Molecular Approaches to Target Heat Shock Proteins ...

Frontiers in Clinical Drug Research - Anti-Cancer Agents, 2015, Vol. 2, 3-47

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CHAPTER 7

Molecular Approaches to Target Heat Shock Proteins for Cancer Treatment Daniel R. Ciocca1, Francesco Cappello2,3, F. Darío Cuello-Carrión1, AndréPatrick Arrigo4 Laboratory of Oncology, Institute of Experimental Medicine and Biology of Cuyo (IMBECU), Technology and Scientific Center (CCT)-National Research Council of Argentina (CONICET) 1

Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy 2

3

Euro-Mediterranean Institute of Science and Technology, Palermo, Italy

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic, Sun Yat-sen University, 54 Xianlie South Road, Guangzhou, Guangdong, 510060, China. Apoptosis, Cancer and Development Laboratory, Lyon Cancer Research Center, INSERM U1052-CNRS UMR5286, Claude Bernard University Lyon 1, Lyon, France 4

Abstract: HSP90 was the first molecular target to inhibit the interaction of this heat shock protein (HSP) with client proteins in cancer cells and tissues. The HSP90 inhibition was attempted to liberate from this chaperone the oncogenic fusion proteins, mutated and activated serine/threonine protein kinases, tyrosine kinases, as well as transcription factors with oncogenic activity, in this manner, the free proteins could be recognized by the proteasome system to be degraded. We should remember here that many HSP family members are overexpressed in different kinds of cancer tissues, these molecules act as chaperones of tumorigenesis. In cancer patients, the first generation of HSP90 inhibitors showed elevated levels of toxicity, which was partially solved with the second-generation of inhibitors that could be intravenously delivered. With the arrival of the third-generation drugs that could be orally administrated, anticancer activities were achieved in clinical trials, however, the results were not as successful as expected due to: 1) limited anti-tumor efficacy, 2) acquisition of drug resistance, 3) difficulty to identify the client protein(s) specifically degraded in response to drug administration. The main problem is the redundancy of chaperones that the cancer cells have, in fact during HSP90 or HSP70 inhibition the heat shock factor (HSF1) could be liberated increasing the levels of other HSPs and in addition, HSF1 can by itself act as an inducer of the multidrug resistance MDR response and is also implicated in HER2 and hormonal responses. These difficulties, rather than decreasing the interest of having the HSPs as molecular targets, are increasing the exploration of new ways to interfere with several HSPs simultaneously and using HSP inhibitors with more “conventional” anticancer drugs. In this article we review, in addition to HSP90, HSP27, HSP70, and HSP60 as targets for anticancer therapy. Address correspondence to Daniel R. Ciocca: Laboratory of Oncology, Institute of Experimental Medicine and Biology of Cuyo (IMBECU), Technology and Scientific Center (CCT)-National Research Council of Argentina (CONICET), Casilla de Correo 855, 5500 Mendoza, Argentina. E-mail: [email protected] *

Atta-ur-Rahman (Ed) All rights reserved-© 2015 Bentham Science Publishers

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Keywords: Cancer, Drug resistance, Heat shock proteins, HSP27, HSP60, HSP70, HSP90, Molecular targets, New anticancer drugs, Therapy.

INTRODUCTION Ferruccio Ritossa (1962, Italy) discovered the heat shock proteins (HSPs) genes when fruit fly Drosophila melanogaster cells were exposed to heat, the polytene chromosomes revealed heat shocked-DNA puffs (sites of increased transcription) given rise to what latter were named as HSPs (Alfred Tissières, Switzerland). These proteins can be induced not only by heat but also by different physiological and pathological conditions; they are present in all living cells and microorganisms and belong to a subset of molecular chaperones that are rapid and abundantly induced by many stresses. Although there are guidelines for the use of a new nomenclature of the human HSP families [1], the old names based on their molecular weight are still amply used Table 1.The HSPs participate in protein homeostasis stabilizing and assisting in the correct folding of nascent polypeptides; they contribute in proteome maintenance (assembly of macromolecularcomplexes, transport and degradation, and refolding or disaggregation of stress-denatured proteins). Therefore the HSPs are involved in the protein quality control (PQC) system [2]. On the other hand, the HSPs are involved in degradation pathways: ubiquitinproteasome system, endoplasmic reticulum-associated degradation (ERAD), autophagy, and in the regulation of apoptosis [2, 3]. In addition, the 96 HSP family members display a considerable degree of functional diversity depending on the protein localization and on the diverse molecules with which they interact (these proteins are in general named “client” proteins) [4]. The HSP family members can be induced constitutively (by genes that are transcribed continually compared to facultative genes which are only transcribed as needed), not induced by stress. Another form is constitutively and induced upon stress; and those that are induced only upon stress [5]. Upon stress, HSF1 is the key transcriptional activator of a) chaperones, b) cochaperones (e.g., those forming part of the family of HSP40/DNAJ proteins that have in common a conserved J domain by which they interact with HSP70, stimulating its ATPase activity) and c) ubiquitin (a system of proteins and enzymes that adding different amount of ubiquitin activate differential pathways leading to protein trafficking, endosomal sorting, endocytosis, histone regulation, DNA repair, cell cycle progression, cell proliferation/apoptosis, and proteolysis). But practically all cellular compartments and metabolic processes are involved in the stress response including other important stress-responsive transcription factors such as NF-κB, p53, and STAT3 [6, 7]. We should remember here that although the HSPs are induced by HSF1, this HSF controls a multitude of processes in stressed and non-stressed cells, and conversely HSPs can be induced by other stimuli for example hormones (HSP27 is an estrogen-regulated protein) [7, 8]. This might has important consequences in cancer such as in prostate cancer were in most of the patients (83%) there was a coordinate expression of HSP27

Targeting Heat Shock

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and ERα (mRNA but not protein), suggesting that HSP27 can act as a surrogate biomarker for estrogen action inducing cell growth in androgen-independent prostate cancer [9]. Table 1. Examples of the nomenclature of the main HSPs. New name

Old names

HSPA

HSP70

HSPA1A (inducible HSP70)

HSP70-1; HSP72; HSPA1

HSPA2

HSP70-2

HSPA5

BIP;GRP78; MIF2

HSPA6

HSP70-6, HSP70B

HSPA7

HSP70-7

HSPA8

HSC70;HSC71; HSP71; HSP73

HSPB

Small HSPs

HSPB1

HSP27;HSP25; CMT2F

HSPB4

Crystalline alpha A; CRYAA

HSPB4

Crystalline alpha B; CRYAB

HSPC

HSP90

HSPC1

HSP90; HSP89; HSP90AA1

HSPC2

HSP90-ALPHA; HSP90AA2

HSPC3

HSP90-BETA; HSP90B

HSPC4

GRP94; GP96; TRA1

HSPD1

HSP60; GroEL

HSPE1

HSP10; chaperonin 10; GroES

HSPH

HSP110

HSPH1

HSP105

HSPH2

HSP110; HSPA4; APG-2

DNAJ

HSP40

DNAJA1

DJ-2; DjA1; HSDJ

Before entering into cancer, we need to describe the concepts of moonlighting proteins, intrinsically disordered proteins and chaperonopathies. Briefly, protein moonlighting are those that through evolution acquired multiple functions expanding the range of the human functional proteome, this is one way to explain the seemingly small number of proteins that are encoded by the human genome (approximately 19000) [10, 11]. Examples of moonlighting proteins are HSPs and also enzymes, receptors, and ion channels. Protein moonlighting are coded by gene sharing, that is by a gene that gains and maintains a second function (without gene duplication and without loss of the primary


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function), this is different to a single gene that can generate different proteins by DNA rearrangement, alternative RNA splicing, or post-translational processing. Gene duplication is responsible for the emergence of novel functions in the moonlighting protein families, therefore is not surprising that the HSPs are perfect examples of proteins that perform a wide range of activities in and/or outside the cells. The second concept of interest is that chaperones are intrinsically disordered proteins (IDPs), they show substantial disordered regions (at least in vitro) behaving as biomolecules that natively do not have a definite 3D structure (they contain long regions that do not form a precise globular structure) and they show propensity to aggregate to form oligomers [12, 13]. The alternative splicing and the post-translational modifications (like phosphorylation) expand the functional repertoire of IDPs; these proteins, according to the localization, may fold into different conformations upon binding different polypeptide clients or protein partners. The conformational changes seem to be necessary to improve the chaperone function, among them we can mention HSP33, HSP60, HSP70, and HSP90. These proteins are greatly influenced by ATP- binding and ATP-hydrolysis and by cochaperones that drive large conformational rearrangements of the chaperones modulating and influencing the protein-client interactions. In addition, there are ATPindependent chaperones (e.g., yeast HSP26) where the activation and client binding is driven by large-scale order-to-disorder transitions and where the client protein/polypeptide release is controlled by disorder-to-order transitions. Within the IDPs we can also mention Parkinson´s protein α-synuclein, Alzheimer´s disease protein tau, p53, BRCA1 and many other disease-associated proteins. Therefore, researchers are interested in IDPs because they are attractive targets for drugs that could act modulate or interfere with protein-protein interactions. The term chaperonopathies was first applied to any condition associated with alteration in the expression, post-translational modification or localization of chaperones, but now has a more restricted use to describe pathological conditions (inherited or acquired) in which mutated chaperones play a determinant etiologic-pathogenic role [4, 14, 15]. There are several diseases that appear when the HSPs are mutated or do not perform their normal task: re-folding of clients proteins or if this is not possible, sending them to degradation using the ubiquitin-proteasome system or using the autophagy-lysosome pathway. When the cells are chronically exposed to toxic protein aggregates, Parkinson’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease, and polyglutamine diseases can be generated; in these diseases the intrinsically misfolded proteins are little or not refolded at all [4, 14, 15]. It has been proposed that in each protein aggregation disease a different group of HSPs is trying to rescue specific types of aggregation [4]. Entering into cancer, Fig.(1) shows the associations between the different chronic stressors, the responses generated by the cells, and the effects that they produce.



Fig. (1). Summary of proposed mechanisms that can be activated when the cells are exposed to different chronic stresses. In the upper layer appears the stressors followed by the primary effects that can be generated, and after that the different stress responses (SR). These responses put the cells in another state more adapted to survive or can lead to chronic diseases including cancer. The chronic psychological stress has been placed because it generates many hormonal and immune changes; in addition, it has been shown that repeated pre-weaning stress in rats (using a chronic unpredictable stress model applied to produce depressive disorders) can change the expression of HSP70 in certain areas of the reproductive tract during early and adult life [16]. Thus, a the social stress environment (even during childhood) can triggers changes to the psychological stress response many years later, linking subsequent changes in stress resiliency to physical health [17]. Oxidative stress is often considered as a causative factor in carcinogenesis, increased production of reactive oxygen species (ROS) is an attribute of malignant cells and is linked to the development of many of the characteristics considered hallmarks of cancer [18]. DNA is constantly subject to numerous insults from endogenous sources (cellular metabolism) and exogenous sources (environmental agents), DNA replication must be tightly regulated to ensure that the genome is accurately duplicated during each cell cycle and when these regulatory mechanisms fail, replicative stress and DNA damage ensue [19]. The HSP response has been involved in DNA repair, some of the HSPs can travel to the nucleus and although it is believed that the HSPs are not capable of repairing the DNA damages by themselves, they might assist to the different DNA repair mechanism (as part of their molecular chaperone capabilities) interacting with DNA repair proteins producing their stimulation and reactivation [20]. However, if the DNA damages cannot be efficiently repaired following the initial chronic stressors (many of which are cancer initiators), HSP increases are fueled in cancer through the proliferation of mutated proteins due to the “mutator phenotype” associated with cancer generating the “addiction to chaperones” hypothesis [21].

There are many stress-responsive pathways/systems leading to network adaptations, in other words, to broad transcriptomic changes that give rise to the mechanistic repertoire that dynamically accommodate the cellular processes to the new environment; the final effects are often associated with cancer causation and other chronic diseases. If the HSP response was not awake during these chronic stresses and cancer is generated, the HSP response can be activated later on. This is seen for example in prostate cancer where at the initial stages the changes in HSP expression are subtle but in most advanced clinical stages there are many HSPs over-expressed and they usually are associated with resistance to different anticancer therapies [22]. However, depending on the type of


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cancer (and their molecular profile and interactions), the HSPs can be over-expressed and strongly associated with disease prognosis and with prediction of response to therapy, but sometimes there is even a decreased expression of certain HSPs or no associations with cancer prognosis [23]. Nevertheless in general we should remember that cancer cells continuously divide and therefore tumor cells are challenged by restricted supplies of nutrients and oxygen, low pH, decreased vascularization, apoptosis or necrosis, and even the macrophages create a more favorable microenvironment for cancer cell development and invasiveness (by secreting cytokines, growth factors and angiogenic factors), all of these can generate or increase the HSP response. Previous reviews have shed light on the involvement of the HSP response with cancer [24 - 32]. Fig.(2) represents the numerous functions where the HSPs are involved.

Fig. (2). Schematic representation of the main events that can produce a HSP response, and the involvements of the HSPs in normal as well as pathological events. Note than under certain circumstances the HSPs can be up-regulated bypassing HSFs.

HSP27 (HSPB1) HSP27 belongs to the family of the small HSPs sharing several common characteristics Table 2. Table 2. Common characteristics of the small HSPs [reviewed in 21]. • small molecular weight (20-40 kDa). • at the level of protein structure, these proteins contain an α-crystalline domain (located in the C-terminal part) and a less conserved N terminal domain (with an hydrophobic WD/PF motif and phospho-serine sites). • ability to oligomerize. • structural heterogeneity. • dynamic plasticity explaining their capacity to interact with various client proteins. • following stress conditions they act as holdase that trap mis-folded proteins avoiding their aggregation (cooperating with HSP70 and HSP90). • ATP-independent molecular chaperone action.



What Happens when HSP27 is Increased/Activated? HSP27 (and other HSPs) participates in the initiation and promotion of cancer, in the regulation of the cellular resistance to apoptosis, acts on the cytoskeleton, is involved in extracellular matrix organization, stimulates metastasis formation and dissemination, and is involved in epithelial to mesenchymal transition (EMT). In cancer stem cells there is increased activation of the p38MAPK, MAPKAPK2, HSP27 pathway, and HSP27 has been implicated in senescence through the activation of the p53 pathway and induction of p21waf. This particular HSP is related with anticancer drug resistance (including chemotherapies, hormonotherapy and radiotherapy). These fundamental actions can be explained by the multitude of interaction with client proteins [reviewed in 21]. Fig.(3) shows the involvement of HSP27 in different signaling pathways. In a recent article the specific associations of HSP27 with resistance to chemotherapies in gynecologic malignancies have been reviewed [33]. The capacity of HSP27 to cooperate with HSP70 and HSP90 to trap miss-folded proteins (to avoid their aggregation) and to escort client proteins (modifying their function, transport, half-lives, etc.) makes it difficult to achieve an effective therapeutic response (e.g., inhibiting HSP90 alone), that is, since they are redundant when one chaperone is inhibited others can replace it. Among the important client proteins of HSP27 we can mention β-catenin, Daxx, cytochrome c, pro-caspase-3, Stat2 and 3, Akt, HDM2, eIF4E, HER2, HDAC6, and PTEN [32]. The complex changes in HSP27 phosphorylation and oligomerization are key parameters to explain the dynamic plasticity of this protein to interact with different client substrates particularly in tumor cells. It has been pointed out that the phosphorylation and oligomeric status of HSP27 are not easy to be studied and for this reason we are relatively far to have a clear understanding of the HSP27 interactome [21, 32]. In addition, a broad inhibition of HSP27-client interaction may prove to be disappointing (as it is been observed for the inhibitors of HSP90), we hypothesize the needing of specific inhibitors of HSP27 with specific clients, with proteins important to cancer initiation, progression or drug resistance. For example, in breast cancer cells and tissues HSP27 interacts with βcatenin, β-catenin is normally located at the cell membrane (in this localization β-catenin coordinates the cell-cell adhesion), but when HSP25 (the rodent analog to human HSP27) was introduced into the cells, a redistribution of β-catenin was observed (translocation to the cytoplasm, in this localization the protein regulates other functions, it may even be located in the nucleus where it can regulate gene transcription) [34]. And the patients where the breast cancer tissues showed cytoplasmic β-catenin rather than membrane expression presented poor prognosis [34, 35]. Therefore, a drug that inhibits HSP27/βcatenin interaction might avoid the translocation of β-catenin to cell compartments that facilitate tumor growth and metastasis.


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Fig. (3). Schematic illustration implying that HSP27 is at the crossroad of important signaling pathways. HSP27 interacts (i) with cytoplasmic (c) beta-catenin [34] leading to poor prognosis of breast cancer patients [34]. In fact membrane beta-catenin has been associated with good prognosis in HER2-positive breast cancer patients [35]. After phosphorylation HSP27 can be translocated to the nucleous (N) where it interacts with MSH2 [233]. Moreover, HSP27 produces down-regulation of PTEN [234], and stabilizes HER2 [235]. Note that some critical other pathways have been omitted for clarity. Some clinical consequences of these interactions have been listed in this figure. The coordinated absence of PTEN/p27 and PTEN/AKT has been associated with lower response to trastuzumab in HER2+ metastatic breast cancer [236], the stabilization of HER2 by HSP27 has been associated with lower response to trastuzumab [235], while MSH2 has been implicated in drug resistance [237]. Therefore, HSP27 targeting is emerging as a critical step in cancer treatment.

Another example might be generating a new(s) antiangiogenic cancer therapy involving HSP27, since in vitro studies revealed that the addition of human recombinant HSP27 to chick chorioallantoic membrane had a pro-angiogenic effect through Toll-like receptor 3 (TLR3) [36]. The authors reported that after rapid internalization of HSP27 with a pool of TLR3, NF-κB was activated leading to VEGF (vascular endothelial growth factor receptor)-mediated cell migration and angiogenesis.

What happens when HSP27 is Decreased? Table 3 shows a summary of the articles of the last 5 years dealing with the decrease of HSP27 content and phosphorylation and the consequences achieved.



Table 3. HSP27 down-regulation and cell line/model Effects and author(s). HSP27 down-regulation and cell line/model Effects and author(s) I. Down-regulation by genetic constructs (references 2010-2014) siRNA, in the doxorubicin-resistant ERp29 (tumor suppressor, regulates mesenchymal-epithelial transition) over-expressing MDA-MB-231 cells, and parental MCF-7 cells. ↓ cell viability, ↑ doxorubicin-induced apoptosis [37]. siRNA, MCF-7 breast cancer cells. Prevented sex steroid receptor trafficking to and signaling from the membrane. Nuclear ERα expression was unaffected [38]. siRNA knockdown, in the highly migratory metastatic HNSCC cell line. ↓ metastatic behaviors, cell invasion and migration [39]. Down-regulation in prostate cancer cells. ↓ eukaryotic translation initiation factor 4E (eIF4E). HSP27 → resistance to androgen ablation and chemotherapy [40]. OGX-427 (second-generation modified antisense oligonucleotide complementary to HSP27), pancreas MiaPaCa-2 cancer cells grown both in vitro and xenografted in mice. Inhibited proliferation, induced apoptosis and enhanced gemcitabine chemosensitivity (involving the eukaryotic translation initiation factor 4E) [41]. siRNA, in a gastric tumour cell line. Completely abolished the effect of acidosis (acidosis → prolong cell survival after doxorubicin treatment) [42]. shRNA constructs. MDA-MB231/B02 human breast cancer cell line in mice with bone metastasis. ↓ cell migration and invasion in vitro and in vivo [43]. ↓ several putative client proteins [44]. siRNA administrated to DLD-1/5-FU cells (colon cancer cells resistant to 5-fluorouracil). Restored the sensitivity to 5-FU (implicating the phosphorylated HSP27 forms) [45]. Knockdown of the adaptor protein p66shc (regulator of mitochondrial production of oxidative stress, phosphorylated p66Shc binds and abrogates HSP27 function), renal proximal tubular cells, cisplatin treatment. Accelerated cisplatin-dependent disruption of the actin cytoskeleton [46]. Down-regulation of HSP27 in angiogenic tumor cells, xenograft tumors. ↓ endothelial cell proliferation, ↓ secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. → long-term tumor dormancy in vivo [47]. Knockdown of HSP27, breast cancer stem-like cells (BCSCs). Potentiate the BCSC targeting effect of 17AAG [48]. OGX-427 administration to rat subpleural/pulmonary fibrosis models. Snail proteasomal degradation → inhibition TGF-β1-induced epithelial-to-mesenchymal transition [49]. siRNA transfected into leukemia cells. ↑ chemosensitivity and apoptosis [50].


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(Table 3) contd.....

siRNA transfected to hepatocellular carcinoma cells. ↑ sensitivity of 17AAG. The sensitivity of cells to 17AAG was associated with the level of HSP27 expression [51]. Locked nucleic acid1 and shRNA administration to head-and-neck cancer cells and tumor xenografts in nude mice. Radiotherapy. ↑ radio sensitivity in vitro and in vitro (implicating HSP27 in double strand break repair) [52]. siRNA administered alone or in combination with sub-therapeutic concentrations of 17-AAG (HSP90 inhibitor) or staurosporine (non-selective kinase inhibitor), glioblastoma cells. ↑ chemo sensitivity [53]. CMPD1 or MAPKAPK2 (MK2) knockdown, or overexpression of non-phosphorylatable mutant HSP273A → decreased HSP27 phosphorylation, HeLa cells. ↑ TNF-α induced apoptosis (interacting with TAK1 and regulating TAK1 post-translational modifications) [54]. II. Down-regulation by natural/synthetic chemical compounds (references 2009-2014) Quercetin (plant flavonoid that suppress HSPs) or siRNA (↓ HSP27 and HSP40), on chemotherapeutic drugs treatment in hepatoma Hep3B and HepG2 cells. Potentiates 5-fluorouracil and carboplatin effects [55]. Quercetin or siRNA-mediated HSP27 gene silencing, breast cancer stem-like cells (BCSCs). ↓ intracellular aldehyde dehydrogenase activity (ALDH+) population, mammosphere formation and cell migration. ↓expression of snail, ↓ vimentin and ↑ E-cadherin [56]. Quercetin or HSP27 knockdown, using a drug-resistant sphere model of oral squamous cell carcinoma. Combination of quercetin and cisplatin → reduced tumor growth and decrease drug resistance via p38 MAPK-Hsp27 axis [57]. Quercetin or siRNA-mediated HSP27 gene silencing. Formation of vascular-like channels (VM) that lack of endothelial cells, breast cancer stem/progenitor cells. Suppression of VM capability [58]. Locked nucleic acids are oligonucleotides that can be synthesized as nuclei acid analogues in which the ribose ring is “locked” by a methylene bridge (connecting the 2'-O atom and the 4'-C atom), they show high affinity binding to complementary RNA resulting in superior potency when used for antisense inhibition. 1

So far the main way to decrease the HSP27 content has been achieved by genetic manipulations by the use of RNA interference (RNAi) transfecting siRNAs or shRNAs. A review article [59] deals with the design of the constructs, transfection efficiency and other major issues for siRNA techniques. The challenge for the clinical use of siRNA and miRNA relies on the development of appropriate carriers mainly for systemic delivery like biodegradable nanoparticles, lipid nanoparticles, bacteria, and attenuated viruses (reviewed elsewhere) [60, 61]. Peptide aptamers are described below in the HSP70 section. All of the articles presented in Table 3 agree that the decrease of HSP27 is producing beneficial effects to interfere with the development of cancer cells, that the decrease in



HSP27 increases the sensitivity to other chemotherapeutic compounds and even to radiation therapy. Special interest has been generated in OGX-427, an HSP27 antisense oligonucleotide developed by Martin E. Gleave (licensed to OncoGenex Technologies, Vancouver, Canada). Besides the two articles mentioned in Table 3, there are others that also show what is happening after OGX-427 administration in vitro and in vivo tumor models [62 66]. In essence these articles show that OGX-427 administration induces apoptosis, autophagy, interferes with EMT, that HSP27 is interacting with proteins involved in DNA repair and alternative splicing, and that targeting HSP27 increases therapy sensitivity to other anticancer therapies (including chemotherapies and HSP90 inhibitors). The synergist enhanced effects obtained when combined with HSP90 inhibitors might be important in the clinic since these inhibitors per se can induce in tumor cells and tissues the expression of HSPs, among them HSP27, in a dose- and time-dependent form. OGX427 (Apatorsen) is currently being tested in seven randomized Phase 2 clinical trials including different tumor types: advanced metastatic bladder cancer, non-small cell lung cancer, metastatic pancreatic cancer, and prostate cancer. These are exciting times for seeing the turn of event targeting HSP27.

HSP60 HSP60 has been classically described as a mitochondrial molecule with specific functions related to mitochondrial biogenesis and homeostasis. It works together with its cochaperonin HSP10, assisting in the correct folding of other important mitochondrial proteins [67]. However, it is now clear that HSP60 can be found also in extramitochondrial sites such as the cytosol [68], intracellular vesicles [69] and plasma membrane [70], and its concentration in extramitocondrial sites can increase particularly during the pathophysiology of some human diseases including autoimmune diseases and cancer [71,72]. Fig.(4) shows the main HSP60 localizations.

Fig. (4). In normal cells, HSP60 is localized mainly inside mitochondria, with a very low concentration in the cytosol. Vice versa, in tumor cells it accumulates in cytosol and reaches the plasma membrane. In cytosol, HSP60 may bind pro-caspase 3 (pC3), thus inhibiting apoptosis cascade. From plasma membrane, Hsp60 is internalized via lipid rafts in multivesicular bodies (now shown) and in turn secreted into exosomes.


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Concerning oncogenesis, several papers showed that extramitochondrial HSP60 can have some direct extrachaperoning roles in tumor transformation and progression. For instance, (i) cytosolic HSP60 is involved in caspase-3 cleavage and apoptosis activation in non-tumor [73,74] and tumor [68,75] cells; and (ii) cytosolic HSP60 is secreted by tumor cells by both the classic (Golgi’s) and alternative (exosomal) pathways [76], in turn having a putative role in modulating the antitumor immune response. Due to these extrachaperoning functions extramitochondrial HSP60 can be considered as a moonlighting protein. The list of tumors in which HSP60 has been found increased and/or secreted has grown in the last years, the list includes large bowel, prostate, liver, stomach, uterus and ovarian cancers. However, HSP60 levels may be reduced during carcinogenesis in other anatomical sites (for unknown reasons) such as in cancer of the oral cavity, bronchial airways and bladder. It has been postulated that smoke habits could be related to HSP60 level reduction in these sites, but very recent evidences showed that cigarette smoke extracts do not reduce HSP60 levels, at least in bronchial airway cells [77]. Reviews on the implications of HSP60 in cancer have been presented elsewhere [15, 78 - 80]. The cancer type(s) in which HSP60 helps the tumor cells to grow and survive has been defined as “chaperonopathies by mistake”, the chaperone favors the disease rather than protect the organism from it [81]. In these cases a negative chaperonotherapy [82] is an option, namely, the chaperone must be blocked in the tumor cells, so these will die. This type of chaperonotherapy should aim at, for example, finding molecular antagonists able to interact with HSP60 and inhibit the HSP60/pro-caspase-3 complex formation [68,75] and, thereby, activate apoptosis in tumor cells without interfering with HSP60 functions in normal cells. A comprehensive review on chaperonopathies and chaperonotherapy has been presented [81]. Despite the role played by HSP60 in tumor progression discussed earlier, only a few studies have been devoted to identify chaperonin targeting drugs. We have already treated this topic in previous publications [15, 81 - 83]. Table 4 shows a list of HSP60 inhibitors. In this paragraph we want to report the most important information to give to the readers an idea about the state-of-the-art on the research targeting HSP60 in cancer therapy. Here is a list of the papers presented in the literature that, to the best of our knowledge, address this point: 1) HSP60 was found to be a target for the immunosuppressant mizoribine, a nucleosidelike molecule containing an imidazole heterocycle that is complexed by HSP60 [84]. 2) Epolactaene is another compound that was discovered to inhibit the chaperoning activity of human HSP60 by covalently binding the Cys442 residue [85]. 3) A hypoxic-inducible factor 1 alpha (HIF-1α) inhibitor carboranylphenoxyacetanilide was recently discovered to bind HSP60 as a primary target, although its role in HSP60



function inhibition was not determined [86]. 4) In the same study, the epolactaene tert-butyl ester (ETB) inhibitor was shown to suppress HIF-1α either by direct or indirect involvement of HSP60 in HIF-1 α accumulation [86]. 5) Other compounds described as HSP60 putative inhibitors are those related to avrainvillamide, which are supposedly involved in the alkylation of cysteine residues through their electrophilic 3-alkylidene-3H-indole 1-oxide moiety. However, no inhibition of HSP60 function was correlated to cysteine interaction with avrainvillamide analogues [87]. 6) In a study on atherosclerosis, several natural products such as sesamol, vanillyl alcohol, trans-ferulic acid, zerumbone, humulene, and caryophylene showed a moderate to significant inhibitory activity of HSP60-induced cell proliferation The activity was more pronounced in compounds possessing a partly unsaturated 11-membered alicyclic ring, although their direct binding to, or interaction with HSP60 has not yet been demonstrated [88]. Table 4. List of HSP60 inhibitors. Compound

Effect

Mechanism

Avrainvillamide

Direct

Alkylation of HSP60 cysteine residues

Caryophylene

Undetermined

Undetermined

Epolactaene

Direct

Binding the Cys442 residue of HSP60

Epolactaene tert-butyl ester inhibitor

Either direct or indirect Undetermined

HIF-1α inhibitor

Undetermined

Undetermined

Humulene

Undetermined

Undetermined

Mizoribine

Direct

Complexing to HSP60

Sesamol

Undetermined

Undetermined

Trans-ferulic acid

Undetermined

Undetermined

Vanillyl alcohol

Undetermined

Undetermined

Zerumbone

Undetermined

Undetermined

The above reported papers show that to date only epolactaene derivatives and pyrazolopyrimidine-based compounds have been proved to bind HSP60. However, since these compounds are able to inhibit HSP60 protein folding activity, they are potentially lethal for normal cells. Hence, further studies have to be made in this direction for the search of new chemical compounds targeting HSP60 with potential antitumor activity without altering its physiological role in normal cells.

HSP70 The 70 kDa heat shock proteins (HSP70s) are expressed in virtually all living organisms.


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The human family of HSP70 proteins is characterized by at least eight polypeptides that share a high level of homology (>80%) but differ in their functions and intracellular localizations [89 - 92]. Six members of the family locate mainly in the cytosol and nucleus while two members are specific to the endoplasmic reticulum and mitochondria. These proteins are characterized by a molecular weight of about 70 kilodaltons and by their conserved ability to act as ATP dependent molecular chaperones. Of interest, recent data suggest that individual HSP70 proteins can have non-overlapping and chaperoneindependent functions essential for growth and survival of cancer cells [92]. New nomenclature has recently been proposed where HSP70 polypeptides are called HSPA polypeptides [1] (Table 1). The human family of polypeptides comprise the heat shock (or stress) inducible HSP70 proteins: HSP70-1 (HSP72, HSPA1) and the recently described HSP70-2 (HSPA2) [93], HSP70-6 (HSP70B, HSPA6) and HSP70-7 (HSPA7). The other members of the family are not stress inducible and have been called HSP70like or HSP70 cognate (Hsc70) polypeptides. They are constitutively expressed in different tissues and cellular organelles and appear assigned to separate biological tasks. Their new names are the following (Bip/GRP78: HSPA5; Hsc70: HSPA8; GRP75/mtHsp70: HSPA9).

HSP70 Functions a). Chaperones Involved in Protein Folding Table 5 shows the main functions of HSP70s. Consequently of their ATP-dependent chaperone activity, HSP70 polypeptides share an ATPase domain (in the N-terminal region), a substrate binding domain (containing a groove) and a C-terminal domain acting as a 'lid' of the groove [94]. Following ATP hydrolysis the refolding of trapped peptide substrates occurs consequently of lid closing [95]. To refold proteins, stress inducible HSP70s [96] usually associate with different co-chaperones, HSP40, HSP90, Hip, Hop and HSP110 [97, 98]. As a consequence of their crucial role in the cell machinery for protein folding these HSPs enhance the resistance of cells to deleterious environmental conditions [91, 99 - 102]. Moreover, the recent literature is filled with reports that describe the considerable importance of HSP70s in maintaining cellular homeostasis. Indeed, in addition of being able to refold aberrantly folded proteins, these polypeptides play key roles in the folding of cytosolic, endoplasmic reticulum and mitochondrial proteins and therefore are involved in the control of many regulatory proteins. They can also participate in the dissolution of protein complexes or in the transport of proteins into subcellular compartments [21, 92, 94, 103]. It is well known that cells have developed sophisticated mechanisms to respond to various stresses, but depending on their strength, different cellular responses are observed. For example a mild stress usually induces a protective heat shock response characterized by an enhanced HSPs expression, in contrast there are more potent stresses that can trigger an apoptotic process and an even stronger



one can lead to necrosis [104]. In that regard, it is interesting to note that HSP70s can protect cells from both necrotic and apoptotic processes. However, more work is needed to determine whether these activities rely on their "stress-like chaperone" properties or to sophisticated interactions which modulate the activity of client polypeptides. Table 5. Main functions of the HSP70 family of proteins. • ATP-dependent molecular chaperone action usually associated with different co-chaperones. • Control of many regulatory proteins. • Transport of proteins into different sub-cellular compartments. • Protect cells from both necrotic and apoptotic processes (but sometimes they have pro-apoptotic functions). • Stimulate signals for the immune system, present tumor associated antigens to antigen presenting cells. • Required for cancer growth and survival.

b). HSP70, Necrosis and Apoptosis Back in early nineties, it was demonstrated that inducible HSP70A1 counteracted the necrotic cell death mediated by oxidative stress [105] or tumor necrosis factor [106]. In the case of oxidative stress, HSP70 was supposed to participate in the elimination of oxidized molecules while in the presence of the pro-inflammatory cytokine TNFα, HSP70 interfered with the signal transduction pathway leading to phospholipase A2 activation [106, 107]. Another intriguing property of HSP70s relates to their roles in controlling apoptosis, for example in thermal or osmotic stress condition, inducible HSP70 interferes with the apoptotic process by inhibiting the activation of caspases 3 and 9 as well as the JNK pathway [108 - 112] (Fig. 5). HSP70 can also negatively alter the apoptotic process by interacting with Apaf-1 (apoptosis protease-activating factor-1) [113] or the proapoptotic polypeptide Bax [114 - 116], hence inhibiting apoptosome formation or cytochrome c release from mitochondria. Downstream of mitochondria, HSP70 is also efficient by interacting with AIF (apoptosis-inducing factor), hence abolishing AIF ability to translocate in the nucleus to induce chromatin condensation [117, 118]. In addition, HSP70 modulates the activity of caspases-activated DNAses [119] and the efficiency of death receptors mediated apoptosis. For example, through association with TRAIL-R1 and TRAIL-R2 receptors, HSP70 interferes with the recruitment and activation of procaspase-8 [120]. Consequently, its suppression by siRNA sensitized cells to TRAILinduced apoptosis by upregulating the expression TRAIL receptors and down regulating c-FLIP-L levels [121]. On the other hand, and contrasting with the earlier observations of Jäättelä [107], HSP70 is pro-apoptotic in human colon cancer cells by differentially regulating TNF(-induced activation of NFκB and JNK [122, 123]. Hence, in spite of having been described as a general inhibitor of apoptosis [38], there are some recent examples where HSP70 has a pro-apoptotic activity [122 - 124].


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c). Other HSP70 Functions Many other intriguing activities of HSP70 polypeptides have been described, one particular example is the maintenance of lysosomal membrane by HSP70-2 (HSP70A2), a member of the HSP70 family essential for spermatogenesis which, as described below, is required for cancer growth and survival [125, 126]. Other interesting observations deal with the fact that, together with antigenic peptides, inducible HSP70 can be expressed at the cell membrane of cancer [127, 128] or virus infected [129] cells. HSP70 can even be found into the extracellular environment where it generates crosstalk with the immune system. Indeed, this protein could act as a source of antigen due to its ability to chaperone peptides or be a stimulating signal for the immune system [130, 131].

Fig. (5). Schematic illustration showing the main pathways of apoptosis where HSP70 is involved. Note that some critical other pathways and other HSPs have been omitted for clarity.

d). HSP70 Functions and Pathologies Important protective role mediated by HSP70 polypeptides have been described in human pathologies. Depending on the cells that are affected and similarly to small HSPs [132], the protection mediated by HSP70 can either be beneficial or deleterious to patients. For example, these HSPs are beneficial in the case of heart and brain ischemia, asthmas, lungs and liver sepsis or degenerative diseases. On the contrary, by promoting tumor development and their resistance to anticancer therapies, these HSPs can be detrimental for patient survival. Consequently, HSP70s are tempting targets for therapeutic interventions in several diseases [104, 132].



HSP70 in Cancer Pathologies Protection against stress or apoptotic process can favor the development and/or maintenance of pathologies characterized by an aberrant proliferation of deleterious cells. Among them one can cite breast, endometrial and uterine cervical cancer cells which display high levels of HSP70 expression, particularly the inducible form HSP70A1 [21, 23, 24, 133]. In these cells, HSP70 confers survival advantage, promotes tumor cell invasion and resistance to chemotherapeutic drugs [21]. In ovarian cancer, despite its limited up-regulation, HSP70 also appears to play a deleterious role associated with a poor prognosis [134, 135]. However, in this type of cancer, HSP70 has not yet been demonstrated to be responsible for the poor response to therapy or a reliable prognostic marker [135, 136]. What are the precise mechanisms by which HSP70 polypeptides are required for tumor development? It is well-known that HSP70 expression can be associated with poor tumor cell differentiation, lymph node metastasis, apoptosis inhibition and increased cell proliferation. HSP70 is thus conferring cancer cell survival advantage as well as resistance to chemotherapeutic agents, all these factors facilitate tumor cell invasion [21]. In spite of the fact that this point is still a matter of discussion, recent observations suggest that at least two members of the HSP70 family, HSP70A1 and HSP70A2, promote cancer cell growth by separate mechanisms [137]. Up until recently, most of the experimental and clinico-pathological data were dealing with uncharacterized HSP70 polypeptides (probably mainly the major stress-inducible HSPA1). None of the other family members were carefully analyzed due to the lack of experimental tools to differentiate between them, limitation recently overcome by the RNA interference technology that can allow targeted knockdown of the individual HSP70 family members.

HSP70A1 The first major observation, performed by Jäättelä in 1995, showed that the overexpression of HSP70A1 conferred tumorigenicity to mouse fibrosarcoma cells [138]. Then, and as described above, it was shown that this protein, which is often expressed in primary tumors, is an efficient inhibitor of apoptosis that can also counteract the cytotoxicity of drugs aimed at destroying cancer cells. In addition, HSP70A1 can interact and probably stabilize the half-life of oncogenic mutants of the tumor suppressor p53; hence favoring the survival of cancer cells [139]. Defects in apoptosis signaling pathways can trigger tumor initiation, apoptosis normally eliminates cells prone to undergo malignant transformation. Then these defects can enhance tumor progression and metastasis by allowing tumor cells to survive the bloodstream transit and growth in ectopic tissue sites. Finally, apoptosis inhibition may render cancer cells resistant to therapies. Hence, HSP70A1 has been considered as a survival protein for cancer cells together with some anti-apoptotic members of the Bcl-2 protein family, HSP27 and


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survivin [140]. Although the role of HSP70 in metastatic cancers still appear complex [133], a recent report suggests that this protein is involved, via the p38/MAPK pathway, in the EMT [141]. Several studies also pointed to the role played by HSP70 in the immune response towards cancer cells. For example, it was shown, using colon cancer cells injected in nude mice (or syngeneic rats), that a reduced level of HSP70 expression, by promoting cell death in vivo, could generate a tumor specific immune response [142]. Indeed, subcutaneous injection of cells with reduced levels of HSP70 expression induced tumors that rapidly regressed in syngeneic rats but grew in nude mice. Moreover, these rats were protected against a further challenge with cells expressing a normal level of HSP70. The next step was the discovery of the association of HSP70 with plasma membrane and its ability to be present outside of cells where it could act as a chemokine/danger signal [128, 143 - 148]. HSP70 is released within exosomes by cancer cells and acts as a carrier for immunogenic peptides. These HSP70/Bag-4 surface-positive tumor exosomes stimulate migratory and cytolytic activity of NK cells that induces apoptosis in tumors through granzyme B release, a phenomenon blockable by HSP70-specific antibody [149, 150]. HSP70 surface-positive tumor exosomes were also described to induce proinflammatory cytokine release and to up-regulate CD83 expression by naive dendritic cells [145]. Moreover, a CD8-specific T cell response is induced following receptormediated uptake of HSP70-GP96 peptide complexes by antigen-presenting cells and their representation by MHC class I molecules [151]. On the other hand, HSP70 tumor-derived exosomes (TDEs) are also known to contribute to cancer development consequently of their interaction with MDSCs (Myeloid-derived suppressor cells), a population of immature myeloid cells that suppress T cell activation. This interaction determines the suppressive activity of the MDSCs via activation of the transcription factor Stat3 in a Toll-Like Receptor (TLR2/MyD88) -dependent manner and through autocrine production of IL-6 [152]. At least three TLRs, TLR2 and TLR4 function as receptors that couple the binding of HSP60, HSP70 and GP96 to NFκB activity [153, 154]. In addition, the cellsurface protein CD14 (which couples LPS exposure to TLR4 activation) is also required for HSP70-mediated induction of TNF-α, IL-1β and IL-6 [155]. However, the precise role played by HSP70 in antigen presentation and cross-presentation remains controversial. On one way, it is well agreed that HSP70 can present tumor associated antigens to antigen presenting cells through MHC class I and class II molecules and thus activate anti-tumor T cells. On the other side of the coin, HSP70 can recruit cells responsible for the generation of complex innate and adaptive immune responses towards tumor cells, a field which is still a matter of intense research [31, 156].

HSP70A2 As mentioned above, one interesting example refers to HSP70-2 (HSP70A2), a protein essential for spermatogenesis, which shows a cancer-associated translocation to the



lysosomal compartment [125, 126, 157] where it promotes cell survival by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced death. Indeed, in cancer cells, lysosomal membrane is instable and requires the presence of HSP70A2 which, by binding to BMP (bis-monoacylglycero-phosphate), enhances the activity of acid sphingomyelinase, an important enzyme that hydrolyzes sphingomyelin to avoid membrane permeabilization and the release of cathepsin enzymes [137, 158]. Consequently, depletion of HSP70A2 results in lysosomal membrane permeabilization and cathepsin-dependent cell death [126]. Moreover, these studies identified LEDGF (lens epithelium-derived growth factor) as a polypeptide regulated by HSP70A2 essential for lysosomal stability in human cancer. LEDGF, by stabilizing lysosomal membrane, increases the tumorigenic potential of human cancer cells and protects them against the cytotoxic effects induced by anticancer agents that trigger the lysosomal cell death pathway. Of interest, LEDGF, which is now known as an oncogenic protein that controls a caspase-independent lysosomal death pathway, shows an increased level in human breast and bladder carcinomas similarly to HSP70A2.

Targeting HSP70 in Cancers HSP70 polypeptides, which are marginally constitutively expressed in non-transformed cells, are highly overexpressed in many tumor cells and at least in the case of HSP70A1 and HSP70A2 have an important role in tumor development. Moreover, many reports point to the fact that their silencing can be cytotoxic to tumor but not normal cells [159]. Hence, a great interest has recently emerged to develop HSP70 inhibitors for cancer therapy [160]. They have the potential to open new treatments for cancers resistant to therapies, via activating the classical apoptosis pathway.

Small Interfering RNA (RNAi) and Abrogation of HSP70 Expression Approaches Decreasing the level of HSP70 has been proposed to improve the efficiency of cancer therapeutic treatments. In fact, several proteomic analyses have revealed that an efficient treatment is often associated with decreased levels of HSP70, as for example the treatment response of neoadjuvant aromatase inhibitors (AI) in ER-positive postmenopausal breast cancer patients [161]. RNAi approaches have been tested with good results, but most of these studies were performed using cultured cells [159]. In that respect, an interesting study dealing with leukemia cells tested the abrogation of HSP70 induction as a strategy to increase antileukemia activity of 17-allylamino-demethoxy geldanamycin (17-AAG), a HSP90 chaperone inhibitor which destroys its association with many client proteins, including the transcription factor HSF1. Treatment of human acute myelogenous leukemia cells with 17-AAG is known to trigger HSP70 overexpression which in turn confers resistance to 17-AAG mediated apoptosis. This resistance was abrogated by treating cells with HSP70 siRNA or KNK437, a benzylidine


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lactam inhibitor of HSP70 induction [115]. A similar analysis was recently performed using HSP70 siRNA in nanocarriers [162]. Hence, this field of research is improving particularly in the way to deliver HSP70 siRNA to tumor cells. The next step will be to test its efficiency in cancer patients.

Chemical Drugs Abolishing HSP70 Functions In contrast to HSP90 [163 - 165], up until recently only few and not very efficient compounds were known to interfere with HSP70 chaperone activity and its ability to interact with client polypeptides. Intense focus was then placed on the search for HSP70 inhibitors [166]. Historically dihydropyrimidines were among the first compounds known to modulate the ATPase activity of HSP70A1. In that respect, 15-deoxyspergualin, showed significant antitumor activity in mice but, in a phase II trial it was inefficient against metastatic breast cancer resistant to front-line chemotherapy [167]. A secondgeneration derivative, such as MAL3-101 showed antimyeloma effects acting on both primary tumor cells and on bone marrow endothelial cells from myeloma patients [168]. Other drugs belonging to the imidazol and rhodocyanine families have been tested. For example, apoptozole which inhibits the ATPase activity of HSP70-1 (HSP70A1) and HSP70-8 (HSC70, HSPA8) and induced apoptosis in human embryonic carcinoma and lung cancer cells [168]. MKT-077, which belongs to the rhodocyanine family of compounds, preferentially inhibited the ADP-bound form of HSP70A9 (GRP75) and HSP70A8 [169]. In spite of MKT-077 antitumor activity in preclinical studies using human tumor xenografts in nude mice [170], a phase I trial revealed functional renal impairment necessitating early termination of the study [171]. Other interesting compounds belong to the phenylethynesulfonamide family. Among them, 2phenylethynesulfonamide (PES) interacts selectively with HSP70 and disrupts its association with its cochaperones and substrate proteins [172]. In a mouse model of Mycinduced lymphomagenesis, PES was found to enhance survival and suppressed tumor development [172]. Cell cultures analysis also revealed that PES stimulates cell death triggered by protein aggregation, impaired autophagy and lysosomal function, hence suggesting that it could disrupt the activities of HSP70 polypeptides involved in different cell signaling pathways [172], including the autophagy-lysosome system and the proteasome pathway [173]. In addition, PES was clearly shown to disrupt the HSP70/HSP90 purinosome chaperone system resulting in the inactivation of purine biosynthesis as well as many other HSP70/HSP90 client substrates essential for tumor development [174]. Structural analysis revealed that PES interacts with the substratebinding domain (SBD) of HSP70 and that the C-terminal helical ‘lid’ (amino acids 573616) is necessary for the interaction [175]. Moreover, these studies allowed the discovery of a PES derivative, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which displayed an increased efficiency compared to plain PES. Interestingly, this new anti-cancer compound has several mechanisms of action: it can impair the activity of the Anaphase Promoting Complex/Cyclosome and induce G2/M arrest and promote genomic instability in cancer



cells [175]. Structural studies aimed at optimizing the potency and therapeutic efficacy of HSP70 modulators allowed to the discovery of new inhibitors that bind to the unique allosteric pocket within the conserved C-terminus SBD of HSP70 polypeptides [176]. Other recent inhibitors, such as VER-155008, bind to the nucleotide binding domain (NBD) of HSP70 and are ATP-competitive inhibitors that interfere with the allosteric control between NBD and SBD [177]. However, none of the SBD and NBD inhibitors were isoform-specific suggesting that they interacted with all the different HSP70 polypeptides. Another study compared the efficiency of the three major HSP70 inhibitors that are studied today: PES-Cl, MKT-077, and Ver-155008. Inhibition of autophagy and reduced levels of HSP90 client proteins was common to the three inhibitors but, surprisingly, only PES-Cl was able to induce a G2/M arrest [178]. Another problem raised by these studies concerns the lack of agreement on the phenotypic effects of HSP70 inhibitors on cancer cells. One explanation could be that, and in contrast to HSP90 [179 - 181] and HSP27 [32, 44, 182, 183], no reliably-identified "clients" of HSP70 have yet been reported; a phenomenon that may result of the relatively nonspecific behavior of HSP70 to control protein folding.

Aptamers, as Tools to Discover Peptidomimetic HSP70 Inhibitor Drugs Peptide aptamers, which are short peptides of random sequence inserted into a scaffold protein, have shown specific interaction with proteins and modulation of their activity, as for example in the case of oncogenes, transcription factors, cell cycle regulators and HSP27 [184 - 188]. Several peptide aptamers have been isolated that interacted with either the peptide-binding domain or the ATP-binding domain of HSP70. Both inhibited HSP70 chaperone activity and enhanced the sensitivity of cells to anticancer drugs. Regression of subcutaneous tumors after local or systemic injection was observed concomitantly with macrophages and T lymphocytes recruitment into the tumor bed [189]. As in the case of HSP27 [187, 188], aptamers probably interfered with HSP70 conformational state and inhibited its interaction with pro-cancerous clients proteins. Aptamer interacting site modeling could be an interesting tool to approach the structural organization of future neo-synthesized inhibitors. On the other hand, screenings aimed at discovering peptidomimetic drugs that interfere with HSP70-aptamer interaction may lead to the discovery of new chemical inhibitors [190].

Targeting Cell Surface and Extracellular HSP70: Antibody, Vaccines and Exosomes Disruption As mentioned above, membrane-bound or extracellular HSP70 acts as carriers for immunogenic peptides [150, 151, 191]. On the cell surface of human and mouse tumors only the C-terminal domain of HSP70A1 bearing a 14-mer peptide, termed "TKD," is detectable and can stimulate the activity of NK cells. An antibody (cmHsp70.1 mAb) that recognized TDK was found to trigger antibody-dependent cellular cytotoxicity of


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membrane HSP70 positive mouse tumor cells by unstimulated mouse spleen cells. Adding TKD to the effector cells further enhanced tumor killing. In vivo, reduced tumor growth, infiltrations of NK cells, macrophages and granulocytes as well as enhanced overall survival was observed after injections of the cmHsp70.1 mAb into mice bearing CT26 tumors [127]. These observations clearly show that membranes of tumor cells bearing HSP70 are tumor-specific target structures. This property was also used to generate autologous therapeutic antitumor vaccines for vaccinating cancer patients. In a particular study performed by Dr. Ciocca’s lab, vaccine was prepared by combining hydroxyapatite (HA) ceramic particles with tumor cell membrane proteins (including HSP70, HSP27, and GP96 and their chaperoned proteins/peptides). HA particles were used as vector to present the tumor self- antigens and adjuvants to antigen-presenting cells (APCs) and for their ability to survive for at least one week after being administered under the skin before being phagocytized. The vaccine, which was well tolerated, induced positive tumor rejection in certain patients by activating the T-cell response [192]. Antibody(ies)-mediated response after vaccination also play a major role in the antitumoral response, a phenomenon that can be up-regulated by abolishing the suppressive tumor microenvironment. Ongoing studies with HSP-based anticancer vaccines are promising, particularly in melanoma patients and in patients with renal clear cell cancer (without advanced disease) [31]. Towards extracellular HSP70, recent observations have enlighted its ability to restraining tumor immune surveillance. Indeed, once release from tumor cells, HSP70 associated with tumor-derived exosomes (TDEs) can interact with MDSCs, a phenomenon that contribute to cancer development by triggering their expansion and ability to suppress T cell activation in an HSP70/TLR2-dependent manner. In that respect, studies aimed at reducing the level of exosomes released from mouse tumors by dimethyl amiloride upregulated the antitumor efficacy of the chemotherapeutic drug cyclophosphamide. A similar effect was observed in cancer patients [152].

HSP90 In eukaryotes, members of the HSP90 family are found in the cytoplasm, in the nucleus and in organelles; in the human five isoforms of HSP90 has been identified differing in domain structure, cellular location and substrate specify, for example there are two cytosolic HSP90 isoforms: an inducible (HSP90α) and a constitutive form (HSP90β) [1,21,193]. Examples of organelle-specific HSP90 forms are: TRAP1 (tumor necrosis factor receptor-associated protein 1) in mitochondria [194], HSP90C in chloroplasts [195], GRP94 (94 kDa glucose- regulated protein) and isoform HSP90N in endoplasmic reticulum [196] [197]. In addition, HSP90 can be secreted and found on the surface of cells [198,199]. Cochaperone molecules such as Aha1, Cdc37, Hop, PP5 and p23 contribute to maintain the normal chaperone function of HSP90, and post-translational modifications of HSP90 and its cochaperones are essential for their functions. HSP90



participates in cellular homeostasis and tightly command environmental stress responses by assisting correct folding and maturation processes of client proteins which include several tumor-related proteins. Of interest here is to mention that HSP90 has an extended substrate binding interface that crosses domain boundaries, exhibiting specificity for proteins with hydrophobic residues spread over a large area regardless of whether they are disordered, partly folded, or even folded [reviewed in 193]. This specificity principle ensures that clients preferentially bind to HSP70 early on in the folding pathway, but downstream folding intermediates bind HSP90. HSP90 is at the crossroads of late folding and early degradation trajectories [193]. Therefore, HSP90 can help tumor cells to maintain the malignant status supporting important oncoproteins required for tumor development including signal-transduction proteins (Bcr-Abl, B-Raf, Alk, Akt), tyrosinekinase receptors (mutated EGFR, HER2, c-Kit, IGFR1), transcription factors (p53, HIF1α, steroids receptors), cell-cycle regulatory proteins (Rb, Cyclin D, CDK4), antiapoptotic proteins (Bcl-2, Apaf1, survivin) and many others, in a similar manner to normal cells (telomerase hTERT, Fak1, MMP2) [180, 200 - 203]. Hence, HSP90 appeared as an attractive molecular target for cancer therapy and HSP90 inhibitors are being investigated as cancer chemotherapeutic drugs. Recent studies revealed that HSP90 inhibitors have the capability to target multiple hallmark cancer processes such as: lack of sensitivity to anti-growth and apoptosis signals, increased angiogenesis, and tissue invasion and metastasis formation [204]. The first-generation of HSP90 inhibitors were benzoquinone ansamycins including natural products like geldanamycin and radicicol and the synthetic derivatives of geldanamycin: tanespimycin (17-AAG), alvespimycin (17-DMAG), retaspymicin hydrochloride and KF58333. However, the clinical efficacy of these compounds has been very poor owing to several disadvantages including limited solubility, formulation troubles, potential multidrug efflux activities, and hepatotoxicity [205,206]. Furthermore, as monotherapeutic agents these molecules displayed modest efficacies in the clinical setting [207,208] indicating that they may be useful only in combination with other anticancer drugs. In an attempt to reverse these restrictions several second-generation HSP90 inhibitors are under study. Among them various synthetic small molecules inhibitors such as ganetespib and NVP-AUY992, which are engaged in active clinical trials [209] (Table 6). HSP90 inhibitors have shown early promising results in specific molecular subgroups of solid tumours including ALK-rearranged non-small-cell lung cancer and HER2-positive breast cancer and some haematological malignancies such as multiple myeloma) [210]. In breast cancer the HSP90 inhibitors have not been approved for use in the clinic yet, the main activity of these compounds has been noted in metastatic HER2-positive breast cancer, for a complete and exhaustive review please refer to Zagouri et al. [211]. The authors suggest that HSP90 inhibitors may also play a beneficial role in the treatment of triple negative and aromatase-inhibitor breast cancer resistance subtypes. Since


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ganetespib affects EGFR signaling cascades (reducing the levels of EGFR), it is not surprising to note synergistic killing with radiation and cisplatin in gastric cancer xenograft tumors [212]. Table 6. Clinical trials evaluating selected inhibitors of HSP90. Inhibitor Tumour type

Treatment arms

Phase

Advanced NSCLC NCT01798485

Ganetespib–docetaxel vs. docetaxel

III


Ganetespib

II

Advanced NSCLC ALK+ NCT01031225

Ganetespib

II

Advanced NSCLC ALK+ NCT01562015

Ganetespib–crizotinib

II

Metastatic breast cancer NCT01579994

Ganetespib

II

Advanced breast cancer NCT01560416

Fulvestrant–ganetespib vs. fulvestrant

II

Advanced or metastatic b.c. HER2+ NCT02060253

Ganetespib–paclitaxel–trastuzumab

I

Ganetespib

Recurrent ovarian, fallopian tube or primary Ganetespib plus weekly paclitaxel peritoneal cancer NCT01962948

II

Malignant pleural mesothelioma NCT01590160

Cisplatin–pemetrexed–ganetespib vs cisplatin–pemetrexed–placebo

I/II

Metastatic ocular melanoma NCT01200238

Ganetespib

II

AML and high-risk MDS NCT01236144

Chemotherapy (daunorubicin–cytarabine–etoposide) plus AC220 or plerixaforor and ganetespib

I/II


NVP-AUY922

II

Advanced EGFR-m+ NSCLC NCT01646125

NVP-AUY922 vs. docetaxel or pemetrexed

II

Advanced NSCLC with exon 20 insertion mutations in EGFR NCT01854034

NVP-AUY922

II

ALK+ NSCLC NCT01752400

NVP-AUY922

II

Advanced HER2+ breast cancer NCT01271920

NVP-AUY922–trastuzumab

IB/II

Metastatic pancreatic adenoca NCT01484860

NVP-AUY922

II

Refractory GIST NCT01404650

NVP-AUY922

II

Advanced GIST NCT01389583

NVP-AUY922

II

Relapsed and refractory lymphoma NCT01485536

NVP-AUY922

II

Myeloproliferative neoplasms NCT01668173

NVP-AUY922

II

NVP-AUY922



(Table ) contd.....

Inhibitor Tumour type

Treatment arms

Phase


Retaspimycin–docetaxel

II

KRAS-mutant advanced NSCLC NCT01427946

Retaspimycin–everolimus

IB/II

Retaspimycin (IPI-504)

Adapted [209]. Clinical trial code that can be searched at https://clinicaltrials.gov/ Abbreviations used: NSCLC = non-small-cell lung cancer; b.c. = breast cancer; EGFR-m = EGFR-mutated; ALK = anaplastic lymphoma kinase; HER2 = human epidermal growth factor receptor 2; AML = acute myelogenous leukemia; MDS = myelodysplastic syndrome; adenoca = adenocarcinoma; GIST = gastrointestinal stromal tumour. a

In breast cancer cell lines relevant interactions of HSP90 with molecules related to hereditary breast cancer have been described, cells with mutations in the BRCA1 gene have reduced DNA repair mechanisms and are relatively sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Johnson et al. [213] showed an HSP90-mediated stabilization of a BRCA1 C-terminal domain. The stabilized BRCA1 mutant protein was essential for conferring both PARP inhibitor as well as cisplatin resistance. These authors showed that treatment of resistant cells with the HSP90 inhibitor 17dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibitors. For other implications HSP90 in drug resistance please see a recent review [33]. The HSP90 inhibitors are showing a broad spectrum of anticancer activity, for example in suppressing squamous carcinogenic progression in a mouse model of esophageal cancer [214], in head and neck squamous cell carcinomas [215] and in patients with advanced solid tumors [216]. Contrasting results have been reported on the immune system, HSP90 inhibitors share the ability to increase the expression of differentiation antigens as well as MHC Class I antigens, without interference on T cell function, which could be important in the design of immunotherapies against cancer [217]. However, HSP90 inhibitors have potent detrimental effects on stimulated CD4⁺ T cells making them not suitable for the treatment with immunotherapeutic approaches aimed to induce dendritic cell/T cell activation [218]. The HSP90 inhibitors that target the N-terminus of the protein can trigger a survival mechanism in cancer cells activating the heat shock response, this response is not activated by HSP90 inhibitors that modulate the C-terminus domain [219]. However, others claim that C-terminal inhibitors as well as HSP90 co-chaperone disruptors may prevent cancer cell proliferation similarly to N-terminal inhibitors destabilizing HSP90 client proteins without affecting the induction of other HSPs [220]. This is an area of intense research, we suggest the reader to consult recent reviews and upcoming articles [221 - 225].


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Liu et al. [226] conducted in ovarian cancer data-mining meta-analyses (merging results from multiple siRNA screens) to identify gene targets showing significant inhibition of cancer cell growth. These authors found several cancer-related genes that were client proteins of HSP90. Based on these findings ganetespib was evaluated against epithelial ovarian cancer using it as a single agent and using it in combination with cytotoxic and targeted therapeutic agents. The authors concluded that ganetespib is augmenting standard epithelial ovarian cancer therapies. Hendrickson et al. [227] evaluated in patients with advanced ovarian and peritoneal cancer the efficacy and biological effects of the gemcitabine/tanespimycin combination. The levels of HSP90 and HSP70 were examined in PBMC (peripheral blood mononuclear cells) and in paired tumor biopsy lysates to assess the effect of tanespimycin on tumor cells. In this two-cohort phase II clinical trial the patients were grouped according to prior gemcitabine therapy. All participants received tanespimycin 154 mg/m 2 on days 1 and 9 of cycle 1 and days 2 and 9 of subsequent cycles. Patients also received gemcitabine 750 mg/m2 on day 8 of the first treatment cycle and days 1 and 8 of subsequent cycles. The tanespimycin/gemcitabine combination induced a partial response in 1 gemcitabine naïve patient and no partial responses in gemcitabine resistant patients. Stable disease was seen in 6 patients (2 gemcitabine naïve and 4 gemcitabine resistant). Limited up-regulation of HSP70 was noted in the immunoblotting and little or no change in the levels of most client proteins (in PBMC and paired tumor samples). Therefore, although well tolerated, in patients with advanced epithelial ovarian and primary peritoneal carcinoma the tanespimycin/gemcitabine combination exhibited limited anticancer activity. This treatment did not induce a significantly down-regulation of the HSP90 client proteins.

CONCLUDING REMARKS HSPs are essential molecular chaperones that assist the correct folding of nascent polypeptides and stress-accumulated misfolded proteins hence preventing their aggregation and lethal damage. Towards cell death processes, HSP27, HSP70 and others are efficient to counteract apoptosis, necrosis as well as cell death mediated by lysosome breakage. A strong protection of cells is therefore generated by the expression of these proteins. This can be beneficial for patients suffering from pathologies characterized by cells that are dying and need to be protected, i.e. degenerative diseases. On the other side, in cancer pathologies characterized by a mitotic escape of deleterious cells, HSP polypeptides appear to have a reverse effect. Indeed, in addition of being up-regulated, these proteins and particularly HSP70A1 and HSP70A2 display a strong efficacy to sustain the aggressivity of cancer cells. Therapeutic approaches were therefore planned to deplete the HSPs or inactivate their protective functions. RNAi technologies as well as drugs that broadly inhibit their chaperone activity were tested alone or in combination with other anti-cancer drugs. However, to date none of these approaches has successfully completed a phase III trial. We should remind readers that by using drastic and broad inhibitors and/or RNAi all HSP clients are knocked out [the “bad” (pathological) ones as



well as the “good” ones] therefore, the therapeutic approaches targeting HSPs will only be successful if one day we succeed in targeting the bad clients and not the good ones, otherwise we will always be facing side-effects deleterious to patients. The recent discovery that HSP70 can be membrane-bound and extracellularly located has complexified therapeutic researches aimed at targeting these proteins. Indeed, in addition to protecting cells HSP70 can also act as danger signals and elicit complex immune responses mediated either by the adaptive or innate immune system. Progresses have been made to better understand the complex HSP roles as carriers for immunogenic peptides, inducers of cytokine release or NK cells activators. Based on their membrane bound localization, recent studies and ongoing clinical trials suggest that the vaccine approach could be successful. In addition, the discovery that extracellular HSP70 can restrain immune surveillance open up a new road of investigation aimed at reducing the level of exosomes released by tumors that interact with Myeloid-derived suppressor cells. In that respect, the potent effect of dimethyl amiloride to up-regulate the antitumor efficacy of chemotherapeutic drugs appears promising to find new research avenues aimed at abolishing the suppressive tumor microenvironment. On the other hand, recent studies revealed that constitutively expressed HSPs have immense cellular implications consequently of their interaction with many client polypeptides, whose number is nowadays growing exponentially. Many proteins interacting with HSP90 have already been characterized, but their number is still growing (up-dated HSP90 client proteins: http://www.picard.ch/Hsp90Int/index.php). Small HSPs rank second since more and more proteins are reported to interact with these HSPs [32, 44, 132, 183]. These observations already lead to a preliminary proteomic analysis of small HSP client proteins [228]. The studies dealing with HSP60 and HSP70 are still less successful consequently of the only small number of reliably-identified clients. So, the crucial question (at least in the case of HSP90 and small HSPs) is what molecular approaches should be tested to identify client proteins as an approach to improve chaperonotherapeutic molecular targeting and efficacy? In that regard, the first approaches that have been tested referred to HSPs expression invalidation (RNAi approaches) or to the use of drugs that inhibit HSPs chaperone activity. Some of these approaches proved to be efficient in rodents [43, 49, 229, 230]. However, they probably will be disappointing in long term treatments of humans, as already revealed by the unsuspected side effects of HSP90 anti-chaperones drugs in clinical trials resulting in lack of FDA recognition [165, 231]. The main reason for this failure results of the complete disruption of HSP90 protein interactomes by anti-chaperone drugs, hence knocking-down the pro-cancerous as well as the friendly and useful client proteins of HSP90. Future and efficient chaperonotherapeutic molecular targeting of HSPs will therefore be based on hyper-specific drugs or techniques targeting only the subset of pathological HSPs-client interactions without altering HSPs interactions with essential clients [232]. This will represent a huge task for the years to come. Indeed, comprehensive dynamic HSPs-


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protein interactomes must first be known before pertinent approaches can be established to destroy HSP specific interactions with pathological clients, in particular in cancer pathology. We believe that the bases are set out, that in the near future we will see synergist enhanced antitumor effects when the established anticancer drugs combined with HSP inhibitors could be administered to cancer patients.

CONFLICT OF INTEREST The authors confirm that this chapter content has no conflict of interest.

ACKNOWLEDGEMENT Supported by the National Research Council (PIP CONICET) of Argentina.

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