by increasing expression of heat shock response genes. Pyrrolidine dithiocarbamate (PDTC) is a small thiol compound that exerts in vivo and in vitro anti-inflammatory properties through mechanisms that remain unclear. Here we report that PDTC induced the release of monomeric HSF1 from the molecular chaperone heat shock protein 90 (Hsp90), with concomitant increase in HSF1 trimer formation, translocation to the nucleus, and binding to promoter of target genes in human HepG2 cells. siRNA-mediated silencing of HSF1 blocked BAG3 gene expression by PDTC. The protein levels of the co-chaperone BAG3 and its interaction partner Hsp72 were stimulated by PDTC in a dose-dependent fashion, peaking at 6 h. Inhibition of Hsp90 function by geldanamycin derivatives and novobiocin elicited a pattern of HSF1 activation and BAG3 expression that was similar to PDTC. Chromatin immunoprecipitation studies showed that PDTC and the inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin enhanced the binding of HSF1 to the promoter of several target genes, including BAG3, HSPA1A, HSPA1B, FKBP4, STIP1 and UBB. Cell treatment with PDTC increased significantly the level of Hsp90α thiol oxidation, a posttranslational modification known to inhibit its chaperone function. These results unravel a previously unrecognized mechanism by which PDTC and related compounds could confer cellular protection against inflammation through HSF1-induced expression of heat shock response genes.

Immune expression and inhibition of HSP90 in uveal melanoma

Abstract Purpose: To examine the immunohistochemical profile of Hsp90 in uveal melanoma and the effects of Hsp90 inhibitor 17‐AAG, in uveal melanoma cell lines. Methods: Hsp90 expression was studied in 44 sections of human uveal melanoma and in five uveal melanoma cell lines (92.1, OCM‐1, MKTBR, SP6.5 and UW‐1). Sulfurhodamine‐B based proliferation assay was assessed with a range of concentrations of 17‐AAG. Changes in cell migration and invasion were evaluated in vitro in the presence or absence of 17‐AAG. Cell cycle fractions were determined by flow cytometry and the caspase 3 protease activity was detected using the BD ApoAlertTM Caspase Colorimetric Assay. Expression of intracellular proteins was determined by Western blot analysis. Results: Immunohistochemical expression of Hsp90 was identified in 68 % of the paraffin embedded sections and was significantly associated with largest tumor dimension. A statistically significant reduction on proliferation rate of uveal melanoma cell lines was observed with drug concentrations of 100 μM to 1 μM and also a reduction of their migratory and invasive capabilities. Exposure to 17‐AAG induced accumulations of cells in G1 and loss of cells in S phase and also a significant increase in Caspase 3 protease activity. The cytotoxic effect of 17‐AAG was associated with decreased levels of phospho‐AKT after 24 h exposure to the drug. Conclusions: The immunohistochemical expression of Hsp90 in uveal melanoma indicates worse prognosis. Inhibition of HSP90 function by 17‐AAG had an effect on cell motility and invasive potential, inducing cell cycle arrest and promoting apoptosis. Further trials in uveal melanoma models should be undertaken to study the effect of 17 AAG to target Hsp90 in vivo.

Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G2/M arrest in glioblastoma cell lines

Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G(2)/M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G(2)/M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50(cdc37), an Hsp90 co-chaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.

Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels

Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. Here, we have investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. We next show that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, we show that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells.

Thrombopoietin Enhances Expression of Vascular Endothelial Growth Factor (VEGF) in Primitive Hematopoietic Cells through Hif-1α.

The development of hematopoietic stem cells (HSCs) is orchestrated by numerous hematopoietic cytokines, growth factors and chemokines. For the most part these proteins are secreted from bone marrow microenviromental cells, including fibroblasts, endothelial cells and stromal cells, or are displayed on their cell surfaces. HSCs have also been shown to produce numerous cytokines and growth factors. Interestingly, secreted cytokines have also been reported to induce the production of additional cytokines from neighboring cells, suggesting that cytokines drive networks of other cytokines to support hematopoietic cell development. Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis and vasculogenesis, also plays an important role in HSC development, where it acts in an intracellular autocrine fashion to promote cell survival. The secretion of VEGF from endothelial or smooth muscle cells is regulated by extracellular stimuli, inflammatory cytokines and hypoxia. In contrast, it is not clear whether synthesis of VEGF in HSCs is regulated by extracellular signals. Because several early acting cytokines, including TPO, affect VEGF-A expression in hematopoietic cell lines, we hypothesized that TPO could be a regulator of a HSC VEGF autocrine loop. We found that TPO induces VEGF transcripts in primitive marrow derived sca-1+/c-kit+/Gr-1− hematopoietic cells, and that VEGF transcripts are reduced in these cells when derived from Tpo−/− mice. Additional studies determined that TPO induces VEGF expression by increasing the nuclear levels of its primary transcription factor, both in the TPO-dependent primitive hematopoietic cell line UT-7/TPO and in purified sca-1+/c-kit+/Gr-1−marrow cells. Elevation of HIF-1α by TPO is achieved by two different mechanisms; augmented protein synthesis and enhanced stabilization. The latter mechanism is dependent on heat shock protein 90 (Hsp90), as inhibition of Hsp90 function by the specific inhibitor geldanamycin inhibited the TPO-dependent stabilization of HIF-1α. In additional studies we also established that VEGF expression was important for the favorable TPO effect on primitive hematopoietic cells, as blockade of the VEGF receptor with a specific inhibitor, SU5416 (0.1mM), substantially blunted TPO induced growth of sorted single sca-1+/c-kit+/Gr-1−marrow cells in serum-free cultures. Importantly, this inhibitor does not affect TPO induced phosphorylation of Jak2, ERK and AKT in UT-7/TPO cells even at a 100-fold higher concentration. Along with our previous findings that TPO affects Hox transcription factors that regulate HSC proliferation, these data contribute to our growing understanding of the mechanisms by which a hormone can influence HSC development.

Inhibition of Heat Shock Protein 90, a Novel RET/PTC1-associated Protein, Increases Radioiodide Accumulation in Thyroid Cells

RET/PTC1 is a rearranged form of the RET tyrosine kinase commonly seen in papillary thyroid carcinomas. It has been shown that RET/PTC1 decreases expression of the sodium/iodide symporter (NIS), the molecule that mediates radioiodide therapy for thyroid cancer. Using proteomic analysis, we identify hsp90 and its co-chaperone p50cdc37 as novel proteins associated with RET/PTC1. Inhibition of hsp90 function with 17-allylamino-17-demothoxygeldanamycin (17-AAG) reduces RET/PTC1 protein levels. Furthermore, 17-AAG increases radioiodide accumulation in thyroid cells, mediated in part through a protein kinase A-independent mechanism. We show that 17-AAG does not increase the total amount of NIS protein or cell surface NIS localization. Instead, 17-AAG increases radioiodide accumulation by decreasing iodide efflux. Finally, the ability of 17-AAG to increase radioiodide accumulation is not restricted to thyroid cells expressing RET/PTC1. These findings suggest that 17-AAG may be useful as a chemotherapeutic agent, not only to inhibit proliferation but also to increase the efficacy of radioiodide therapy in patients with thyroid cancer.

Hsp90 as a therapeutic target in prostate cancer

Prostate cancers are hormone-dependent malignancies that respond to drugs that reduce circulating testosterone levels or prevent binding of this ligand to the androgen receptor (AR). While effective, these approaches are not curative and, in almost all cases, progression to a castration-resistant state is eventually observed. The mechanisms underlying the development of hormone resistance are poorly defined but several molecular changes are commonly associated with this process. Since a common element of these resistance mechanisms is restoration of AR signaling, agents that target AR expression represent an attractive treatment option for prostate cancer patients with disease progression following castration. Prior to ligand binding, AR exists in a complex with heat shock protein 90 (Hsp90) and other co-chaperones. The AR-Hsp90 interaction maintains AR in a high-affinity ligand-binding conformation, which is necessary for efficient response to hormone. 17-Allyamino-17-demethoxygeldanamycin (17-AAG) is an inhibitor of the Hsp90 chaperone protein. Inhibition of Hsp90 function causes the proteasomal degradation of proteins that require this chaperone for maturation or stability. Hsp90 clients include several proteins of potential importance in mediating prostate cancer progression, including wild-type and mutated AR, HER2, and Akt. In murine models of prostate cancer, 17-AAG causes the degradation of these client proteins at nontoxic doses and inhibits the growth of hormone-naive and castration-resistant tumors. These data suggest that inhibitors of Hsp90 may represent a novel strategy for the treatment of patients with prostate cancer and clinical trials to test this hypothesis are currently ongoing.

437 A multicenter phase II trial to evaluate gefitinib (‘Iressa’, ZD1839) (500 mg/day) in patients with metastatic breast cancer after previous chemotherapy treatment

Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells.