Among the heat shock proteins (HSP), HSP27, HSP70 and HSP90 are the most studied stress-inducible HSPs, and are induced in response to a wide variety of physiological and environmental insults, thus allowing cells to survive to lethal conditions based on their powerful cytoprotective functions. Different functions of HSPs have been described to explain their cytoprotective functions, including their most basic role as molecular chaperones, that is to regulate protein folding, transport, translocation and assembly, especially helping in the refolding of misfolded proteins, as well as their anti-apoptotic properties. In cancer cells, the expression and/or activity of the three HSPs is abnormally high, and is associated with increased tumorigenicity, metastatic potential of cancer cells and resistance to chemotherapy. Associating with key apoptotic factors, they are powerful anti-apoptotic proteins, having the capacity to block the cell death process at different levels. Altogether, the properties suggest that HSP27, HSP70 and HSP90 are appropriate targets for modulating cell death pathways. In this review, we summarize the role of HSP90, HSP70 and HSP27 in apoptosis and the emerging strategies that have been developed for cancer therapy based on the inhibition of the three HSPs.
Introduction
Apoptosis
HSF1
Targeting HSP27
Targeting HSP70
Targeting HSP90
Conclusions
Stress or heat shock proteins (HSPs) are a family of highly conserved proteins induced in response to a wide variety of physiological and environmental insults such as hypoxia, hyperoxia, exposure to UV light and chemicals, viral agents, surgical stress, nutritional deficiencies (e.g. glucose deprivation), emotional and mechanical stress, or other stresses, thus helping maintain cellular homeostasis under stress or allowing the cell to survive to lethal conditions (
Mammalian HSPs have been classified into six families according to their molecular size: HSP100, HSP90, HSP70, HSP60, HSP40 and small HSPs (15 to 30 kDa) including HSP27. Family members of HSPs are expressed either constitutively or regulated inductively, and are present in different subcellular compartments (
Cancer cells, with higher metabolic requirements and more abundant signal transduction pathways than normal cells, thereby have a higher need of chaperones than non-transformed cells to maintain cancer cells survival. In addition, by commanding over the folding and stabilization of relevant oncoproteins, HSPs stand at the crossroads of multiple important oncogenic pathways. Inhibition of HSPs hereby offers the unique advantage of depleting multiple oncoproteins while simultaneously attacking several pathways necessary for tumor progression (
Mainly, two pathways of apoptosis can be distinguished, although crosstalk between the two signal transducing cascades is present: the intrinsic or mitochondrial pathway and the extrinsic or death receptors pathway. The two signal-transducing cascades meet at the point of caspase-3, an effector caspase that leads to the typical morphologic and biochemical changes of the apoptotic cell.
The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of proapoptotic molecules normally confined to the intermembrane space. Outer mitochondrial membrane permeabilization leads to the release of caspase activators under control of the Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene) family of proteins. Bcl-2 proteins include antiapoptotic members such as Bcl-2 and Bcl-xL, multi-domain proapoptotic members mainly Bax and Bak (
The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of caspases, starting with the receptor-proximal caspase-8 or caspase-10 in the death-inducing signalling complex (DISC). Caspase-8 either directly activates the downstream cascade of caspases or cleaves Bid into an active truncated form named tBid that connects the extrinsic to the intrinsic apoptotic pathways through mitochondria permeabilization (
The rapid induction of HSPs in response to multiple stress is collectively referred to as the heat shock response (HSR)(
HSP27 (HSPB1) belongs to a member of the small heat shock proteins (sHSP). The primary structure of HSP27 is highly homologous to other members of the sHSP family, containing the conserved α-crystallin domain and differing in the C- and N-terminal regions. HSP27 is expressed in all human tissues, including astrocytes and primary neuronal cells but mainly in skeletal, smooth and cardiac muscles (
HSP27 has a strong protective effect on cells. High levels of HSP27 have been observed in many cancer types, and the tumorigenic potential of HSP27 has been observed in experimental models (
Numerous studies describe that HSP27 inactivates the caspase cascade through its binding with caspase-3 and cytochrome
High intracellular levels of HSP27 can inhibit caspase activation by interfering upstream of the mitochondria (
Large oligomers of HSP27 have also been described to display anti-oxidant property, which is related to its ability to maintain glutathione in its reduced (non-oxidized) form to abolish the production of the potentially lethal burst of intracellular reactive oxygen species (ROS) that can occur (
The function of HSP27 and the role that it plays in cancer were recently reviewed (
The strong cyto-protective function of HSP27, together with the fact that this protein is overexpressed in most cancers, makes this chaperone an attractive target in cancer therapy. Depletion of HSP27 in various animal models induces the regression of tumors (
The antisense oligonucleotide OGX-427 is the only known specific inhibitor of HSP27 that can be safely administered in patients and is currently in phase II clinical trials (
Less specific, the chemical molecule RP101 (also known as bromovinyldeoxyuridine, BVDU, brivudine) was reported to improve the efficacy of chemotherapy in pancreatic cancer through its interaction with HSP27 (
A strategy of peptide aptamers has also been used to target HSP27. Protein aptamers, small amino acid sequences, are designed to bind to a specific protein domain, thus inhibiting its function (
Different kind of inhibitors, which have been experimentally tested, like the flavonoid quercetin and the diterpene triepoxide, triptolide (
HSP70 refers to a family of chaperone proteins that are 70 kDa. The HSP70 human genome superfamily consists of at least 13 members (
HSC70 is ubiquitously expressed in practically all organs and tissues. Under normal conditions, it functions as ATP-dependent molecular chaperone that assists the folding of newly synthesized polypeptides, the assembly of multi-protein complexes and the transport of proteins across cellular membranes (
All of the proteins share homology and contain two distinct functional domains: a C-terminal peptide-binding domain (PBD) and the N-terminal ATPase domain (ABD), which were connected through a hydrophobic linker and both domains are important for substrate binding and stabilization. The PBD, which include a carboxyl-terminal chaperone EEVD motif, is responsible for substrate binding and refolding. The ABD, containing the ATPase pocket and binding J-domain-containing proteins, such as HSP40 that regulate the HSP70 ATPase activity, in turn, facilitates the release of the client protein after ATP hydrolysis. A conserved proline in the ATPase domain is essential to alternate HSP70 conformations in response to ATP binding and hydrolysis (
HSP70 chaperone activity is regulated by distinct co-chaperones, e.g. Hip, CHIP or Bag-1. These co-chaperones bind to HSP70 and modulate its chaperone function by increasing or decreasing HSP70 affinity for substrates through the stabilization of the ADP or ATP bound state of HSP70. They can be classified into three groups. i) The J-domain co-chaperones, like HSP40, are a relatively large group that binds to the HSP70 ABD and stimulate the low ATPase activity of this chaperone (
Similar to HSP27, HSP70 is also abundantly expressed in many tumor forms and is accompanied by increased cell proliferation, metastases and poor response to chemotherapy. Constitutively high expression of HSP70 enhances the ability of the cancer cells to survive to a range of lethal conditions. The cytotoxic effect of HSP70 down-modulation is particularly strong in transformed cells yet undetectable in normal, non-transformed cell lines or primary cells (
HSP70 can regulate apoptosis at the different levels from death receptors signaling to executors of cell death program affecting both upstream and downstream of the death-associated mitochondrial events.
At the level of death receptors, HSP70 can bind to the death receptors DR4 and DR5, thereby inhibiting the TNF-α-related apoptosis-inducing ligand (TRAIL)-induced assembly and activity of death inducing signaling complex (DISC) (
At the premitochondrial level, HSP70 inhibits stress-activated kinases, such as apoptosis signal regulating kinase 1 (Ask1). In NIH3T3 cells, it was shown that downregulation of HSP70 facilitates H2O2-induced Ask1 activation and subsequent apoptosis (
HSP70 has also been shown to affect some transcription factors involved in the expression of the Bcl-2 family. Bcl-2 family of proteins, playing a critical role in the regulation of apoptosis through controling the release of caspase activators, are transcriptional targets of the tumor suppressor protein p53. The transcription of Bcl-2 is repressed by p53, whereas that of Bax is induced. HSP70 can form stable complexes with mutated p53, thus inducing apoptosis in response to DNA damage. HSP70 can also cover the nuclear localization sequence of p53, thereby preventing its nuclear import (
At the mitochondrial level, HSP70 blocks heat-induced apoptosis by binding to Bax to prevent its translocation to the mitochondria (
At the post-mitochondrial level, downstream of the release of cytochrome
HSP70 can prevent cell death under caspase inactivation. That is HSP70 can also prevent caspase-independent pathways (
Moreover, HSP70 can also rescue cells from a later phase of apoptosis. During the final phases of apoptosis, the main characteristic is nuclear condensation and fragmentation, and the chromosomal DNA fragmentation is digested by the DNase CAD (caspase activated DNase) following activation by caspase-3. It has been reported that HSP70 has an important influence on the enzymatic activity and proper folding of CAD, which also depends on its cochaperones: HSP40 and the inhibitor of CAD(ICAD), suggesting that HSP70 plays a role in maintaining DNA integrity (
HSP70 has been shown to promote cancer cell viability by safeguarding lysosomal integrity. In cysteine cathepsin-dependent death, HSP70 acts to inhibit lysosomal membrane permeabilization, thereby preventing the release of lysosomal constituents into the cytosol, which contains a group of proteases that are involved in apoptosis (
HSP70 is a crucial negative regulator of the mitochondrial pathway of apoptosis that can block cell death at several levels from death receptors signaling to executors of cell death program affecting both upstream and downstream of the death-associated mitochondrial events.
Despite the critical role of HSP70, as discussed above, in protein regulation and cancer progression, tremendous efforts have produced few advances in hsp70 inhibitors.Here, we explore HSP70 inhibitors though three basic categories: small molecule inhibitors, protein aptamers, and antibody treatments, also, the targets of drugs - targeting PBD, targeting ABD and targeting HSP70 co-chaperones are also discussed.
Neutralization of HSP70 functions could be achieved with peptides that mimic a domain of the AIF which is required for HSP70 binding. The AIF-derived peptides were designed carrying the AIF region from amino acid 150 to 228, which was previously defined as required for HSP70 binding in itsPBD and lack AIF pro-apoptotic function (
15-Deoxyspergualin (15-DSG), a natural immunosuppressive agent, disrupting HSP70-ATP interaction through binding to HSP70 and stimulating its ATPase activity, was the first compound described by Nadeau
VER-155008 is an adenosine-derived compound. It functions to inhibit the chaperone activity of HSP70 and other family members by binding the ATPase domain. Although further studies are necessary to determine its specificity and potency, some
Azure C, methylene blue and myricetin have been identified as inhibitors of HSP70 through a high-throughput screening for ATP turnover mediated by human HSP70, but their specificity for inducible HSP70 family has not yet been analyzed (
MKT-077, a cationic rhodacyanine dye analog, can also bind to the ABD of Hsp70 (
Apoptozole was discovered to induce apoptosis in the human embryonic carcinoma cell line while looking for small molecules that induced apoptosis in the imidazole compound library (
Sphingolipids, another group of HSP70 ABD binding agents, can bind and specifically inhibit HSP70 ATPase activity
HSP90, a highly abundant chaperone protein expressed by all eukaryotic cells, belongs to another important class of the HSP family (
As a molecular chaperone, like HSP27 and HSP70, HSP90 helps nascent proteins adopt their biologically active conformations, correct the conformation of misfolded proteins, and helps incorrigibly misfolded proteins to be removed and degraded by the ubiquitin-proteo-some system (
HSP90 functions as part of a multichaperone complex via association with a variety of co-chaperones and client proteins that rely on the complex for acquiring active conformation. It facilitates the maturation, stability, activity and intracellular sorting of more than 200 client proteins (
Because these oncogenic proteins substantially rely on the function of HSP90 for their maturation and/or stabilization, as well as regulation of their activated states (
Over 20 co-chaperones regulate HSP90 activity mainly through the modulation between the interconvertion of the ATP- and ADP-bound states. Some of these inhibit HSP90 ATPase activity, thus to be involved in client loading or the formation of mature HSP90 complexes, such as HSP70/HSP90 organizing protein (HOP), cell division cycle protein 37 (CDC37) and p23. Whereas, others enhance it, such as activator of HSP90 ATPase 1 (AHA1) and cyclophilin-40 (Cpr6 and Cpr7), hence leading to their use as activators of the HSP90 conformational cycle (
Many of HSP90 client proteins hold important functions in the development and promotion of cancer, as described above, thus, Hsp90 has a putative role in numerous cancers and deserves to be an attractive target for therapeutics. Targeting HSP90 as a therapeutic approach in treating cancer began with geldanamycin (GM), which exhibits antiproliferative activity by binding to the ATP-binding site of HSP90 and thereby preventing its function. However, GM has limited therapeutic potential owing to its hepatotoxicity. The discovery of GM sparked much interest in the inhibition of HSP90 as a strategy for the treatment of cancer, resulting in intense efforts from both industry and academic research institutes to develop clinically viable HSP90 inhibitors (
As illustrated above, the rationale for using HSP90 inhibitors in cancer therapy is well established. Pursuing after new easily administrable HSP90 inhibitors and their evaluation in clinical trials is a goal for many pharmaceutical companies. However, no HSP90 inhibitor has been FDA-approved to date. Lessons learned in oncology clinical trials give us strategies and future directions that may enhance therapeutic benefit and accelerate the drug approval process for safe and efficacious HSP90 inhibitors.
There have been hints that these inhibitors are minimally effective and with more side-effects as single agents against various cancer cell line, and that they may show tremendous promise when used as combination and dual treatment agents (
Inhibition of HSP90 activates the heat shock response, which compensatorily induces expression of several heat shock factors, including heat shock factor 1 (HSF1), members of the HSP70 family and HSP27, which are protective proteins that could counteract the pro-cell death effects of HSP90 inhibitors (
Since targeted therapy was introduced into cancer treatment, it has brought hope, and it is increasingly believed that combination therapies targeting parallel signaling pathways that regulate iconic processes that are absolutely necessary for cancer cell survival and proliferation may provide better cancer therapy. As described above, many of HSP90 client proteins are involved in critical cellular functions, thus, the development of these HSP90 inhibitors may require close developing client protein inhibitors, e.g., RAF inhibitors (
To enhance the effectiveness of HSP90 inhibitors, combinatorial targeting of HSP90 cochaperones and/or of post-translational modifications that influence HSP90’s function is a potentially attractive approach (
In addition, a better understanding of the HSP90 inhibitors is still the key. For HSP90 has various isoforms and each has different functions, specific inhibition for HSP90 inhibitors is expected. It has been suggested that HSP90α plays an essential and unique role in embryo cell differentiation, and its inhibition blocks macrophagic differentiation in the already formed animal (
Clinical trial designs may ultimately be critical in determining if one HSP90 inhibitor has any clear clinical benefit to exploit. Several strategies can be applied to enhance the effectiveness of HSP90 inhibitors. Personalizing treatments is always one of the principles of clinical treatment. Personalizing treatments to match patients’ genetic profiles and targeting specific tumors/tumor types should be recognized as ways to increase the effectiveness of HSP90 inhibitors. As we enter the era of targeted therapy and personalised medicine, development of biomarkers to help to stratify patients, ascertain target inhibition, and monitor or predict response to HSP90 inhibitiors is of vital importance if HSP90 inhibitors are to succeed. It is also possible that the therapeutic schedule of HSP90 inhibitors has not been optimized. Future effective target inhibition would benefit from a valuable method to optimise drug dosing and scheduling.
Owing to the complicated pathogenesis, poor prognosis and resistance to treatments, cancer remains a notoriously unsolved medical issue and desperately requires efficacious drug candidates. By commanding over the folding and stabilization of relevant oncoproteins, HSPs are involved in vital mechanisms of cancerous cells, such as cell proliferation, differentiation, invasiveness, neoangiogenesis, metastasis and immune system recognition. Additionally, they have the added advantage of reducing the likelihood of the tumor acquiring resistance to any single therapeutic strategy. As a consequence, HSPs are emerging as interesting targets in cancer therapy, particularly HSP90, 70 and 27. Owing to the potent anti-apoptotic function of HSPs 27, 70 and 90 as well as their role in drug resistance, it is considered that their deletion may increase tumor cell susceptibility to apoptosis and fight against carcinogenesis or elicit drug sensitivity (
We acknowledge financial support from the National Science and Technology Research Supporting Programme in Chinese Medicine in ‘11th Five-Year Plan’ (2006BAI11B08-01) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and thank for the assistance from Professor Xu Zhang of the Nanjing University of Medicine.
HSP27, 70, 90 in the regulation of apoptosis and proliferation. HSP27, 70 and 90 regulate apoptosis at different levels, from the death receptors signaling to executors of cell death program, affecting both upstream and downstream of the death-associated mitochondrial events. Induction of caspase activation provokes proteasome activation. Also, HSPs facilitate the degradation of proteins by the ubiquitin/proteasome system (see text).
Heat shock factor 1 in the regulation of the expression of HSPs. HSP90 and HSP70 bind to HSF1 in unstressed state to abrogate the transcription function of HSF1 and dissociate from it under the exterior cellular stress to activate HSF1. Then the monomeric HSF1 trimerizes, phosphorylates and translocates to the nucleus. In the nucleus, HSF1 binds
HSP inhibitors in clinical development as mono- or combination-therapy.
Drug (HSP90 inhibitors) | Disease type | Stage of development |
---|---|---|
Geldanamycin analogues | ||
Tanespimycin (17-AAG) | ||
17-AAG | Kidney tumors in Von Hippel-Lindau disease; relapsed or refractory anaplastic large cell lymphoma, mantle cell lymphoma, or Hodgkin’s lymphoma | II |
17-AAG + trastuzumab | Breast cancer | II |
17-AAG + bortezomib | Multiple myeloma | II/III |
17-AAG + gemcitabine | Recurrent advanced ovarian epithelial or peritoneal cavity cancer | II |
17-AAG + bortezomib | Advanced solid tumors or lymphoma | I |
Alvespimycin (17-DMAG) | ||
17-DMAG | Melanoma | I |
Prostate | I | |
17-DMAG + trastuzumab | Breast cancer | I |
17-DMAG (KOS-1022) + trastuzumab | Ovarian | I |
Retaspimycin hydrochloride (IPI-504) | ||
IPI-504 | Hormone-resistant prostate cancer | II |
Relapsed and relapsed refractory multiple myeloma | I | |
Relapsed/refractory stage IIIb, or stage IV NSCLC | I/II | |
IPI-504 + docetaxol | Advanced solid tumors | I |
IPI-504 + everolimus | KRAS mutant NSCLC | I/II |
IPI-504 + trastuzumab | HER2+ breast cancer (study terminated) | II |
IPI-493 | Advanced malignancies; hematologic malignancies(study terminated) | I |
Resorcinol derivatives | ||
Ganetespib (STA-9090) | ||
STA-9090 | Patients with unresectable stage III or stage IV melanoma who received prior tyrosine kinase inhibitor treatment (has 2 arms-mutant V600E BRAF arm and a wild-type BRAF arm); metastatic hormone-resistant prostate cancer previously treated with docetaxel-based chemotherapy; previously untreated metastatic HER2+ or triple negative breast cancer; stage IIIB/IV NSCLC; metastatic ocular melanoma; metastatic or unresectable GIST; refractory metastatic colorectal cancer | II |
Advanced hepatocellular carcinoma; solid tumors, STA-9090 administered twice-weekly | I | |
AML, ALL and blast-phase CML | I/II | |
STA-9090 as second-or third-line therapy | Metastatic pancreatic cancer | II |
STA-9090 + docetaxel | Solid tumors | I |
STA-9090 + dutasteride | Castration-resistant prostate cancer | II |
STA-9090 + fulvestrant | HR+, metastatic breast cancer | II |
AUY922 | ||
AUY922 | Advanced solid malignancies in older patients (≥75 years) | I |
Lymphoma; metastatic pancreatic cancer resistant to first line chemotherapy; NSCLC patients who received 2 previous lines of chemotherapy; refractory GIST | II | |
HER2+ trastuzumab-resistant breast cancer [imaging component using 89Zr-trastuzumab positron emission tomography (PET) to study the effect of HSP90 inhibition on HER2 expression] | I/II | |
ER+ hormone therapy refractory breast cancer (to study the effect of HSP90 inhibition by AUY922 on VEGF using 89Zr-bevacizumab PET) | I/II | |
AUY922 + capecitabine | Patients with advanced solid tumors (active not recruiting) | I |
AUY922 + cetuximab | KRAS wild-type metastatic colorectal cancer | I |
AUY922 + erlotinib hydrochloride | Stage IIIB/IV NSCLC | I/II |
AUY922 + trastuzumab | Patients with HER2+ advanced gastric cancer, who had received trastuzumab plus chemotherapy as first line treatment | II |
AT-13387 | Refractory solid tumors | I |
KW-2478 | ||
KW-2478 + bortezomib | Relapsed or refractory multiple myeloma | I/II |
Purine analogues | ||
BIIB021 (CNF 2024) | ||
BIIB021 | Advanced solid tumors; advanced breast cancer (PK/PD study) | I |
GIST | II | |
BIIB021 + aromasin | Hormone receptor positive metastatic breast cancer | II |
MPC-3100 | Refractory or relapsed cancer | I |
Debio 0932 (CUDC-305) | Advanced solid tumors or lymphoma | I |
PU-H71 | Refractory solid tumor and low-grade non-Hodgkin’s lymphoma; advanced malignancies (metastatic solid tumor, lymphoma) | I |
Other compounds | ||
SNX-5422 | Refractory solid tumor malignancies or non-Hodgkin’s lymphoma | I |
DS-2248 | Advanced solid tumors | I |
XL-888 | ||
XL-888 | Solid tumors | I |
XL-888 + AT13387, abiraterone | Prostate cancer | I |
XL-888 + vemurafenib | Unresectable BRAF mutated stage III/IV melanoma | I |
Source,