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The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.
Prostate cancer remains the second highest contributor to male cancer-related lethality. The transition of a subset of tumors from indolent to invasive disease is associated with a poor clinical outcome. Activation of the epithelial to mesenchymal transition (EMT) genetic program is a major risk factor for cancer progression. We recently reported that secreted extracellular Hsp90 (eHsp90) initiates EMT in prostate cancer cells, coincident with its enhanced expression in mesenchymal models. Our current work substantially extended these findings in defining a pathway linking eHsp90 signaling to EZH2 function, a methyltransferase of the Polycomb repressor complex. EZH2 is also implicated in EMT activation, and its up-regulation represents one of the most frequent epigenetic alterations during prostate cancer progression. We have now highlighted a novel epigenetic function for eHsp90 via its modulation of EZH2 expression and activity. Mechanistically, eHsp90 initiated sustained activation of MEK/ERK, a signal critical for facilitating EZH2 transcriptional up-regulation and recruitment to the E-cadherin promoter. We further demonstrated that an eHsp90-EZH2 pathway orchestrates an expanded repertoire of EMT-related events including Snail and Twist expression, tumor cell motility, and anoikis resistance. To evaluate the role of eHsp90 in vivo, eHsp90 secretion was stably enforced in a prostate cancer cell line resembling indolent disease. Remarkably, eHsp90 was sufficient to induce tumor growth, suppress E-cadherin, and initiate localized invasion, events that are exquisitely dependent upon EZH2 function. In summary, our findings illuminate a hitherto unknown epigenetic function for eHsp90 and support a model wherein tumor eHsp90 functions as a rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
There is an unmet need to develop new, more effective and safe therapies for the aggressive forms of triple negative breast cancers (TNBCs). While up to 20% of women under 50 years of age with TNBC harbor germline mutations in BRCA1, and these tumors are sensitive to treatment with poly(ADP) ribose polymerase inhibitors, a majority of TNBCs lack BRCA1 mutations or loss of expression. Findings presented here demonstrate that by attenuating the levels of DNA damage response and homologous recombination proteins, pan-histone deacetylase inhibitor (HDI) treatment induces 'BRCAness' and sensitizes TNBC cells lacking BRCA1 to lethal effects of PARP inhibitor or cisplatin. Treatment with HDI also induced hyperacetylation of nuclear hsp90. Similar effects were observed following shRNA-mediated depletion of HDAC3, confirming its role as the deacetylase for nuclear HSP90. Furthermore, cotreatment with HDI and ABT-888 induced significantly more DNA strand breaks than either agent alone, and synergistically induced apoptosis of TNBC cells. Notably, co-treatment with HDI and ABT-888 significantly reduced in vivo tumor growth and markedly improved the survival of mice bearing TNBC cell xenografts. These findings support the rationale to interrogate the clinical activity of this novel combination against human TNBC, irrespective of its expression of mutant BRCA1.
Hsp90 is widely overexpressed in cancer cells and believed to be essential for the maintenance of malignant phenotypes. Targeting Hsp90 by small molecules has shown promise in solid and hematologic malignancies, which likely involves degradation of client oncoproteins in a cell-type-specific manner. In this study, we found that structurally unrelated Hsp90 inhibitors induce DNA damage and apoptosis via p53-dependent induction of PUMA, which indirectly triggers Bax activation and mitochondrial dysfunction in colon cancer cells. Deficiency in PUMA, BAX, or p53, at lesser extent, abrogated 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced apoptosis and mitochondrial dysfunction, and enhanced clonogenic cell survival. Furthermore, suppression of p53-dependent p21 induction or enhanced p53 activation synergized with 17-AAG to induce PUMA-dependent apoptosis. Finally, PUMA was found to mediate apoptotic and therapeutic responses to the 17-AAG analog 17-DMAG in xenografts. These results show an important role of the p53/PUMA/Bax axis in Hsp90 inhibitor-induced killing of p53 wild-type cells, and have important implications for their clinical applications.
UNLABELLED - EML4-ALK gene rearrangements define a unique subset of patients with non-small cell lung carcinoma (NSCLC), and the clinical success of the anaplastic lymphoma kinase (ALK) inhibitor crizotinib in this population has become a paradigm for molecularly targeted therapy. Here, we show that the Hsp90 inhibitor ganetespib induced loss of EML4-ALK expression and depletion of multiple oncogenic signaling proteins in ALK-driven NSCLC cells, leading to greater in vitro potency, superior antitumor efficacy, and prolonged animal survival compared with results obtained with crizotinib. In addition, combinatorial benefit was seen when ganetespib was used with other targeted ALK agents both in vitro and in vivo. Importantly, ganetespib overcame multiple forms of crizotinib resistance, including secondary ALK mutations, consistent with activity seen in a patient with crizotinib-resistant NSCLC. Cancer cells driven by ALK amplification and oncogenic rearrangements of ROS1 and RET kinase genes were also sensitive to ganetespib exposure. Taken together, these results highlight the therapeutic potential of ganetespib for ALK-driven NSCLC.
SIGNIFICANCE - In addition to direct kinase inhibition, pharmacologic blockade of the molecular chaperone Hsp90 is emerging as a promising approach for treating tumors driven by oncogenic rearrangements of ALK. The bioactivity profi le of ganetespib presented here underscores a new therapeutic opportunity to target ALK and overcome multiple mechanisms of resistance in patients with ALK-positive NSCLC.
Proteomic analyses of protein-electrophile adducts generally employ affinity capture of the adduct moiety, which enables global analyses, but is poorly suited to targeted studies of specific proteins. We describe a targeted molecular probe approach to study modifications of the molecular chaperone heat-shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl analog of the HSP90 inhibitor geldanamycin enables detection of the native protein isoforms Hsp90α and Hsp90β and their phosphorylated forms. We applied this probe to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. This approach was also applied to measure the kinetics of site-specific adduction of selected Hsp90 residues. A protein-selective affinity capture approach is broadly applicable for targeted analysis of electrophile adducts and their biological effects.
PURPOSE - ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.
EXPERIMENTAL DESIGN - By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.
RESULTS - Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.
CONCLUSIONS - Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.
Thoracic oncologists traditionally have made treatment decisions based upon tumor histology, distinguishing non-small cell lung cancer (NSCLC) from small cell lung cancer (SCLC). However, recent data has revealed that at least one histological subtype of NSCLC, lung adenocarcinoma comprises multiple molecularly distinct diseases. Lung adenocarcinoma subsets now can be defined by specific 'driver' mutations in genes encoding components of the EGFR signaling pathway. Importantly, these mutations have implications regarding targeted therapy. Here, we focus on EGFR mutant NSCLC-a prime example of a clinically relevant molecular subset of lung cancer, with defined mechanisms of drug sensitivity, primary drug resistance, and acquired resistance to EGFR tyrosine kinase inhibitors. Efforts are now being made to overcome mechanisms of acquired resistance. These findings illustrate how knowledge about the genetic drivers of tumors can lead to rational targeted therapy for individual patients.
Chronic stress is increasingly considered to be a main risk factor for the development of a variety of psychiatric diseases such as depression. This is further supported by an impaired negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis, which has been observed in the majority of depressed patients. The effects of glucocorticoids, the main hormonal endpoint of the HPA axis, are mediated via the glucocorticoid receptor (GR) and the mineralocorticoid receptor. The FK506-binding protein 51 (FKBP5), a co-chaperone of the Hsp90 and component of the chaperone-receptor heterocomplex, has been shown to reduce ligand sensitivity of the GR. This study aimed to investigate the function of FKBP5 as a possible mediator of the stress response system and its potential role in the development of stress-related diseases. Therefore, we assessed whether mice lacking the gene encoding FKBP5 (51KO mice) were less vulnerable to the adverse effects of three weeks of chronic social defeat stress. Mice were subsequently analyzed with regards to physiological, neuroendocrine, behavioral and mRNA expression alterations. Our results show a less vulnerable phenotype of 51KO mice with respect to physiological and neuroendocrine parameters compared to wild-type animals. 51KO mice demonstrated lower adrenal weights and basal corticosterone levels, a diminished response to a novel acute stimulus and an enhanced recovery, as well as more active stress-coping behavior. These results suggest an enhanced negative glucocorticoid feedback within the HPA axis of 51KO mice, possibly modulated by an increased sensitivity of the GR. This article is part of a Special Issue entitled 'Anxiety and Depression'.
Copyright © 2011 Elsevier Ltd. All rights reserved.
The analysis of protein modification by electrophiles is a challenging problem. Most reported protein-electrophile adducts have been characterized from in vitro reactions or through affinity capture of the adduct moiety, which enables global analyses but is poorly suited to targeted studies of specific proteins. We employed a targeted molecular probe approach to study modifications of the molecular chaperone heat shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl-geldanamycin probe isolated both isoforms of the native protein (Hsp90α and Hsp90β) from human RKO colorectal cancer cells. Geldanamycin-biotin capture afforded higher purity Hsp90 than did immunoprecipitation and enabled detection of endogenously phosphorylated protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We applied this approach to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. LC-MS/MS analyses of tryptic digests by identified His(450) and His(490) of Hsp90α as having a 158 Da modification, corresponding to NaBH(4)-reduced HNE adducts. Five histidine residues were also adducted on Hsp90β: His(171), His(442), His(458), His(625), and His(632). The rates of adduction at these sites were determined with Hsp90 protein in vitro and with Hsp90 in HNE-treated cells with a LC-MS/MS-based, label-free relative quantitation method. During in vitro and cell treatment with HNE, residues on Hsp90α and Hsp90β displayed adduction rates ranging from 3.0 × 10(-5) h(-1) to 1.08 ± 0.17 h(-1). Within the middle client-binding domain of Hsp90α, residue His(450) demonstrated the most rapid adduction with k(obs) of 1.08 ± 0.17 h(-1) in HNE-treated cells. The homologous residue on Hsp90β, His(442), was adducted more rapidly than the N-terminal residue, His(171), despite very similar predicted pK(a) values of both residues. The Hsp90 middle client-binding domain thus may play a signicant role in HNE-mediated disruption of Hsp90-client protein interactions. The results illustrate the utility of a protein-selective affinity capture approach for targeted analysis of electrophile adducts and their biological effects.