Post-translational modifications of Hsp90 and their contributions to chaperone regulation

AbstractMolecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport, and degradation of proteins as well as in facilitating cell signaling. Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the varied roles of known post-translational modifications of cytosolic and nuclear Hsp90 (phosphorylation, acetylation, S-nitrosylation, oxidation and ubiquitination) in fine-tuning chaperone function in eukaryotes. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90)

Ras, ROS and Proteotoxic Stress: A Delicate Balance

Ras-deregulated cells require reactive oxygen species for proliferation. They survive the resultant proteotoxic stress by maintaining sufficient levels of reduced glutathione and optimally functioning stress response machinery. In this issue of Cancer Cell, De Raedt et al. identify a novel strategy that utilizes this dependency to cause cell death

ErbB2 and NFκB Overexpression as Predictors of Chemoradiation Resistance and Putative Targets to Overcome Resistance in Muscle-Invasive Bladder Cancer

Radical cystectomy for muscle-invasive bladder cancer (MIBC) patients frequently impairs their quality of life (QOL) due to urinary diversion. To improve their QOL, a bladder-sparing alternative strategy using chemoradiation has been developed. In bladder-sparing protocols, complete response (CR) to induction chemoradiation is a prerequisite for bladder preservation and favorable survival. Thus predicting chemoradiation resistance and overcoming it would increase individual MIBC patients' chances of bladder preservation. The aim of this study is to investigate putative molecular targets for treatment aimed at improving chemoradiation response. Expression levels of erbB2, NFκB, p53, and survivin were evaluated immunohistochemically in pretreatment biopsy samples from 35 MIBC patients in whom chemoradiation sensitivity had been pathologically evaluated in cystectomy specimens, and associations of these expression levels with chemoradiation sensitivity and cancer-specific survival (CSS) were investigated. Of the 35 patients, 11 (31%) achieved pathological CR, while tumors in the remaining 24 patients (69%) were chemoradiation-resistant. Multivariate analysis identified erbB2 and NFκB overexpression and hydronephrosis as significant and independent risk factors for chemoradiation resistance with respective relative risks of 11.8 (P = 0.014), 15.4 (P = 0.024) and 14.3 (P = 0.038). The chemoradiation resistance rate was 88.5% for tumors overexpressing erbB2 and/or NFκB, but only 11.1% for those negative for both (P <0.0001). The 5-year CSS rate was 74% overall. Through multivariate analysis, overexpression of erbB2 and/or NFκB was identified as an independent risk factor for bladder cancer death with marginal significance (hazard ratio 21.5, P = 0.056) along with chemoradiation resistance (P = 0.003) and hydronephrosis (P = 0.018). The 5-year CSS rate for the 11 patients achieving pathological CR was 100%, while that for the 24 with chemoradiation-resistant disease was 61% (P = 0.018). Thus, erbB2 and NFκB overexpression are relevant to chemoradiation resistance and are putative targets aimed at overcoming chemoradiation resistance in MIBC

HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts

Niemann Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study we evaluated the effect of several HSP90 inhibitors on NPC

Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 æ from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, cochaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary

Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis

Little is known about the molecular mechanisms that control adrenomedullin (AM) production in human cancers. We demonstrate here that the expression of AM mRNA in a variety of human tumor cell lines is highly induced in a time-dependent manner by reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2. This AM expression seems to be under hypoxia-inducible factor-1 (HIF-1) transcriptional regulation, since HIF-1alpha and HIF-1beta knockout mouse cell lines had an ablated or greatly reduced hypoxia AM mRNA induction. Similarly, inhibition or enhancement of HIF-1 activity in human tumor cells showed an analogous modulation of AM mRNA. Under hypoxic conditions, immunohistochemical analysis of tumor cell lines revealed elevated levels of AM and HIF-1alpha as compared with normoxia, and we also found an increase of immunoreactive AM in the conditioned medium of tumor cells analyzed by RIA. AM mRNA stabilization was shown to be partially responsible for the hypoxic up-regulated expression of AM. In addition, we have identified several putative hypoxia response elements (HREs) in the human AM gene, and reporter studies with selected HREs were capable of enhancing luciferase expression after exposure to DFX. Furthermore, transient coexpression of HIF-1alpha resulted in an augmented transactivation of the reporter gene after DFX treatment. Given that most solid human tumors have focal hypoxic areas and that AM functions as a mitogen, angiogenic factor, and apoptosis-survival factor, our findings implicate the HIF-1/AM link as a possible promotion mechanism of carcinogenesis

Asymmetric Hsp90 N domain SUMOylation recruits Aha1 and ATP-competitive inhibitors

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90's function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs. © 2014 Elsevier Inc

HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation

Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients