Chaperoning the Guardian of the Genome. the Two-Faced Role Of

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BBA - Reviews on Cancer 1869 (2018) 161–174

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BBA - Reviews on Cancer

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Review Chaperoning the guardian of the genome. The two-faced role of molecular T chaperones in p53 tumor suppressor action ⁎ Bartosz Wawrzynowa, Alicja Zyliczb, Maciej Zyliczb, a Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland b International Institute of Molecular and Cell Biology, Warsaw, Poland

ARTICLE INFO ABSTRACT

Keywords: Organized networks of heat shock proteins, which possess molecular chaperone activity, protect cells from Tumor suppressor p53 abrupt environmental changes. Additionally, molecular chaperones are essential during stress-free periods, Heat shock proteins where they moderate housekeeping functions. During tumorigenesis, these chaperone networks are extensively Mutant p53 gain of function remodeled in such a way that they are advantageous to the transforming cell. Molecular chaperones by buffering Chemoresistance critical elements of signaling pathways empower tumor evolution leading to chemoresistance of cancer cells. Carcinogenesis Controversially, the same molecular chaperones, which are indispensable for p53 in reaching its tumor suppressor potential, are beneficial in adopting an oncogenic gain of function phenotype when TP53 is mutated. On the molecular level, heat shock proteins by unwinding the mutant p53 protein expose aggregation-prone sites leading to the sequestration of other tumor suppressor proteins causing inhibition of apoptosis and chemoresistance. Therefore, within this review therapeutic approaches combining classical immuno- and/or chemotherapy with specific inhibition of selected molecular chaperones shall be discussed.

1. Introduction p53 has been shown to be associated with normal development and cell differentiation, including proper regulation of self-renewal and differ- 1.1. The concept of p53 tumor suppressor protein entiation of embryonic stem cells (ESC) and adult stem cells (ASC) [5,6]. Moreover, p53 was shown to inhibit cellular transformation The p53 protein is a pivotal tumor suppressor, termed “guardian of within the population of damaged stem cells by selectively inducing the genome” on account of its role in maintaining genomic stability and apoptosis [7]. inhibiting tumorigenesis of the cell [1,2]. Under normal conditions the A prerequisite for tumor development is often the disruption of level of genotypic wild type p53 (WT p53) protein is tightly controlled normal p53 function. Thus, mutations in the TP53 gene are acutely (maintained at low levels) through proteolysis mediated by the ubi- common in cancer cells [2,8]. > 70% of them are missense mutations, quitin-proteasome pathway, intricate translation control, dynamic which result in a single amino acid substitution clustered in the DNA subcellular relocalization and numerous interactions with other com- binding domain of the p53 protein [8]. These mutations can be di- ponents of transcriptional machinery [3]. Upon stress conditions such chotomized into at least two classes: these which disrupt the global as: DNA damage, oncogene activation, telomere erosion, hypoxia, heat conformation of the DNA binding domain (structural mutations), and shock or ribosomal stress, the p53 protein is stabilized and activated those that affect DNA binding without affecting the conformational (Fig. 1). Following its activation p53 induces a variety of processes stability of the domain (contact mutations). The inactivating missense primarily acting as a transcription factor (through the activation and/or mutations of TP53 are advantageous during cancer development due to repression of specific promoters). Furthermore, it directly interacts with their action as trans-dominant inhibitors of WT p53. Moreover, p53 key regulator proteins or is involved in maturation of miRNA reg- tumor-associated mutants, apart from the canonical loss of tumor sup- ulators. These actions of p53 tumor suppressor protein can lead to cell pressor activity, gain new oncogenic functions (GOF) (Fig. 1). This GOF cycle arrest, initiation of DNA repair processes, metabolic pathway(s) phenotype broadly manifests itself in site-specific regulation of cancer refinement, cellular senescence, autophagy, inhibition of cell invasion metabolism, malignant progression including increased tumorigenesis and metastatic potential culminating in the induction of apoptosis and metastasis [9,10]. Within the GOF spectrum, mutant p53 may (Fig. 1) (for review see [4]). In addition to tumor suppressive activity, operate alone or in complex with other proteins. One such group

⁎ Corresponding author at: International Institute of Molecular and Cell Biology in Warsaw, 4 Ksiecia Trojdena Street, 02-109 Warsaw, Poland. E-mail address: [email protected] (M. Zylicz). https://doi.org/10.1016/j.bbcan.2017.12.004 Received 17 October 2017; Received in revised form 28 December 2017; Accepted 29 December 2017 Available online 31 January 2018 0304-419X/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). B. Wawrzynow et al. BBA - Reviews on Cancer 1869 (2018) 161–174

Fig. 1. Tumor suppressor versus oncogene, chaperoning the p53 protein. Detailed description of the figure is presented in the text (Sections 1.1 and 3). Briefly, due to its labile thermodynamic nature, the p53 polypeptide at physiological conditions is in equilibrium between native - wild type and mutant conformations. Molecular chaperones through transient cyclic bind-unwind- release reactions shift this state, so that p53 can adopt its proper tertiary and quaternary structure and carry out its tumor suppressor role. Chemical chaperones such as divalent cations or small molecule agents have a similar end effect. Nevertheless, this dynamic state can be shifted through internal and external stimuli, as de- picted. Molecular chaperones in this case, through stable non-cyclical binding, stabilize mutant p53, prevent it from proteasomal degradation, stimulate its dominant negative properties and facilitate the formation of non-canonical quaternary arrangement. In effect, being sequestered themselves to these structures. In other words, the involvement of molecular chaperones belonging to the heat shock protein family, is indiscriminate between p53 tumor suppressor and its pro oncogenic gain of function counterpart. The prime difference is the network organization in which these chaperones act.

comprises of molecular chaperones, which belong to the heat shock substrate [21] and by co-chaperone specificity factor DnaJ (HSP40), family of proteins [11–13]. along with the nucleotide exchange factor GrpE [23], or ii) substrate specificity determined by DnaJ co-chaperone [24] were later confirmed in their eukaryotic orthologs [25,26]. Nucleotide exchange factors 1.2. The concept of molecular chaperones (NEFs) not only recycle HSPs bound to client polypeptide [23,27] but also are involved in selective recognition of the client polypeptide The specific three-dimensional fold of proteins governs their biolo- [26,28]. Moreover, they were shown to link the HSPA machinery to gical functions. Protein folding, maintenance of proteome integrity, and protein folding, protein disaggregation or proteolysis [14,15 ,29]. The protein homeostasis (proteostasis) critically depend on a complex net- co-chaperone HOP was demonstrated to link the HSP70 (HSPA) and work of molecular chaperones [14–16]. Most molecular chaperones HSP90 (HSPC) chaperone machines [30]. Apart from the HSPA/HSPC – belong to the class of heat shock proteins (HSPs), originally identified as dependent polypeptide folding, 10 to 15% of nascent polypeptide stress-responsive proteins required managing thermal and other pro- chains require chaperonins to reach proper native conformation teotoxic stresses. Historically the nomenclature for HSPs was desig- (Hsp60; GroEL, GroES in prokaryotes and CCT (TRiC) chaperonins in nated in accordance to their approximate molecular weight and con- eukaryotic cells [15]). sequently the main families of these proteins were distinguished: small The functions of molecular chaperones, belonging to the HSP fa- HSP (sHPS), HSP40, HSP70, HSP90 and HSP60 (chaperonins). The mily, are very broad. Initially the first biologically relevant reaction human genome encodes 11 members of - sHSP (HSPB) with the mole- utilizing molecular chaperones was described on the molecular level for cular weight between 15 and 30 KDa, 13 members of the HSP70 (HSPA) bacteriophage lambda initiation of DNA replication [21,31]. Based on family ranging from 66 to 78 kDa, 5 members of the HSP90 (HSPC) those findings and other data available at the time, such as: i) the family with 85–100 kDa and roughly 14 members of the chaperonin discovery that, HSP upon stress is translocated from the cytoplasm to and related cohort, HSP60 (HSPD1, HSPE1, CCT) with molecular nucleus and subsequently catalyzes the re-assembly of damaged pre- weight around 60 kDa [17]. The most abundant group is the HSP40 ribosomes within the nucleoli, and probably other ribonuclear proteins (DNAJ) or J-like protein family. At present it is known that the human (RNPs) after heat shock [32] and ii) that the BiP protein (HSP70 genome encodes 41 members of DNAJ family [18] with very broad member residing within the ER) was shown to transiently interact with range of molecular weight from 10 kDa (DNAJC19) to 254 kDa heavy chain immunoglobulins [33], Hugh R. Pelham published an ar- (DNAJC13) or 520 kDa for J-like protein, DNAJC29 (sacsin) [19]. ticle in Cell (1986) speculating on the role of heat shock proteins [34]. The first Hsp gene [20] and its protein product [21] were identified Astonishingly, all his predictions turned out to be correct. The data and biochemically characterized as bacterial DnaK, later to be desig- from several contributing laboratories, including our laboratory, has nated as a prokaryotic ortholog of the eukaryotic HSP70 (HSPA) family. firmly supported the conjectures proposed by HR Pelham: i) HSP70 has The amino acid sequence, as well as protein function, of HSP70 (HSPA) a general affinity for polypeptides with hydrophobic amino acid re- family members are highly evolutionary conserved [22]. All key bio- sidues, denatured and abnormal protein(s) [35], ii) HSP70 aids in the chemical properties of bacterial heat shock proteins such as: i) week assembly of specific protein-protein complexes e.g. the preprimosomal ATPase activity of DnaK, which is stimulated by binding to the

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BBA - Reviews on Cancer 1869 (2018) 161–174 complex involved in bacteriophage lambda DNA replication [36], as One possible explanation for the negative correlation is that HSP70 well as the assembly of purified Rubisco enzyme involved in photo- (HSPA1A) can be involved in anti-cancer immunity [62]. synthesis [37], iii) under non-stress conditions constitutive HSC70 For other tumors, a direct correlation was observed between the (HSPA8) recognizes nascent polypeptide chains [38,39], iv) HSP70 and levels of HSPA family members and patient prognosis; e.g., HSPA2 the protein degradation system recognize similar types of substrates elevation correlated with lower survival rates for lung adenocarcinoma [14], v) members of the HSPC (HSP90) family interplay with HSPA (LUAD) and esophageal squamous cell carcinoma patients [63] albeit, (HSP70) proteins in protein assembly (activation of glucocorticoid re- for the same HSP a decrease of its expression (below the median) cor- ceptor [40], vi) members of the HSPA family partially unfold protein related with poor survival for breast invasive carcinoma (BRCA) pa- substrates and thus dissociate preexisting aggregates [26,41,42]or tients [48]. Also, a decrease in HSPA14 expression correlated with protein structures e.g. uncoating clathrin vesicles [43]. In other words, decreased survival rate for LUAD and BRCA alike [48]. For other molecular chaperones not only dissociate existing aggregates within members of the HSPA family like HSPA4, HSPA5, HSPA6, HSPA12A cellular compartments, but also, by passively blocking aggregation, their expression in tumor tissues was associated with poor outcomes they increase the rate of protein folding. Moreover, molecular cha- from HBV-related early-stage hepatocellular carcinoma [64]. In addi- perone assistance is particularly beneficial for proteins for which the tion, overexpression of HSPA9, a gene that encodes mortalin - a mi- native structure(s) do not correspond to the global thermodynamic tochondrial chaperone, was advocated to be a poor prognostic bio- energy minimum. marker for BRCA patients [65]. Moreover, mortalin (MOT2) was shown Currently more than thirty years after Pelham's Cell paper, we know to be involved in cytoplasmic sequestration of WT p53 protein [66], that HSPs belong to a broader class of molecular chaperones, which thus its high expression during cellular transformation is probably regulate a variety of cellular processes during normal and stress con- needed for breast cancers possessing WT p53 [48]. ditions including protein folding, translocation of polypeptide to the The pro-survival effect(s), resulting from HSP70 (HSPA1A) eleva- organelles and outside of the cell, proteolysis, protection of client tion, in cancer cells are attributable to the fact that this molecular proteins from denaturation, dissociation of aggregates. John Ellis de- chaperone: i) is a potent suppressor of apoptosis [54,67] and lisosomal fined molecular chaperones as a class of unrelated families of proteins death [68], ii) is required for tumor growth (the presence of HSP70 that assist the correct non-covalent assembly of other polypeptide- bypasses senescence) [69], HSP70 (HSPA1A) also controls expression of containing structures but are not components of these assembled p21/CDKN1A (Cyclin-Dependent Kinase Inhibitor 1A), a major factor structures when they are performing their normal biological function inducing cell cycle arrest [70]) iii) affects cell signaling (as mentioned [44]. However, a growing body of evidence suggests that molecular before) and iv) assists in the repair and proper folding of damaged or chaperones are biological molecules with evolutionary conserved mutated proteins. functions in helping cells to adapt, survive and proliferate. Among them Within the human cell two cytosolic isoforms of HSP90 (HSPC) are molecular chaperones belonging to the HSP families, which are co- exist, an inducible HSP90α (HSP90AA1) variant and a constitutive opted by cancer cells to evade cell death, bypass senescence and refa- HSP90β (HSP90AB1) form. These are known to collaborate with several shion cell signaling. These HSPs are actively involved in supporting classical co-chaperones, such as: HOP, PP5, p23, AHA1, SGT1, CDC37, highly malignant human cancers preserving them from diverse stress FKBP51/52, CYP40, CHIP, which were shown to regulate the ATPase conditions, including chemotherapy [45–48]. activity and chaperone activity of HSP90 proteins. Additionally, they act as specificity factors for the HSP90 client proteins. The HSP90 in- 2. Molecular chaperones in cancer teractome appears to be smaller than that of HSPA or chaperonins (HSP60) family. Most of them are kinases, E3 ubiquitin ligases, hor- Elevated expression of several molecular chaperones is a common mone receptors and transcription factors [71]. Interestingly, HSP90 feature of both solid tumors and hematological malignancies [49,50]. interacts with a group of specialized co-chaperones, e.g. R2TP, where Increased chaperone activity allows tumor cells to cope with im- its complex with HSP90 is responsible for multi-molecular protein balanced signaling (pathways) that are associated with neoplastic complex assembly. These comprise basic gene expression machinery: transformation and thereby escape apoptotic death that would nor- (1) nuclear RNA polymerases; (2) the snoRNPs, essential to produce mally ensue. Molecular chaperones have been implicated in all char- ribosomes; and (3) mTOR Complex 1 and 2, which control translational acteristic hallmarks of cancer; including self-sufficiency in growth sig- activity and cell growth. The scientific field speculates on the roles of nals, insensitivity to growth-inhibitory signals, evasion of apoptosis and R2TP as a master regulator of cell growth under normal or pathological increased replicative potential [50]. conditions [72,73]. Two AAA+ ATPases belonging to R2TP complex, The HSP70 (HSPA1A) molecular chaperone together with its co- Pontin and Reptin possess broad cellular activities. They are involved in chaperones and HSP90 was found to be necessary for the conforma- transcriptional regulation, chromatin remodeling, DNA damage, sig- tional stability and activity of many: i) oncogenic proteins (e.g. EGFR naling and repair, assembly of macromolecular complexes, regulating and HER2 (ERBB2)); ii) signaling proteins (e.g. AKT (AKT1), RAF1, cell cycle/mitotic progression, and cellular motility. Furthermore, they IKKB (IKBKB), SRC, HIF1A, STAT3) iii) transcription factors (e.g. HSF1 are considered as candidates for promoting tumorigenesis and cancer and p53 (TP53)); iii) cell cycle regulators; iv) chimeric signaling pro- development [74]. Recently the crystal structure of yeast HSP90-R2TP teins (e.g. BCR/ABL+) and hormone receptors (e.g. estrogen and glu- complex was solved [75]. Elevated expression of cytosolic HSP90 pro- cocorticoid receptors) [40,50–56]. teins and their co-chaperones was observed to occur in many cancer In breast cancer, overexpression of HSP70 (HSPA1A) has been cor- cells. However, this expression is tumor-specific[76]. Therefore, the related with metastasis and poor prognosis [57,58]). Ectopic over- activity of HSP90 in various cancer subtypes is different and most expression of HSP70 (HSPA1 A, B) in several cell types increased their probably is modified by a divergent subset of co-chaperones. This leads transformation potential [54,59]. Consistent with these observations, to altered client substrate(s) specificity and aberrant sensitivity to ad- the abrogation of HSP70 expression inhibited tumor cell proliferation ministered drugs during chemotherapy [77]. Lately it has been shown and induced apoptotic response [60,61]. Although HSP70 (HSPA1A)is that TSC1 tumor suppressor is a novel HSP90 co-chaperone, which fa- overexpressed in most human cancers, there are some tumor types with cilitates HSP90-mediated folding of kinase and non-kinase clients (TSC1 low expression of this gene, which exhibit no correlation between and TSC2 proteins form the tuberous sclerosis complex - TSC, a reg- HSP70 (HSPA1A) levels and cancer survival (e.g. renal cancer). More- ulator of mTOR activity) [78]. over, an inverse correlation between HSP70 (HSPA1A) expression and Apart from regulatory cohort of co-chaperones, the function of patient prognosis/survival rate was observed for squamous cell carci- HSP90 proteins is additionally modulated by numerous post transla- noma, cholangiocarcinoma, or oral carcinoma (for review see [54]). tional modifications (PTMs), which include phosphorylation,

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SUMOylation, ubiquitination, acetylation and S-nitrosylation 3. Chaperoning the wild type p53 protein [55,56,79,80]. Recent evidence suggests that inhibition of the enzymes that catalyze HSP90 PTMs can act synergistically with HSP90 in- Biophysical analysis revealed that full-length genotypic wild type hibitors, providing a novel therapeutic strategy to enhance the efficacy p53 (FL WT p53) protein, under physiological conditions, occurs in the of HSP90 inhibitors in cancer cells [81]. form of monomers, dimers and tetramers, with the equilibrium shifted Oncogenesis itself can be considered as a microevolution process in towards dimers [89]. Additionally, from a thermodynamic standpoint, which the hostile environment within the tumor exerts selection pres- the tertiary structure of p53 tumor suppressor is relatively unstable, sure on the population of cancer cells. For example, the induction of with its low melting temperature around 45 °C [90]. The folding process angiogenesis is one of the critical adaptation steps necessary to over- of p53 multi-domain structure is very complex and most probably leads come oxygen and nutrient deficits and promotes tumor progression. to the formation of folding intermediates which ultimately can end up The HIF-1α (HIF1A) transcription factor, a labile master regulator of with wild-type, mutant-like conformations or aggregates. Regarding the angiogenesis, is stabilized by HSP90 and HSP70 [82]. Moreover, severe latter, a different mode of p53 aggregation was suggested. The ex- hypoxia is often found within the stroma surrounding the tumor. Gene periments performed by Wang and Fersht on purified FL WT p53 and its mutations in tumor cells and hyper-activation of intracellular HSP90 different variants show that the aggregation mechanism of WT p53 is and HSP70 chaperone machinery cause constitutive accumulation of different from classical nucleation, growth and formation of amyloid HIF1A. This in turn triggers secretion of HSP90α (HSP90AA1) and most fibrils [91,92]. The authors suggest that the aggregation of purified FL probably HSP70 (HSPA) family members via exosome mediated protein WT p53 and its core domain is very rapid and only concentration de- trafficking. Extracellular HSP90α (HSP90AA1) activates MMP2, LRP-1, pendent. The rate limiting step is the sequential unfolding and combi- tyrosine kinase receptors to promote cell motility and invasion of tumor nation of the two molecules. The presence of fresh un-aggregated mu- cells [83]. These findings suggest an alternative therapeutic approach tant p53 accelerates the aggregation process, through which to target HSP90 in cancer, through selective inhibition of the tumor- occasionally WT p53 can be trapped [91,92]. secreted HSP90α (HSP90AA1) instead of the intracellular HSP90 Under cellular physiological conditions WT p53 exists in a constant (HSP90α and HSP90β)[84]. Nevertheless, this approach is challenged state of thermodynamic equilibrium between wild type (WT) and mu- by the fact that extracellular (and membrane bound) HSP70 and HSP90 tant-like conformation (Fig. 1). Conformational switching between the have been shown to play key role in eliciting antitumor immune re- two states can occur as a result of: i) binding to another protein [93,94] sponses by acting as carriers for tumor-derived immunogenic peptides ii) posttranslational modifications of p53 [95,96] and iii) changes (for review see [85,86]). within the microenvironment. The presence of specific chemical com- In addition, for quite some time now, chaperones from the HSP70/ ponents, imbalance of metal ions level, temperature increase (see HSP90 (HSPA/HSPC) families are presumed to fold mutant oncopro- below) or serum starvation induce mutant-like conformation in geno- teins to the conformation termed wild type-like. Thus, in such a way typic WT p53 cells [97–99]). In addition, mutant-like conformation of they act as a buffer, which aids the induction of adaptive mechanisms p53 can be shifted to wild type conformation through interactions with and facilitates evolutionary processes by creating heritable new traits several chemical compounds [100,101](Fig. 1). [87]. Furthermore, this buffering effect mediated by the HSP70/HSP90 By means of conformation specific immunoprecipitation, we have chaperone machinery can assist pre-malignant cancer cells to accu- shown that within the cell, under heat shock conditions (1 h at 42 °C), mulate passenger and/or driver mutations. The phenotype of which can native conformation of WT p53 is shifted towards mutant-like con- be manifested in the presence of environmental micro-changes within formation. Importantly, during the recovery from heat shock at 37 °C, pre-malignant cancer cells or due to localized cellular stress caused by inhibition of HSP90 and/or HSP70 significantly suppressed the re- chemotherapy or irradiation. The acquisition of cancer cell chemore- folding of WT p53 [102]. Furthermore, the recovery of WT p53 native sistance is one of the biggest challenges in cancer therapy. For example, conformation in the presence of active HSP90 and HSP70 was observed at present the effects of hormonal therapies for advanced estrogen re- to be a reaction with exponential-like kinetics (R2 > 0.99). On the ceptor positive breast cancers are limited by the development of ac- other hand, in the presence of 17-AAG, an inhibitor of HSP90, this required resistance [88]. Recently it has been shown that in the presence covery was not only considerably slower but also its kinetics were linear of low doses of HSP90 inhibitor(s), metastatic estrogen receptor-posi- (R2 > 0.99), indicating that low HSP90 activity may be the rate-lim- tive breast cancer(s) do not develop resistance to hormonal therapy iting factor [102]. The p53 protein is well documented to possess a vast [46]. The mentioned data, regarding the estrogen receptor-positive interactome [103,104]. Interaction with binding partners can also shift breast cancer, additionally suggests that HSP90 by buffering critical the conformational equilibrium of the p53 protein. We discovered that elements of signaling pathways is likely to facilitate cancer cell selec- purified human HSP90β (HSP90AB1) transiently bound to WT p53 and tion, which would be resistant to estrogen therapy. underwent a dynamic cyclical process of association and dissociation to In addition, members of the HSP70/HSP90 (HSPA/HSPC) and p53. That resulted in a conformational equilibrium shift of genotypic HSP40 (DNAJ) co-chaperone families were shown to form tight net- WT p53 towards native conformation allowing binding of full-length works (functionally integrated complexes) with proteins involved in p53 protein to the promoter sequence [12,105](Fig. 1). Binding of protein homeostasis. A thorough comparative proteomic analysis of a HSP90 to p53 partially unfolds this client protein. Upon binding to ATP, large set of tumor specimens has shown, that under stress conditions HSP90 dissociates from the p53-HSP90 complex allowing spontaneous such as malignant transformation, accelerated by MYC transcriptional folding of p53. If during this step p53 protein fails to reach a folded activity, this chaperone network is extensively reshaped leading to the native state capable of binding to the promoter sequence, the cycle is formation of stable multiprotein complexes (called the epichaperome) repeated by rebinding of HSP90 chaperone to p53 [106]. The role of facilitating tumor survival, irrespective of tissue of origin or genetic HSP90 in binding of WT p53 to the promoter sequence was further background [47]. Thus, the notion that HSP network(s) should be ad- confirmed by chromatin immunoprecipitation assays (ChIP), measuring dressed and targeted during cancer treatment is extensively gaining the amount of the p21/CDKN1A promoter bound to the p53 protein. momentum in the field. Under non-stress conditions, inhibition of HSP90 (and to some extent Given the vast scope of the data linking molecular chaperones to also HSP70) reduced the amount of p21/CDKN1A promoter bound to tumorigenesis, from here on out this review will focus on the role of p53. ChIP data analysis also revealed that when heat shock was pro- molecular chaperones on the activity of wild type and mutant p53 longed, the HSP70 system had a larger impact on WT p53 DNA-binding tumor suppressor protein. capacity than HSP90 did alone. Those findings were confirmed and expanded on by utilizing a reconstituted chaperone dependent p53 – promoter sequence binding assay with the use of purified proteins

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Fig. 2. HSF1 in cancer cells drives transcriptional programs, which promote not only expression of HSPs but also support highly malignant cancers. HSF1 in cancer cells was shown to regulate the expression of genes involved in the inhibition of apoptosis, activation of protein translation, anabolic metabolism, protein folding, cell adhesion, proliferation, invasion and metastasis. In unstressed cells, HSF1 is sequestered by HSP90 and most probably by other chaperones like HSC70 (HSPA8), keeping synthesis of heat shock proteins suppressed. During proteotoxic stress and other signals HSF1 is liberated from the complex with HSP90 and its transcriptional activity is triggered. These events depend on the phosphorylation of HSF1 (pS326) and most probably on the sequestration of HSP90/HSC70 by partially unfolded proteins produced during stress. Phosphorylated HSF1 forms a homotrimer and is translocated to the nucleus, where it acts as a transcription factor which recognizes heat shock elements (HSE) within the promoter sequences of HSP genes. Moreover, during tumorigenesis, HSF1 is believed to recognize other, not yet identified transcriptional elements, inducing non HSP-dependent mechanisms involved in survival and metastasis of cancer cells. In cancer cells HSF1 is activated by: i) intrinsic proteotoxic stress (when partially unfolded proteins appear, HSF1 is liberated from the complex with HSP); ii) posttranslational modifications, including phosphorylation by mTOR on S326, the mTOR pathway is frequently upregulated in cancer by oncogenic PIK3CA and PTEN mutations which may also contribute to HSF1 activation; iii) HER2 and EGFR signaling pathways; iv) mutants p53 gain of function. Mutant p53 was shown to interact with activated HSF1 in the nucleus and induced HSF1 transcriptional activity. A novel gain of function mechanism of mutant p53 (mut p53) was proposed, whereby cancer cells which overexpressed mut p53 acquired superior tolerance to proteotoxic stress. Mutant p53 via constitutive stimulation of EGFR and ERBB2 signaling was shown to hyperactivate the MAPK and PI3K cascades, which induced stabilization and phosphoactivation of HSF1 on S326. Moreover, mut p53 protein via direct interaction with activated pS326 HSF1 facilitated HSF1 recruitment to its specific DNA-binding elements and stimulated transcription of heat-shock proteins, including HSP90. In turn, induced HSP90 stabilized its oncogenic clients including EGFR, ERBB2 and mut p53, thereby further reinforcing oncogenic signaling. Thus, mut p53 initiates a feed forward loop that renders cancer cells more resistant to adverse conditions, providing a strong survival advantage. Mutant p53 in cancer cell can also interact with other transcription factors like TAp63, TAp73, SP1, SREBP and independently of HSF1 induce inhibition of apoptosis, genetic instability, proliferation, invasion and metastasis.

[102]. At physiological temperature 37 °C, predominantly HSP90β sequence, ii) the refolding of heat inactivated luciferase and iii) the (HSP90AB1), in an ATP-dependent reaction, restored binding of WT folding of WT p53 in cells where degradation of WT p53 is abrogated. p53 to the CDKN1A promoter sequence. Under heat shock conditions These results strongly suggest that MDM2 protein possesses a cha- (42 °C) predominantly HSP70/HSP40 chaperone machinery was re- perone-like activity towards WT p53 and can shift the equilibrium to- quired for binding of WT p53 to the promoter sequence. Under heat wards native WT p53 conformation [107]. Nevertheless, regulation of shock conditions, the presence of HOP and HSP90 were additional ef- the decision fork between the negative (E3 ligase activity) and positive fective catalysts of that reaction [102]. regulator (folding of WT p53) still remains elusive. Upon genotoxic In addition, we have shown that purified full length MDM2 E3 stress the ATM kinase was shown to phosphorylate MDM2 at S395. The ubiquitin ligase can substitute HSP90 in activating WT p53 for binding resulting allosteric change within the protein prevents MDM2 from to the promoter sequence and also in a chaperone-dependent luciferase targeting p53 for degradation and at the same time prompts an increase refolding assay [107]. Analogously to HSP90 chaperone activity, the of p53 mRNA bound by MDM2. In effect, these events stimulate the rate binding of ATP to MDM2 is required for this reaction [107]. The MDM2 of p53 mRNA translation and temporarily suppress the E3 ubiquitin K454A mutant deficient in ATP binding, yet active as a E3 ubiquitin ligase activity of MDM2 towards p53 [108]. We can speculate that, ligase, does not promote: i) the binding of WT p53 to the promoter during stress, phosphorylation of MDM2 not only results in the

165 B. Wawrzynow et al. BBA - Reviews on Cancer 1869 (2018) 161–174 stimulation of p53 translation but also may enhance folding of newly folding surveillance [118]. This hypothesis relies heavily on the de- synthesized p53 tumor suppressor. We cannot exclude the possibility scribed mechanism of HSF1 activation, which involves the reversal of that MDM2 also possesses chaperone activity towards RNA and/or is its inhibition by HSPs such as HSP70 and HSP90 [119,120](Fig. 2). involved in proper assembly of the p53 mRNA-ribosomal complex. The Increased concentration of HSP90 shifts the equilibrium towards for- interaction of MDM2 with several ribosomal proteins was demonstrated mation of the HSF1-HSP90 complex consequently leading to the in- [109], highlighting indirect pathways in which MDM2 controls the rate hibition of the homotrimerization and phosphorylation of HSF1 [119]. of p53 synthesis. Until recently the common assumption had been that the effects of Our attempts to shift the conformational equilibrium towards native HSF1 on cancer survival and development were mediated primarily by like conformation of several cancer derived p53 missense mutants HSPs chaperone activities. To expand on this notion, the Lindquist la- (R273H, R248Q, R249S, R175H) by implementing molecular chaper- boratory has shown that in cancer cells HSF1, besides managing the ones were, mildly successful. Only in the case of p53 R249S variant, canonical heat shock response (HSR), also regulates the expression of molecular chaperones HSP90β (HSP90AB1), HOP, HSP40 (DNAJB1 or many genes that are HSP independent but are actively involved in DNAJA1) and HSP70 (HSPA1A) or HSC70 (HSPA8) partially restored supporting highly malignant human cancers by inhibition of apoptosis, its binding to the promoter sequence [102]. activation of protein translation, anabolic metabolism, protein folding, A spatial mechanism, established through evolutionary selection, to cell adhesion, proliferation, invasion and metastasis [45](Fig. 2). rescue proper development of mouse embryos, which harbor mutation Interestingly, tumor cells also commander normal physiological in TP53, was described recently [5]. The authors therein postulate that pathways and programs in the stroma (these include: cancer associated embryonic stem cells (ESCs) (but not somatic iPS cells) despite har- fibroblasts, endothelial cells, immune cells end extracellular matrix boring mut p53 remain pluripotent and maintain genomic integrity. (ECM) components), which form a microenvironment essential for Within this cellular background mut p53 conformation is shifted to- tumor progression and metastasis. Activation of HSF1 in stromal cells wards the WT form, which results in a transcriptionally active p53. reprograms them along with cancer associated fibroblasts to facilitate Using MS-based proteomics a specific interactome that bound to the angiogenesis, ECM organization, adhesion and migration. In early-stage WT-like conformation of mut p53 was identified. These binders fall into breast and lung cancer, high stromal HSF1 activation is strongly cor- three main groups that can be directly associated with conformational related with poor patient outcome [121]. Stromal HSF1 activation also change and stability of p53: i) molecular chaperones CCT (TRiC) tweaks the transcriptional program of cancer cells in order to further complex, HSP40 (DNAJ), and HSC70 (HSPA8); ii) ubiquitin-related promote their malignant phenotype. In stromal cells, HSF1 activates proteins and iii) regulators of posttranslational modifications such as transcription of the TGFB (TGFB1) and SDF1 (CXCL12) genes. The protein kinases AURORA (AURKA) or MAPK1 [5]. Independently of protein products of these genes, TGFB and SDF1, are secreted by those findings it was shown that CCT complex interacted with WT p53 stromal cells allowing crosstalk between stromal and cancer cells in [110]. Moreover, inhibition of that interaction increased mutant-like developing tumors [121]. conformation of WT p53, which could behave like tumor-driven mut The pro-oncogenic role of HSF1 in cancer development and metas- p53 [110]. Recently, CCT was suggested to be involved in oncogenesis tasis is substantiated by the findings that depletion of HSF1 remarkably [111]. reduced survival and metastatic potential of cancer cells [45,122]. Those findings were strongly supported by in vivo studies: HSF1 null 4. The interplay between HSF1 and p53 mice developed normally but were profoundly resistant to tumorigenesis. HSF1 deficiency increased median overall survival of knock-in mut Selection and micro-evolution of cancer cells depends on the in- p53 mice [123]. duction of pro-survival adaptive signals, known as the heat shock re- Given the pluripotent potential of HSF1 transcription factor in sponse (HSR). This program, driven by HSF1 transcription factor, in- safeguarding the proteome against misfolding and aggregation induced duces in several types of cancer cells massive overproduction of by proteotoxic stress due to heat, ROS, hypoxia, acidosis, DNA damage numerous heat shock proteins (HSPs). This event is maintained by: i) and aneuploidy, HSF1 has been described as the guardian of the pro- the release of powerful repression of HSP70 promoter sequence(s) teome [124] by analogy to p53 tumor suppressor termed, as mentioned caused by WT p53 [112], ii) missense mutation(s) in the TP53 gene earlier in this review, the guardian of the genome [1]. leading to the synthesis of altered mut p53s which directly (or in- directly) interact with HSF1 and facilitate HSF1 recruitment to specific 5. HSPs role in mut p53 oncogenic gain of function phenotype HSE sequences [113], iii) the fact that mut p53 enhances EGFR recycling to the cell surface of cancer cells [114], thus activating HSF1 Mutant p53 gain of function (GOF) was first demonstrated in 1984 through MAPK and PI3K phosphorylation cascades [113](Fig. 2). where induction of mut p53 was shown to transform cell lacking p53 Moreover, through a regulatory feed forward loop extensive over- [125]. The broad spectrum of GOF for mut p53 was described in several production of HSPs in cancer cells: i) activates protein kinases [AKT reviews [7,9,113,126,127]. The GOF phenotypes orchestrated by mut (AKT1), RAF (BRAF)] and EGFR as well as HER2 (ERBB2) receptors p53 were shown to: i) disrupt cell cycle control and enhance pro- involved in hyperactivating phosphorylation cascades directed towards liferation; ii) drive epithelial-to-mesenchymal transition (EMT), in- HSF1 [113,115], ii) increases the stability of mut p53 [13,55]. As crease cell motility, tumor invasion and metastasis; iii) inhibit DNA mentioned above, overproduction of HSPs in cancer cells not only aids repair which stimulated genome instability; iv) control nutrient supply in the repair and proper folding of damaged or mutated proteins, but by regulating angiogenesis and glycolysis; v) promote inflammation; vi) also inhibits cell death, bypasses senescence and alters cell signaling attenuate cell death and induce drug resistance; vii) generate cancer and glucose metabolism [45,54,116](Fig. 2). Mutant p53 can manifest stem cells and subsequent acquisition of drug resistance by cancer cells its gain of function (GOF) phenotype (inhibition of apoptosis, genetic and viii) induce and maintain angiogenesis. A critical future of GOF instability, proliferation, invasion and metastasis) independently of phenotypes is the stabilization of mut p53, manifested by gross accu- HSF1 (Fig. 2). mulation of mut p53 in cancer cells [13,127–131]. One of the proposed hypotheses in the field addressing the aber- The abundance of both WT and mut p53 is tightly controlled by rantly high concentration of HSPs in cancer describes this phenomenon degradation mediated by the ubiquitin proteasome pathway. Originally as “an addiction to chaperones” [117]. Tumors are perceived to be in identified as a transrepressor of p53 mediated transcription, the MDM2 some way similar to mildly shocked cells, which incorporate oncogene protein was subsequently shown to control the steady state levels of p53 overexpression and mutation, polyploidy and overamplified levels of in unstressed cells by acting as a RING finger domain E3 ubiquitin ligase translation generating an abnormally high demand for intracellular [132,133], which results in the polyubiquitination of p53 ensuing 26S

166 B. Wawrzynow et al. BBA - Reviews on Cancer 1869 (2018) 161–174 proteasomal degradation. In turn WT p53 transcriptional activity is proteins [12,135]. We suggest that this complex represents a folding responsible for upregulating the expression of MDM2. Such an auto- intermediate of mut p53 trapped by molecular chaperones (Fig. 3). The regulatory feedback loop does not exist in the case of mut p53, which mut p53 protein is recognized by HSP70/HSP40/HSP90 chaperone devoid of canonical transcription factor potential, cannot induce the machinery. As it has been previously shown both chaperones, HSP90β expression of MDM2. In some cancers, despite the presence of mut p53, (HSP90AB1) [106] and HSP70 (bacterial DnaK) [136] exhibit unfoldase high levels of MDM2 are maintained resulting from MDM2 gene am- activity. The human chaperones, HSP90 and HSP70 upon binding to plification and/or by hyper regulated TGFB/SMAD2/3 and RAS/RAF/ mut p53, partially unfold this protein substrate and facilitate its spon- MEK/ERK oncogenic pathways, which, independently of p53, can ac- taneous folding to conformations with a divergent energy minimum tivate MDM2 transcription [134]. (Fig. 4). The probability for the unfolded mutated p53, during the re- In mutant homozygotic Trp53 R172H knock-in mice (equivalent to folding process, to reach wild type conformation (global minimum of R175H in humans), MDM2 was responsible for efficient degradation of energy) is very low [102]. The kinetics of the mut p53 refolding reac- mut p53 in normal, non-cancer tissue(s) [128]. In the same animal, in tion indicates that molecular chaperones HSP70 and probably HSP90, cancer tissue(s), mut p53 was stabilized by unknown factor(s). More- during ATP-dependent unfolding, induce transient exposure of p53 sites over, the authors hypothesize that the stabilization of mut p53 is a responsible for interactions with other proteins (like TAp73) and also prerequisite for its GOF phenotype [128]. Recently we, alongside HSPs. This in turn can lead to the inhibition of the refolding process and others, have shown that elevated levels of HSP90 [55,127]) or HSP70 formation of mut p53-TAp73-HSPs complexes, which in the presence of and HSP90 [13] are the factors which stabilize mut p53. It has also been proteasomal inhibition form pseudo-aggregates [13](Fig. 3). shown before that in cancer cells, where endogenous levels of HSPs are We have shown that in non-cancer mouse embryonic fibroblasts already elevated (see previous section), mut p53 is recognized as a MDM2-dependent ubiquitination and subsequent degradation of mut misfolded, aberrant protein by the HSP machinery, prompting the for- p53 protein is partially inhibited by increasing concentration of exo- mation of a stable multi-chaperone complex [11,12,48]. This multi- genous HSP70 (HSPA1A). This phenomenon correlated well with the chaperone complex was reconstituted in vitro using highly purified appearance of HSP70-dependent folding intermediates, which upon

Fig. 3. Folding intermediates and aggregation of mut p53. The quality control mechanism driven by HSPs is co-opted by cancer cells. The molecular chaperones that participate in the folding of mutated p53 intermediates stabilize the interactions with p73. This results in the inhibition of mut p53 degradation and p73-mediated induction of apoptosis. When cancer cells overexpress MDM2, then HSPs are replaced from the intermediate complexes comprising mut p53-p73-HSPs. The amyloid-like aggregates containing mut p53, p73, MDM2 are formed, which enhances cancer cell chemoresistance profoundly.

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mut p53. However, regardless of the phosphorylation state, for both of the chaperones, the multichaperone p53 R175H-HSP40-HSP70-HOP- HSP90 complex was previously reconstituted in vitro from bacterially expressed proteins [12]. Interestingly, the equilibrium between aggregated and non-aggregated mut p53 undergoes a change when MDM2 is ectopically overproduced [13](Figs. 3, 4). MDM2 overproduction shifts this equilibrium towards aggregation and formation of a stable amyloid-like structures which in non-transformed cells occasionally merge into an aggresome [13]. In cancer cells, where endogenous HSP70 (HSPA1A) levels are already elevated (see previous section), mut p53 protein forms nuclear pseudo-aggregates without the addition of exogenous HSP70 (HSPA1A). Formation of these aggregates is inhibited by overexpression of a dominant negative HSP70 K71S suggesting that p53 pseudo-aggregates are the intermediates of ATP dependent folding reaction. However, when MDM2 is overproduced, these pseudo-aggregates are converted into amyloid-like structures [13]. Mutant p53 protein was shown to form aggregates in some cancers cell lines in the nucleus, perinuclear regions and in the cytoplasm [143]. Moreover, amyloid-like structures containing mut p53 were detected in cancer biopsies and were localized both in the cytoplasm and the nucleus [144–148], for reviews see [149,150]. According to the data published by the Schymkowitz laboratory, structurally destabilized p53 mutants in cancer cells induce co-ag- Fig. 4. The thermodynamic landscape of mut p53 folding. Folded mut p53 protein gregation of WT p53 and its paralogs p63 and p73. A peptide motif reaches the local energy minimum. Upon energy dependent unfolding, catalyzed by HSPs, the released intermediate mut p53 form can spontaneously fold to a different con- surrounding I254 within the primary structure of p53 was proposed to formation, with a divergent energy minimum. During this unfolding process the ag- be the aggregation-nucleating sequence in cells [143]. Additionally, it gregation prone sites of mut p53 are exposed. Elevated levels of MDM2 shift this equi- was proposed that the aggregation propensity of this sequence could be librium towards aggregation of mut p53 with MDM2. Sequestration of other proteins, like suppressed by mutagenesis, which in turn abrogated the GOF pheno- α α– p73 tumor suppressor, in such aggregates, diminishes p73 dependent apoptosis, thus type and restored WT p53 activity and its paralogs as well [143]. Re- making cancer cell resistant to chemotherapy. cently, it was shown that peptides designed to inhibit mut p53 amyloid- like nuclear structure formation, rescued p53 function in cancer cell proteasomal inhibition form the dynamic cytoplasmic spots containing lines and in organoids derived from high-grade serous ovarian carci- aggregate-prone mut p53 and several molecular chaperones including nomas [151]. However, the Fersht laboratory argued that peptides HSP70 (HSPA1A) and HSP90 [13]. Interestingly, those pseudo-ag- designed to inhibit formation of p53 amyloid-like structure(s) did not gregates containing mut p53 did not form when overexpressed HSP70 inhibit aggregation of mut p53 but caused rapid cell death by p53-in- (HSPA1A) was replaced with its constitutive HSC70 (HSPA8) paralog. dependent mechanism(s) [152]. We cannot exclude the possibility that Furthermore, we have shown that mut p53 half-life substantially de- these peptides could release, for example, TAp73 from mut p53-cha- creased after treatment of human non-small lung carcinoma (NSCLC) perone complex and induce the TAp73α dependent apoptosis of cancer cells expressing p53 R175H with HSP90 and HSP70 inhibitors [13]. cells, as was suggested by [48]. The presence of amyloid-like structures HSP90 was demonstrated to protect mut p53 from MDM2 and also from containing mut p53 in pathological tissue samples (biopsies) and the CHIP-mediated degradation [55]. Apart from MDM2 several other E3 aggregation of mut p53 were both shown to be linked to lower rate of ubiquitin ligases like; CHIP, PIRH2 (RCHY1), ARF-BP1 (GGA1) and loss of heterogeneity and patient survival. Therefore the clinical re- COP1 (RFWD2) have been identified to effectively ubiquitinate mut p53 levance of p53 aggregation is justified. Nevertheless, the correlation [55,137–142]. Interestingly, partial inhibition of mut p53 degradation between the presence of amyloid-like structures containing mut p53 in the presence of HSP70 (HSPA1A) occurred also when expression of and elevated levels of MDM2 oncoprotein in such clinical material was CHIP E3 ubiquitin ligase was inhibited by specific siRNA [13]. This never tested. Hence, this avenue of investigation requires further study. suggests that the potential of CHIP mediated degradation of mut p53 is The broad spectrum of mut p53 GOF phenotype is possible due to minor in comparison to MDM2, at least in some cancer cells. The ex- the potential of these altered proteins to directly interact and alter periments performed by the Vojtesek laboratory demonstrated that (repress and/or hyperactivate) the transcriptional activity of several phosphorylation of C-terminal domains of HSP70 and HSP90 α/β by transcription factors: TAp63, TAp73, E2F1, SP1, NFkB, Ets-1, NF-Y, CK1, CK2 or GSK3-β protein kinases, in the presence of HOP co-cha- VDR, SREBP-2 (SCAP) Top BP1, [126,127,153,154] (see Fig. 2). We, perone stimulated multichaperone complex formation with client pro- alongside others, have shown that the interaction of TAp63 or TAp73 teins, thus impeding their degradation [56]. The level of p53 R175H with mut p53 depends on type of the missense mutation in TP53. The client protein decreased when H1299 cells were co-tranfected with p53 p53 contact mutants displayed a weak interaction towards TAp63 or R175H along with non-phosphorylable alanine mutant of HSP70 and TAp73. In contrast, the p53 conformational mutants displayed very HSP90α (HSP90AA1) but was stabilized in the presence of phospho- high affinity towards TAp63 or TAp73 and yield stable complexes mimetic mutants [56]. Although one can argue that H1299 cells already comprising mut p53-TAp63 and/or mutp53-TAp73 [48,153,155]. In- contain elevated levels of endogenous HSP70 and HSP90 and the interestingly, genetically WT p53 could also interact with TAp63α and troduction of phospho-mimetic mutants created a very heterogenous TAp73α under certain conditions. Heat shock [48] or phosphorylation population of both endogenous and exogenous chaperones, the authors of WT p53 at S269 [95,156] induce mutant-like conformation of p53 demonstrated that HSP70 and HSP90 exist predominantly as C-terminal which possesses high affinity towards TAp63α or TAp73α. phosphorylated forms in primary tumor tissues compared with non- Ectopic overexpression of HSP70 (HSPA1A) was shown to dissociate neoplastic tissue. These findings suggest that, in cancer cells, phos- the conformational mutant p53 from the mut p53 – TAp63α complex phorylated molecular chaperones by attracting the HOP co-chaperone yielding an increase in TAp63-mediated transcriptional activity [13]. to mut p53 form a multichaperone complex and consequently stabilize However, that was not the case for the conformational mutant of the

168 B. Wawrzynow et al. BBA - Reviews on Cancer 1869 (2018) 161–174 mut p53 – TAp73α complex. HSP70 (HSPA1A) and HSP40 (DNAJB1) Interestingly, when both events occurred in parallel (TP53 mutation proteins further stabilized the mut p53 – TAp73α interaction and sub- and elevation of MDM2), the survival rate decreased significantly, sequently a multi-chaperone complex (containing also HSP90) formed especially in the early stages of cancer development [48]. Similar, but in the nucleus [48]. Consecutive double immunoprecipitation experi- not as pronounced, effect was seen for lung, ovarian and head and neck ments (Two-step Co-IP) revealed that molecular chaperones HSP70 cancers (unpublished data). Interestingly, at least in the case of breast (HSPA1A), HSP40 (DNAJB1) and HSP90α/β (HSP90AA1/AB1) inter- cancer patients with mutated TP53 and MDM2 overexpression de- acted simultaneously with the mut p53-TAp73α complex. A limited creased survival rate strongly correlated with elevated levels of HSP40 portion of HSC70 (HSPA8) also co-immunoprecipitated with mut p53- (DNAJ) heat shock proteins - DNAJB1 and DNAJB6 [48]. The in- TAp73α complex [48]. This nuclear multi-chaperone complex seques- volvement of these specificity factors for the HSP70 (HSPA) family in tered the transcriptional activity of TAp73α and in consequence in- carcinogenesis with limited mechanistic insight has been already re- hibited TAp73α-dependent drug-inducible apoptosis. This phenomenon ported [164–166]. Surprisingly, such a correlation was not observed for strongly augmented cancer cell chemoresistance. Nevertheless, the de- prominent members of HSPA family, namely HSPA1A (HSP70) and crease of apoptotic response could be partially reversed by means of HSPA8 (HSC70), with the exception of HSPA6 and HSPA12B. This TAp73α derived, short interfering mutant p53 peptides (SIMPs) [157] phenomenon most likely is due to the redundancy of substrate speci- which released TAp73α from the mut-p53–multi-chaperone complex ficity for those molecular chaperones, which are directed by different [48]. Similarly, inhibition of the HSP70 (HSPA1A) activity by the DNAJ specificity factors. Furthermore, compensation effects resulting dominant negative HSP70 K71S or D10S, or inhibition of HSP40 from differential gene expression within the HSPA family should not be (DNAJB1) expression by specific siRNA results in the dissociation of ruled out [167]. The HSPA6 protein is known to be an HSP40-in- TAp73α from the complex with mut p53, and induction of TAp73α- dependent molecular chaperone [168]. HSPA12B was shown to facil- dependent apoptosis. itate lung tumor growth [169]. Elevated expression of endoplasmic Interestingly, elevated levels of MDM2 released chaperones from reticulum resident – HSPC4 (HSP90B1, GP96, GRP94, Endoplasmin), a mut p53-TAp73α complex and induced the formation of a three-body known oncogenic factor involved in breast cancer metastasis [170], mut p53-TAp73α-MDM2 complex, which further intensified cancer cell correlated with low survival rate of mut TP53/high MDM2 breast chemoresistance [48]. In this regard HSP70 (HSPA1A), in an ATP-de- cancer patients. In addition, high expression of HSPC4 (HSP90B1) was pendent reaction, works as a molecular chaperone. At first, it stabilizes recently shown to be associated with cancer cell polarity and migration the intermediate interaction between TAp73α and p53 R175H. Sec- [171]. On the other hand, it is worth noticing that the low survival rate ondly, after MDM2-dependent reaction, it is released from the complex of mut TP53/high MDM2 breast cancer patients strongly correlated and a new mut p53-TAp73α-MDM2 complex is formed (Fig. 3)[48].The with low expression levels of certain molecular chaperones and co- sequestration of TAp73α in the mut p53-TAp73α-MDM2 complex could chaperones HSP90β (HSP90AB1), DNAJB4 [48]. be rescued by Nutlin-3, a cis-imidazoline analog, mostly known as a In view of these results it is tempting to speculate that the group of selective inhibitor of MDM2-p53 interaction [48,158]. One can con- cancers, which possess structural p53 mutants and overexpress MDM2 sider that Nutlin-3 acts here not only as an antagonist of mut p53- will boast elevated resistance to classical chemotherapy. For those pa- MDM2 interaction(s) but additionally possesses an agonist effect on this tients, classical chemotherapeutic approach should be supplemented network of protein interactions. By releasing, trapped in the complex, with drugs, which release the sequestered TAp73α and induce TAp73α- TAp73α can activate the drug induced apoptosis, and also by releasing dependent apoptosis of cancer cells [48]. Two other approaches are also the mut p53 can allow for its degradation. A recent paper from Ball and discussed and tested: i) development small compounds capable of re- Hupp laboratories highlights the agonist property of Nutlin-3 that restoring wild-type function of mut p53 protein (for review see [172]); ii) sults in the activation of WT p53 by increasing chromatin-bound elimination of the stabilized mut p53 proteins by the use of HSP in- MDM2-p53 protein complexes. The refined mode of WT p53 ubiquiti- hibitors. Recently, proof-of-concept of this second approach was pre- nation after administration of Nutlin-3 in A375 cancer cells caused sented using novel mut p53 mouse model system expressing p53 in- enhanced monoubiquitination of WT p53 versus polyubiquitination. A activatable R248Q structural hotspot mutant (FloxQ) [130]. After pool of monoubiquitinated p53 is transferred to the nucleus and tightly ablation of mut p53 expression, extension of animal survival occurred associated with chromatin. In consequence, instead of undergoing the and advanced tumors underwent apoptosis. Moreover, long-term steps leading to degradation, monoubiquitinated WT p53 is stabilized HSP90 inhibition significantly extended the survival of Trp53 (R248Q/ and activated [159]. − or R172H/R172H) mice but not their corresponding Trp53 (−/−) Recent use of MDM2 inhibitors from the Nutlin chemical family was knock-down variants [130]. It would be interesting to test, whether the reported to be a promising new therapeutic strategy for human tumors potent HSP90 inhibitor (Ganetespib) used in those experiments did not retaining wild-type p53 status. Induction of the TP53 target genes abrogate WT tumor suppressor p53 activity required for tumor sup- CDKN1A, BAX and PUMA in chronic lymphocytic leukemia (CLL) after pression. As we have previously shown in a cell line model, the activity treatment with Nutlin-3 occurred mainly in samples possessing WT p53 of HSP90 is required for transcriptional activity of p53 tumor sup- [160]. In addition, Nutlin-3a was also shown to sensitize WT TP53 and pressor [105,106]. The concept that an HSP90 inhibitor would block MDM2 amplifying sarcoma cell lines to TNF-α cytotoxic action [161]. the maturation of the oncogenic ‘drivers’ without harming normal cells Moreover, the data presented within the Tracz-Gaszewska [48] paper and diminish the chemoresistance was methodically suggested before supports the notion that Nutlin-3 could be considered as a clinical drug [173–176]. Collectively, the data presented in these studies highlights in mut p53 background. Furthermore, MDM2 was shown to differently the observations that high levels of HSP90 inhibitors administered modulate binding of mutant p53 to TAp63α and TAp73α [162]. The during clinical trials were well tolerated but only transiently destabi- formation of a p53 R175H-TAp73α-MDM2 complex was proposed, yet lized oncogenic kinases. HSP90 client proteins returned to baseline le- no evidence for the three-body complex was presented. vels within a few days of drug administration. High doses of HSP90 By implementing the TCGA GDAC data-sets [163], we were able to inhibitors, used in those trials, also activated HSF1 transcription factor show that breast cancer patients treated with chemotherapy, bearing [46] .responsible for the induction of other HSPs and specific genes TP53 mutation(s) along with MDM2 overexpression were significantly involved in metastasis and cancer cell proliferation (see previous sec- predisposed to decreased overall survival. The Kaplan-Meier survival tion). The use of HSP90 inhibitor(s) in low dosage which would not curves indicated the best prognosis for the breast cancer patients as- induce proteotoxic stress and HSF1-dependent heat shock response (no sociated with WT TP53 and no overproduction of MDM2. Mutation(s) HSP70 expression) would be beneficial in preventing the development in TP53 alone, as well as MDM2 overexpression alone resulted in the of acquired resistance to hormone therapy [46] decrease of the survival rate of patients treated with chemotherapy.

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6. Concluding remarks crowding and have the propensity to sequester other functional proteins/transcription factors. We have recently shown that HSPs facilitate During the course of evolution, cellular organisms were compelled the formation of a functionally inactive mut p53-TAp73α subcomplex, to develop protective mechanisms, which would render them resistant effectively decreasing the apoptotic response of breast and lung cancer to sudden external environmental changes including rapid temperature cells [48]. MDM2 oncoprotein expressional elevation in this scenario elevation. One of these shielding mechanisms is the induction of the further induced rearrangement of the subcomplex leading to formation heat-shock protein synthesis, possessing molecular chaperone activities. of a stable mutp53-TAp73α-MDM2 complex, which yields a further Hence, it should not be surprising that the genes encoding heat-shock decrease of apoptotic response and bolsters chemoresistance. Moreover, responsive proteins are stringently conserved [177]. Their induction by in the case of breast cancer patients who bear TP53 mutations and the host cell during stress (temperature based, metabolic, etc.) provides overexpress MDM2, elevation in expression of certain members from abuffer effect and enables the cell to adapt and survive the strenuous the HSP family inversely correlated with their survival rate [48]. period. Additionally, of note is the fact that the mentioned molecular The data obtained in the last several years, indicates the HSPs act as chaperones are also essential during stress-free periods, where they a network(s) in order to provide the functional physiological activity to moderate housekeeping functions including maintaining equilibrium thermodynamically liable client proteins. During tumorigenesis, these between protein aggregation and polypeptide folding. Through passive chaperone networks are extensively reshaped and remodeled so that aggregation blockage and/or active partial unfolding of the client they become advantageous to the proliferating cell, i.e. misregulation of protein, molecular chaperones shift the equilibrium towards properly stress signaling cascades, receptor blockage or hyper-activation, effec- folded, functional products. The role of molecular chaperones in this tive apoptosis and senescence evasion. Thus, a synergistic approach process is extremely important for proteins that during their folding combining classical chemotherapy and specific inhibition of selected cycle have not reached their global potential energy minimum. Such HSP members is tempting. On one hand, such an approach may prove proteins are quite numerous in the eukaryotic cell, including several e ffective against combating cancer cells, on the other it may be dis- signal receptors, stress signaling kinases and transcription factors, of astrous to normal non-cancer cells where the housekeeping functions of which p53 tumor suppressor is a prime example. As summarized and HSPs are paramount for their proper function, maintenance and sur- highlighted in this review, molecular chaperone and co-chaperone vival. At present, several approaches were proposed to address this members from the HSPA, DNAJ, HSPC families are indispensable for issue. One bases on the use of HSP inhibitors in low sub pharmacoki- proper activity of WT p53 as a transcription factor and tumor sup- netic doses in so that to not induce the HSF1-mediated heat shock re- pressor [12,102,105,106]. sponse [45]. The other bases on a cancer specific approach and the use Unfortunately, this quality control mechanism driven by HSPs was of high efficacy, selective inhibitors designated against specificity fac- co-opted by cancer cells [45]. These, by extensively overexpressing tors from the HSP40 (DNAJ) family [48]. In addition, Parrales et al. has HSPs, bypass growth inhibition signals, metabolic starvation, forgo suggested statins, cholesterol-lowering drugs, as degradation inducers apoptosis induction and senescence, up regulate and fine-tune the sig- for conformational or misfolded p53 mutants with minimal effects on naling network(s) driving the proliferative potential of the cell and wild-type p53 (WT p53) and DNA contact mutants. Mechanistically cellular motility. All these events catalyze tumor growth and metastatic statins work via the mevalonate pathway - HSP40/CHIP axis leading to onset. As it turns out, the same molecular chaperones belonging to the CHIP-mediated nuclear export, ubiquitination and degradation of mut HSP subset, necessary for WT p53 in reaching its tumor suppressor p53. Interestingly DNAJA1 knockdown was shown to induce CHIP- potential, are the drivers of the oncogenic GOF phenotype, when TP53 mediated mut p53 degradation, while its overexpression antagonized is mutated and the resulting protein bearing hotspot missense mutation statin-induced mut p53 degradation [166]. New opportunities, for de- (s) adopts a mutant conformation. The same members of the HSP fa- signing advanced chaperone inhibitors, have recently opened upon the milies that are key in proper folding of WT p53 for it to reach native discovery that in almost 50% of cancers, chaperone networks are re- conformation, in the case of mut p53 (with hot spot missense muta- shaped to form stable multiprotein complexes named the epicha- tions) stabilize the polypeptide in a folding intermediate state liable to perome. This structure is manifested by enhanced physical interactions form pseudo-aggregates [13]. These intermediary protein species are between the HSP90 and HSP70 machinery. Furthermore, a significant quite hazardous to the cell, as they frequently evade proteolysis, are correlation was found between the epichaperome abundance (but not resistant to compartment seclusion, locally intensify molecular of total HSP90) and the sensitivity to the HSP90 drug PU-H71 [47].

Table 1 Molecular chaperones activities in normal and cancer cells.

Diﬀerentiated post-mitotic cells with WT TP53 Proliferating cancer cells with missense mutation in TP53 (mut p53)

Lower expression of HSPs, transient induction of their expression, which is triggered by HSF1-dependent expression of HSPs at elevated and constitutive levels and other genes HSF1 transcription factor upon stress [178]; involved in survival of highly malignant cancer cells [50,118,124,178]; Folding and activation of proteins damaged during stress conditions or semi-folded Folding, activation and stabilization of oncogenes, selective signaling proteins proteins [15]; including stress kinases and receptors [54,127]; WT p53 suppresses heat shock promoters decreasing HSPs expression [112]; Mut p53 does not suppress heat shock promoters and enhances expression of HSPs [112]; High level of HSPs, triggered by stress conditions, inhibits HSF1 activity and suppresses More extensive phosphorylation of HSF1 and its interaction with mut p53 additionally the expression of HSPs [119,120]; enhance HSPs and other genes expression [113,127]; Dephosphorylation of HSP70 and HSP90 at their C-termini induces preferential binding Phosphorylation of HSP70 and HSP90 at their C-termini induces preferential binding of CHIP co-chaperone (E3 ubiquitin ligase) [56]; of HOP co-chaperone [56]; Transient interactions of HSPs with WT p53 intensify its tumor suppressor activity Formation of HOP-dependent mut p53-HSPs multichaperone complexes stabilizes mut [102]; p53 [12,13,55] and supports the manifestation of its oncogenic function [127,128]; HSPs prevent protein aggregation and dissociate already preformed complexes HSPs induce aggregation of mut p53. These aggregates can sequester other factors, like (aggregates) [41,179]; tumor suppressor TAp73 and in the presence of elevated level of MDM2 induce formation of amyloid-like structures [13,48]; In non-transformed cells the network of chaperones is limited and the ‘epichaperome’ Formation of multi-chaperone complex – the ‘epichaperome’ facilitates tumor survival: complexes are not formed. Stress conditions induce the p53 tumor suppressor inhibition of apoptosis, senescence, DNA repair and autophagy, induction of response: induction of apoptosis, cell cycle arrest, senescence, DNA repair and proliferation and metastasis [47]. autophagy [47].

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