extracellular hsp90: an emerging target for cancer therapy

8/13/2019 Extracellular HSP90: An Emerging Target for Cancer Therapy

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Current Signal Transduction Therapy, 2009, 4, 51-58 51

1574-3624/09 $55.00+.00 2009 Bentham Science Publishers Ltd.

Extracellular HSP90: An Emerging Target for Cancer Therapy

Katerina Sidera1and Evangelia Patsavoudi1,2,*

1Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece; 2Department of Biomedical Instrumentation

Technology, Technological Educational Institute of Athens, Greece

Abstract: Cancer is a genetic disease which progresses from benign to malignant stages through the steady acquisition of

genomic mutations in key cell-regulatory genes, namely oncogenes, tumor-suppressors, and stability genes. In many ways

cancer is considered as a disease of de-regulated signal transduction. Oncogenic mutations frequently lead to over-

expression and/or constitutive activation of signal transduction components, allowing cancer cells to override the control-

ling mechanisms of signalling networks and acquire the cancer-associated traits known as the six hallmarks of cancer.

The molecular chaperone HSP90 is viewed as a key player in the subversion of normal cells toward transformation, since

many of its client proteins are linked to signalling pathways, commonly de-regulated during tumorigenesis. Consequently,

over the past years HSP90 has emerged as a promising and exciting target for the development of cancer chemotherapeu-

tics and already several HSP90 inhibitors are under clinical evaluation.

Recently, a pool of HSP90 was identified at the cell surface, where it was shown to be involved in signalling pathways

leading to cell motility and invasion. Independent studies suggest that surface HSP90 could be a promising target for the

development of effective anti-metastatic strategies. Thus a need for the development of novel cell-impermeable HSP90

inhibitors is emerging.

Key Words: Extracellular HSP90, cell invasion, signal transduction, cancer therapeutics.

INTRODUCTION

Cancer has become in recent years less of an enigma;after decades of rapid advances in cancer research, severallines of evidence indicate that during the course of tumorprogression, cancer cells gradually acquire a number of dy-namic genetic alterations [1]. In 2000, Hanahan and Wein-berg [2], proposed that the complex array of phenotypes dis-played by cancer cells may be organized in six cancer-associated traits. These include the cellular capacities to pro-

liferate indefinitely (immortalization), to become independ-ent of extracellular growth or anti-growth signals, to evadeapoptosis, to induce a self-sustained supply of nutrients andoxygen (angiogenesis) and ultimately, to invade and metas-tasize to distant sites. These cancer-associated hallmarkcharacteristics actually reflect genetic alterations in multiplesafeguard genes of the cell, namely oncogenes and tumor-suppressor genes. These genes are responsible for the regula-tion of signal transduction molecules, including among oth-ers receptor tyrosine kinases (RTKs), products of the Rasand raf genes, and protein kinases of the mitogen-activatedprotein kinase (MAPK), phosphoinositide 3-kinase (PI3K)and Akt. These molecules in normal cells control the tightcoordination of diverse processes such as cell survival, pro-

liferation, growth, differentiation and motility [3, 4].

The broad term signal transduction refers to the flow ofbiological information from the cells micro-environment(extracellular matrix, neighbouring cells) via detection sys-tems such as surface receptors and through intermediatemolecules, inside the cell. The latter include a wide array of

*Address correspondence to this author at the Department of Biochemistry,Hellenic Pasteur Institute, 127, Vas. Sofias Av.,11521 Athens, Greece; Tel:+30 2106478871; Fax: +30 2106423498; E-mail: [email protected]

intracellular elements interacting through cascades of chemical signals and ultimately controlling the activities of specific intracellular effectors, resulting in the tight coordinationof fundamental processes of cellular life [5]. Signallingpathways are not isolated from each other, but instead theyare interconnected, forming a labyrinth of intersecting andoverlapping networks, enhancing thus the robustness anddiversity of signalling and permitting fine-tuning and amplification or attenuation of the output [6].

Advances in the understanding of aberrant signallingpathways in various types of cancers have directly promotedthe development of targeted therapies-that is the development of drugs that influence the action and/or activity of aspecific signalling molecule. Elucidation of the roles omany kinase signalling pathways in cancer, including growthfactor receptors and their downstream effectors, along withthe identification of kinases as an attractive drug-target classhas led to the expectation of innovating cancer-treatmenstrategies with more specific mechanisms of action thanconventional chemotherapeutic agents. However, the cruciachallenge to select appropriate targets/approaches in cancetherapeutics still remains.

HSP90: MORE THAN A FOLDING TOOL

Heat shock proteins (HSPs) were first discovered in 1962[7, 8] as a small set of highly conserved proteins whose expression increased, as a response to elevated temperatureSince then, the biology of HSPs has been studied extensivelyand it is generally accepted that these proteins have evolvedas a cellular protection response to a multitude of stresseincluding heat, alcohol, hypoxia, oxidative stress, acidosisnutrient deprivation, and inflammation [9-11]. FuthermoreHSPs function as central components of the molecular chaperone machinery, responsible for the folding, stability, deg


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52 Current Signal Transduction Therapy,2009, Vol. 4, No. 1 Sidera and Patsavoud

radation and transportation of a multitude of proteins andconsequently affecting diverse cellular processes [12-14].Among the members of the family, the 90-kDa heat shockprotein (HSP90), has recently emerged as a molecule of par-ticular interest due to the fact that many of its "client" pro-teins, including mutant p53, HER-2, HIF-1, Akt/PKB c-Raf -1, hTERT and CDK4, are members of the most-wanted listof proteins considered responsible for the multiple hallmark

traits of malignancy (see Table 1) [15-18].

Table 1. HSP90 Clients and the Multiple Hallmarks of

Cancer

Cancer-associated Cellular Traits HSP90 Client Proteins

Self sufficiency in growth signals MEK

SRC tyrosine kinases

Steroid hormone receptors

Insensitivity to anti-growth signals Akt

CDK2,CDK4

HIF1a

RTKsSRC tyrosine kinases

Steroid hormone receptors

Evading apoptosis Akt

RTKs

Survivin

Tissue invasion and metastasis MMP2

Limitless replicative potential CDK2, CDK4

Telomerase

Sustained angiogenesis VEGFR2

HIF1a

HSP90 is a highly conserved and essential stress protein,expressed throughout the eukaryotic lineage [19-22]. Highereukaryotes possess multiple HSP90 homologues, includingthe highly conserved HSP90 and HSP90 isoforms (86%amino acid conservation) which are mainly cytoplasmic [23],GRP94 in the endoplasmic reticulum [24], and TRAP1 in themitochondrial matrix [25]. Although the HSP90 bacterialhomolog HtpG is typically nonessential [26], eukaryotesrequire a functional cytoplasmic HSP90 for survival underall conditions tested [27]. Indeed whereas the term heat-shock protein is perfectly appropriate for other HSPs, it isreally somewhat of a misnomer for HSP90. In most, if not allcell types, HSP90 is actually a constitutively expressed mo-

lecular chaperone, already extremely abundant prior to cellu-lar stress and is typically induced only a few-fold understress conditions [21]. More specifically, HSP90 is one ofthe most abundant proteins (1- 2 %) in the cytoplasm of un-stressed cells where it performs housekeeping functions,controlling the stability, maturation, activation, intracellulardisposition and proteolytic turn-over of a plethora of proteinsgenerally termed as client proteins [28-30]. HSP90 exertsits molecular chaperone activity by conformational cycles ofbinding and release which are dependent upon its ATPaseactivity [20, 31-34]. This ATP-driven conformational cycle

is regulated by specific co-chaperones, such as HSP70, Hopimmunophilins, cdc37 and p23, that complex with HSP90and assemble into the HSP90 chaperone machinery, in ordeto assist the loading and release of client proteins [20, 22, 3132, 35].

The broad clientele of HSP90 extends to more than 100proteins (see Dr Picards web site for an up-to-date list) andincludes molecules that are structurally and functionally diverse. Among themfeaturemultiple signal transduction molecules such as transcriptional factors, members of the Srckinase family, serine/threonine kinases, and growth factoreceptors. These key-components of the cells signallingmachinery are often activated, mutated and/or over-expressed in cancer cells and are considered responsible for theacquisition of the malignant phenotype [4, 18]. Subsequently, HSP90 is viewed as a key player in the subversionof normal cells towards transformation and therefore an exciting new target for the development of innovating molecular cancer therapeutics [16, 17, 36-42].

HSP90: THE CANCER CHAPERONE

HSP90 plays a pivotal role in the acquisition and mainte-nance of the malignant phenotype. Its expression in malignant cells is 2- to 10- fold higher than in normal cells [3643-45]. These higher expression levels are coupled to multiple fundamental oncogenic pathways and indicate a cruciarole associated with the development and maintenance of themalignant phenotype as well as the acquisition of drug resistant phenotypes.

The abundance of HSP90 in tumours, in part reflects anappropriate cyto-protective stress response to the hostile hypoxic, acidotic and nutrient-deprived tumor microenvironment. Consequently, the increased activities of this molecular chaperone allow tumour cells to cope and adapt to envi-ronmental changes as well as to the imbalanced signalling

associated with neoplastic transformation, and thereby escape apoptosis.

Malignant transformation involves the over-expressionand/or mutation of multiple HSP90-dependent key-regulatorof cellular growth. Almost 50 proteins known to play important roles in the control of cell cycle and growth, includingreceptor protein kinases and transcription factors have beenidentified as oncogenic clients of HSP90 [17, 23, 46, 47]HSP90 is indispensable for maintaining these proteins in anactive conformation, and thereby it is considered to drive thecell to self-sufficiency in growth signals. Moreover, HSP90confers survival advantages to cancer cells through its association with important elements of apoptosis such as survivin

[48], the Akt-survival pathway [49] and the Raf/MAPKgrowth regulatory pathway [50]. Finally HSP90 plays important anti-apoptotic roles by interfering with the intrinsiccaspase apoptotic pathway [51]. In addition to permittingautonomous growth and facilitating cell survival with respecto stressful environmental challenges, HSP90 also allowtumor cells to tolerate genetic alterations, including mutations of critical signalling molecules that would otherwise belethal [52, 53]. It not only moderates the impact of potentially lethal mutations in cancer cells, but also stabilizes andpermits the accumulation of mutant proteins, therefore func


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Extracellular HSP90 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1 53

tioning as a capacitor of evolution [54]. Thus, HSP90 mightalso serve as a biochemical buffer for the genetic instabilityfrequently found in cancers. As a result of this buffering ca-pacity, phenotypic diversity within the tumour cell popula-tion increases and the evolution of invasive, metastatic anddrug resistant phenotypes accelerates [18, 55].

To summarize, one can say that HSP90 participates inalmost all the key processes of oncogenesis, such as self-sufficiency in growth signals and stabilization of mutantproteins. The involvement of this molecular chaperone in theacquisition and maintainance of the transformed phenotypeis particularly interesting and suggests that inhibiting HSP90may have a coordinated effect on all of the key alterations onwhich cancer cells depend for their growth and survival.Consequently it is no wonder that HSP90 has emerged as apromising and exciting target for the development of cancertherapeutics.

HSP90 INHIBITORS: TARGETING SIGNAL TRANS-

DUCTION IN CANCER

The earliest inhibitor of HSP90 was the natural product

geldanamycin (GA) which is a member of the family ofansamycin antibiotics. This agent has featured in the litera-ture for several years as an anti-tumor agent, and was origi-nally isolated based on its ability to promote a significantdecrease in the activity of oncogenic tyrosine kinases such asv-Src and ErbB-2 [37, 56, 57]. In 1994, Whitesell et al. [57]reported that ansamycins and GA in particular, exerts itsfunction by acting as a nucleotide mimetic and by bindingspecifically to the ATPase domain of HSP90, resulting ininhibition of its chaperone function, and consequently in theubiquitination and degradation of client proteins by the pro-teasome pathway [38, 58-60]. Despite their promising activ-ity as anti-cancer agents, these antibiotics proved to havelimited clinical potential, because of their high liver toxicity

and/or cellular instability [61]. However, subsequent derivati-zation of GA, yielded analogues with reduced liver toxicitythat retained the potent anti-tumor activity of the parentcompound. One such example is the analogue 17-allylamino-17-demethoxygeldanamycin (17AAG) which has alreadyentered clinical trials. [62-65]. At present, several derivativesof natural products or fully synthetic small-molecule drugs

that target HSP90 have been discovered as potential anticancer agents (see Table 2) [37, 55]. These drugs are considered as unique in that, although they are directed against aspecific molecular target, they simultaneously inhibit multiple signalling pathways by inactivating, destabilizing, andeventually leading to degradation of numerous chaperonedependent client proteins [66, 67]. As a consequence they arecompetent to mount a multi-pronged assault on cancer cells

Moreover, although initially there were concerns that HSP90targeted drugs would attack proteins expressed both in normal and malignant cells and thus would lack specificity andcause damage to normal tissues, these fears were provedunfounded. Interestingly, exclusively tumour cells wereshown to exhibit sensitivity to HSP90 inhibition, thus lending credence to the feasibility of selectively targeting cancetissues via the pharmacological modulation of HSP90 function [68, 69]. Even more remarkably, HSP90 inhibitors sensitise tumour cells to the cytotoxic effects of a variety ofstandard therapeutic agents. Consequently they are likely tohave broad utility in combination therapy. At present, structurally unique HSP90 inhibitors are undergoing preclinicaand clinical evaluation. These anti-HSP90 drugs show grea

promise through their potential to block a wide spectrum othe main pathways of autonomous tumor growth.

EXTRACELLULAR FUNCTIONS OF HSP90

While HSP90 was originally discovered and perceived aan intracellular molecular chaperone, more than two decadelater, a number of studies challenged this view. In 1986, Ul-rich et al. [70], identified HSP90 as a tumor-specific antigenlocalized on the surface of mouse cells. Since then, severaresearchers have reported the presence of this moleculachaperone on the cell surface. However these studies did noattribute precise functions to this pool of the molecule andthe best studied cases of extracellular HSP90 concerned innate and adaptive immunity [71]. More specifically, HSP90

has been documented as a key player in antigen processingand presentation during immune responses. It has been detected on the membrane and/or culture media of antigenpresenting cells (APCs) [72] and in surface-peptide complexes [73], where it was reported to play crucial roles inanti-tumor and anti-viral responses through interaction withvarious HSP receptors on APCs, such as CD40, CD91 and

Table 2. HSP90 Inhibitors

Name Chemical Class Binding Site

GA Benzoquinone ansamycin N-terminal ATP-binding pocket

17-AAG GA-derivative N-terminal ATP-binding pocket

17-DMAG GA-derivative N-terminal ATP-binding pocket

Radicicol Macrolide N-terminal ATP-binding pocket

PU24FC1 Purine scaffold N-terminal ATP-binding pocket

CCT018159 Pyrazole N-terminal ATP-binding pocket

Radamycin Hybrid N-terminal ATP-binding pocket

Novobiocin Noviosylcoumarin crosslinker C-terminus


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ment of signalling complexes that modulate the actin cy-toskeleton and mediate the extension of membrane protru-sions at the leading edge [94, 95]. Several molecules havebeen described as being tightly involved in the re-organi-zation of the actin cytoskeleton of a cell. These include amongothers adhesion molecules, like integrins and selectins,transmembrane receptor tyrosine kinases (EGF-R, ErbB-2),phospholipids, focal adhesion kinases (FAKs) and GTPases

[96-98].

The understanding of the exact molecular mechanismsunderlying the role of surface HSP90 in so complex proc-esses such as cell motility and invasion is a major challengefor the investigators. Which are the extracellular substrate(s)

composing the clienteleof this extracellular moleculachaperone?

Eustace et al. [79], focused on MMP-2, an enzymewhose activity in the extracellular matrix is essential for celmigration and invasion, as shown by both in vitroand in vivomodel systems [99, 100]. Extracellular HSP90was shownto associate with, and activate MMP-2, promoting thus theinvasion of fibrosarcoma cells (Fig. 2A). Furthermore, functional inhibition of the molecule by GA covalently affixed tocell-impermeable beads, inhibited cancer cell invasion. Extracellular interaction of HSP90 with MMP-2, associatedwith tumor cell invasion was recently confirmed by Yang eal. [87].

Fig. (2). Schematic representation of 4 proposed models concerning the involvement of extracellular HSP90 in cell migration and

invasion.

A. HSP90 is identified extracellularly, in association with MMP2 and this interaction is suggested to be necessary for the maturation of the

enzyme, which in turn is crucial for fibrosarcoma cell invasion [79].

B. Surface HSP90 is shown to participate in melanoma and prostate cancer cell invasion through regulation of focal adhesion formation,

including ECM-induced c-src/integrin association and re-organization of the actin cytoskeleton [85].

C.Surface HSP90 is shown to be involved in breast cancer cell invasion, stimulated by the presence of HRG [81]. The model suggests that

HSP90 exerts its function through an interaction with the extracellular domain of ErbB2, essential for the activation of the downstream signal

transduction pathways leading to actin cytoskeletal re-arrangement and cell invasion.

D.TGFa is shown to trigger keratinocytes to secrete HSP90 in the culture medium through the exosome pathway. Secreted HSP90 in turn

acts as a pro-motility factor for skin cells, through its cell surface receptor LRP-1/CD91 [86].


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On the other hand, Tsutsumi et al. [85], suggested thatsurface HSP90 participates in cancer cell invasion throughthe signalling processes involved in the regulation of focaladhesion formation (Fig. 2B). Focal adhesions are siteswhere clusters of integrin and associated proteins such asFAK, c-Src and GTPases mediate adhesion links to the actincytoskeleton [95, 101]. By exploiting the function blockingproperties of a cell-impermeable HSP90 inhibitor named

DMAG-N-oxide, the authors speculate that surface HSP90 isinvolved in cell motility and invasion processes, throughparticipation in leading-edge actin polymerization and focaladhesion formation. More specifically, they suggest that cellsurface HSP90 is involved in the ECM-induced c-Src/integrinassociation and the re-organization of the actin cytoskeleton.

In another recent study, surface HSP90 involvement inbreast cancer cell invasion was attributed to a functional in-teraction of this molecule with the extracellular domain ofHER-2 [81], a protein whose intracellular kinase domain isalready known to interact with HSP90 [102-104]. This ex-tracellular interaction was shown to be crucial for maintain-ing the receptor in an active conformation, able to form het-

erodimers with other ErbB family members and thus activatethe downstream signal transduction pathways leading to cellmotility and actin re-arrangement (Fig. 2C). Disruption ofthis extracellular interaction by the cell-impermeable mAb4C5 resulted in reduced activation of HER-2 and decreasedformation of ErbB heterodimers, accompanied by impaireddownstream kinase signalling, leading to inhibiton of actincytoskeletal re-arrangement and reduced cell invasion.

Finally, Cheng et al. [86], demonstrated that extracellularHSP90a promoted migration of both epidermal and dermalcells through the cell surface receptor LPR-1/CD91, whichwas found to mediate HSP90a signalling. Furthermore, theauthors linked EGFR activation by TGFa to the exosomepathway leading to the secretion of HSP90a (Fig. 2D).

The present datasupportthe idea that extracellular HSP90,interacts with a wide-range of molecules, some of them mostprobably implicated in signal transduction processes leadingto cell motility, invasion and metastasis.

PERSPECTIVES

The discovery of HSP90 on the surface of cancer cells, incombination with accumulating evidence reporting its in-volvement in invasion and metastasis commences a new andexciting era in the field of cancer therapeutics. Cell surfaceHSP90 provides a novel and promising molecular target forthe development of effective anti-metastatic drugs. Althoughcompounds targeting HSP90 already exist, the need for fur-

ther development of cell-impermeable HSP90 inhibitors isemerging. In this context, the delineation of the pathwaysthrough which this protein acts and the basis for its varyingactions extracellularly , remains a great challenge.

ABBREVIATIONS

17AAG = 17-dimethylaminoethylamino geldanamycin

APCs = antigen-presenting cells

FAK = Focal Adhesion Kinase

GA = Geldanamycin

HRG = Heregulin

HSPs = Heat shock proteins

mAb = Monoclonal antibody

MAPK = Mitogen-activated protein kinase

PI3K = Phosphoinositide 3-kinase

RTKs = Receptor Tyrosine KinasesREFERENCES

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Received: July 10, 2008 Revised: August 27, 2008 Accepted: September 09, 2008

extracellular hsp90: an emerging target for cancer therapy

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