Contribution of chaperones to STAT pathway signaling

Abstract

Aberrant STAT signaling is associated with the development and progression of many cancers and immune related diseases. Recent findings demonstrate that proteostasis modulators under clinical investigation for cancer therapy have a significant impact on STAT signaling, which may be critical for mediating their anti-cancer effects. Chaperones are critical for protein folding, stability and function and, thus, play an essential role in the maintenance of proteostasis. In this review we discuss the role of chaperones in STAT and tyrosine kinase (TK) protein folding, modulation of STAT and TK activity, and degradation of TKs. We highlight the important role of chaperones in STAT signaling, and how this knowledge has provided a framework for the development of new therapeutic avenues of targeting STAT signaling related pathologies.

Keywords:

• chaperone
• chaperonin
• heat shock proteins
• JAK
• RTK
• SRC
• STAT
• tyrosine kinase

Abbreviations

Seventeen-AAG	=	17-N-Allylamino-17-demethoxygeldanamycin
AD HIES	=	Autosomal Dominant Hyper-IgE Syndrome
DMAG	=	17-allylamino-de-methoxy geldanamycin
EFGR	=	Epidermal Growth Factor Receptor
ERp57	=	Endoplasmic reticulum protein 57
ES cells	=	Embryonic stem cells
GA	=	Geldanamycin
H2O2	=	Hydrogen peroxide
Hip	=	HSP70-interacting protein
Hop	=	HSP70-HSP90 organizing complex
HSC	=	Heat shock cognate
HSE	=	Heat shock element
Hsf1	=	Heat shock factor
HSP	=	Heat shock protein
IL	=	Interleukin
IFN	=	Interferon
JAK	=	Janus Kinase
LPS	=	Lipopolysaccharide
mtHSP90	=	Mitochondrial HSP90
NAC	=	nascent polypeptide-associated complex
pY-Stat	=	phosphotyrosylated signal transducer and activator of transcription
RTK	=	receptor tyrosine kinases
SH2 domain	=	Src Homology 2 domain
SOCS	=	suppressor of cytokine signaling
STATs	=	signal transducers and activators of transcription
TK	=	tyrosine kinases
TOM/TIM	=	Translocase of the outer/ inner mitochondrial membrane
TRiC	=	tailless-complex polypeptide-1 ring complex
VSMC	=	Vascular smooth muscle cells
WT	=	Wild type

Introduction

Chaperone proteins play a prominent role in the maintenance of proteostasis, or cellular protein homeostasis,^1-4 by modulating myriad cellular processes including protein synthesis, complex assembly, protein degradation, and protein trafficking.^5,6 Chaperones facilitate cellular adaptation in response to stress,⁷ and also are actively involved in the modulation of major biological processes under normal conditions, including cell proliferation, differentiation and apoptosis.^8-10 Extensive data now reveal the foremost role chaperones play in modulating the cell signaling machinery,¹¹ including the de novo synthesis and folding of key cell signaling players, stabilization of the active conformations of these proteins, targeting and translocation of protein complexes into the nucleus, and disposing of used or misfolded proteins via proteasomal degradation.^10,11 In this review we highlight recent findings that contribute to our understanding of how chaperones impact cell-signaling pathways involving signal transducers and activators of transcription (STATs). This review is motivated by the flurry of recent investigations in this area and by studies demonstrating that the anticancer effects of drugs targeting chaperones, such as HSP90, are mediated, at least in part, through their impact on the STAT signaling pathway.

The STAT family of latent cytosolic transcription factors consists of 7 members (Stat1, Stat2, Stat3, Stat4, Stat5A, Stat5B, and Stat6).¹² Additional key components of the STAT signaling apparatus include: cytokines and growth factors, cell surface receptors, tyrosine kinases (TKs), receptor tyrosine kinases (RTKs), tyrosine phosphatases, and suppressor of cytokine signaling (SOCS) proteins. STAT proteins are located predominantly within the cytoplasm dimerized N-terminal-to-N-terminal (head-to-head) The canonical STAT signaling pathway is initiated when cytokines or growth factors bind the extracellular portion of specific cell surface receptors inducing receptor dimerization or oligomerization, resulting in juxtapositioning and activation of receptor-associated (JAK and Src) or receptor-intrinsic tyrosine kinases through kinase-mediated transphosphorylation. Dimerized STAT proteins are then recruited to activated receptor complexes via their Src homology 2 (SH2) domain and are phosphorylated at specific tyrosine residues by the activated kinase. The phosphotyrosylated (pY-) STATs undergo a conformational change, dimerizing C-terminal to C-terminal (tail-to-tail). Activated STAT dimers accumulate in the nucleus where they bind to promoters and modulate expression of genes that influence a wide variety of cellular processes.¹³

As components of the STAT signaling apparatus are often large, multi-domain proteins with complex topologies, and as STAT signal transduction depends on sophisticated protein structural rearrangements,¹² it is not surprising that chaperones play a major role in STAT signaling. It is clear from the findings reviewed here that chaperones greatly influence the maturation and activation of STATs, TKs, and RTKs, and that further explorations of this area will yield new information useful for modulating chaperone activity in a variety of disease states.

STAT Proteins and Chaperones

Overview

STAT proteins are highly dependent on elaborate cellular machinery composed of chaperones for biogenesis and optimal activity, as depicted in Figure 1. Here we review findings that reveal that STATs require interaction with the eukaryotic chaperonin protein, tailless-complex polypeptide-1 (TCP-1) ring complex (TRiC; also know as chaperonin containing TCP-1, CCT) for de novo folding. Multiple additional chaperones play a role in optimizing STAT protein activity, especially heat shock protein (HSP) 90. There are many exciting potential clinical applications of manipulating STAT-HSP90 interaction in the development of improved treatment approaches for human diseases such as cancer and atherosclerosis. Of all the STAT proteins, Stat3 interactions with chaperone proteins are most well characterized, and these interactions are summarized in Figure 2A.

Figure 1. STAT proteins interact with multiple chaperones to achieve nascent folding, activation, nuclear (and possibly mitochondrial) translocation, DNA binding, and possibly degradation.

Figure 2. Interactions of chaperone proteins with Stat3 panel (A) and Jak2 panel (B). Stat3 requires interaction with TRiC for optimal biogenesis (portion 1). As most TRiC clients also interact with nascent polypeptide-associated complex (NAC), HSP70/HSP40, and prefoldin, we hypothesize that these chaperones are also involved in de novo Stat3 folding. Inactive Stat3 is found in large complexes in the cytoplasm containing chaperone proteins HSP90 and ERp57 (portion 2). Stat3 is recruited to activated cytokine receptors, and HSP90 is required for Stat3 interaction with JAK kinases, Stat3 phosphorylation, and dimerization of activated Stat3 (portion 3). It is thought that HSP90 stabilizes activated Stat3, making Stat3 phosphorylation and tail-to-tail dimerization more favorable. HSP90/Hop is required for Stat3 nuclear translocation (portion 4). HSP90/Hop co-localizes with Stat3, stabilizing Stat3 while it is bound to DNA (portion 5). Although data are limited, others have hypothesized that mtHSP90 is involved, in conjunction with TOM/TIM, with Stat3 mitochondrial translocation (portion 6). ERp57 activity within the ER decreases Stat3 activity in the cytoplasm (portion 7). The activity of other chaperone proteins, such as HSP70 and HSP22, is associated with increased Stat3 activity (not pictured). In panel B, tyrosine kinases are classic HSP90 clients that require interaction with HSP90 for de novo folding and maturation as well as activation. HSP70 plays an important role in the degradation of tyrosine kinases, which can occur at any stage of tyrosine kinase maturation or after activation. In the presence of HSP90 inhibitors tyrosine kinases are preferentially degraded.

STAT Proteins and TRiC

Chaperonins are a structurally conserved class of oligomeric, double-ring, high molecular weight, ATP-dependent chaperones with the unique ability to fold proteins that cannot be folded by simpler chaperone systems.⁵ TRiC/CCT is a 1 MDa complex composed of 8 homologous subunits arranged in 2 octameric rings, forming a cage that facilitates protein folding.^14,15 TRiC is not induced by stress but instead participates in the de novo folding of > 10% of cytosolic proteins.¹⁴ Our lab has recently shown that Stat3 is a TRiC substrate, and that Stat3 requires interaction with TRiC for biogenesis and optimal function within human cell lines.¹⁶ Furthermore, manipulating the interaction between Stat3 and TRiC affects Stat3 activity: knock down of TRiC using shRNA reduces Stat3 sensitivity to IL-6-mediated activation, while increasing Stat3 affinity for TRiC through the addition of an extra TRiC binding domain improves in vitro Stat3 activity. In addition to Stat3, we have demonstrated that Stat1 requires interaction with TRiC for biogenesis (Fig. 3).

Figure 3. TRiC binds Stat1/Stat3 co-translationally and is required for Stat1/Stat3 synthesis in RRLs. In panel (A), TRiC was immunoprecipitated from rabbit reticulocyte lysate with a combination of antibodies to CCT2 and CCT5 (Anti-TRiC) or with a nonspecific control antibody (Human IgG) following translation of the indicated proteins in the presence of³⁵S-methionine. Immunoprecipitates were separated by SDS-PAGE and autoradiographed. Half of each IP reaction prior to precipitation was run separately on SDS-PAGE and autoradiographed (Input). In panel (B), Stat1/Stat3 was translated in TRiC-depleted RRLs or following the addition of purified bovine TRiC in increasing amounts in the presence of³⁵S-methionine followed by SDS-PAGE and autoradiography. The results shown are representative of 3 experiments.

TRiC client proteins usually require interaction with ribosome-associated chaperones for stabilization during translation and for facilitation of TRiC binding. The nascent polypeptide-associated complex (NAC) is assumed to aid in the co-translational folding of most peptides.^17,18 The HSP70/40 complex and prefoldin are cytosolic chaperones that act on newly synthesized proteins, aiding de novo protein folding and targeting newly synthesized substrates to the TRiC complex.^17,19 As we have shown that Stat1/3 are TRiC substrates, it is possible that Stat1/3 also interact with NAC, HSP70/40 and prefoldin during de novo folding.

STAT Proteins and HSP90

HSP90 is a highly conserved molecular chaperone that serves as a proteostasis buffer under environmentally stressful conditions and facilitates the maturation and activation of > 100 client proteins, many of which are critical for cell signaling and adaptive responses to stress. HSP90 exists as 2 isoforms in the cytosol; HSP90α is induced by stress and is commonly upregulated in cancer cells while HSP90β is expressed constitutively at high concentrations in most tissues.²⁰ HSP90 utilizes energy garnered from ATP hydrolysis and interacts with more than 20 co-chaperones—HSP70, HSP40, HSP70-interacting protein (Hip), HSP70-HSP90 organizing complex (Hop), CDC37, p23, and immunophilins—to help client proteins fold.^21-24

Physical association between HSP90 and Stat1/3 was initially reported in plasma membrane cholesterol-rich rafts [with caveolin-1, cytokine receptors (gp130), JAK1/JAK2, and Tyk2] and cytosolic complexes (with ERp57).²⁵ The DNA binding domain of Stat3 was shown, using domain deletion mutations and immunoprecipitation studies, to directly interact with the N-terminal ATPase domain of HSP90, and that HSP90 inhibitors, like geldanamycin (GA), inhibit this interaction by binding to the same HSP90 domain.^26,27 More recently, surface plasmon resonance spectroscopy showed that the high-affinity, specific interaction between the N-terminal domain of HSP90β and Stat3 requires a functional Stat3 DNA binding domain, and that this interaction has no preference for inactive vs. activated Stat3.²⁷ Domain deletion mutations and immunoprecipitation studies localized the Stat5 HSP90 binding domain to the C-terminal half of the coiled-coil domain.²⁸

Results using HSP90 inhibitors strongly suggest that STAT-HSP90 interactions contribute to optimal STAT activity, and that this contribution becomes essential when cells undergo increased stress. For example, treatment of Hep3B cells with GA results in reduced interleukin (IL)-6-induced-Stat3 signaling at 37°C and markedly decreased signaling when the cells are stressed at 39.5°C.²⁵ Overexpression of HSP90 in cells treated with GA restores IL-6-induced-Stat3 activity to normal levels.²⁶ Similarly, GA blocks the activation and phosphorylation of IL-2-induced Stat5 in an IL-2 dependent T cell line.²⁸

STAT proteins are not considered traditional HSP90 clients in that they do not require HSP90 for de novo folding; multiple studies show that HSP90 inhibitors decrease STAT activity through reduction of pY-STAT levels with minimal to no effect on total STAT protein levels.^29-32 Moulick et al. recently proposed that HSP90 promotes optimal STAT activity by serving as a scaffolding molecule, stabilizing STAT proteins in their active, phosphotyrosylated configuration.³³ In human chronic myelogenous leukemia cells treated with novel HSP90 inhibitor PU-H71, pY-Stat5 activity was reduced shortly after HSP90 inhibition, and this reduction could not be attributed to a change in kinase or phosphatase activity. Furthermore, binding to HSP90 appeared to change the conformation of Stat5, as Stat5 was more susceptible to trypsin cleavage when bound to HSP90. To investigate whether HSP90 binding results in a more favorable Stat5 configuration for phosphorylation and activation, the authors used a pulse-chase experiment after cells were treated with the phosphatase inhibitor, orthovanadate. Cells exposed to the HSP90 inhibitor demonstrated rapid decrease in pY-Stat5 concentration compared with unexposed cells, suggesting that Stat5 is more likely to remain phosphorylated when bound to HSP90.³³

Evidence also suggests that HSP90, along with the co-chaperone Hop, remains bound to dimerized pY-STATs, which targets and facilitates their translocation into the nucleus and stabilizes their interaction with DNA. Stat3-HSP90 interaction was shown to be essential for leukemia inhibitory factor (LIF)-induced, Stat3-mediated self-renewal of mouse embryonic stem (ES) cells.^34,35 Hop co-precipitates with HSP90 and Stat3 in ES cells, and Hop knockdown decreases pY-Stat3 nuclear translocation.³⁶ Using these observations, Setati et al. initially suggested that HSP90/Hop interacts with Stat3 at activated cytokine receptors, facilitating pY-Stat3 dimerization, nuclear targeting, nuclear translocation, and DNA binding.³⁴ Howard et al. also reported that Stat1 and HSP90 are both recruited to the plasma membrane in alveolar macrophage cells after stimulation with interferon (IFN)-γ, and that inhibition of HSP90 with 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) resulted in inhibition of Stat1 phosphorylation and DNA binding.³⁷ Finally, HSP90 has been shown to support Stat5-mediated transcription in that HSP90 inhibition led to decreased Stat5 DNA binding; quantitative chromatin immunoprecipitation assays demonstrated that HSP90 and Stat5 co-localize at critical Stat5 target promoters.³³

To summarize available data thus far, HSP90 clearly influences STAT protein activity through direct interaction; through this interaction, HSP90 stabilizes STAT dimers into a configuration that is more favorable for phosphorylation and activation. HSP90 (with co-chaperone Hop) then translocates with activated STAT dimers into the nucleus where it continues to stabilize STAT dimers bound to DNA.

Stat3/5 are constitutively upregulated in many cancers, and multiple studies in human cancer cell lines and animal models have demonstrated that HSP90 inhibition leads to decreased Stat3/5 signaling, thus highlighting the potential promise of this treatment strategy. HSP90 inhibitor STA-1474 potently decreased constitutive Stat3 phosphorylation in osteosarcoma cell lines with minimal effect on total and pY-Stat3 in nonmalignant canine osteoblasts.²⁹ Multiple myeloma cells treated with HSP90 inhibitors had decreased pY-Stat3 and Stat3 activity³⁰ and chronic lymphocytic leukemia cells treated with NVP-AUY922 and fludarabine had decreased Stat3 activity and increased apoptosis even when cultured on CD40L-stromal layer mimicking the supportive environment of the bone marrow.³⁸ Leukemia cells (with mutated FLT3 kinase) treated with 17-AAG had decreased Stat5 activity with increased cell cycle arrest and apoptosis.³⁹ Human pancreatic cancer cells treated with 17-AAG were found to have decreased Stat3/5 activation and signaling, as well as disruption of a novel IL-6/Stat3/HIF-α-mediated autocrine activation loop required to facilitate transcription of targets such as VEGF.⁴⁰ A subcutaneous xenograft tumor model and an orthotopic tumor model of pancreatic cancer cells both demonstrated potent reduction of tumor growth and tumor vascularization after treatment with HSP90 inhibitor 17-allylamino-de-methoxy geldanamycin (DMAG), and these effects were mediated by a reduction of IGF-IR and Stat3 signaling.⁴⁰ A recent review by Kim et al. summarizes the development of HSP90 inhibitors for clinical use in selectively inhibiting tumor growth.⁴¹

HSP90 inhibitors are also being studied to block STAT signaling in other disease processes such as atherosclerosis. The use of 17-DMAG in hyperlipidemic ApoE^−/− mice decreased the size of atherosclerotic lesions and decreased lipid and macrophage content of lesions, correlating with decreased pY-Stat3 and NF-κB signaling. The authors concluded that HSP90 inhibitors reduce inflammation in atherosclerosis.⁴²

As discussed above, Stat1/3/5 have been shown to require HSP90/Hop for nuclear translocation. These STAT proteins also are found in the mitochondria. Although this finding remains controversial, Stat3 has been shown to reduce oxidative stress and influence the electron transport chain,⁴³ leading to the question of whether Stat3 and/or other STAT proteins require chaperoning for mitochondrial translocation.⁴⁴ Mitochondrial HSP90 (mtHSP90), mtHSP60, and mtHSP70 are involved in the transport of multiple mitochondrial transcription factors such as p53, IRF3, CREB, NF-κB, and MEF2D, and some authors have hypothesized that mitochondrial chaperone proteins TOM/TIM (translocase of the outer/inner mitochondrial membrane) work with mtHSP90 to aid Stat3 entry into the mitochondria.⁴⁵ Although data addressing this hypothesis is limited, Stat3 does co-immunoprecipitate with TOM20 in rat left ventricular protein extracts.⁴⁶ Other authors have reported that GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) is involved in the transport of Stat3 into the mitochondria.^47,48

STAT Proteins and Other Chaperones

HSP70

The human HSP70 family includes at least 12 proteins, including stress-induced HSP70 (HSP70–1A and HSP70–1B), constitutively expressed heat shock cognate (HSC) protein 70, glucose-regulated protein (GRP) 78 (Bip, primarily located in the ER), and mitochondrial HSP75 (mtHSP70).⁴⁹ The HSP70 family contributes to multiple biochemical functions in the cell, including nascent protein folding, protein homeostasis, protein translocation into the nucleus, ER and mitochondria, and trafficking of misfolded proteins to the ubiquitin/ proteasome machinery for degradation. Both HSC70 and HSP70 activity require interaction with multiple co-chaperones, such as J-domain protein HSP40, BAG family, Hip, Hop, HSPBP1, and CHIP (carboxyl-terminus of HSC70 interacting protein).⁴⁹

HSP70 has been shown through immunoprecipitation to directly interact with multiple STAT proteins, including Stat1/3/5B in normal rat kidney interstitial fibroblast (NRK-49F) cells; this interaction was increased when cells were stressed by exposure to advanced glycation end-product.⁵⁰ Although data are limited, a few reports suggest that manipulating HSC70/HSP70 activity impacts STAT protein activity within cells. Knock down of HSP70 (HSP70–1A and HSP70–1B) using siRNA further decreased constitutive Stat3 activity in an acute myeloid leukemia cell line (HEL) treated with arsenic trioxide and 17-DMAG.⁵¹ Also, increasing HSP70 activity with geranylgeranylacetone in a rat model of intracerebral hemorrhage resulted in increased Stat3 phosphorylation, decreased neuronal apoptosis, and improved functional recovery.⁵²

HSP22

HSP22 is only expressed in heart and skeletal muscle, and is upregulated during cardiac stress. In an HSP22 knockout mouse model, there was decreased Stat3 signaling in response to overload stress, as well as decreased Stat3 levels and activity within mitochondria.⁵³ These findings support the hypothesis that HSP22 is critical for nuclear and mitochondrial Stat3 activity in cardiomyocytes. Preemptive overexpression of HSP22 also protected swine myocardium from post-ischemic or H₂O₂-mediated apoptosis through increased STAT and NF-κB signaling.⁵⁴

ERp57

The endoplasmic reticulum (ER) is involved in the response to cell stress and contains multiple chaperone proteins that are generally thought to perform quality control of newly synthesized glycoproteins and regulation of the Unfolded Protein Response (UPR). ERp57 (or glucose-regulated protein 58, Grp58) is a thiol disulfide oxidoreductase and chaperone protein that is found in the ER and predominantly acts as part of a complex (with calnexin and calreticulin) involved in the quality control of the secretory pathway. ERp57 is also found in the cytoplasm, and has been shown to interact with Stat3 within plasma membrane rafts and within the cytoplasm.^55-60 Stat3 and ERp57 have also been found to be associated in the nucleus bound to DNA in M14 melanoma cells and IL-6-stimulated HepG2 hepatoma cells.⁶¹ The effect of Stat3-ERp57 interaction on Stat3 activity is controversial. When recombinant ERp57 was added in excess there was decreased pY-Stat3 in S100 cytosol, and when ERp57 was overexpressed in hepatoma Hep3B cell lines, both constitutive and IL-6-induced Stat3 activity was reduced.⁶⁰ The authors hypothesized that ERp57 may function to sequester activated pY-Stat3 in cytokine-stimulated cells leading to decreased Stat3 activity. However, anti-ERp57 antibodies decreased activated Stat3 DNA binding in melanoma and HepG2 cell lysates stimulated with IL-6, suggesting that ERp57 may be a critical component of the DNA-bound Stat3 complex.⁶¹ Finally, ERp57-deficient cells have significantly increased Stat3 activity. This increased activity was restored to normal by expression of the ERp57 within the ER but not within the cytoplasm ERp57; ERp57-dependent modulation of Stat3 activity was further enhanced by interactions in the lumen of the ER between ERp57 and calreticulin.⁶²

Tyrosine Kinases and Chaperones

Overview

Unlike STAT proteins, kinases are considered classic HSP90 clients, requiring interaction with HSP90 and co-chaperones for de novo folding to achieve maturation i.e. their native folded state. Additionally HSP90 also has been shown to modulate kinase activity post-maturation i.e., to facilitate mature kinase conformational transitions between active and inactive forms, which are coupled to the HSP90 ATPase cycle.^63,64 In addition, others have suggested that HSP90 regulates mature kinase activity by sequestration, a process by which mature kinases in active or metastable conformation are rendered inactive when bound to the chaperone and released from the chaperone complex only when the kinase activity is needed.^65,66 Since HSP90 has a diverse group of client proteins, it depends on multiple co-chaperones to recruit specific substrates. One such polypeptide is the kinase specific adaptor CDC37, which is known to play a major role in modulating kinase function. Tyrosine kinase interactions with chaperone proteins are summarized in Figure 2B. Here, we will focus our discussion primarily on kinases involved in STAT signaling.

Src Kinases and Chaperones

Src kinases constitute a family of 8 members including Src, Yes, Fyn, and Fgr (that form the SrcA subfamily), Lck, Hck, Blk, and Lyn (that form the SrcB subfamily) and Frk. Src-family protein kinases are involved in a wide variety of cellular activities including cell differentiation, motility, proliferation, and survival.⁶⁷ Although some of the Src kinases also play a prominent role in non-STAT signaling networks, there is an abundance of biochemical evidence showing that most Src kinases are capable of specifically activating STATs, especially Stat3 and to some extent Stat5.^68-72

The first oncogene discovered, v-Src, is one of the earliest HSP90 kinase substrates described. In an attempt to pinpoint the inhibitory mechanism behind benzoquinone ansamycins toward tyrosine kinase induced oncogenic transformations, Whitesell et. al. discovered that v-Src was a client of HSP90. These studies demonstrated that the antibiotic geldanamycin (GA) was a pharmacological inhibitor of HSP90 chaperone, thereby identifying the basis for GA's anti-oncogenic activity.^73,74 It is now well established that the oncogenic transformation ability of v-Src is due to its unrestrained tyrosine kinase catalytic activity toward multiple substrates, including Stat3/5, which is highly dependent on HSP90.^75-77 The proto-oncogene counterpart of v-Src, c-Src, also was shown to depend on HSP90 for activity. Interestingly, when compared to its oncogenic mutant counterpart, c-Src kinase showed markedly weaker binding and less sensitivity to HSP90 inhibition,⁷⁸ suggesting that each has a different requirement for HSP90 chaperone activity. In addition, Koga et al. showed that HSP90 binds to c-Src, and sequesters it in an intermediate state between active and inactive conformations; in contrast to v-Src, HSP90 inhibition was accompanied by activation of c-Src and its downstream targets after dissociation from HSP90. Because of the potential toxicity that might arise from HSP90 inhibition in normal tissues, the benefit of targeting HSP90 activity for therapeutic purposes may not be straightforward. Thus, this approach warrants further studies.^65,79

HSP90 and CDC37 also modulate the activity of other Src kinase family members such as Hck, Lck, Fyn, and Lyn. GA treatment of macrophages abrogates lipopolysaccharide (LPS)-induced macrophage cell adhesion and results in a concomitant decrease in the concentration of Hck, and to lesser extent Fyn and Lyn, all well established effector molecules downstream of LPS.^80,81 Similar observations have been made with Lck.⁸² Additionally, the specific catalytic activity of the kinases is markedly diminished in the presence of HSP90 inhibitors, implying that in the absence of chaperone activity, the kinases do not achieve their native functional configuration. To further examine the role of HSP90 pre- and post-maturation, pulse chase experiments were performed on wild type (WT) kinases, demonstrating that mainly nascent kinases are prone to degradation (short half-life) in the presence of HSP90 inhibitors, while the mature kinases exhibit a longer half-life.⁸⁰ These results suggest that WT kinases rely on HSP90 primarily for maturation.

Interestingly, however, the impact of HSP90 activity on kinase function after maturation appears to be conditional. This has been demonstrated through stability assays. Constitutively active mutants of Hck and Fyn remain dependent on HSP90 for stability post-maturation, whereas WT kinases do not.^80,83 This observation suggests that either the mutations are destabilizing and require increased HSP90 activity, or that the constitutively active (open) kinase conformation may be inherently unstable, thus more dependent on chaperone activity for stability than the inactive kinase (closed).⁸⁰ If the latter is true, it implies that WT kinases may require HSP90 transiently during activation (while in the open conformation). Therefore, it is possible that the role of chaperones does not stop at nascent kinase folding, as under certain circumstances HSP90 remains actively engaged to maintain stability and modulate kinase activity post-maturation.

HPS90 is intimately associated with its co-chaperones, they represent the major way in which HSP90 activity is modulated within the cell and have been shown to target specific substrates to HSP90. Although there are over 20 HSP90 co-chaperones known to date, CDC37 stands out as the predominant co-chaperone in kinase biology. Indeed, disruption of CDC37/HSP90 association by siRNA depletion or inhibition of its activator, CK2 kinase, in human multiple myeloma cells, results in the degradation of client kinases, including c-Src, thereby decreasing levels of constitutively active Stat3 and other downstream effectors.⁸⁴ Interestingly, some HSP90 client kinases have been reported to phosphorylate key sites on the chaperone, implying a link between HSP90 regulatory mechanism and the kinase activity of its substrates.^85,86 These studies showed that kinases such as pp60 v-Src and c-Src phosphorylate HSP90 at specific sites affecting its activity and substrate specificity.^87,88 How this impacts downstream STAT signaling is not clear, however, a recent study indicates that phosphorylation of the C-terminal end of HSP70 and HSP90 results in the preferential binding to co-chaperone HOP; this then leads to increased protein folding activity of HSP90. The same study showed an association between phosphorylation of HSP90 and increased proliferation rates in neoplastic cells.⁸⁹

JAK Kinases and Chaperones

JAK kinases make up the predominant non-receptor kinases involved in STAT activation. There are 4 known JAKs—Jak1, Jak2, Jak3 and Tyk2.⁹⁰ The available sequence and structural information, show that JAK kinases bear a structural organization similar to other kinases.⁹¹ Thus, it is not surprising that HSP90 is also essential for JAK kinase folding and maturation. Indeed, the cellular protein levels of all 4 JAK kinases are markedly reduced when cells are exposed to HSP90 inhibitors, suggesting that JAK kinases are dependent on HSP90 for de novo folding and maturation. This, as expected, is accompanied by the loss of STAT phosphorylation, especially Stat1/3/5/6.⁹²

The involvement of HSP90 in the regulation of normal JAK kinase activity was suggested by findings that HSP90 chaperone is necessary for IFN I and II signaling. IFN-γ, is a key player in both innate and adaptive immunity. It has been established that the canonical IFN-γ- signaling occurs through JAK/STAT pathway. Recent studies indicate that HSP90/CDC37 protein complex is a molecular chaperone for Jak1/2 and is important to the normal immune response. Co-immunoprecipitation experiments reveal a physical association between JAK kinases and the CDC37/HSP90 complex that is disrupted when cells are treated with HSP90 inhibitors. Additionally, studies examining IFN-γ-induced T-cell activation, showed that chemical inhibition of HSP90 or siRNA knockdown of HSP90 and CDC37 results in depletion of Jak1/2; which accordingly led to diminished pY-Stat1, decreased expression of IFN-γ-inducible genes and consequently, impaired T cell activation, suggesting that JAK kinases are bona fide HSP90 clients.⁹³

As mentioned above, it is clear that HSP90 is essential for maintaining the proper functioning of JAK signaling, thereby enabling appropriate responses to cytokine stimuli. However, when overexpressed in certain cell lines, HSP90 has been shown to result in JAK kinase dysregulation, thereby leading to inappropriate JAK/STAT signaling. For instance, in classical Hodgkin's lymphoma, HSP90 is overexpressed and thought to support an oncogenic signaling network in which Stat3, Stat5 and Stat6 are constitutively activated. Inhibition of HSP90 leads to JAK kinase depletion, reduced phosphorylation of Stat3/5/6 and proliferation of cHL cells.⁹⁴

Another way in which HSP90 contributes to aberrant JAK/STAT signaling, is by stabilizing constitutively active oncogenic mutations. It has recently been demonstrated that mutation V617F in Jak2 kinase is the genetic cause for several myeloproliferative disorders.^95-97 This mutation has been shown to abolish JAK kinase autoinhibition resulting in a constitutively active kinase that, in turn, constitutively actives Stat3/5, leading to transformation and proliferation of these cells. As has been observed in other kinases, mutant and aberrantly activated JAK kinases found in association with malignant phenotypes, also appear to be highly dependent on HSP90 for stability. Pharmacological inhibition of HSP90 in cells containing Jak2 oncogenic mutations results in Jak2 degradation, diminished constitutive Stat5 activation, and reduced proliferation.^98,99 Although both wild type and mutant JAK kinases to some extend depend on HSP90 chaperone for stability, data on Jak2 V617F seems to mirror observations made on other kinases showing that constitutively active mutant kinases are more dependent on HSP90 than their WT counterparts, a difference that could be potentially exploited for therapeutic benefit.¹⁰⁰ In addition to V617F, other interesting Jak2 mutations such as E864K, Y913C and G935R, confer resistance to JAK kinase inhibitors; however, when cells harboring the resistance mutations are exposed to both HSP90 and kinase inhibitors, the resistance is overcome. Indeed, in the same studies, cytotoxicity assays show that the cells having the resistant mutations were more sensitive than WT cells to HSP90 inhibition using AUY22.¹⁰¹ Similarly, inhibition of HSP90 activity has also been shown to be effective in cases where cells are able to overcome Jak2 inhibition and sustain aberrant JAK/STAT signaling by co-opting other kinases to trans-activate Jak2.¹⁰² These examples, while not exhaustive, highlight the importance of HSP90 in the development and maintenance of many pathological states mediated by aberrant JAK/STAT signaling, including both solid and hematologic malignancies,⁹⁹ along with the therapeutic potential of pharmacological inhibition HSP90 as potential treatment for such malignancies.

Receptor Tyrosine Kinase and Chaperones

As might be expected, receptor tyrosine kinases (RTK) pose protein-folding challenges for cells, because, in addition to the proper folding and maturation of the chaperone-dependent kinase domain, they also have to be targeted to the membrane. There is abundant evidence showing that molecular chaperones play an important role in the folding and maturation of many membrane proteins, particularly RTK, in addition to facilitating degradation and modulating the activities of mature receptors.¹⁰³ Biochemical and genetic studies in mammalian cells have provided great insight into the role of HSP90 and HSP70 in the biogenesis and maturation of RTK. The use of HSP90-specific inhibitors as chemical probes has contributed immensely to the identification of the majority of RTK that are HSP90 clients. Several RTK that are involved in the STAT signaling network,^103-106 including EGFR, MET, PDGF, IGF-IR and FGFR,^40,107,108 have been identified as HSP90 clients.

Studies of the ErbB family of RTK especially highlight the critical role of chaperones in the modulation of STAT signaling. The epidermal growth factor receptor (EGFR) is the most studied of these perhaps because of its prominent role in many human cancers. EGFR and other ErbB family members have been found in association with a handful of molecular chaperones and co-chaperones, most prominently HSP90 and CDC37. Like the non-receptor kinases discussed above, it appears that all studied RTK, including the ErbB family, are dependent on HSP90 at the nascent stage.¹⁰⁹ However, ErbB2 and the majority of the active mutants in the ErbB family appear to remain engaged and require chaperones throughout their lifespan, including nascent protein folding, trafficking, modulating activity of the mature receptor, and assisting in the degradation process.^110,111The contribution of chaperones to EGFR/ErbB biology has been excellently reviewed elsewhere.¹¹² Consequently, we will only highlight a few key studies demonstrating the direct effects of inhibiting HSP90 on STAT signaling. For example, when HSP90 activity in cells is inhibited with the HSP90 inhibitor, SNX-2112, both WT and mutant EGFR fail to fold properly and are in turn degraded, thereby preventing EGF activation of pY-Stat3.¹¹³ Certain mutations on exon 20 of ErbB2 and EGFR are of great clinical interest because they result in constitutive activation of the receptors and confer resistance to EGFR tyrosine kinase inhibitors. Analysis of these mutants shows a strong dependence on HSP90, and, as expected, the mutants were not sensitive to the kinase inhibitor gefitinib. However treatment with 17-AAG alone, and in combination with gefitinib, abolished the unrestrained EGFR activity and persistent phosphorylation of its downstream signal transducers Stat3 and Akt.¹¹⁴ These studies again emphasize the therapeutic potential of HSP90 inhibitors in the treatment of aggressive malignancies.

Recent studies reveal that molecular chaperones also exert their activity extracellularly. HSP90/CDC37 and HSP70 and its co-chaperones have been found in extracellular complexes with cell surface receptors including EGFR, HER2 and matrix metalloproteinase 2. Extracellular HSP90 and HSP70 are thought to be involved in polypeptide complex assembly and in facilitating cell invasion signaling pathways. HSP90 has been shown to be essential for HRG-induced HER-2 activation and its downstream signaling events that lead to cytoskeletal rearrangements necessary for cell invasion. Thus, inhibition of extracellular chaperone activity results in diminished cancer cell migration and invasion.^115,116,117 In addition, recent work looking at the mechanism of heat stress-induced transactivation of EGFR suggests a role for secreted HSP70 in the initial stages of heat shock-mediated signaling. The ligand independent activation of EGFR and subsequent downstream activation of Stat3 observed during the initial stages of heat stress response in a squamous carcinoma cell line was shown to be mediated by extracellular HSP70.¹¹⁸

Contribution of Chaperones to Degradation of STAT Pathway Proteins

Part of the therapeutic rationale behind HSP90 inhibition is based on observations that molecular chaperones collaborate with the ubiquitin/proteosome system during polypeptide triage¹¹⁹ such that when cells are treated with HSP90 inhibitors, traditional HSP90 client proteins, including kinases, are shuttled to the proteasome for degradation. Several studies have implicated HSP70 as having a major role in this degradation process,^120,121 likely serving as a sensor for stressed proteins and determining the fate of the proteins.¹²² The emerging view is that, depending on the circumstances, HSP90 and HSP70 are involved in either the stabilization or degradation of client polypeptides. Exactly how the decision to stabilize versus degrade proteins is reached is not clear, but evidence from several labs has begun to provide insight into a possible mechanism. For example, it has been observed that GA treatment of cells induces a change in the composition of cofactors in the HSP90/HSP70 complex to include polypeptides with E3 ligase activity. This, in turn, results in the ubiquitination of client proteins within the chaperon complex.^119,123 Indeed, with regards to v-Src, An et al. elegantly demonstrated that the composition of the complex of HSP90 and cofactors changes from Hsp90/p23/p50 to Hsp90/Hsp70/Hop in the presence of GA, which results in increased proteosomal degradation of v-Src.¹²⁴ Others have reported similar dynamics involving other chaperone co-factors in conjunction with HSP70.¹²⁵ It is also clear that although HSP90 is the predominant chaperone involved in the modulation of kinase activity, other chaperones such as HSP70 and co-chaperones/co-factors p23, p50, Hop, CHIP and HSP40 also play an important supporting role.

Results are not as straightforward in defining the role of chaperones in STAT protein degradation. As stated above, STAT proteins are not classic HSP90 clients, in that they do not require HSP90 for de novo folding or maturation. Multiple studies with HSP90 inhibitors demonstrate a reduction of pY-Stat3/5 with minimal to no effect on total Stat3.^29-32 Howard et al. did show that inhibition of HSP90 with 17-AAG resulted in inhibition of Stat1 phosphorylation which lead to Stat1 insolubility and ultimately degradation via the proteasome, but the role of HSP70 or other chaperones in this degradation process was not explored.³⁷ As discussed above regarding the kinases, although HSP90 is the predominant chaperone involved in the modulation of STAT protein activity, HSP70 activity has been associated with increased STAT protein activity – especially when cells are undergoing increased stress. Further studies are needed to clarify the potential role that chaperones play in the degradation of STAT proteins.

STAT Proteins and Regulation of Chaperones

While the focus of this review is to highlight the dependence of STAT signaling pathways on chaperone proteins, we should acknowledge that it is also well recognized that STAT signaling pathways play an important role in the induction of HSP expression and activity as well. It is clear from the reports summarized below that STAT signaling can upregulate the HSPs that are responsible for optimizing STAT protein activity, thereby serving as a positive feedback mechanism for STAT protein activity.

Stephanou, et. al., initially reported that Stat1/3 are involved in the regulation of HSP90 and HSP70 by showing that IL-6-induced Stat3 signaling increased expression of HSP90β and that IFN-γ-activated Stat1 signaling induced expression of HSP70 and HSP90β.^126-130 Additional evidence for this relationship includes the following: Hydrogen peroxide (H₂O₂) activates Stat1/3 in vascular smooth muscle cells (VSMC) leading to HSP70 expression.¹³¹ Thrombin-induced HSP70/HSP90 expression in VSMC also appears to be mediated through the JAK/STAT pathway (Stat1/2/3).¹³² HSP105β, a member of the HSP70 family that is expressed during stress and functions in suppressing protein aggregation, has been shown to induce HSP70 expression through Stat3 in mammalian cells.¹³³ Stat3 activation via IL-6 administration as a resuscitation adjuvant in a rat model of trauma-hemorrhagic shock leads to upregulation of HSP70 expression in the liver, which contributes to prevention of hepatocyte apoptosis.¹³⁴ Finally, sodium 4-phenylbutyrae (4PBA), a drug that corrects trafficking and function of the mutant ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, appears to increase HSP70 expression, at least in part, through Stat3 activation.¹³⁵

Additional HSPs also appear to be induced by Stat1/3/5 signaling. HSP27 is induced by stress and participates in stabilizing proteins, inhibiting apoptosis, mediating thermotolerance, and regulating cell differentiation. Constitutively activated Stat3 in breast cancer cells upregulates HSP27, which may play a part in the mechanism by which Stat3 contributes to oncogenesis.¹³⁶ Stat3/5B both appear to be mediators of thrombin-induced HSP27 expression in VSMC.¹³⁷ Stat1 also appears to induce HSP27 in cells after exposure to heat or chemical stress; cells infected with viral pathogens have dramatically reduced levels of Stat1 leading to decreased HSP27, and therefore more readily undergo apoptosis after exposure to heat.¹³⁸ HSP60/HSP10 comprise the mitochondrial chaperonin complex that stabilizes mitochondrial proteins. Upregulation of HSP60/10 in a rat model of the post-ischemic brain appeared to be mediated through Stat3 signaling,¹³⁹ as was IFN-γ-induced expression of HSP60/HSP10 in C6 astroglioma cells.¹⁴⁰

Finally, Stat1/3 have also been shown to directly interact with heat shock factor (Hsf) 1, the transcription factor most notably responsible for inducing HSPs in response to stress. After stimulation with IFN-γ, Stat1 and Hsf1 form a DNA binding complex that binds to the heat shock element, leading to enhanced activation of transcription.¹²⁸ The effect of Stat3-Hsf1 interaction on Stat3 or Hsf1 signaling remains controversial. It has been reported that Stat3 and Hsf1 are antagonistic with regard to HSP90β gene expression.^126,127 Stat3-Hsf1 complexes in gastric carcinoma cells infected with Helicobacter pylori appeared to be transcriptionally inactive; infected cells became depleted of HSP70 and eventually underwent apoptosis.¹⁴¹ In contrast, interaction of Stat3 with Hsf1 also has been shown to result in increased Stat3 activity: G-CSF-stimulated Stat3 signaling mediated cardio-protection by decreasing cardiomyocyte apoptosis in a mouse heart ischemia/reperfusion model; decrease in cardiomyocyte apoptosis was shown to be critically dependent on the association of Stat3 and Hsf1.¹⁴²

Conclusions

Chaperone proteins play a substantial role in STAT signaling; they are clearly involved in the de novo folding and maturation of STAT proteins and kinases, in the optimization of STAT protein and kinase activity, and in the degradation of kinases. STAT proteins require interaction with TRiC for biogenesis and optimal function, and while STAT proteins are not traditional HSP90 clients, in that they do not require HSP90 for de novo folding or maturation, they do depend on HSP90 for activation, nuclear translocation, and DNA binding. STAT protein activity is also positively regulated by a number of additional chaperones, especially in cells experiencing increased stress. Kinases, on the other hand, are true HSP90 clients as they depend on HSP90 and co-chaperones for de novo folding, maturation, and activation.

The potential therapeutic implications of the relationship between chaperones and STAT signaling pathways are substantial (Table 1). Stat3, for example, plays a major role in many disease states involving dysregulated or dysfunctional apoptotic signaling pathways. Stat3 activity is decreased when cells undergo oxidative stress during ischemia/reperfusion (from trauma-hemorrhagic shock, myocardial infarction, or intracerebral hemorrhage) leading to increased pathologic apoptosis. Up-regulating chaperone activity in these conditions has been associated with increased Stat3 activity, decreased apoptosis, and improved functional recovery.

Table 1. Current and future strategies for modulating Stat3 activity for therapeutic benefit through targeting of chaperones.

Disease state	Therapeutic modulation of Stat3	Potential strategy
- Cancer	Decrease Stat3 activity	- HSP90 inhibitors (currently being used/ studied)
- Inflammatory Bowel Disease		- HSP70 inhibitors (currently being studied)
- Asthma		- Inhibitors of Stat3-TRiC interaction
- Cachexia
- Atherosclerosis
Ischemia/reperfusion injury	Increase Stat3 activity	- Upregulation of HSF1, HSP70, and HSP90 (currently being studied)
- Inhibitors of Stat3-TRiC interaction- Trauma/hemorrhagic shock		- Upregulation of Stat3-TRiC interaction
- Myocardial infarction
- Intracerebral hemorrhage
Infection

Stat3 activity is aberrantly increased in a number of human pathologies as well, including > 50% of all cancers and atherosclerosis. HSP90 inhibitors, and to a lesser extent HSP70 inhibitors, have been used to decrease Stat3 activity and increase apoptosis of cancer cells in multiple cancer systems and models. Because HSP90 inhibitors affect other components of the Stat3 pathway in cancer cells besides Stat3, as summarized above, increased cancer cell apoptosis following HSP90 inhibitor treatment likely is due to the effects of reduced HSP90 activity on more than just Stat3 alone. HSP90 inhibitors also have been reported to decrease Stat3 activity and inflammation contributing to atherosclerosis in a mouse model of coronary artery disease. HSP90 inhibitors may be a therapeutic option for additional diseases that result from increased inflammation secondary to Stat3 activity, such as inflammatory bowel disease, cachexia, asthma and fibrosis. Decreasing Stat3-TRiC interaction could also represent a novel therapeutic approach to these disease states. We are excited about the therapeutic potential of manipulating the interaction between chaperones and STAT signaling pathways, while also recognizing that more research clearly is needed to understand the full nature of these interactions and to best exploit them for the benefit of patients.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Jane Bocchini for her help in Adobe Illustrator, especially with Figures 1 and 2.

Funding

Supported, in part, by grants RP110291 (to DJT) from the Cancer Prevention and Research Institute of Texas, PN1EY016525 (to DJT) from the NIH/NIE, T32 AI055413 (to DJT and CEB) from the NIH/NIAID, and a Stanley A. Plotkin Sanofi Pasteur Pediatric Infectious Diseases Society Fellowship Award (to CEB).