Cdc37 as a Co-chaperone to Hsp90.

The co-chaperone p50/Cdc37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Analysis of the structure of Hsp90-Cdc37-kinase complexes demonstrates the way in which Cdc37 interacts with and controls the folding of a large proportion of intracellular protein kinases. This co-chaperone thus stands at the hub of a multitude of intracellular signaling networks. Indeed, the influence of Cdc37 reaches beyond the housekeeping pathways of protein folding into the regulation of a wide range of cellular processes. This co-chaperone has attracted attention as a potential intermediate in carcinogenesis. Cdc37 is an attractive potential target in cancer due to (1) high expression in a number of tumor types and (2) control of multiple signaling pathways. These properties indicate (3) a potential for selectivity due to its elevated expression in malignant cells and (4) robustness, as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than less versatile oncoproteins. Cdc37 may also be involved in other aspects of pathophysiology and has been shown to be secreted in exosomes. Protein aggregation disorders have been linked to age-related declines in molecular chaperones and co-chaperones. Cdc37 also appears to be a potential agent in longevity due to its links to protein folding and autophagy, and it will be informative to study the role of Cdc37 maintenance/decline in aging organisms.

Extracellular Hsp90 and protection of neuronal cells through Nrf2.

Heat shock protein 90 (Hsp90), although one of the most essential intracellular chaperones, can also play key roles in the extracellular milieu. Here, we review the properties of extracellular Hsp90 in cellular homeostasis in the heat shock response (HSR), focusing on cells of the central nervous system. Hsp90 can be secreted by microglia as well as other cell types by non-canonical pathways of secretion. The chaperone may then influence the behavior of distant cells and can for instance protect neuronal cells from the oxidative burst accompanying phagocytosis by microglia of beta-amyloid fibrils. A mechanism involving activation of the transcription factor Nrf2, and induction of the antioxidant response is reported. We review the potential role of extracellular Hsp90, Nrf2 and transcellular chaperone signaling in the non-cell-intrinsic HSR.

The functions and regulation of heat shock proteins; key orchestrators of proteostasis and the heat shock response.

Cells respond to protein-damaging (proteotoxic) stress by activation of the Heat Shock Response (HSR). The HSR provides cells with an enhanced ability to endure proteotoxic insults and plays a crucial role in determining subsequent cell death or survival. The HSR is, therefore, a critical factor that influences the toxicity of protein stress. While named for its vital role in the cellular response to heat stress, various components of the HSR system and the molecular chaperone network execute essential physiological functions as well as responses to other diverse toxic insults. The effector molecules of the HSR, the Heat Shock Factors (HSFs) and Heat Shock Proteins (HSPs), are also important regulatory targets in the progression of neurodegenerative diseases and cancers. Modulation of the HSR and/or its extended network have, therefore, become attractive treatment strategies for these diseases. Development of effective therapies will, however, require a detailed understanding of the HSR, important features of which continue to be uncovered and are yet to be completely understood. We review recently described and hallmark mechanistic principles of the HSR, the regulation and functions of HSPs, and contexts in which the HSR is activated and influences cell fate in response to various toxic conditions.

Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90ß.

Because of their importance in maintaining protein homeostasis, molecular chaperones, including heat-shock protein 90 (Hsp90), represent attractive drug targets. Although a number of Hsp90 inhibitors are in preclinical/clinical development, none strongly differentiate between constitutively expressed Hsp90ß and stress-induced Hsp90α, the two cytosolic paralogs of this molecular chaperone. Thus, the importance of inhibiting one or the other paralog in different disease states remains unknown. We show that the natural product, gambogic acid (GBA), binds selectively to a site in the middle domain of Hsp90ß, identifying GBA as an Hsp90ß-specific Hsp90 inhibitor. Furthermore, using computational and medicinal chemistry, we identified a GBA analog, referred to as DAP-19, which binds potently and selectively to Hsp90ß. Because of its unprecedented selectivity for Hsp90ß among all Hsp90 paralogs, GBA thus provides a new chemical tool to study the unique biological role of this abundantly expressed molecular chaperone in health and disease.

Heat Shock Proteins Are Essential Components in Transformation and Tumor Progression: Cancer Cell Intrinsic Pathways and Beyond.

Heat shock protein (HSP) synthesis is switched on in a remarkably wide range of tumor cells, in both experimental animal systems and in human cancer, in which these proteins accumulate in high levels. In each case, elevated HSP concentrations bode ill for the patient, and are associated with a poor outlook in terms of survival in most cancer types. The significance of elevated HSPs is underpinned by their essential roles in mediating tumor cell intrinsic traits such as unscheduled cell division, escape from programmed cell death and senescence, de novo angiogenesis, and increased invasion and metastasis. An increased HSP expression thus seems essential for tumorigenesis. Perhaps of equal significance is the pronounced interplay between cancer cells and the tumor milieu, with essential roles for intracellular HSPs in the properties of the stromal cells, and their roles in programming malignant cells and in the release of HSPs from cancer cells to influence the behavior of the adjacent tumor and infiltrating the normal cells. These findings of a triple role for elevated HSP expression in tumorigenesis strongly support the targeting of HSPs in cancer, especially given the role of such stress proteins in resistance to conventional therapies.

Overexpressed HSF1 cancer signature genes cluster in human chromosome 8q.

BACKGROUND: HSF1 (heat shock factor 1) is a transcription factor that is found to facilitate malignant cancer development and proliferation. In cancer cells, HSF1 mediates a set of genes distinct from heat shock that contributes to malignancy. This set of genes is known as the HSF1 Cancer Signature genes or simply HSF1-CanSig genes. HSF1-CanSig genes function and operate differently than typical cancer-causing genes, yet it is involved in fundamental oncogenic processes. RESULTS: By utilizing expression data from 9241 cancer patients, we identified that human chromosome 8q21-24 is a location hotspot for the most frequently overexpressed HSF1-CanSig genes. Intriguingly, the strength of the HSF1 cancer program correlates with the number of overexpressed HSF1-CanSig genes in 8q, illuminating the essential role of HSF1 in mediating gene expression in different cancers. Chromosome 8q21-24 is found under selective pressure in preserving gene order as it exhibits strong synteny among human, mouse, rat, and bovine, although the biological significance remains unknown. Statistical modeling, hierarchical clustering, and gene ontology-based pathway analyses indicate crosstalk between HSF1-mediated responses and pre-mRNA 3' processing in cancers. CONCLUSIONS: Our results confirm the unique role of chromosome 8q mediated by the master regulator HSF1 in cancer cases. Additionally, this study highlights the connection between cellular processes triggered by HSF1 and pre-mRNA 3' processing in cancers.

Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter.

The heat shock response (HSR) is a cellular mechanism for counteracting acute proteotoxic stress. In eukaryotes, transcriptional activation of the HSR is regulated by heat shock factor 1 (HSF1). Activation of HSF1 induces the expression of heat shock proteins (HSPs) that function as molecular chaperones to fold and maintain the three-dimensional structure of misfolded proteins. The regulation of the degree and duration of the HSR is controlled by multiple biochemical mechanisms that include posttranslational modification of HSF1 and numerous protein-protein interactions. In this chapter, we describe a method to evaluate the activation and deactivation of the HSR at the transcriptional level using a short half-life luciferase reporter assay. This assay can be used to further characterize the HSR or as a screen for small molecule inducers, amplifiers, or repressors.

Heat shock proteins in cell signaling and cancer.

Heat Shock Proteins (HSPs) and their co-chaperones have well-established roles in regulating proteostasis within the cell, the nature of which continues to emerge with further study. To date, HSPs have been shown to be integral to protein folding and re-folding, protein transport, avoidance of protein aggregation, and modulation of protein degradation. Many cell signaling events are mediated by the chemical modification of proteins post-translationally that can alter protein conformation and activity, although it is not yet known whether the changes in protein conformation induced by post-translational modifications (PTMs) are also dependent upon HSPs and their co-chaperones for subsequent protein re-folding. We discuss what is known regarding roles for HSPs and other molecular chaperones in cell signaling events with a focus on oncogenic signaling. We also propose a hypothesis by which Hsp70 and Hsp90 may co-operate to facilitate cell signaling events that may link PTMs with the cellular protein folding machinery.

SCAND1 Reverses Epithelial-to-Mesenchymal Transition (EMT) and Suppresses Prostate Cancer Growth and Migration.

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many carcinomas depend on EMT activation, partial EMT, or hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGFß signal and EMT-inducing transcription factors, such as ZEB1/2, in tumor cells. However, reverse EMT factors are less studied. We demonstrate that prostate epithelial transcription factor SCAND1 can reverse the cancer cell mesenchymal and hybrid E/M phenotypes to a more epithelial, less invasive status and inhibit their proliferation and migration in DU-145 prostate cancer cells. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and the transcriptional co-repression of target genes. We found that SCAND1 expression correlated with maintaining epithelial features, whereas the loss of SCAND1 was associated with mesenchymal phenotypes of tumor cells. SCAND1 and MZF1 were mutually inducible and coordinately included in chromatin with hetero-chromatin protein HP1γ. The overexpression of SCAND1 reversed hybrid E/M status into an epithelial phenotype with E-cadherin and ß-catenin relocation. Consistently, the co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression was negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBRs, in prostate adenocarcinoma specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, in a mouse tumor xenograft model, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer. Kaplan-Meier analysis showed high expression of SCAND1 and MZF1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that SCAND1 induces expression and coordinated heterochromatin-binding of MZF1 to reverse the hybrid E/M status into an epithelial phenotype and, inhibits tumor cell proliferation, migration, and metastasis, potentially by repressing the gene expression of EMT drivers and the MAP3K-MEK-ERK signaling pathway.

HSF1: Primary Factor in Molecular Chaperone Expression and a Major Contributor to Cancer Morbidity.

Heat shock factor 1 (HSF1) is the primary component for initiation of the powerful heat shock response (HSR) in eukaryotes. The HSR is an evolutionarily conserved mechanism for responding to proteotoxic stress and involves the rapid expression of heat shock protein (HSP) molecular chaperones that promote cell viability by facilitating proteostasis. HSF1 activity is amplified in many tumor contexts in a manner that resembles a chronic state of stress, characterized by high levels of HSP gene expression as well as HSF1-mediated non-HSP gene regulation. HSF1 and its gene targets are essential for tumorigenesis across several experimental tumor models, and facilitate metastatic and resistant properties within cancer cells. Recent studies have suggested the significant potential of HSF1 as a therapeutic target and have motivated research efforts to understand the mechanisms of HSF1 regulation and develop methods for pharmacological intervention. We review what is currently known regarding the contribution of HSF1 activity to cancer pathology, its regulation and expression across human cancers, and strategies to target HSF1 for cancer therapy.

MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer.

Cell division control 37 (CDC37) increases the stability of heat shock protein 90 (HSP90) client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SREZBP-CTfin51-AW1-Number 18 cDNA (SCAN) transcription factor family-MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1/ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced the tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in the CDC37 gene, negatively regulated the CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1.

Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter.

The heat shock response (HSR) is a cellular mechanism for counteracting acute proteotoxic stress. In eukaryotes, transcriptional activation of the HSR is regulated by heat shock factor 1 (HSF1). Activation of HSF1 induces the expression of heat shock proteins (HSPs) that function as molecular chaperones to fold and maintain the three-dimensional structure of misfolded proteins. The regulation of the degree and duration of the HSR is controlled by multiple biochemical mechanisms that include posttranslational modification of HSF1 and numerous protein-protein interactions. In this chapter, we describe a method to evaluate the activation and deactivation of the HSR at the transcriptional level using a short half-life luciferase reporter assay. This assay can be used to further characterize the HSR or as a screen for small-molecule inducers, amplifiers, or repressors.

Role of Heat Shock Factors in Stress-Induced Transcription.

Heat shock proteins (HSP) are rapidly induced after stresses such as heat shock and accumulate at high concentrations in cells. HSP induction involves primarily a family of heat shock transcription factors (HSF) that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo.

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.

Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants.

The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90ß, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90ß with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90ß, while HSP90ß bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

Englerin A stimulates PKCθ to inhibit insulin signaling and to simultaneously activate HSF1: pharmacologically induced synthetic lethality.

The natural product englerin A (EA) binds to and activates protein kinase C-θ (PKCθ). EA-dependent activation of PKCθ induces an insulin-resistant phenotype, limiting the access of tumor cells to glucose. At the same time, EA causes PKCθ-mediated phosphorylation and activation of the transcription factor heat shock factor 1, an inducer of glucose dependence. By promoting glucose addiction, while simultaneously starving cells of glucose, EA proves to be synthetically lethal to highly glycolytic tumors.

The role of heat shock factors in stress-induced transcription.

Heat shock proteins (HSPs) are rapidly induced after stresses, such as heat shock, and accumulate at high concentrations in cells. HSP induction involves a family of heat shock transcription factors that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo.