Abstract
The heat shock proteins (Hsps) are a family of highly conserved proteins involved in the regulation of numerous cellular processes including those associated with cancer. Inhibiting the function of these Hsps, specifically Hsp70 and Hsp90, is a major strategy used in the development of new cancer therapies. Numerous Hsp90 inhibitors have been evaluated in the clinic, and while some have experienced success, many have produced disappointing results. One reason explaining their failure is that they induce a cytoprotective response that protects cancer cells from the negative effects of Hsp90 inhibition. In order to maximise the therapeutic outcomes, dual inhibition of Hsp70 and Hsp90 can be employed to overcome cell rescue mechanisms induced by monotherapies. In this chapter, we discuss dual inhibition of Hsp70 and Hsp90 using small molecules and evaluate the potential of this strategy for the development of cancer therapeutics.
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1 Introduction
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the maintenance of protein homeostasis in the cell [1–3]. Hsp90 assists in the folding, stabilisation, activation and degradation of numerous cellular proteins. Hsp90 interacts with over 400 client proteins [4], many of which are associated with cancer [1]. Upon activation, these client proteins assist in disease progression, which makes Hsp90 a regulator of many disease-causing pathways. Consequently, Hsp90 inhibition has emerged as a promising strategy for the treatment of diseases involving aberrant protein structure and function, including cancer.
Hsp90 exists as a homodimer, where each monomer contains an amino (N-) terminus, a middle domain and a carboxy (C-) terminus (Fig. 1). The N-terminus contains an ATP-binding site, the middle domain contains binding sites for client proteins and co-chaperones, and the C-terminus serves as the dimerisation domain and also contains binding sites for co-chaperones. It is well understood that clinical Hsp90 inhibitors or “classical inhibitors” target the N-terminal ATP-binding site of Hsp90, impacting Hsp90’s protein folding cycle (Fig. 1).
Hsp90’s protein folding cycle and the mechanism of classical Hsp90 inhibition. (a) Unfolded client proteins are delivered to Hsp90 by Hsp40 and Hsp70 via interactions with the co-chaperone Hsp70/Hsp90 organising protein (HOP). (b) Hsp90 utilises ATP hydrolysis to change conformation. (c) Fully folded client protein is released
The first Hsp90 inhibitor, geldanamycin, was identified in 1994, and its derivative tanespimycin (17-AAG) entered the clinic as a cancer therapeutic in 1999 [5] (Fig. 2). Both of these analogs are from the ansamycin class of compounds. Since 1995, Hsp90 inhibitor drug candidates have been steadily entering the clinic, with a total of 15 different drugs being tested as monotherapies in clinical trials since 1999 [6, 7]. The ansamycin molecules are reported to inhibit ATP from binding to Hsp90 (Fig. 1b). However, recent evidence shows that they are highly promiscuous and, as such, are likely targeting many proteins, not just Hsp90, which would explain their failure in clinical trials.
The two classes of classical Hsp90 inhibitors: ansamycin analogs and ATP-like molecules
More recent Hsp90 inhibitor drug candidates mimic the ATP molecule (Fig. 2). Several of these molecules are still in clinical trials and are being used to treat many cancer types including breast cancer, non-small cell lung cancer (NSCLC), melanoma, renal cell carcinoma (RCC), multiple myeloma (MM), gastrointestinal stromal tumour (GIST), castrate-resistant prostate cancer (CRPC) and several types of leukaemia [6, 7]. Clinical inhibitors block ATP from binding, leading to the inhibition of protein folding. However, clinical trial results showed that when used as single agents, these Hsp90 inhibitors were not highly efficacious and have generated disappointing patient outcomes.
As a single agent, 17-AAG had significant side effects [8] and was subsequently dropped from clinical trials. Three other recent monotherapy regiments in clinical trials involved (1) 17-DMAG to treat CRPC, melanoma or acute myeloid leukaemia; (2) ganetespib to treat breast cancer and NSCLC; and (3) IPI-504 to treat NSCLC and GIST [9, 10] (Fig. 2). However, patient’s responses were modest, where 17-DMAG caused a response in 2 of 28 patients (7%) HER2+ breast cancer patients, ganetespib caused a partial response (PR) in 2 of 22 (9%) breast cancer patients, and IPI-504 caused a PR in 5 of 76 (7%) NSCLC patients and 1 in 36 (3%) GIST patients [7, 10, 11]. 17-DMAG was also reported to cause a complete response (CR) in a single patient that had CRPC, three CR in acute myeloid leukaemia and one PR in melanoma [8, 11]. Thus, Hsp90 inhibitors have shown positive clinical benefit for patients, although the low response rate is a significant concern. Classical Hsp90 inhibitors continue to enter into clinical trials as both single and combination therapies. Currently, there are 32 active studies evaluating the effects of these drugs on numerous cancer types (Table 1).
The low response rate coupled with hepatotoxicity, ocular toxicity and in one case mortality has caused the suspension of most clinical trials using Hsp90 inhibitors as single-agent chemotherapeutics [12, 13]. The limited effectiveness of these Hsp90 inhibitors appears to be due to several key factors. The first is that the ATP-binding site, where these clinical molecules interact with Hsp90, has a binding pocket that is similar to several classes of proteins, specifically DNA polymerases and tyrosine kinases [14]. Thus, classical inhibitors appear to bind to other proteins in addition to Hsp90, thereby producing off-target effects [15–25] and potentially contributing to the observed toxicity associated with these drugs.
Second, resistance and anti-apoptotic pathways are activated immediately upon patient treatment with these clinical Hsp90 inhibitors. This resistance is a result of the specific types of Hsp90 inhibitors activating the cellular heat shock response (HSR) [26]. The HSR is an evolutionary response that is triggered when the cell is under stress and was first discovered by subjecting cells to high temperatures. Triggering a HSR induces high levels of heat shock proteins (Hsps), which are responsible for refolding the aggregated and misfolded proteins that accumulate in the stressed or rapidly growing cell, and they aid in protein degradation [27]. The HSR facilitates cell survival by activating resistance mechanisms and anti-apoptotic pathways [28, 29].
Specifically, cellular stress leads to releasing heat shock factor 1 (HSF-1) from Hsp90 (Fig. 3b) [30–34]. Transport of HSF-1 into the cytoplasm is inhibited leading to a build-up of HSF-1 in the nucleus [35]. HSF-1 then forms a trimer in the nucleus, which is extensively phosphorylated [36]. The HSF-1 trimer binds to specific sequences known as heat shock elements (HSE) in DNA promoters and induces transcription of genes encoding for itself and multiple cellular chaperones, including heat shock protein 27 (Hsp27), heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70), in order to rescue the cell from the accumulating unfolded proteins [37, 38] (Fig. 3c). In the absence of stress, promoters for these genes are occupied and unavailable [39, 40]. The mRNAs encoding for inducible and constitutive Hsp70 (HSPA1A and HSPA8, respectively) are produced during the HSR, as well as mRNA that encodes for HSF-1, Hsp40 and Hsp27 (Fig. 3d). These Hsps attempt to rescue the cell from the unfolded protein that is accumulating. The high levels of Hsps refold the aggregated and misfolded proteins that accumulate in the stressed cell, and selected Hsps can also aid in protein degradation [27].
Depiction of the widely accepted model of heat shock and the induction of the heat shock response (HSR). (a) Stress from heat shock or clinical Hsp90 inhibitors triggers an accumulation of unfolded proteins in the cell. (b) The Hsp90 protein complex collects these unfolded proteins, which causes the release of HSF-1 from the protein complex. (c) HSF-1 then forms an active trimer, which translocates to the nucleus and binds to DNA. (d) The mRNA of the heat shock proteins is transcribed from the DNA. (e) The mRNA is then translated into the heat shock proteins, which can then facilitate folding of the previously accumulated unfolded proteins
Similar to the stress caused by high temperatures, the excessive growth of cancer creates stress in cells, and thus cancer cells produce high levels of Hsps. These Hsps maintain protein folding and protein degradation and repair the large quantity of proteins required for rapid cell division, as well as stabilising mutated oncoproteins [27, 41]. This high level of Hsps, including Hsp90, is why Hsp90 inhibitors are a promising treatment for cancer. However, inhibiting Hsp90 function using the clinical inhibitors is well established to produce high levels of Hsp70; indeed, Hsp70 is often used as a pharmacodynamic marker to determine if Hsp90 is being inhibited by classical inhibitors [42–44].
Hsp70 assists in the delivery of specific clients to Hsp90 [45], as well as functioning as an independent chaperone that facilitates protein translocation; stabilises anti-apoptotic proteins; plays a key role in cellular resistance; and prevents apoptosis [46–49]. Thus, inducing high levels of Hsp70 (>6-fold over background) such as those observed when treating cells and patients with the classical Hsp90 inhibitors is problematic for killing cancer cells. Indeed, the high production of Hsp70 likely plays a key role in the disappointing clinical results [20–24]. In response to these poor results for classical inhibitors, two strategies have been employed.
The first approach, which is the development of Hsp90 inhibitors that target sites other than the ATP-binding site of Hsp90, offers alternative mechanisms for blocking Hsp90’s activity. Specifically, inhibiting activity at the C-terminus of Hsp90 does not induce a HSR, nor lead to the upregulation of the Hsps [20–24, 50–55]. Thus, this approach may succeed as a single treatment as it does not produce the anti-apoptotic or resistance observed with the classical inhibitors. The second approach, which is already being used in the clinic, is a combination of classical Hsp90 inhibitors with other forms of therapy. This second approach is discussed in this chapter.
Dual inhibition is a rapidly developing area, and there are a large number of clinical trials and patents being reported in this field. Of the 32 active clinical trials, 17 are studying the effects of Hsp90 inhibitors with one or more other drugs (www.clinicaltrials.gov database). Recent patents include the use of combination treatments utilising Hsp90 inhibitors with Hsp27 or Hsp70 inhibitors (patent number WO-2007041294). Yukimasa patented results using a classic Hsp90 inhibitor (KW-2478) in combination with an Hsp70, Hsp27 or BCL2 cancer treatment drug (patent number WO-2007028387). Kyowa Hakko Kogyo patented the treatment of a classical inhibitor 17-AAG being used in combination with a kinase inhibitor such as gefitinib or a proteasome inhibitor such as bortezomib (patent number WO-2008108386). Astex Therapeutics has patented the drug combination of the classical inhibitor AT9283 with cyclin-dependent kinase inhibitors or aurora kinase inhibitors (WO2008044045). Patent activity on dual inhibitors shows that this line of investigation is being vigorously pursued.
2 Hsp90 Inhibition-Based Combination Treatment
Hsp90 is vital for most cancer cells because of its pivotal role in modulating protein conformation and maturation [56–60]. To date, more than 400 proteins are regulated by or associated with Hsp90, and as such they are called Hsp90 client proteins [4]. About half of these clients are critical for cancer cell growth [61], including transmembrane tyrosine kinases (HER2 and EGFR), metastable signalling proteins (Akt, K-ras and Raf-1), mutated signalling proteins (p53 and v-Src), chimeric signalling proteins (Bcr-Abl), cell cycle regulators (Cdk4 and Cdk6) and steroid receptors (androgen, oestrogen and progesterone receptors) [62–67]. When mutated or deregulated, these clients promote cancer growth. Cancer cell proliferation and survival [68, 69] are facilitated by Hsp90 by maintaining tumours and homeostasis and helping cells to adapt to unfavourable or stressful microenvironments that include heat, hypoxia, free radical production, radiation and chemotherapy [68–71].
Because of its key roles in tumour development, Hsp90 has emerged as a promising target for cancer therapy [63, 72–74]. Inhibiting Hsp90 has involved targeting all three domains: the N-, middle and C-domains as a paradigm of network-oriented drug discovery [63, 71, 75, 76]. Indeed, success at suppressing cancer cell growth has been reported in both preclinical and clinical studies [5, 50, 51, 54, 61, 72–74, 77–80]. Although there are currently 32 clinical trials involving Hsp90 inhibitors, only three unique structures are involved in these studies and are being tested on patients. All three target the N-terminal ATP-binding site of Hsp90 [54] and more than half of these clinical trials are using the compounds in conjunction with other therapies [81–84].
Given Hsp90’s central regulating role in cancer development and its close relationship with numerous key oncogenic proteins, studies are now exploring if Hsp90 inhibitors can sensitise tumours to other chemotherapeutic agents. Developing combination therapies using Hsp90 inhibitors and other types of anticancer agents with a distinct mechanism of action is one avenue that is currently being investigated. Encouragingly, Hsp90 inhibition-based combination treatments of cancer have proven to be more effective and more successful than monotherapies in clinical trials, indicating a promising future for anticancer treatment. In this section, we focus on the investigation and achievement of combination treatments based on direct Hsp90 and Hsp70 dual inhibition.
2.1 Dual Hsp90 and Hsp70 Inhibition
The disappointing clinical results of Hsp90 inhibitors are likely connected to induction of the HSR, which upregulates Hsp70 and Hsp27 as well as HSF-1 [74, 85, 86]. Induction of Hsp70 produces the undesirable effect of counteracting the efficiency of Hsp90-based treatment, and it has been identified as a hallmark of N-terminal Hsp90 inhibitors [87–97] (Fig. 4a). The C-terminal modulators, which do not target the ATP site on Hsp90, do not induce HSF-1 nor the HSR [50, 51, 54, 77–80, 98] (Fig. 4b and c). Herein we discuss two approaches to dual inhibition of Hsp70 and Hsp90 including combining small-molecule inhibitors of both Hsp70 and Hsp90 and combining Hsp70 silencing with Hsp90 inhibitors.
Hsp90 inhibitors in clinical trials. (a) Structures of current Hsp90 inhibitors in clinical trials, all targeting the ATP-binding site at the N-terminus. (b) Structures of Hsp90 inhibitors targeting alternative sites on Hsp90. (c) Diagram of Hsp90 showing the binding locations of each inhibitor. (d) The interactions shown (ATP binding and TPR-containing co-chaperones) are modulated by the inhibitors
There are several rescue mechanisms that are induced with the HSR. First, Hsp90 is induced and can still perform its protein folding and regulatory role. Second, Hsp70 is also induced and may compensate for some of Hsp90’s inhibited functions by assisting in protein folding, preventing protein aggregation and regulating protein complex assembly or disassembly [99, 100]. Third, Hsp70 actively participates in the protection of cancer cells from both extrinsic and intrinsic apoptosis [99]. Ectopic overexpression or induced endogenous levels of Hsp70 promote cancer cell survival by effectively inhibiting lysosomal membrane permeabilization [49], death receptor pathway [48], mitochondria-initiated signalling for caspase-dependent apoptosis [46, 47, 101–104] as well as AIF-associated caspase-independent apoptosis [105, 106].
Evidence of Hsp70’s critical role in apoptosis was seen when silencing Hsp70 expression using antisense oligonucleotides or ectopic transfection produced extensive apoptotic cancer cell death [48, 107, 108]. Furthermore, Hsp70 inhibition triggers an antitumour immune response by blocking the Hsp70-induced activation of myeloid suppressive cells (MDSC), which have the capacity to suppress both the cytotoxic activities of natural killer (NK) and NKT cells and the adaptive immune response mediated by CD4+ and CD8+ T cells [109–113]. All of these factors make dual inhibition of Hsp90 and Hsp70 an optimal cancer therapy.
Using a combination treatment of Hsp90 and Hsp70 inhibitors may not only neutralise the issues associated with N-terminal Hsp90 inhibition, but it may also amplify their anticancer efficiency based on their multiple and independent mechanisms of action. Encouragingly, Hsp70 silencing using siRNA (small interfering RNA), shRNA (small hairpin RNA) or cDNA (complementary DNA in the reversed orientation) of Hsp70 has proven to successfully and synergistically potentiate Hsp90-based anticancer treatment in both solid tumours and leukaemia [48, 89, 90, 95]. However, only a few scientific studies on dual inhibition using small-molecule inhibitors have been published, mainly because only a limited number of compounds effectively modulate Hsp70’s function [54, 90, 114]. MAL3-101, Pifithrin-μ and VER-155008 (Fig. 5c) are the only three drugs that specifically target Hsp70 and show synergism or an additive effect in combination treatment with Hsp90 inhibitors both in vitro and in vivo [89, 93, 115–117].
Hsp70 inhibitors. (a) Diagram of the structure of Hsp70 indicating the binding sites of inhibitors: VER-155008, Pifithrin-μ and ADD70. (b) Hsp70 inhibitor MAL3-101 binds at the interface between the Hsp70/Hsp40 complexes. (c) Structures of Hsp70 inhibitors
VER-155008 binds to the ATP-binding site in Hsp70, blocking Hsp70’s access to ATP and halting Hsp70’s function by denying it energy to perform (Fig. 5a). VER-155008 was developed through a structure-based X-ray crystallographic design [118]. It is also the first molecule to target the ATP-binding domain of Hsp70 protein [116]. Treatment of cancer cells with VER-155008 showed antiproliferative activity in many types of human cancer cells, including colon cancer [116], breast cancer [118], multiple myeloma [89] and acute myeloid leukaemia [117]. As expected, VER-155008 shows synergistic or additive combination effects with Hsp90 inhibitors in preclinical cancer treatments [89, 116, 117].
Pifithrin-μ binds to the substrate-binding domain of Hsp70 and blocks other substrates from effectively interacting with that site (Fig. 5). Pifithrin-μ specifically targets the inducible isoform of Hsp70, without binding to the constitutive Hsp70 or to Hsp90 [119]. It interferes with the C-terminal substrate-binding domain of Hsp70 and disrupts its association with client proteins, causing cell cycle arrest and significant apoptosis at low micromolar concentrations. This leads the loss of Hsp70 function, as it can no longer interact with substrates.
Like Pifithrin-μ, MAL3-101 (Fig. 5) binds to Hsp70 at the substrate-binding site. It blocks Hsp70’s essential cellular function by inhibiting the ability of Hsp40 co-chaperones to stimulate Hsp70 ATPase activity [120, 121] (Fig. 5b). Hsp40 docks to Hsp70 during substrate transfer of unfolded client proteins (Fig. 1); thus, inhibiting this binding event halts the transfer of unfolded proteins and impacts protein homeostasis, thereby inducing cell death.
2.1.1 Combination of VER-155008 and NVP-AUY922 in Multiple Myeloma Treatment
VER-155008-based Hsp70 inhibition has been relatively successful in the treatment of multiple myeloma (MM) [89]. VER-155008 significantly decreased the cellular viability in MM cell lines, including INA-6, MM.1S, L363, KMS11 and JJN-3. The sensitivity of MM cells to VER-155008 differed between cell lines with IC50 values from 2.5 to 17 μM, and the drug concentrations that induce near complete cell demise in all studied cell lines are between 10 and 30 μM. VER-155008-induced Hsp70 inhibition led to apoptosis in MM with substantial accumulation of apoptosis-inducing factor (AIF) in the nucleus and with increased cleavage of pro-caspases 9/3 and the caspase substrate poly(ADP-ribose) polymerase 1 (PARP 1). Additionally, VER-155008 treatment simultaneously degraded many Hsp90 client proteins involved in a number of oncogenic signalling pathways including Ras/Raf/MAPK, JAK/STAT3, PI3K/Akt and the IKK/NFkB pathways.
When a dual inhibition approach of VER-155008 and NVP-AUY-922 was implemented into INA-6, MM.1S and primary MM cells, a synergistic mode of action was observed [89]. Specifically, the combination treatment with two inhibitors significantly enhanced apoptosis induction, where the combination effect on INA-6 cells and MM.1S cells was synergistic and additive, respectively, with combination indices (CI) less than 1 for all effect levels calculated [89, 90] (Table 2).
2.1.2 Combination of VER-155008 and 17-DMAG in Leukaemia Treatment
Acute myeloid leukaemia (AML) is a biologically heterogeneous malignancy characterised by bone marrow infiltration of immature leukaemic blasts [122]. Hsp90 has emerged as a potent therapeutic target in AML, and the Hsp90 inhibitor 17-DMAG is effective in killing AML cells in vitro preclinical tests [117]. However, Hsp90 inhibition showed limited antileukaemic effects in phase I clinical trials [11, 123]. One major reason for this is the compensatory Hsp70 upregulation, which is induced by HSF-1 [74, 95, 124]. This is supported by the observation that, in a manner observed with other Hsp90 inhibitors that target the ATP pocket, 17-DMAG-based AML treatment increased Hsp70 and Hsp90 production [11].
However, combination treatment with Hsp90 and Hsp70 inhibitors to neutralise the induced Hsp70 proteins shows promise for human AML [117]. In primary human AML cells from 19 unselected patients, the Hsp70 inhibitor VER-155008 itself showed significant antileukaemic activity at 10 μΜ, causing a dose-dependent inhibition of cancer cell proliferation, where growth was inhibited by 72%. Additionally, Hsp90 inhibitor 17-DMAG was used to treat cells at 50 nM, which also resulted in growth inhibition of 58%. The combination treatment with both inhibitors in primary AML cells decreased cell growth to 82%, indicating an additive growth inhibition effect on AML cells. Moreover, although both VER-155008 (10 μM) and 17-DMAG (50 nM) were able to cause an inhibition in AML colony formation for most patients, the strongest and most significant decrease in colony number was observed when the two drugs were combined.
VER-155008-mediated Hsp70 inhibition in AML cells did not induce any compensatory increase in other Hsps; in fact it caused a significant reduction of both Hsp70 and Hsp90 expressions when used alone. In contrast, 17-DMAG-mediated Hsp90 inhibition resulted in a significant increase in Hsp70 and Hsp90 levels. When VER-155008 was used in combination with 17-DMAG, Hsp90 and Hsp70, expression levels increased to the same level as when cells were treated with 17-DMAG alone [117]. Thus, in contrast to silencing Hsp70 using siRNA, chemical inhibition of Hsp70 by VER-155008 fails to regulate the Hsp70 and Hsp90 protein increase that is induced by Hsp90 inhibition. These data explain why the combined effect of VER-155008 and 17-DMAG is only additive and not synergistic. Furthermore, it is possible that given that VER-155008 targets an ATP-binding pocket, it may have off-target effects.
2.1.3 Combination of VER-155008 and SM122 in Colon Cancer Treatment
SM122 (Fig. 4b) is a unique Hsp90 inhibitor that modulates the C-terminus and does not induce a HSR or produce an accumulation of Hsp70 in the human colon cancer cell line (HCT116). Recent work by Wang and McAlpine investigated the effects of combining SM122 with Hsp70 inhibitor VER-155008 on chaperone-mediated protein folding and the induction of apoptosis, compared to a combination of 17-AAG and VER-155008 [20, 21]. Synergistic effects for both SM122/VER-155008 and 17-AAG/VER-155008 treatments were observed in multiple cell lines including HCT116, human lung adenocarcinoma epithelial cells (A549), human cervical cancer cells (HeLa) and human pancreatic cancer cells (MiaPaca-2). In addition to showing synergism, both combination treatments displayed tumour-specific effects with an acceptable therapeutic window. Analysis of chaperone-mediated protein folding was achieved using a rabbit reticulocyte lysate (RRL)-based luciferase-refolding assay. Individually, SM122 and 17-AAG have a similar impact on protein folding, where they both have an IC50 value of ~2 μM. However, the most effective inhibition of protein folding was observed when Hsp90 and Hsp70 were concomitantly inhibited. Combinations of 20 μM VER-155008 with increasing concentrations of either SM122 or 17-AAG showed very strong synergism. Interestingly, while 17-AAG and SM122 have very different GI50 values of 50 nM and 8 μM in HCT116 cells, respectively, they inhibit protein folding at a similar concentration [20, 21].
Combination treatments of SM122 or 17-AAG with VER-155008 both showed synergism in their ability to kill multiple cancer cell types and had a similar impact on protein folding. However, each combination induced apoptosis via a unique mechanism [20, 21]. HCT116 cells were treated with 50 μM VER-155008 and either SM122 or 17-AAG at two- to threefold over their GI50. Apoptosis was induced in 75% of the cells treated with SM122/VER-155008, while only 50% apoptosis was induced in cells treated with 17-AAG/VER-155008. Cell death occurred via a caspase 3/7-dependent pathway with PARP-1 cleavage in both dual treatments. Interestingly, while the 17-AAG/VER-155008 treatment showed a better capacity to activate caspase 3/7, SM122/VER-155008 induced higher levels of early and late apoptosis [20, 21]. This data suggests that the primary mechanism through which 17-AAG/VER-155008 induces cell death is via caspase pathways; however, SM122/VER-155008 triggers cell death through additional pathways simultaneously, which may be beneficial in reducing the chance of tumour cells developing resistance.
The individual mechanisms by which SM122 and 17-AAG trigger cell death explains the differences in the apoptosis observed as they induce apoptosis via different cellular pathways when used in combination with VER-155008 [20–24]. Each drug combination has distinct impacts on HSR pathways. Evaluating the impact of SM122 and 17-AAG with VER-155008 on mRNA transcription, translation and protein expression levels of Hsps provided evidence of their individually unique mechanism of action [20, 21]. Activation of the HSR is characterised by an accumulation of Hsps including Hsp70, Hsp40 and Hsp27. When HCT116 cells were treated with 17-AAG and VER-155008 individually, Hsp70 mRNA levels increased by 45- and 250-fold, respectively. In contrast, SM122 produced a twofold decrease in Hsp70 mRNA [20]. These data show that Hsp70 inhibition and N-terminal Hsp90 inhibition triggers the HSR at a transcriptional level.
When HCT116 cells were treated with 17-AAG/VER-155008 and SM122/VER-155008, Hsp70 mRNA levels increased by 3,500- and 1,500-fold, respectively [20, 21]. The SM122/VER-155008 treatment did not trigger the HSR as rapidly as 17-AAG/VER-155008, which is likely because SM122 suppresses and/or delays the transcription of Hsp70 mRNA when used in combination with VER-155008.
These phenotypic differences between SM122 and 17-AAG are also observed at the translational level, where SM122/VER-155008 synergistically inhibits protein translation, while 17-AAG/VER-155008 has no impact on translation [20]. Evaluation of heat shock protein expression levels (Hsp27, Hsp70 and Hsp40) showed that treating HCT116 cells with 17-AAG, VER-155008 or 17-AAG/VER-155008 in combination produced a large increase in Hsp70, Hsp40 and Hsp27. As discussed earlier, cells treated with SM122 decreased these protein levels [20, 21]. Dual treatment with SM122 and VER-155008 produced higher protein levels than when cells were treated with SM122 alone; however, the levels were no higher than cells treated with VER-155008 alone, showing that SM122 did not contribute to the rescue mechanism.
These results show that C-terminal modulators and N-terminal Hsp90 inhibitors have distinct mechanisms when used in combination with an Hsp70 inhibitor. Dual treatments are synergistic and induce rapid cell death in numerous cancer cell lines far more effectively than monotherapies. Thus, dual therapies have great potential as cancer treatment regimens, particularly those involving C-terminal Hsp90 modulators like SM122, which has the added benefit of reducing the HSR and limiting the ability of the cancer cell to rescue itself following treatment.
2.1.4 Combination Treatment of MAL3-101 with 17-AAG
MAL3-101 binds to an interface between Hsp40 and Hsp70, thereby impacting Hsp40-mediated stimulation of Hsp70’s ATPase activity [120, 125]. Using MAL3-101 to inhibit Hsp70 alone has successfully treated preclinical MM primary tumour cells and endothelial progenitor cells (EPCs) obtained from MM patients [126]. Specifically, MAL3-101 treatment led to the inhibition of proliferation and survival in NCI-H929 cells with an IC50 value of 8.3 μM at 40-h exposure using an MTS assay. Cell cycle analysis showed that after 48-h treatment, MAL3-101 caused a 2.5-fold decrease at G2/M phase, with a nearly threefold increase at sub-G0/G1 phase in NCI-H929 cells, which indicated an activation of an apoptotic pathway. Confirmation by FACS analysis showed that cells treated with MAL3-101 increased apoptosis, cleavage of caspase-3 and PARP in a time-dependent manner.
In contrast to MM cells being treated with VER 155008 and 17-DMAG inhibitors, treatment of MM cells with MAL3-101 and 17-AAG led to apoptosis [126]. Specifically, in NCI-H929 MM cell line, 10 μM of MAL3-101 significantly decreased the IC50 of 17-AAG from 400 to 30 nM. The isobologram analysis of 10 μM MAL3-101 and 17-AAG with five different concentrations (25, 50, 100, 500 and 1,000 nM) showed tremendous synergistic effect, with combination index (CI) values from 0.008 to 0.12, where CI < 0.1 is “very strong synergism” and 0.1 ≤ CI ≥ 0.3 is “strong synergism” (Table 2). These data support the hypothesis that VER-155008 may have off-target effects and is not only targeting Hsp70, whereas MAL3-101s may have a more selective impact on Hsp70’s activity.
2.1.5 Combination Treatment of PFT-μ with 17-AAG
Pifithrin-μ (PFT-μ) has been identified as a potent Hsp70 inhibitor specifically targeting the inducible isoform of Hsp70, without binding to Hsp90 [119]. It interferes with the C-terminal substrate-binding domain of Hsp70 and disrupts its association with client proteins, causing cell cycle arrest and significant apoptosis at low micromolar concentrations in acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and primary AML blasts [119]. Importantly, normal haematopoietic cells and stromal cells exhibited a remarkably high resistance to PFT-μ compared to leukaemic blasts [115]. In bone marrow stromal cells (BMSC), the median IC50 value was ~4-fold higher than that in leukaemic blast cancer cells [115], which indicates that a therapeutic index can be achieved using PFT-μ.
Combination treatment with PFT-μ and the Hsp90 inhibitor 17-AAG showed synergism in reducing cell viability of all studied acute leukaemia cells including NALM-6, TOM-1 and KG-1a. Among the three cell lines, KG-1a was the least sensitive to PFT-μ and 17-AAG individual treatment, with 81% and 72% cell viability after exposure to 10 μM of PFT-μ and 5 μM of 17-AAG, respectively. However, this cell line had the most significant response to treatment by both inhibitors, showing only 29% cell viability upon treatment with these two concentrations. For the NALM-6 leukaemia cell line, their viability when treated with PFT-μ and 17-AAG monotherapies was 70% (2 μM of PFT-μ) and 70% (2 μM of 17-AAG), respectively, versus 42% when used in combination. For the TOM-1 cell line, viability was 85% (3 μM of PFT-μ) and 57% (1 μM of 17-AAG) when using monotherapies versus 36% when treating cells with both drugs.
2.1.6 Combination Treatment of AIF-Derived Peptide with 17-AAG
ADD70 is a designed peptide constructed from the amino acid residues in the AIF protein that bind to Hsp70 (amino acids 150–228) (Fig. 6). ADD70 sensitises cancer cells to apoptosis induction by capturing and neutralising the endogenous Hsp70 protein in the cytosol. ADD70 does not exert any apoptotic effects by itself [105, 127, 128]. ADD70 displayed significant anti-tumorigenic and anti-metastatic properties, as well as the ability to enhance cancer cell immunogenicity by facilitating the induction of a tumour-specific immune response, which increased the number and cytotoxic activity of CD8+ tumour-infiltrating T cells [127].
Apoptosis-inducing factor is 613 amino acids in length. The region between amino acids 150 and 228 is essential for AIF to bind to Hsp70. ADD70 is a truncated version of AIF, where it only contains amino acids 150–228
The expression of ADD70 showed an additive effect when 17-AGG was used in the rat colon cancer ProB cells and mouse melanoma cancer B16F10 cells. These two distinct models of tumours were developed in syngeneic rodents. The additive effect observed when using ADD70 and 17-AAG appears to be related to the reduction of inducible Hsp70 protein by ADD70, where low levels of Hsp70 protein allowed AIF-mediated caspase-independent apoptotic pathways (Fig. 6) to induce pro-apoptotic functions [105, 106, 129]. Inducing AIF-mediated apoptosis is unique to ADD70 and is not seen with any of the small molecules described above. It is thought that, since ADD70 contains the AIF sequence that binds to Hsp70, ADD70 disrupts the AIF-Hsp70-binding event inducing apoptosis via the AIF pathway. Release of AIF facilitates apoptosis and an indirect induction of the Apaf-1-mediated caspase-dependent apoptosis (Fig. 6).
Interestingly, ADD70 significantly enhanced the chemosensitizing effect of 17-AAG on cisplatin-mediated chemotherapy [127]. For example, the combination of 17-AAG and cisplatin only showed additive anticancer effects on several cancer cells. However, in the presence of ADD70, the impact of cisplatin on cell death was strongly enhanced in both cell lines, indicating that the expression of ADD70 can efficaciously potentiate the chemosensitizing effect of 17-AAG. Thus, the study of ADD70 and 17-AAG provided evidence that simultaneous targeting Hsp70 and Hsp90 can effectively provide anticancer therapy.
2.1.7 Combining Hsp70 Silencing with Hsp90 Inhibition in Human Solid Tumours
Constitutive heat shock cognate 70 (Hsc70) and inducible heat shock protein 72 (Hsp72) are two major cytoplasmic isoforms of the Hsp70 multigene family, and they have different expression patterns in mammalian cells. In non-tumour tissues, Hsc70 is abundantly and ubiquitously expressed, whereas Hsp72 is present at relatively low levels. However, under stressed conditions, Hsp72 is overexpressed, while Hsc70 is minimally impacted [89, 95, 130–132]. Selectively knocking down either Hsp72 or Hsc70 isoform using siRNA had no impact on cell proliferation in multiple cancer cells [95]. However, silencing Hsp72 significantly enhanced the antiproliferative effect of 17-AAG-mediated Hsp90 inhibition on colon cancer HCT116 cells, inducing a fivefold increase in apoptosis [95]. In contrast, when Hsc70 was silenced, there was no improved apoptosis or response to 17-AAG in any cancer cell line [95].
The differential effects of selective Hsp70 isoform silencing on the combination treatment with 17-AAG indicate that although both Hsc70 and Hsp72 can bind to Hsp90, both are induced after 17-AAG-mediated Hsp90 inhibition [92, 133–136]. Hsp72 appears to play the most important role in maintaining cell viability. These data are supported by recent evidence that 17-AAG induces 80–100-fold increases in Hsp72 mRNA levels, but only ~6-fold increase in Hsc70 [21, 23]. Thus, it appears the cell protection effects are primarily produced by an increase in Hsp72, and this isoform is heavily induced by 17-AAG. The protective effects can be silenced by knocking down Hsp72, and indeed this is the most effective route for enhancing Hsp90 inhibitors [48, 137]. Furthermore, coupling treatment of Hsp72/Hsc70 inhibition with 17-AAG also shows no effect on non-tumour cells. This observation suggests that inhibiting Hsp72 in combination with an Hsp90 inhibitor may offer a reasonable treatment with a potential therapeutic window [138].
3 Conclusions
Highlighted in this chapter are examples that indicate Hsp90 inhibition is a more effective treatment when used in combination with other chemotherapies. Successfully combining Hsp90 inhibitors with other chemotherapy drugs including molecules that target Hsps produces rapid apoptosis and cell death, which can avoid resistance and cancer metastasis. Specifically, combining Hsp90 and Hsp70 inhibitors produces large increases in apoptosis and potency of up to 92% compared to using single inhibitors. A major reason for this combination being so effective is that inhibiting Hsp90 using classical inhibitors increases the production of Hsp70 protein, which is pro-survival. We also describe how using C-terminal Hsp90 inhibitors is more effective than a classical N-terminal inhibitor when used as a dual therapy. Thus, targeting multiple points in the cell protection mechanism known as the HSR is likely to produce a highly effective new therapeutic approach (Fig. 7).
(a) Using N-terminal inhibitors (i.e. classical inhibitors) promotes production of Hsp70 and the heat shock response, whereas using a C-terminal modulator inhibits co-chaperones from binding to Hsp90 and induces cell death. (b) Inhibiting both Hsp90 and Hsp70 stops the function of both proteins simultaneously, blocking the rescue response and inducing massive cancer cell death
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Buckton, L.K., Wang, Y., McConnell, J.R., McAlpine, S.R. (2015). Evaluating Dual Hsp90 and Hsp70 Inhibition as a Cancer Therapy. In: McAlpine, S., Edkins, A. (eds) Heat Shock Protein Inhibitors. Topics in Medicinal Chemistry, vol 19. Springer, Cham. https://doi.org/10.1007/7355_2015_96
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