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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochim Biophys Acta. Author manuscript; available in PMC Mar 1, 2013.
Published in final edited form as:
PMCID: PMC3263322
NIHMSID: NIHMS320695

TRAP-1, THE MITOCHONDRIAL Hsp90

Abstract

Protein folding quality control does not occur randomly in cells, but requires the action of specialized molecular chaperones compartmentalized in subcellular microenvironments and organelles. Fresh experimental evidence has now linked a mitochondrial-specific Heat Shock Protein-90 (Hsp90) homolog, Tumor Necrosis Factor Receptor-Associated Protein-1 (TRAP-1) to pleiotropic signaling circuitries of organelle integrity and cellular homeostasis. TRAP-1-directed compartmentalized protein folding is broadly exploited in cancer and neurodegenerative diseases, presenting new opportunities for therapeutic intervention in humans.

Keywords: Mitochondria, apoptosis, permeability transition pore, chaperone, tumor growth

INTRODUCTION

Molecular chaperones of the Hsp90 gene family are considered indispensable regulators of protein folding quality control in virtually every organism [1]. Their structure-function properties, regulation by sequential cycles of ATP hydrolysis, and requirement for hosts of co-chaperones in maintaining the stability, maturation and subcellular trafficking of disparate client proteins have been the subject of intense interest, and are reviewed elsewhere in this monographic contribution. There has also been growing appreciation that chaperone control of proteostasis is often subverted in human diseases, including cancer [2], and may provide therapeutic opportunities [3]. What has also emerged, adding further complexity to this network, is the role of Hsp90-like chaperones compartmentalized in organelles and discrete subcellular microenvironments [4]. As specialized resident chaperones of the endoplasmic reticulum (ER) [5], as well as mitochondria [6], organelle-specific Hsp90 molecules are being recognized not only as subcellular orchestrators of protein folding quality control, but also as hubs for far-reaching signaling pathways of cellular homeostasis.

Much of our knowledge regarding organelle-specific chaperoning comes from the ER-compartmentalized Hsp90-like molecule, Glucose-Regulated Protein 94 (Grp94) [5]. Recognized for its selectivity of client protein recognition and association with ER late folding intermediates, Grp94 functions as a pivotal regulator of ER proteostasis. However, it is now clear that Grp94 also controls Ca2+ balance, proteotoxic stress response, embryonic development, stem cell maintenance, host defense, as well as cell adhesion [710]. We also know that at least some of these pathways are subverted in human diseases, especially cancer, where increased expression of Grp94 has been linked to tumor cell proliferation, metastasis and drug resistance [11].

Conversely, our understanding of Hsp90-like molecule(s) localized to mitochondria has long remained more elusive, complicated by the unique organelle architecture, and the role of chaperones in both mitochondrial preprotein import and intra-organelle proteolysis [12]. Fresh experimental evidence has now brought the mitochondrial-compartmentalized Hsp90-like chaperone, Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP-1) [6], to the forefront of pivotal pathways of mitochondrial integrity, oxidative cell death, organelle-compartmentalized protein folding, and transcriptional responses to proteotoxic stress. In addition, aberrant deregulation of TRAP-1 function has been noted in cancer and neurodegenerative disorders, with potential far-reaching consequences for disease progression and therapeutic intervention.

TRAP-1 Structure-Function

TRAP-1 was first identified as an Hsp90-like chaperone while screening for proteins associated with the cytoplasmic domain of the type 1 Tumor Necrosis Factor Receptor-1 (TNFR1) using the yeast two-hybrid system [13]. A positive clone bound to the NH2-terminal domain of TNFR-1 in vitro, and was designated TNFR1-Associated Protein-1 (TRAP-1) [13]. In an independent yeast two hybrid screen, an Hsp90-like protein of ~75 kD, and designated Hsp75, was also identified that bound the Retinoblastoma (Rb) protein [14]. Functionally, this Hsp75 protein was implicated in subcellular trafficking of Rb during mitosis, as well as heat shock [14]. Sequence analysis later determined that TRAP-1 and Hsp75 were identical molecules.

In terms of tissue distribution, a ~2.4 kb TRAP-1 mRNA was originally detected in normal tissues, but more highly represented in tumor cell lines [13]. By sequence alignment, a TRAP-1 cDNA showed 34% identity and 60% homology with members of the Hsp90 gene family [13]. Current annotations in public databases indicate that a TRAP-1 mRNA of 2225 nucleotides is transcribed from a single locus on chromosome 16p13 in humans. Transcription from this locus is heterogeneous, and gives rise to at least 14 independent mRNA species, with extensive nucleotide polymorphism. At least 6 TRAP-1 protein variants (TRAP-1 1–6) are predicted, characterized by different splicing patterns, amino acid additions, or deletions. Translation of the main TRAP-1 mRNA generates a precursor protein of 704 amino acids, containing a putative mitochondrial import sequence of 59 amino acids, which is removed upon organelle import. A mature TRAP-1 protein of 645 amino acids contains an ATP binding pocket that conforms to the Bergerat ATP-binding fold found in Hsp90, gyrases and MutL in which ATP binds with an unusual kinked conformation with the γ-phosphate group oriented towards the NH2 terminus [15] (Table 1). Sequence analysis suggests that several amino acids in TRAP-1 may be potential targets for post-translational modifications, including acetylation and phosphorylation, whereas the molecule lacks a COOH-terminus MEEVD sequence, which is characteristic of cytosolic Hsp90 (Table 1). Structurally, and similar to yeast Hsp90, TRAP-1 forms a tight homodimer with melting temperature of 55°C [16], thus considerably more stable than Grp94, which denatures at approximately 30°C. Also different from Grp94, TRAP-1 ATPase activity is induced by ~200 fold in response to heat shock. Phylogenetic analysis suggests that TRAP-1 originated from a common ancestor that also gave rise to cytosolic and ER Hsp90s. However, its divergent nucleotide and protein features suggest that TRAP-1 may be a more distant paralogue of this class [17].

Accordingly, there are important functional differences between TRAP-1 and other Hsp90 molecules (Table 1). For instance, TRAP-1 does not bind co-chaperones, p23 or Hop (p60), and cannot substitute for Hsp90 to enable the progesterone receptor hormone-binding state, i.e. receptor maturation [6]. Conversely, similarly to Hsp90, TRAP-1 ATPase activity is inhibitable by the small molecule pocket antagonists, Geldanamycin or radicicol, which have been extensively used to probe Hsp90’s role in protein folding [6] (Table 1).

The ATPase cycle of TRAP-1 has been recently studied in some detail [16]. Differently from Grp94, which remains in an open configuration upon nucleotide binding, but reminiscent of human Hsp90, ATP binding shifts TRAP-1 conformation to a predominantly closed configuration, albeit with slower kinetics than Hsp90 [16]. In this context, TRAP-1 binds nucleotide approximately 10 times tighter than human or yeast Hsp90. However, ATP binding to TRAP-1 is insufficient to commit to nucleotide hydrolysis, and is instead followed by reopening of the chaperone configuration, which occurs faster than the rate of hydrolysis. This has prompted a two-step model for TRAP-1 ATPase cycle with ATP binding followed by irreversible hydrolysis, quantitatively comparable or higher than that of Hsp90 [16].

Subcellular localization studies conducted with newly produced antibodies confirmed sequence predictions that TRAP-1 is indeed a mitochondrial-compartmentalized Hsp90-like chaperone [6, 18]. TRAP-1 organelle import requires the predicted NH2-terminal mitochondrial localization sequence, and appears evolutionary conserved, as a putative TRAP-1 homolog in Drosophila was also localized to mitochondria [6]. In terms of sub-organelle topography, immunogold electron microscopy and biochemical studies suggested that TRAP-1 predominantly accumulates in the mitochondrial matrix [18], with a fraction that may also distribute to the intermembrane space, as more recently proposed [19]. In certain tissues, an extramitochondrial localization of TRAP-1 has also been noted [18], but its potential function was not further investigated. How an almost exclusive mitochondrial localization of TRAP-1 could be reconciled with its potential association with TNFR1 [13], or Rb [14] has so far remained unclear.

TRAP-1 cytoprotection against oxidative stress and mitochondrial cell death

The first function assigned to TRAP-1 was protection against mitochondrial apoptosis (Figure 1). There is now a general consensus that mammalian cells utilize two main circuitries to commit suicide by programmed cell death: an extrinsic pathway centered on the recognition and signaling properties of death receptor molecules at the cell surface [20], and an intrinsic or mitochondrial pathway, centered on the sudden induction of organelle dysfunction by various apoptotic stimuli, and culminating with the release of apoptogenic proteins, most notably, cytochrome c, in the cytosol [21]. There is extensive functional crosstalk between these two pathways, and both converge on the activation of a caspase cascade, ultimately responsible for dismantling the cell’s architecture [22]. In studying the anti-tumorigenic properties of a non-ATP competitive tyrosine kinase inhibitor, the natural compound β-hydroxyisovalerylshikonin (β-HIVS), Masuda and collaborators found that tumor cells treated with this agent or a DNA-damaging chemotherapeutic, VP-16, exhibited decreased expression of TRAP-1, which was associated with enhanced mitochondrial apoptosis [23]. Silencing of TRAP-1 by small interfering RNA (siRNA) reproduced the same phenotype, pointing to a protective role of this chaperone on mitochondrial integrity [23].

Figure 1
TRAP-1 cytoprotection

A similar conclusion was reached in independent studies looking at cell death pathways activated during innate immunity, a host defense mechanism against viral infection and, potentially, oncogenic transformation. One of the lesser studied mediators of this response is Granzyme M, a serine protease stored in granules of effector cell populations, and released in the extracellular environment during target cell killing [24]. Mechanistically, Granzyme M acts on the mitochondria, inducing loss of transmembrane potential, swelling of the matrix, generation of reactive oxygen species (ROS), and discharge of cytochrome c [25]. It turns out that Granzyme M cleavage of TRAP-1 within mitochondria contributed to this cell death response. This proteolytic event caused loss of TRAP-1 ATPase activity, associated with increased production of ROS, cytochrome c release and enhanced apoptosis [25].

Other evidence suggests that TRAP-1 cytoprotection may be important to help cells cope with oxidative stress, and thus thwart the ensuing ROS-mediated apoptosis (Figure 1). Accordingly, changes in TRAP-1 levels induced by anti-tumor agents, β-HIVS or VP-16 were prevented by a ROS scavenger [23], and treatment of normal hepatocytes with the iron chelator, deferoxamine decreased TRAP-1 expression, while concomitantly elevating ROS production in these cells [26]. Mirroring these results, stable expression of TRAP-1 attenuated the effects of deferoxamine, reducing ROS production and the appearance of markers of oxidative stress [26]. The idea of TRAP-1 as a stress-responsive cytoprotective chaperone, whether following oncogene expression, ROS production or DNA damage, gained further support from independent studies. Accordingly, microarray analyses identified TRAP-1 as one of the target genes upregulated by the Myc oncogene [27], and tumor cells rendered chronically resistant to cisplatin or other chemotherapeutic agents consistently showed an increase in TRAP-1 expression levels [28]. Functionally, these TRAP-1-positive cells become resistant to oxidative stress (H2O2)-induced apoptosis, with decreased expression of oxidative markers, and reduced activation of apoptotic and non-apoptotic pathways [29]. How modulation of TRAP-1 levels is controlled under conditions of cellular stress is still largely unexplored. Although some of these mechanisms are likely transcriptional, very little information is available concerning the requirements of TRAP-1 gene expression under basal or stress conditions.

But what are the regulators of TRAP-1 in buffering ROS production in mitochondria and opposing oxidative cell death? A clue to this question came from studies looking at neuronal cell death in neurodegenerative diseases, in particular Parkinson’s disease (PD) [30]. It had long been appreciated that selective loss of dopaminergic neurons in the substantia grigia is a hallmark of PD [30]. There is also evidence from postmortem analyses of primary human specimens of mitochondrial defects in these patients, especially deficiencies of complex I [31]. One of the key molecular players in this pathway was uncovered in genetic studies of patients with recessively inherited PD, which identified hosts of inactivating mutations in a mitochondrial-localized serine-threonine kinase, designated PTEN-Induced Putative Kinase, or PINK1 [32]. Similar mutations were later found in late-onset PD patients, suggesting that deregulation of PINK1 function due to acquired mutation(s) could function as a disease-driver in neurodegeneration. Functionally, PINK1 had been linked to an evolutionary-conserved anti-apoptotic pathway [33], but the requirements of this response, and the potential target substrate(s) of its kinase activity, if at all involved in cytoprotection, had not been identified. Against this backdrop, Pridgeon and colleagues found that PINK1 cytoprotection required TRAP-1, and, in fact, depended on an active mechanism of PINK1 phosphorylation of TRAP-1 within mitochondria, in vivo [19]. In turn, PINK1-phosphorylated TRAP-1 opposed mitochondrial dysfunction and suppressed oxidative stress-induced apoptosis [19] (Figure 1). Importantly, variants of PINK1 that carried the inactivating mutations found in PD patients were unable to phosphorylate TRAP-1, and to reverse apoptosis [19]. On this basis, PINK1 phosphorylation seems to provide a necessary step to enable TRAP-1 cytoprotection and preserve mitochondrial integrity, at least in model of neuronal cells, but the identity of potential downstream target(s) of TRAP-1 had remained elusive [19], a point that came into better focus from results obtained in tumor cells.

Exploitation of TRAP-1 cytoprotection in cancer

Although there had been suggestions that TRAP-1 cytoprotection was somehow exploited in cancer, the actual distribution of this chaperone in normal versus malignant tissues in vivo had not been formally investigated. Recent studies addressed this question, and found that TRAP-1 levels were consistently elevated in primary human tumor specimens, while present at very low levels, and sometimes undetectable in the corresponding normal tissues, in vivo [34]. In prostate cancer, TRAP-1 differential expression proved an attractive biomarker of disease, being abundantly represented in prostatic intraepithelial neoplasia (PIN), all Gleason grade primary tumors, and metastatic disease to bone and lymph nodes, while largely undetectable in normal prostatic epithelium or benign prostatic hyperplasia [35]. Unexpectedly, these studies also identified a previously unrecognized pool of Hsp90 localized to mitochondria [34]. Similar to the data with TRAP-1, also the pool of mitochondrial Hsp90 was differentially expressed in tumors, in vivo, while undetectable in the corresponding normal tissues [34].

Additional studies aimed at identifying potential molecular partners of mitochondrial Hsp90 and TRAP-1 in tumor cells [34]. Here, biochemical evidence demonstrated that both of these two chaperones physically associated with cyclophilin D (CypD), a matrix peptidyl prolyl isomerase (PPI), which is a physical component of the organelle permeability transition pore (PTP) [34]. There is a general consensus that opening of the mitochondrial PTP is a key molecular prerequisite for induction of mitochondrial apoptosis [21]. Biochemically, this process, also called mitochondrial permeability transition, comprises a cascade of events, which include sudden increase in mitochondrial permeability to solutes, swelling of the matrix, dissipation of transmembrane potential, remodeling of the cristae, and ultimately rupture of the outer membrane with release of apoptogenic cytochrome c into the cytosol [21].

Despite the broad acceptance of this model, the actual molecular composition of the PTP is still a matter of debate [36], as proteins long associated with CypD, including the voltage-dependent anion channel (VDAC) [37], and the adenine nucleotide translocator (ANT) [38], do not seem to participate in apoptosis, at least based on the negative phenotype of knockout studies. There is also uncertainty as to whether the model of a rigid mitochondrial PTP faithfully recapitulates how organelle cell death is actually initiated [21]. In fact, a more dynamic model has been proposed, in which aggregated and/or misfolded proteins cluster at the outer mitochondrial membrane as a result of stress conditions, including Ca2+ imbalance, and have the ability to conduct solutes, thus initiating permeability transition [39] (Figure 2). These controversies aside, knockout studies have unambiguously demonstrated that CypD is indeed required for PTP opening and oxidative stress-induced apoptosis [40, 41]. This concept has also been validated in animal studies, as deletion of CypD and abrogation of its pro-apoptotic properties protected against aberrantly increased cell death in various disease models [42], including neurodegeneration [43], in vivo. Against this backdrop, a functional interaction between TRAP-1 and CypD [34] fits well with the ability of this chaperone to modulate oxidative stress responses, and antagonize ROS-mediated cellular damage [19, 23, 25]. Conceptually, this fits well also with a proposed CypD regulation of mitochondrial permeability transition [21] mediated by protein misfolding at the outer membrane (Figure 2), a model that postulates a critical role of chaperone-mediated protein (re)folding in antagonizing cell death [39].

Figure 2
Regulation of mitochondrial permeability pore opening by an organelle-specific chaperone network

However, available evidence is still insufficient to conclude that CypD is the first mitochondrial client protein of TRAP-1, refolded in a closed PTP configuration by the chaperone ATPase activity. For instance, the structure-function relationship of a TRAP-1-CypD complex has not been characterized, so that it is unclear whether this recognition conforms to the paradigm of an Hsp90-client protein interaction. On the other hand, recent studies were carried out to address the requirement of TRAP-1 ATPase activity in regulating CypD-dependent mitochondrial permeability transition [21]. Surprisingly, despite the wealth of structurally diverse small molecule Hsp90 antagonists in (pre)clinical development [3], none of these compounds was shown to have the ability to accumulate in mitochondria, and trigger permeability transition [44]. Conversely, a synthetic peptidomimetic inhibitor of Hsp90 ATPase activity, Shepherdin [45], which was structurally modeled on the Hsp90-survivin binding interface [46], readily accumulated in mitochondria [34]. It is still unclear what enables Shepherdin to penetrate inside mitochondria, but domain swapping experiments suggested that this property was likely mediated by the highly positively charged Antennapedia cell penetrating moiety placed at Shepherdin’s NH2-terminus, which was also responsible for its distribution to all submitochondrial compartments [34]. Functionally, Shepherdin inhibition of TRAP-1, and likely also mitochondrial Hsp90 ATPase activity triggered rapid collapse of mitochondrial integrity in tumor cells [34], with loss of transmembrane potential, release of cytochrome c, caspase activation and extensive cell death [34, 45]. Importantly, this cell death pathway required CypD PPIase function [34, 45], suggesting that opening of the PTP was an obligatory molecular prerequisite in cell death induction [21]. Conversely, Shepherdin did not affect normal cells [34, 45], in keeping with the low level of TRAP-1 and mitochondrial Hsp90 in organelles of normal tissues [34].

Altogether, these data support a model in which TRAP-1 antagonizes CypD pore-forming properties, potentially by protein (re)folding [39], and blunts the main cell death pathway maintained by CypD [21], namely oxidative apoptosis [40, 41] (Figures 1, ,2).2). In cancer, such adaptive response may be important to endow tumor cells with the ability to thrive in unfavorable environments, i.e. rich in ROS. This is consistent with the high levels of TRAP-1 (as well as of mitochondrial Hsp90) found in various types of cancer, in vivo [34], suggesting that this pathway is not only broadly exploited for disease maintenance, but may also favor the acquisition of additional malignant traits, in vivo. Accordingly, over-expressed TRAP-1 in a fraction of colorectal cancer cases has been associated with multi-drug resistance via inhibition of mitochondrial apoptosis [28], and genome-wide association studies also in colorectal cancer identified a 72-gene signature, comprising TRAP-1, which correlated with drug resistance and disease progression [47]. Mechanistically, recent proteomics data in non-tumor cell types identified TRAP-1 as a bona fide hypoxia-regulated gene, whose expression is significantly increased under conditions of low oxygen tension, even though the genetic requirements of this response, whether transcriptional or post-transcriptional, have not been further elucidated [48].

Although CypD [34], and PINK1 [19], are pivotal for TRAP-1 cytoprotection, the existence of additional regulator(s) of this pathway has been intensely pursued. Using a proteomics approach, a novel TRAP-1-interacting molecule was recently identified as a mitochondrial-localized, lower molecular weight isoform of the Ca2+ binding protein, Sorcin [49]. This is a ubiquitously expressed member of the penta EF gene family of Ca2+-dependent regulators of protein interactions, and its binding to TRAP-1 has been implicated in increasing the stability of the chaperone presumably against proteolytic degradation in mitochondria [49]. Functionally, this was also associated with inhibition of apoptosis and development of a multi-drug resistant phenotype in colon cancer cells [49] (Figure 1). In an independent study, a biochemical approach of size exclusion chromatograpy of normal or tumor mitochondrial extracts identified the mitochondrial-localized chaperonin, Hsp60 as an additional CypD-associated molecule, in vivo [50]. Experiments with recombinant proteins confirmed that Hsp60 bound CypD directly, and that this interaction was also required to antagonize CypD-dependent mitochondrial apoptosis [50] (Figure 2). Similar to the paradigm of other mitochondrial chaperones that interact with CypD, Hsp60 was also differentially over-expressed in organelles of tumor cells, compared to normal tissues, where no biochemical interaction between “normal” Hsp60 and CypD was demonstrated [34]. In terms of disease relevance, Hsp60 inhibition of CypD-mediated permeability transition was important for tumor maintenance, as stable silencing of Hsp60 in model glioblastoma cell types inhibited intracranial tumor growth and prolonged animal survival, in vivo [34].

Targeting TRAP-1 for subcellularly-compartmentalized cancer therapeutics

Taken together, the data above suggest the existence of not just one molecule, but of a network of mitochondrial-localized molecular chaperones, potentially regulated by post-translational modifications, i.e. phosphorylation [19], and stabilized by Ca2+ sensing proteins [49] (Figure 1), which antagonizes CypD-dependent pore-forming properties [21], and oxidative cell death [40, 41] in tumors, potentially via ATPase-directed protein (re)folding (Figure 2). Because of its nearly ubiquitous differential expression in cancer, as opposed to normal tissues, these properties may make this mitochondrial chaperone network a potentially attractive target for cancer therapy [3].

Proof-of-concept results obtained with Shepherdin supported this model [34, 45], and reiterated the feasibility of selective targeting of Hsp90 chaperones specifically in specialized subcellular compartments. To further test this concept, and circumvent the limitations of peptidomimetics in drug development, a new class of small molecule Hsp90 inhibitors selectively engineered to accumulate in mitochondria was recently synthesized. These compounds, designated Gamitrinibs (GA mitochondrial matrix inhibitors) have a combinatorial design, in which the ATPase inhibitory component of 17-allylaminogeldanamycin (17-AAG) is fused through a linker region to a “mitochondriotropic” moiety, provided by either 1–4 tandem repeats of guanidinium or, alternatively, tryphenylphosphonium [44].

Accordingly, Gamitrinibs readily accumulated in mitochondria of normal or tumor cells, and inhibited the ATPase activity of TRAP-1 (and likely of mitochondrial Hsp90), inducing collapse of mitochondrial integrity, and all the biochemical hallmarks of PTP opening [44] (Figure 3). When tested in a preclinical model of prostate cancer, Gamitrinibs indistinguishably induced apoptosis of androgen-dependent or –independent prostate cancer cells [35], and killed chemoresistant prostate cancer cells that over-expressed the P-glycoprotein transporter, independently of pro- or anti-apoptotic Bcl-2 proteins, as general orchestrators of mitochondrial cell death [51]. Conversely, Gamitrinibs had no effect on normal cell types, including normal prostatic epithelium [35, 51]. Consistent with a selective, “mitochondriotropic” mechanism of action, Gamitrinibs did not inhibit the chaperone activity of cytosolic Hsp90, with no difference in the level of expression of client proteins, Akt or Chk1, or of the Heat Shock Factor (Hsf)-regulated chaperone, Hsp70 [44, 51]. When analyzed in xenograft or genetic mouse models of prostate cancer, Gamitrinibs had encouraging anticancer activity and tolerability, inhibiting localized or bone metastatic disease, with no overt organ or systemic toxicity, in vivo [51, 52].

Figure 3
TRAP-1 control of cell survival and stress response

Altogether, the results obtained with Shepherdin [34, 45], and, now, with Gamitrinibs [51, 52], argue that TRAP-1 ATPase activity is required to maintain tumor cell viability, and that genetically hetergeneous tumors may become “addicted” to this survival mechanism (Figure 3). But is the effect of these agents truly related to changes in the protein folding environment in mitochondria? To begin probing this question, more recent experiments were carried out with sub-optimal concentrations of Gamitrinibs, enough to alter ATPase-directed protein folding within the organelle’s confines, but insufficient to trigger irreversible collapse of mitochondrial integrity and cell death. When used under these conditions, the Gamitrinib variant containing triphenylphosphonium as mitochondriotropic moiety [44] initiated a complex signaling pathway in tumor cells, characterized by accumulation of unfolded, i.e. insoluble proteins in mitochondria, and expression of organelle stress markers [53]. These are features of an unfolded protein response (UPR), a cellular and transcriptional program that has been well-characterized in response to ER damage [54, 55], but far less defined when it comes to mitochondria [12] (Figure 3).

Reminiscent of an ER UPR [54, 55], tumor cells treated with low dose Gamitrinib also underwent dramatic changes in gene expression profile, with upregulation of stress response transcription factor, CCAAT Enhancer Binding Protein (C/EBPβ), and its dimerization partner C/EBP homology protein, CHOP, and increased expression of several molecular chaperones, including Hsp70 [53] (Figure 3). A third phenotype observed under these conditions was a dramatic activation of autophagy [53] (Figure 3), a process of self-digestion of subcellular organelles that aims at maintaining cellular energy levels under stress conditions [56]. The role of autophagy in cancer is still debated, as to whether this functions as a cell death mechanism, a cell survival phenotype, or both [57]. In the case of Gamitrinib, activation of autophagy was clearly a compensatory survival mechanism, as its genetic or pharmacologic inhibitors converted non-cytotoxic concentrations of Gamitrinib into effective tumor cell killing regimens [53].

One of the transcriptional networks that was shut down by low dose Gamitrinib was NFκB, which resulted in a concomitant loss of several NFκB downstream target genes [53]. There has been intense interest in NFκB as a survival pathway almost ubiquitously exploited in tumors, and the possibility that targeting its function may enhance the efficacy of anticancer therapies has been intensely pursued [58]. Accordingly, combining low, non-cytotoxic concentrations of Gamitrinib with a cell death inducer normally antagonized by NFκB, i.e. Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL) [59], dramatically enhanced anticancer activity, killing disparate tumor cell types, and inhibiting intracranial glioblastoma growth in mice, with no detectable toxicity, in vivo [53]. These findings are consistent with other data linking changes in TRAP-1 expression to modulation of gene transcription pursued in recent molecular profiling studies [60]. Here, tumor cell types that were differentially induced or silenced for TRAP-1 expression exhibited upregulation of proliferation-associated genes, or regulators of cell motility and metastatic spread, respectively [60].

Outstanding questions and future directions

Originally considered a somewhat elusive member of the Hsp90 gene family, TRAP-1 has now emerged as a novel and potentially pivotal regulator of mitochondrial integrity. Its link to the control of apoptosis is now fairly established, especially in the context of oxidative stress, and some of its key molecular partners in this pathway, for instance CypD [34], and PINK1 [19], have been identified. It is also clear that the TRAP-1 pathway of cytoprotection is subverted in human diseases, most notably, cancer and neurodegeneration. Despite the wealth of new data, many open questions still remain. For instance, targeting TRAP-1 expression or function triggers a mitochondrial UPR that globally reprograms the cellular transcriptome [53, 60]. But how are potential signals of proteotoxic stress generated within mitochondria relayed to the nucleus to control gene expression? The answer to this question is presently unknown, but a number of scenarios can be envisioned, including production of second messengers, for instance Ca2+ imbalance [61], or soluble peptides generated from mitochondrial proteotoxicity [62], that can ultimately affect nuclear gene expression under these conditions. In a fanciful scenario, it is also possible that proteotoxic signals originating from mitochondria may spread to activate the potent, multi-pronged ER-stress sensing machinery [54, 55] to further amplify nuclear gene expression. Such concept of an inter-organelle signaling network has been proposed earlier [63], and this putative subcellular cooperation may be well suited to generalize the cellular response to stress. After all, mitochondria and ER share extensive areas of physical contact [64], populated by diverse classes of chaperones ideally positioned to process proteotoxic stress signals, and maximize compensatory gene expression programs [61].

Second, targeting TRAP-1 triggers massive activation of autophagy as a compensatory response likely to eliminate damaged mitochondria, and more recent data have shown that the TRAP-1-phosphorylating kinase, PINK1 [19], is also an indispensable regulator of this process [65]. But how is autophagy activated after TRAP-1 targeting? We know that ER stress is linked to activation of autophagy, and that this process is also important for cell survival [66], but the circuitries linking TRAP-1 ATPase activity, control of mitochondrial protein folding and stimulation of autophagy are not known.

And, finally, is TRAP-1 as good as a cancer therapeutic target as some of the preclinical data with Shepherdin [45], or Gamitrinibs [44, 51, 52] may suggest? Despite the intense interest in exploiting the nodal properties Hsp90 for cancer therapeutics [2], the results of clinical trials with structurally diverse small molecule Hsp90 inhibitors have not been particularly overwhelming [3]. Can it be that this is because the compounds currently in the clinic do not penetrate mitochondria [44], and, therefore, leave unscathed the pivotal survival role of compartmentalized Hsp90 and TRAP-1? And, if so, does this mean that we have to contemplate the additional concept of “subcellular targeting” in drug development, as only ATPase antagonists directed to subcellular compartments [67] can unlock the potential of molecular chaperones as universal drug targets, and produce durable responses in cancer patients [68]? Given the fast pace of research in this exciting field of investigation, the answers to some of these questions will undoubtedly be forthcoming.

HIGHLIGHTS

  • TRAP-1 is a mitochondrial-specific Hsp90 chaperone
  • Antagonizes mitochondrial apoptosis and oxidative stress
  • Regulates the mitochondrial permeability transition pore
  • Controls the protein folding environment in mitochondria
  • Couples to the cellular stress response and gene expression

Acknowledgments

This work was supported by NIH grants CA140043, CA78810 and CA118005.

ROLE OF THE FUNDING SOURCE

The funding source had no involvement in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

Footnotes

CONFLICT OF INTEREST

The authors declare that no conflict of interest exists.

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