Biochimica et Biophysica Acta 1835 (2013) 211–218

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Review

Hsp90: Still a viable target in prostate cancer

Margaret M. Centenera a, Alyssa K. Fitzpatrick a, Wayne D. Tilley a,b, Lisa M. Butler a,b,c,⁎a Dame Roma Mitchell Cancer Research Laboratories and Adelaide Prostate Cancer Research Centre, University of Adelaide and Hanson Institute, Adelaide, 5000, Australiab Freemasons Foundation Centre for Men's Health, University of Adelaide, 5000, Australiac Centre for Personalised Cancer Medicine, University of Adelaide, 5000, Australia

⁎ Corresponding author at: Dame RomaMitchell CancerofMedicine, University of Adelaide andHanson Institute, Ad8 8222 3270; fax: +61 8 8222 3217.

E-mail addresses: margaret.centenera@adelaide.edualyssa.fitzpatrick@student.adelaide.edu.au (A.K. Fitzpatrwayne.tilley@adelaide.edu.au (W.D. Tilley), lisa.butler@

0304-419X/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbcan.2012.12.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 October 2012Received in revised form 17 December 2012Accepted 20 December 2012Available online 31 December 2012

Keywords:Hsp90InhibitorsProstate cancerClinical trials

Heat shock protein 90 (Hsp90) is amolecular chaperone that regulates thematuration, activation and stability ofcritical signaling proteins that drive the development and progression of prostate cancer, including the androgenreceptor. Despite robust preclinical data demonstrating anti-tumor activity of first-generation Hsp90 inhibitorsin prostate cancer, poor clinical responses initially cast doubt over the clinical utility of this class of agent. Recentadvances in compound design and development, use of novel preclinical models and further biological insightsinto Hsp90 structure and function have now stimulated a resurgence in enthusiasm for these drugs as a thera-peutic option. This review highlights how the development of new-generation Hsp90 inhibitors with improvedphysical and pharmacological properties is unfolding, and discusses the potential contexts for their use either assingle agents or in combination, for men with metastatic prostate cancer.

© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2112. Heat shock protein 90 as an anti-cancer target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2123. The first generation: ansamycin-based Hsp90 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2124. Non-ansamycin based Hsp90 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2145. Other classes of Hsp90 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

5.1. C-terminal Hsp90 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2145.2. Mitochondrial targeted Hsp90 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

6. Combinatorial approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2156.1. Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2156.2. Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2156.3. Molecular agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2156.4. Heat shock response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

7. Clinical markers of Hsp90 inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2158. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

1. Introduction

Prostate cancer remains a significant cause ofmorbidity andmortalityin the western world [1]. The standard of care for men with non-organ

Research Laboratories, Disciplineelaide, 5000, Australia. Tel.:+61

.au (M.M. Centenera),ick),adelaide.edu.au (L.M. Butler).

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confined or metastatic prostate cancer is androgen deprivation therapy(ADT), which exploits the dependence of prostate epithelial cells onandrogen signaling, mediated by the androgen receptor (AR). Althoughthere is an excellent initial response to ADT, manifested as a decreasein serum prostate specific antigen (PSA) levels and reduced tumorburden, these therapies inevitably fail with the emergence of an aggres-sive and incurable form of disease known as castrate-resistant prostatecancer (CRPC). While the treatment options for men with metastaticCRPC were previously limited to docetaxel-based chemotherapy, whichextends survival by only a few months [2], the past 2 years has seen a

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radical change in the therapeutic landscape for this disease. The im-munotherapy sipuleucel-T [3], the chemotherapeutic cabazitaxel [4],the androgen biosynthesis inhibitor abiraterone acetate [5] and theAR antagonist MDV3100 [6] have all received FDA approval in thepost-docetaxel setting. Further, the radioisotope alpharadin [7] hasshown efficacy in phase III studies and is on track for approval. Whilethese new agents have increased the therapeutic options for cliniciansand their patients, it is recognized that achieving lasting suppressionof CRPC growth remains a serious challenge and requires new treat-ment strategies.

2. Heat shock protein 90 as an anti-cancer target

Heat shock proteins are molecular chaperones that facilitate nor-mal cellular homeostasis by regulating the folding of newly translatedor denatured proteins, preventing protein misfolding or aggregation,stabilizing proteins in a conformation ready for ligand activationand assisting with the transport of proteins across membranes [8].Heat shock proteins are up-regulated in response to cellular stressesor toxic insults that increase the degree of protein misfolding or aggre-gation and threaten cell survival such as hypoxia, acidosis, nutrient dep-rivation and geneticmutation or overexpression [9,10]. As these stressesare characteristic of the tumor cell environment, the upregulation ofheat shock proteins, known as the heat shock response, is commonlyobserved in cancer cells and is believed to support malignant transfor-mation [11–13].

Hsp90 is the most abundant heat shock protein within the cell andit exists as part of a dynamic multi-chaperone complex. In mammals,Hsp90 is encoded by two genes, resulting in two very similar (≅85%identical at the amino acid level) protein isoforms, Hsp90α andHsp90β, that are thought to be functionally similar, but with evidencefor tissue- and substrate-specificity [14]. Residing largely in the cyto-plasm, Hsp90 has an N-terminal ATP-binding pocket, a middle domainfor binding to client proteins and a C-terminal homodimerization do-main (Fig. 1A) [15]. The chaperone activity of Hsp90 is dependentupon iterative rounds of ATP hydrolysis. In the ATP-bound state,Hsp90 acquires a mature conformation that allows it to participate inclient protein folding and stabilization (Fig. 1B) [15]. The chaperonedoes not covalently modify its client, but participates in a series ofintricate loading and unloading events in coordination with a rangeof co-chaperones including Hsp70, Hsp40, HOP and p23 (Fig. 1B)[16]. Hsp90 has over 200 client proteins (see http://www.picard.ch/downloads for a curated list) including steroid receptors, transcriptionfactors and protein kinases and is therefore critical to many cellularfunctions and to cell survival. Additionally, one of the key reasons whyHsp90 is a desirable anti-cancer target is thatmany of its client proteinsare known oncoproteins [17]. Someof these clients aremore dependenton Hsp90 function in cancer cells compared with normal cells [18],thereby affording the potential for Hsp90 inhibitors to exhibit tumorselectivity.

Hsp90 inhibition is of particular significance for prostate canceras Hsp90 is overexpressed in prostate cancer cells compared withnormal prostate epithelium and thus provides a potential selectivetarget [19]. Further, many Hsp90 client proteins have known rolesin prostate carcinogenesis including HER2, EGFR, CDK4, AKT and im-portantly the AR. The critical role of the AR in prostate carcinogenesisand in the progression to CRPC is well established [20]. Further, theAR drives growth of CRPC through a number of mechanisms that inti-mately rely on Hsp90 for cell survival, particularly AR overexpressionand gain-of-function AR gene mutations [21]. By promoting degrada-tion of AR and other proteins that influence AR signaling or prostatetumorigenesis, Hsp90 inhibition provides a distinct and multifacetedapproach to targeting prostate cancer cells compared with currentagents such as abiraterone acetate that targets androgen synthesisor MDV3100 that inhibits androgen binding. Importantly, the broadinhibitory action of Hsp90 inhibitors appears to be effective in cells

expressing spliced variants of the AR that are devoid of the ligandbinding domain and therefore resistant to conventional AR antagonists[22].

3. The first generation: ansamycin-based Hsp90 inhibitors

The first Hsp90 inhibitors to be studied were geldanamycin andradicicol, both naturally occurring antibiotics [23,24]. These agents pos-sess a higher affinity for the N-terminal ATP-binding pocket of Hsp90than the natural ligand, and upon binding, prevent the chaperonefrom cycling between its ATP and ADP bound states [25]. Unable tobindATP, Hsp90 cannot perform its chaperone function and client pro-teins are degraded via the ubiquitin proteasome pathway [26,27]. Toaugment this process, drug-bound Hsp90 recruits E3 ubiquitin ligasessuch as CHIP (carboxy-terminus of hsp70-interacting protein) [28,29].Both geldanamycin and radicicol were unsuitable for clinical use dueto animal studies showing unacceptable levels of hepatotoxicity ordrug instability, respectively [30,31]; thus, the geldanamycin deriva-tive 17-allylamino-17-demethoxygeldanamycin (17-AAG) was the firstHsp90 inhibitor to undergo clinical assessment.

As the first-in-class agent, 17-AAG underwent extensive preclinicalinvestigation. In prostate cancer cell lines and animal models, 17-AAGcaused proteasomal degradation of client proteins AR, HER2 and Aktamong others, cell cycle arrest and substantive growth inhibition ofboth androgen-sensitive and androgen-insensitive xenograft tumors[32,33]. The initial phase I studies of 17-AAGwere conducted in patientswith advanced solid malignancies [34–37]. In one study that includedprostate cancer patients, no partial or complete responses were ob-served, but one patient experienced disease stabilization for elevencycles of 17-AAG treatment and a 25% reduction in PSA levels [37].These clinical results were seen as promising and 17-AAG was sub-sequently evaluated in a multicenter phase II trial in patients withmetastatic CRPC. No responses were observed in terms of PSA or byradiological imaging and the authors concluded that investigation into17-AAG as a single agent treatment for CRPC should be discontinued(Table 1) [38]. The limited success of 17-AAG in this and other phaseII trials in solid malignancies has been attributed to its intrinsic chem-ical structure [39]. 17-AAG contains a benzoquinone moiety that con-tributes to hepatotoxicity of the agent and provides a mechanismfor acquired cellular resistance. 17-AAG is metabolized by the en-zymes NAD(P)H quinine oxoreductase (NQ01) [40] and cytochromeP450 3A5 (CYP3A5) [41], thus the level of expression or polymorphism/mutation of either gene causes variability in pharmacokinetics and resultin drug resistance. 17-AAG is also susceptible to efflux by P-glycoprotein[40], and is poorly soluble thus further limiting its application to clinicalpractice [42].

To circumvent the drawbacks of 17-AAG, the analogue 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) wasdesigned to have increased water solubility and oral bioavailability[43]. Replacing the 17-allylamino moiety with 17-diethylaminoethylamino also reduced susceptibility to NQO1 [44]. Until recently, itappeared that 17-DMAG would suffer the same fate as 17-AAG, withminimal efficacy observed in initial Phase I clinical trials. However in2011, a phase I study in patients with advanced solid malignanciestreated with weekly 17-DMAG reported objective responses in twoCRPC patients. A complete response, confirmed by PSA and radiologicalimaging, was sustained in one patient for 124 weeks, and stable diseaseobserved for 59 weeks in the other [45]. The encouraging outcome ofthis trial has been attributed to the unique study design aimed at de-fining a biologically active dose, rather than the maximum tolerateddose that is standard in Phase I studies.

Another derivative, IPI-504, is a hydroquinone salt-derived formof 17-AAG that is highly water-soluble and does not require organicsolvents such as DMSO [46]. Despite promising preclinical activity,in a phase II study in patients with CRPC, no patient achieved tumorresponse or ≥50% post-treatment decline in PSA levels, and two of

Fig. 1. Hsp90 protein structure and ATPase cycle. (A) Schematic diagram of the Hsp90 protein structure. The N-terminal domain (NTD) is responsible for ATP-binding and is linkedto the middle domain (MD) by a charged linker. The C-terminal domain (CTD) is essential for homodimerization and is an interaction site for certain co-chaperones. Hsp90 interactswith client proteins through regions that span the MD and CTD. (B) In its “open” ADP-bound state, Hsp90 is preferentially bound by HOP, a co-chaperone that delivers partiallyfolded proteins to HSP90 via Hsp70/40/HIP. When Hsp90 cycles from its ADP- to ATP-bound state it releases the bound co-chaperones, and converts into its “closed” form. Bindingof p23 stabilizes Hsp90 in the conformation required for client protein folding. After the client protein is released, Hsp90 cycles back to its ADP-bound state making it available forthe next chaperone cycle.

213M.M. Centenera et al. / Biochimica et Biophysica Acta 1835 (2013) 211–218

the nineteen patients died as a result of treatment-related adverseeffects, including hepatotoxicity and ketoacidosis (Table 1) [47]. Thetoxicity of IPI-504, along with its lack of clinical benefit, precludesits further investigation as a single agent for CRPC.

Table 1Clinical evaluation of Hsp90 inhibitors as monotherapy for castrate-resistant prostate cance

Agent Phase Patient groups Starting Dosage Responses

Tanespimycin(17-AAG)

II Chemotherapytreated (n=15)

300 mg/m2 i.v., days 1, 8,15 of 28 day cycle

No evaluablobjective disgrade 3 adv

Retaspimycin(IPI-504)

II Chemotherapynaïve (n=4),docetaxel treated(n=15)

400 mg/m2 i.v., days 1, 4,8, 11 of 21 day cycle

1 evaluable ppartial resporesponses orhad treatme2 patient dea

In summary, although extensive efforts have beenmade to improvethe pharmacology of the ansamycin class of Hsp90 inhibitors, unless adiscrete sensitive patient population can be identified, it appears thatthey will remain limited by their characteristic benzoquinone group,

r.

Conclusions Ref

e patients achieved PSA response orease response. 9/15 patients haderse events.

Further investigation assingle agent for CRPC notwarranted

[38]

atient (chemotherapy-naïve) achievednse with 48% PSA decline, no otherantitumor activity observed. 18/19 patientsnt-related adverse events, 11 were ≥grade 3,ths attributed to treatment.

Further investigation as singleagent for CRPC not warranted

[47]

214 M.M. Centenera et al. / Biochimica et Biophysica Acta 1835 (2013) 211–218

which has been held responsible for the liver toxicities associated withthese agents in clinical trial [48].

4. Non-ansamycin based Hsp90 inhibitors

Given the dose-limiting toxicity associated with the ansamycinbased inhibitors, considerable effort has been made to rationallydevelop synthetic Hsp90 inhibitors based on the crystal structuresof Hsp90 bound to ATP/ADP, 17-AAG, geldanamycin or radicicol, orthrough the identification of novel compounds via large scale drugscreens [49]. Synthetic inhibitors have the potential advantages ofimproved pharmacokinetic profiles, increased water solubility, easeof administration, evasion of resistance mechanisms and activity overa wider range of tumors [48]. In addition, there is the opportunity todevelop Hsp90 isoform-specific inhibitors that could provide furtherselectivity for certain client or tissue targets [50]. At least thirteendifferent synthetic Hsp90 inhibitors are currently under evaluation inclinical trials (clinicaltrials.gov), and these can generally be categorizedinto two structural classes, the purines and the resorcinols; for anextensive review see [49]. Relatively few Hsp90 inhibitors have specif-ically been evaluated for their potential as prostate cancer therapeutics,these are detailed below.

SNX-2112 is administered orally as its prodrug SNX-5542. It has sim-ilar antitumor effects as 17-AAG but has greater potency in a rangeof prostate cancer cell lines in vitro and in vivo, most notably thosethat express the AR [51]. In previous animal studies, 17-AAG has beenshown to stimulate osteoclastogenesis and promote tumor growthwithin bone through disruption of the Hsp90-cSrc complex [52]. Whilea tumor-promoting effect of Hsp90 inhibition on bony metastases hasnot been demonstrated in patients, the authors proposed that a majoradvantage of SNX-2112 was its significant inhibition of RANK ligand(RANKL) osteoclast differentiation [51]. SNX-2112 was well toleratedin its first phase I trial and disease stabilization occurred in 47% of pa-tients [53]. Despite these findings, and its potential for use in patientswith existing bony metastases, the development of SNX-2112 hasbeen discontinued due to ocular toxicity [53].

AUY922 is an isoxazole resorcinol that exhibits nanomolar affinityand selectivity for Hsp90 [54]. It is 5-fold more selective for Hsp90than other Hsp90 family members such as GRP94 and TRAP-1, andis 50-fold more selective compared with ATPase-dependent kinases[54]. Unlike the ancamycin based agents, AUY922 is not metabolizedby CYP3A4 or NQO1/DT-diaphorase enzymes and is not susceptible toefflux by the multidrug resistance protein P-glycoprotein, whichmeans that AUY922 can be maintained at higher concentrations incancer cells. AUY922 is a potent inhibitor of cellular proliferation,cell cycle progression, tumor cell invasion and angiogenic endothelialcell functions in a variety of cell lines [54–58]. In xenograft cancermodels, including the PC-3 model of prostate cancer, AUY922 hasdemonstrated target inhibition along with reductions in primarytumor growth and vascularization. Eccles et al. (2008) demonstratedthat AUY922 also reduced the number and size of lymph node metas-tases, which was the first in vivo evidence that Hsp90 inhibitors caninhibit both local disease and haematogenous metastases. We recentlyreported on the marked anti-proliferative and pro-apoptotic activitiesof AUY922 in primary human prostate tumors cultured ex vivo [55],providing evidence that new generation Hsp90 inhibitors can inducesignificant responses in the context of the normal tumor microenviron-ment where the complex tissue architecture and signaling remain in-tact, including stromal interactions that may be required for or impacton drug action. AUY922 has been well tolerated in phase I clinical trialsand achieved disease stabilization in a subset of patients [59,60], withPhase II trials continuing (clinicaltrials.gov).

Another resorcinol derived Hsp90 inhibitor currently under clini-cal investigation is STA-9090 [61,62]. The triazolone-containing res-orcinol is much smaller than most other Hsp90 inhibitors, and as aconsequence can bind to Hsp90 while the chaperone is in its “closed”

conformation [62], which is in contrast to the ancamycin-based inhib-itors that can only bind while Hsp90 is in its “open” conformation.There is speculation that this trait may contribute to the high potencyof STA-9090. At low nanomolar concentrations, STA-9090 inducesdegradation of many Hsp90 client proteins and induces death in avariety of cancer cell lines, including DU-145 prostate cancer cells[62,63]. The chemical structure of STA-9090 makes the compoundhighly lipophilic thereby improving penetration and distribution withintumors [62]. STA-9090 is one of only two compounds currently beingevaluated exclusively in patientswithmetastatic CRPC (clinicaltrials.gov).

AT13387 is the other compound being evaluated in a clinical trialin men with CRPC, and specifically in those who have failedtreatment with abiraterone acetate (clinicaltrials.gov). AT13387 is adihydroxybenzamide that was designed following a fragment-baseddrug screen [64]. AT13387 has demonstrated potent anti-proliferativeactivity in a panel of 30 tumor cell lines that included 4 different pros-tate cancer cell lines [65]. In animal models, AT13387 showed wide-spread tissue distribution and a longer duration of depletion of Hsp90client protein than has been reported for other small molecular inhib-itors of Hsp90 [65]. Target modulation has been reported in the firstPhase I study performed in patients with refractory solid tumors [66].

5. Other classes of Hsp90 inhibitors

This review has focused on agents that target the N-terminus ofHsp90 as these are the only category of Hsp90 inhibitor that hasentered clinical trial to date. Alternative strategies currently underpreclinical evaluation include those that target the C-terminus ofHsp90 or mitochondrial Hsp90.

5.1. C-terminal Hsp90 inhibitors

The coumarin antibiotic, novobiocin, targets a site in the carboxylterminus of Hsp90 that interacts with co-chaperones such as Hsc70and p23, as well as ATP [67]. Since novobiocin has low affinity forHsp90, various analogues such as F-4 and KU174 have been identifiedthat elicit enhanced anti-tumor activity compared with 17-AAG inprostate cancer cell lines [68,69]. In contrast to N-terminal inhibitors,C-terminal Hsp90 inhibitors do not induce a heat shock response, asreduction in Hsp70, Hsc70 and HSF-1 is observed [68,69]. This is aparticularly attractive characteristic as the role of the heat shock re-sponse is to enhance cell survival and suppress apoptotic cell death. Is-sues withmetabolism and clearance have prevented KU174 from beingevaluated in themouse, however a pilot study in the PC3-MM2 rat pros-tate cancermodel showed some efficacy of KU174 on tumor growth andthis class of inhibitors will be interesting to observe as their develop-ment continues [69].

5.2. Mitochondrial targeted Hsp90 inhibitors

In contrast to most normal tissues, the mitochondria of tumor cellsexpress the Hsp90 family member TNF receptor associated protein1 (TRAP1) and therefore represent a potential cancer-specific target[70]. The geldanamycin mitochondrial matrix inhibitor gamitrinibwas designed with this in mind, and contains a benzoquinone back-bone derived from 17-AAG with a mitochondrial targeting moiety[71]. Gamitrinib has undergone considerable pre-clinical evaluationfor prostate cancer, and induces prostate cancer cell death throughacute mitochondrial dysfunction including loss of membrane poten-tial and release of cytochrome c [72], which are hallmarks for theinitiation of apoptosis. Systemic administration of gamitrinib causedinhibition of localized and metastatic prostate cancer growth in xeno-graft models [72], and in the TRAMP mouse model of prostate cancer[73]. No effect on other organs or on PIN or local inflammation withinthe prostate was observed in either study. At the time of publishingthis article, gamitrinib had not yet entered clinical trials.

215M.M. Centenera et al. / Biochimica et Biophysica Acta 1835 (2013) 211–218

6. Combinatorial approaches

6.1. Chemotherapy

Despite the failure of 17-AAG as a single agent therapy in phaseII trials, interest remains in combining 17-AAG at lower doses withother cytotoxic drugs. Cytotoxic chemotherapeutic agents induceDNA damage, which activates cell replication checkpoints. Key tocheckpoint regulation is the Chk1 signaling pathway, whereby Chk1is activated to arrest the damaged cell in the G2/S phases of cellularreplication. Since Chk1 is an Hsp90 client, inhibition of Hsp90 limitsthe capacity for DNA repair and sensitizes the cells to chemotherapy[74]. Phase I dose-escalating trials of 17-AAG in combination withgemcitabine and/or cisplatin in a range of advanced solid malignanciesincluding prostate cancer demonstrated anti-tumor activity at tolerabledoses when 17-AAG was combined with gemcitabine and this combi-nation was recommended for phase II studies [75]. In a Phase I trial of17-AAG plus docetaxel, 25% of patients with prostate cancer exhibiteda PSA decline of ≥20% [76]. A correlation between 17-AAG dose andreduced docetaxel clearance was observed however the DMSO formu-lation of 17-AAG precluded dose escalation above 500 mg/m2, whichhas prompted trials of docetaxel with alternative Hsp90 inhibitors[77,78].

6.2. Radiation

By the same rationale outlined above for chemotherapy, Hsp90inhibitors can enhance the sensitivity of tumor cells to DNA damageinduced by radiation therapy. Various Hsp90 client proteins, includ-ing Raf-1, Akt and HER2, are involved in protecting the cell againstradiation-induced cell death. Exposure to Hsp90 inhibitors causesa loss of these proteins and hence an increase in radiosensitivity viaincreased cell cycle arrest and apoptosis, reviewed in [79]. Multiplestudies have shown the efficacy of combining 17-AAG or 17-DMAGwith ionizing radiation on prostate cancer cell growth in vitro andin vivo [80–82].

6.3. Molecular agents

Combinationwith other targetedmolecular agents can also sensitizetumor cells to Hsp90 inhibition. The tumor necrosis factor-α-relatedapoptosis inducing ligand (TRAIL) is a potent inducer of apoptosisin most prostate cancer cell lines but not LNCaP cells [83]. However,LNCaP cells can be sensitized to TRAIL by the addition of low-dosegeldanamycin [84]. Combination of Hsp90 inhibitors with an ARantagonist may be a valuable avenue in prostate cancer therapy asAR antagonists such as bicalutamide require Hsp90 to facilitate AR nu-clear translocation [85]. MDV3100 would be a particularly interestingcandidate for use in combination with Hsp90 inhibitors as the antago-nist is proposed to sequester the AR in the cytoplasm [86], a featurethat is likely to involve Hsp90 and thereby maintain the AR as a targetfor Hsp90 inhibition.

6.4. Heat shock response

The heat shock response is mediated by the heat shock transcrip-tion factor 1 (HSF-1), which is released in response to cellular stressand binds to heat shock elements within the promoters of stress-responsive genes including Hsp70, Hsp27 and Clusterin [87]. As expo-sure to Hsp90 inhibitors is a toxic insult it similarly induces the heatshock response [88], and there is concern that the protective functionof heat shock proteins could counterbalance the cytotoxicity of Hsp90inhibition. While induction of the heat shock response has some clin-ical utility, such as measurement of the levels of Hsp70 in peripheralblood mononuclear cells (PBMCs) as a pharmacodynamic marker ofHsp90 inhibition in clinical trials [36,89–91], it has also been cited as

a mechanism of cellular resistance and an underlying mechanism forthe failure of ansamycin antibiotics as single-agent therapy in clinicaltrials [92]. Hsp70 has been shown to inhibit conformational change andmitochondrial localization of the pro-apoptotic protein Bax, preventinginitiation of the mitochondrial pathway of apoptosis [93]. Similarly,Hsp27 canmediate resistance through increased synthesis of glutathi-one [94]. Inhibition of Hsp70, Hsp27 or Clusterinwith small interferingRNA or antisense oligonucleotides significantly increases the potencyof Hsp90 inhibitors [94–96], and suggests that clinically targeting theheat shock response may provide additional therapeutic benefit incombinationwith N-terminal Hsp90 inhibitors. Alternatively, the devel-opment of agents with reduced activation of the heat shock response,such as the C-terminal inhibitors, could also be advantageous.

7. Clinical markers of Hsp90 inhibition

The use of Hsp70 as a marker to monitor efficacy of Hsp90 inhibi-tion in clinical trials is based on robust association with biological ac-tivity of these agents in vitro. However increasing evidence suggeststhat Hsp70 induction is not sufficient to predict clinical activity ofthese agents. For example, in clinical trials of 17-DMAG in patientswith advancedmalignancies, Hsp70 levelsmeasured in PBMCs showedno correlation with clinical response [90,91]. Similarly, our recent pre-clinical studies in an ex vivo human prostate cancer model providedcompelling evidence that modulation of Hsp70 levels is not indicativeof anti-tumor response to Hsp90 inhibition [55]. A critical priority forthe development of these agents is the identification of more informa-tive biomarkers that indicate biological efficacy and/or sensitivity. Tothis end, archived tumor specimens from 8 phase I/II clinical trials atMemorial Sloan Kettering Cancer Center were recently used to identifybiomarkers that may predict sensitivity to Hsp90 inhibitors, and there-by improve patient selection and monitoring of treatment [97]. Usingpreclinical models that incorporate human prostate tumors, either inan ex vivo or in vivo xenograft setting, will facilitate these endeavoursby utilizing patient-matched samples of treated and untreated tissuesfor comparison, and enabling more informative analysis of emergingagents that have not yet reached the clinic.

8. Conclusion

Hsp90 inhibitors will likely have the greatest clinical benefit incancers that are driven by proteins that are Hsp90 clients, a notionsupported by the positive results seen in clinical trials for Her2-positivebreast cancer [98] and Alk-driven non small cell lung carcinoma[99,100]. Appreciation for the ongoing role of the AR signaling axis inthe progression of CRPC clearly places prostate cancer in this category.The ansamycin antibiotic class of Hsp90 inhibitors that includes17-AAG has been extensively evaluated in CRPC but failed to provideconvincing evidence of anti-tumor activity in clinical trials, and isnow being primarily assessed in combinatorial-based approaches.The array of new generation synthetic agents showing promisingresults in Phase I/II studies suggests that with further improvementsin agent formulations, pharmacokinetics, and understanding of thebiology of Hsp90 and its isoforms, there is reason for optimism in theuse of Hsp90 inhibitors to (i) achieve more durable suppression ofCRPC growth and (ii) minimize drug resistance. The identification ofbiologically relevant markers that can be used as surrogate endpointsof efficacy represents a critical need for the translation of Hsp90 inhib-itors into clinical practice.

Acknowledgements

This work was supported by grants from the National Health andMedical Research Council (ID 627185 to L.M. Butler and W.D. Tilley),Cancer Australia/Prostate Cancer Foundation of Australia (ID 627229to L.M. Butler and W.D. Tilley), the Prostate Cancer Foundation of

216 M.M. Centenera et al. / Biochimica et Biophysica Acta 1835 (2013) 211–218

Australia (ID 2711 to L.M. Butler and M.M. Centenera), the RoyalAdelaide Hospital Research Committee (to M.M. Centenera, L.M. Butlerand W.D. Tilley). The Adelaide Prostate Cancer Centre is supportedby an establishment grant from the Prostate Cancer Foundation ofAustralia (ID 2011/0452). LM Butler holds a senior research fellowshipfrom the Cancer Council of South Australia.

References

[1] A. Jemal, R. Siegel, J. Xu, E. Ward, Cancer statistics, 2010, CA Cancer J. Clin. 60(2010) 277–300.

[2] I.F. Tannock, R. de Wit, W.R. Berry, J. Horti, A. Pluzanska, K.N. Chi, S. Oudard, C.Theodore, N.D. James, I. Turesson, M.A. Rosenthal, M.A. Eisenberger, Docetaxelplus prednisone or mitoxantrone plus prednisone for advanced prostate cancer,N. Engl. J. Med. 351 (2004) 1502–1512.

[3] P.W. Kantoff, C.S. Higano, N.D. Shore, E.R. Berger, E.J. Small, D.F. Penson, C.H.Redfern, A.C. Ferrari, R. Dreicer, R.B. Sims, Y. Xu, M.W. Frohlich, P.F.Schellhammer, Sipuleucel-T immunotherapy for castration-resistant prostatecancer, N. Engl. J. Med. 363 (2010) 411–422.

[4] J.S. de Bono, S. Oudard, M. Ozguroglu, S. Hansen, J.P. Machiels, I. Kocak, G. Gravis,I. Bodrogi, M.J. Mackenzie, L. Shen, M. Roessner, S. Gupta, A.O. Sartor, Prednisoneplus cabazitaxel or mitoxantrone for metastatic castration-resistant prostatecancer progressing after docetaxel treatment: a randomised open-label trial,Lancet 376 (2010) 1147–1154.

[5] J.S. de Bono, C.J. Logothetis, A. Molina, K. Fizazi, S. North, L. Chu, K.N. Chi, R.J.Jones, O.B. Goodman Jr., F. Saad, J.N. Staffurth, P. Mainwaring, S. Harland, T.W.Flaig, T.E. Hutson, T. Cheng, H. Patterson, J.D. Hainsworth, C.J. Ryan, C.N.Sternberg, S.L. Ellard, A. Flechon, M. Saleh, M. Scholz, E. Efstathiou, A. Zivi, D.Bianchini, Y. Loriot, N. Chieffo, T. Kheoh, C.M. Haqq, H.I. Scher, Abiraterone andincreased survival in metastatic prostate cancer, N. Engl. J. Med. 364 (2011)1995–2005.

[6] H.I. Scher, K. Fizazi, F. Saad, M.E. Taplin, C.N. Sternberg, M.D. Miller, R. de Wit, P.Mulders, K.N. Chi, N.D. Shore, A.J. Armstrong, T.W. Flaig, A. Flechon, P.Mainwaring, M. Fleming, J.D. Hainsworth, M. Hirmand, B. Selby, L. Seely, J.S. deBono, Increased survival with enzalutamide in prostate cancer after chemotherapy,N. Engl. J. Med. 367 (2012) 1187–1197.

[7] H.I. Scher, K. Fizazi, F. Saad, M.-E. Taplin, C.N. Sternberg, K. Miller, R. De Wit, P.Mulders, M. Hirmand, B. Selby, J.S. De Bono, for the AFFIRM Investigators, EffectofMDV3100, an androgen receptor signaling inhibitor (ARSI), on overall survival inpatients with prostate cancer postdocetaxel: results from the phase III AFFIRMstudy, J. Clin. Oncol. 30 (2012) LBA1.

[8] J. Trepel, M. Mollapour, G. Giaccone, L. Neckers, Targeting the dynamic HSP90complex in cancer, Nat. Rev. Cancer 10 (2010) 537–549.

[9] L.Whitesell, S.L. Lindquist, HSP90 and the chaperoning of cancer, Nat. Rev. Cancer 5(2005) 761–772.

[10] S.K. Calderwood, M.A. Khaleque, D.B. Sawyer, D.R. Ciocca, Heat shock proteins incancer: chaperones of tumorigenesis, Trends Biochem. Sci. 31 (2006) 164–172.

[11] C. Dai, L. Whitesell, A.B. Rogers, S. Lindquist, Heat shock factor 1 is a powerfulmultifaceted modifier of carcinogenesis, Cell 130 (2007) 1005–1018.

[12] J.N. Min, L. Huang, D.B. Zimonjic, D. Moskophidis, N.F. Mivechi, Selective sup-pression of lymphomas by functional loss of Hsf1 in a p53-deficient mousemodel for spontaneous tumors, Oncogene 26 (2007) 5086–5097.

[13] A.S. Sreedhar, E. Kalmar, P. Csermely, Y.F. Shen, Hsp90 isoforms: functions,expression and clinical importance, FEBS Lett. 562 (2004) 11–15.

[14] A.K. Voss, T. Thomas, P. Gruss, Mice lacking HSP90beta fail to develop a placentallabyrinth, Development 127 (2000) 1–11.

[15] L.H. Pearl, C. Prodromou, Structure and mechanism of the Hsp90 molecularchaperone machinery, Annu. Rev. Biochem. 75 (2006) 271–294.

[16] S.K. Wandinger, K. Richter, J. Buchner, The Hsp90 chaperone machinery, J. Biol.Chem. 283 (2008) 18473–18477.

[17] M. Scaltriti, S. Dawood, J. Cortes, Molecular pathways: targeting hsp90—whobenefits and who does not, Clin. Cancer Res. 18 (2012) 4508–4513.

[18] A. Haupt, G. Joberty, M. Bantscheff, H. Frohlich, H. Stehr, M.R. Schweiger, A.Fischer, M. Kerick, S.T. Boerno, A. Dahl, M. Lappe, H. Lehrach, C. Gonzalez, G.Drewes, B.M. Lange, Hsp90 inhibition differentially destabilises MAP kinase andTGF-beta signalling components in cancer cells revealed by kinase-targetedchemoproteomics, BMC Cancer 12 (2012) 38.

[19] M.R. Cardillo, F. Ippoliti, IL-6, IL-10 and HSP-90 expression in tissue microarraysfrom human prostate cancer assessed by computer-assisted image analysis,Anticancer Res. 26 (2006) 3409–3416.

[20] C.D. Chen, D.S. Welsbie, C. Tran, S.H. Baek, R. Chen, R. Vessella, M.G. Rosenfeld,C.L. Sawyers, Molecular determinants of resistance to antiandrogen therapy,Nat. Med. 10 (2004) 33–39.

[21] K.K. Waltering, A. Urbanucci, T. Visakorpi, Androgen receptor (AR) aberrationsin castration-resistant prostate cancer, Mol. Cell. Endocrinol. 360 (2012) 38–43.

[22] S. He, C. Zhang, A.A. Shafi, M. Sequeira, J. Acquaviva, J.C. Friedland, J. Sang, D.L.Smith, N.L. Weigel, Y. Wada, D.A. Proia, Potent activity of the Hsp90 inhibitorganetespib in prostate cancer cells irrespective of androgen receptor status orvariant receptor expression, Int. J. Oncol. 42 (2013) 35–43.

[23] L. Whitesell, E.G. Mimnaugh, B. De Costa, C.E. Myers, L.M. Neckers, Inhibitionof heat shock protein HSP90-pp 60v-src heteroprotein complex formation bybenzoquinone ansamycins: essential role for stress proteins in oncogenic trans-formation, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 8324–8328.

[24] S.V. Sharma, T. Agatsuma, H. Nakano, Targeting of the protein chaperone,HSP90, by the transformation suppressing agent, radicicol, Oncogene 16 (1998)2639–2645.

[25] S.M. Roe, C. Prodromou, R. O'Brien, J.E. Ladbury, P.W. Piper, L.H. Pearl, Structuralbasis for inhibition of the Hsp90 molecular chaperone by the antitumor antibi-otics radicicol and geldanamycin, J. Med. Chem. 42 (1999) 260–266.

[26] C. Esser, S. Alberti, J. Hohfeld, Cooperation of molecular chaperones with theubiquitin/proteasome system, Biochim. Biophys. Acta 1695 (2004) 171–188.

[27] A.J. McClellan, S. Tam, D. Kaganovich, J. Frydman, Protein quality control: chaperonesculling corrupt conformations, Nat. Cell Biol. 7 (2005) 736–741.

[28] P. Connell, C.A. Ballinger, J. Jiang, Y. Wu, L.J. Thompson, J. Hohfeld, C. Patterson,The co-chaperone CHIP regulates protein triage decisions mediated by heat-shockproteins, Nat. Cell Biol. 3 (2001) 93–96.

[29] C.A. Ballinger, P. Connell, Y. Wu, Z. Hu, L.J. Thompson, L.Y. Yin, C. Patterson, Identi-fication of CHIP, a novel tetratricopeptide repeat-containing protein that interactswith heat shock proteins and negatively regulates chaperone functions, Mol. Cell.Biol. 19 (1999) 4535–4545.

[30] J.G. Supko, R.L. Hickman, M.R. Grever, L. Malspeis, Preclinical pharmacologic evalu-ation of geldanamycin as an antitumor agent, Cancer Chemother. Pharmacol. 36(1995) 305–315.

[31] S. Soga, Y. Shiotsu, S. Akinaga, S.V. Sharma, Development of radicicol analogues,Curr. Cancer Drug Targets 3 (2003) 359–369.

[32] D.B. Solit, F.F. Zheng, M. Drobnjak, P.N. Munster, B. Higgins, D. Verbel, G. Heller,W. Tong, C. Cordon-Cardo, D.B. Agus, H.I. Scher, N. Rosen, 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptorand HER-2/neu and inhibits the growth of prostate cancer xenografts, Clin. CancerRes. 8 (2002) 986–993.

[33] C.R. Williams, R. Tabios, W.M. Linehan, L. Neckers, Intratumor injection of theHsp90 inhibitor 17AAG decreases tumor growth and induces apoptosis in aprostate cancer xenograft model, J. Urol. 178 (2007) 1528–1532.

[34] R.K. Ramanathan, D.L. Trump, J.L. Eiseman, C.P. Belani, S.S. Agarwala, E.G.Zuhowski, J. Lan, D.M. Potter, S.P. Ivy, S. Ramalingam, A.M. Brufsky, M.K.Wong, S. Tutchko, M.J. Egorin, Phase I pharmacokinetic-pharmacodynamicstudy of 17-(allylamino)-17-demethoxygeldanamycin (17AAG, NSC 330507), anovel inhibitor of heat shock protein 90, in patients with refractory advancedcancers, Clin. Cancer Res. 11 (2005) 3385–3391.

[35] M.P. Goetz, D. Toft, J. Reid, M. Ames, B. Stensgard, S. Safgren, A.A. Adjei, J. Sloan,P. Atherton, V. Vasile, S. Salazaar, A. Adjei, G. Croghan, C. Erlichman, Phase I trialof 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer,J. Clin. Oncol. 23 (2005) 1078–1087.

[36] U. Banerji, A. O'Donnell, M. Scurr, S. Pacey, S. Stapleton, Y. Asad, L. Simmons, A.Maloney, F. Raynaud, M. Campbell, M. Walton, S. Lakhani, S. Kaye, P. Workman, I.Judson, Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino,17-demethoxygeldanamycin in patients with advanced malignancies, J. Clin.Oncol. 23 (2005) 4152–4161.

[37] J.L. Grem, G. Morrison, X.D. Guo, E. Agnew, C.H. Takimoto, R. Thomas, E. Szabo, L.Grochow, F. Grollman, J.M. Hamilton, L. Neckers, R.H. Wilson, Phase I and pharma-cologic study of 17-(allylamino)-17-demethoxygeldanamycin in adult patientswith solid tumors, J. Clin. Oncol. 23 (2005) 1885–1893.

[38] E.I. Heath, D.W. Hillman, U. Vaishampayan, S. Sheng, F. Sarkar, F. Harper, M. Gaskins,H.C. Pitot, W. Tan, S.P. Ivy, R. Pili, M.A. Carducci, C. Erlichman, G. Liu, A phase II trial of17-allylamino-17-demethoxygeldanamycin in patients with hormone-refractorymetastatic prostate cancer, Clin. Cancer Res. 14 (2008) 7940–7946.

[39] G. Chiosis, E. Caldas Lopes, D. Solit, Heat shock protein-90 inhibitors: a chronicle fromgeldanamycin to today's agents, Curr. Opin. Investig. Drugs 7 (2006) 534–541.

[40] L.R. Kelland, S.Y. Sharp, P.M. Rogers, T.G. Myers, P. Workman, DT-Diaphorase ex-pression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin,an inhibitor of heat shock protein 90, J. Natl. Cancer Inst. 91 (1999) 1940–1949.

[41] M.J. Egorin, D.M. Rosen, J.H. Wolff, P.S. Callery, S.M. Musser, J.L. Eiseman, Metab-olism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murineand human hepatic preparations, Cancer Res. 58 (1998) 2385–2396.

[42] E.A. Sausville, J.E. Tomaszewski, P. Ivy, Clinical development of 17-allylamino,17-demethoxygeldanamycin, Curr. Cancer Drug Targets 3 (2003) 377–383.

[43] M. Hollingshead, M. Alley, A.M. Burger, S. Borgel, C. Pacula-Cox, H.H. Fiebig, E.A.Sausville, In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), awater-soluble geldanamycin deriv-ative, Cancer Chemother. Pharmacol. 56 (2005) 115–125.

[44] S.Y. Sharp, L.R. Kelland, M.R. Valenti, L.A. Brunton, S. Hobbs, P. Workman, Estab-lishment of an isogenic human colon tumor model for NQO1 gene expression:application to investigate the role of DT-diaphorase in bioreductive drug activa-tion in vitro and in vivo, Mol. Pharmacol. 58 (2000) 1146–1155.

[45] S. Pacey, R.H. Wilson, M.I. Walton, M. Eatock, A. Hardcastle, A. Zetterlund, H.T.Arkenau, J. Moreno-Farre, U. Banerji, B. Roels, H. Peachey, W. Aherne, J.S. deBono, F.I. Raynaud, P. Workman, I.R. Judson, A phase I study of the heat shockprotein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patientswith advanced, solid tumors, Clin. Cancer Res. 17 (2011) 1561–1570.

[46] J.R. Sydor, E. Normant, C.S. Pien, J.R. Porter, J. Ge, L. Grenier, R.H. Pak, J.A. Ali, M.S.Dembski, J. Hudak, J. Patterson, C. Penders, M. Pink, M.A. Read, J. Sang, C.Woodward, Y. Zhang, D.S. Grayzel, J. Wright, J.A. Barrett, V.J. Palombella, J.Adams, J.K. Tong, Development of 17-allylamino-17-demethoxygeldanamycinhydroquinone hydrochloride (IPI-504), an anti-cancer agent directed againstHsp90, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 17408–17413.

[47] W.K. Oh, M.D. Galsky, W.M. Stadler, S. Srinivas, F. Chu, G. Bubley, J. Goddard, J.Dunbar, R.W. Ross, Multicenter phase II trial of the heat shock protein 90 inhib-itor, retaspimycin hydrochloride (IPI-504), in patients with castration-resistantprostate cancer, Urology 78 (2011) 626–630.

217M.M. Centenera et al. / Biochimica et Biophysica Acta 1835 (2013) 211–218

[48] T. Taldone, W. Sun, G. Chiosis, Discovery and development of heat shock protein90 inhibitors, Bioorg. Med. Chem. 17 (2009) 2225–2235.

[49] K. Jhaveri, T. Taldone, S. Modi, G. Chiosis, Advances in the clinical development ofheat shock protein 90 (Hsp90) inhibitors in cancers, Biochim. Biophys. Acta 1823(2011) 742–755.

[50] C.T. Chan, R.E. Reeves, R. Geller, S.S. Yaghoubi, A. Hoehne, D.E. Solow-Cordero, G.Chiosis, T.F. Massoud, R. Paulmurugan, S.S. Gambhir, Discovery and validation ofsmall-molecule heat-shock protein 90 inhibitors throughmultimodality molecularimaging in living subjects, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) E2476–2485.

[51] F. Lamoureux, C. Thomas,M.J. Yin, H. Kuruma, L. Fazli, M.E. Gleave, A. Zoubeidi, A novelHSP90 inhibitor delays castrate-resistant prostate cancer without altering serum PSAlevels and inhibits osteoclastogenesis, Clin. Cancer Res. 17 (2011) 2301–2313.

[52] A. Yano, S. Tsutsumi, S. Soga, M.J. Lee, J. Trepel, H. Osada, L. Neckers, Inhibitionof Hsp90 activates osteoclast c-Src signaling and promotes growth of prostatecarcinoma cells in bone, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 15541–15546.

[53] A. Rajan, R.J. Kelly, J.B. Trepel, Y.S. Kim, S.V. Alarcon, S. Kummar, M. Gutierrez, S.Crandon, W.M. Zein, L. Jain, B. Mannargudi, W.D. Figg, B.E. Houk, M. Shnaidman,N. Brega, G. Giaccone, A phase I study of PF-04929113 (SNX-5422), an orally bio-available heat shock protein 90 inhibitor, in patients with refractory solid tumormalignancies and lymphomas, Clin. Cancer Res. 17 (2011) 6831–6839.

[54] S.A. Eccles, A. Massey, F.I. Raynaud, S.Y. Sharp, G. Box, M. Valenti, L. Patterson, A.de Haven Brandon, S. Gowan, F. Boxall, W. Aherne, M. Rowlands, A. Hayes, V.Martins, F. Urban, K. Boxall, C. Prodromou, L. Pearl, K. James, T.P. Matthews,K.M. Cheung, A. Kalusa, K. Jones, E. McDonald, X. Barril, P.A. Brough, J.E.Cansfield, B. Dymock, M.J. Drysdale, H. Finch, R. Howes, R.E. Hubbard, A.Surgenor, P. Webb, M. Wood, L. Wright, P. Workman, NVP-AUY922: a novelheat shock protein 90 inhibitor active against xenograft tumor growth, angio-genesis, and metastasis, Cancer Res. 68 (2008) 2850–2860.

[55] M.M. Centenera, J.L. Gillis, A. Hanson, S. Jindal, R.A. Taylor, G. Risbridger, P.D.Sutherland, H.I. Scher, G.V. Raj, K.E. Knudsen, T. Yeadon, W.D. Tilley, L.M.Butler, Evidence for efficacy of new Hsp90 inhibitors revealed by ex vivo cultureof human prostate tumors, Clin. Cancer Res. 18 (2012) 1–9.

[56] N. Gaspar, S.Y. Sharp, S.A. Eccles, S. Gowan, S. Popov, C. Jones, A. Pearson, G. Vassal,P. Workman, Mechanistic evaluation of the novel HSP90 inhibitor NVP-AUY922 inadult and pediatric glioblastoma, Mol. Cancer Ther. 9 (2010) 1219–1233.

[57] M.R. Jensen, J. Schoepfer, T. Radimerski, A. Massey, C.T. Guy, J. Brueggen, C.Quadt, A. Buckler, R. Cozens, M.J. Drysdale, C. Garcia-Echeverria, P. Chene,NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activityin preclinical breast cancer models, Breast Cancer Res. 10 (2008) R33.

[58] A. Monazzam, P. Razifar, S. Ide, M. Rugaard Jensen, R. Josephsson, C. Blomqvist, B.Langstrom, M. Bergstrom, Evaluation of the Hsp90 inhibitor NVP-AUY922 inmulticellular tumour spheroids with respect to effects on growth and PET traceruptake, Nucl. Med. Biol. 36 (2009) 335–342.

[59] T.A. Samuel, C. Sessa, C. Britten, K.S. Milligan, M.M. Mita, U. Banerji, T.J. Pluard, P.Stiegler, C. Quadt, G. Shapiro, AUY922, a novel HSP90 inhibitor: final results of afirst-in-human study in patients with advanced solid malignancies, ASCO Meet.Abstr. 28 (2010) 2528.

[60] Y. Onozawa, T. Doi, K. Yamazaki, J. Watanabe, N. Fuse, T. Yoshino, M. Akimov, M.Robson, N. Boku, A. Ohtsu, Abstract B98: AUY922, a novel HSP90 inhibitor: finalresults of a phase I dose escalation study in Japanese patients with advancedsolid malignancies, Mol. Cancer Ther. 10 (2011) B98.

[61] J.K. McCleese, M.D. Bear, S.L. Fossey, R.M. Mihalek, K.P. Foley, W. Ying, J.Barsoum, C.A. London, The novel HSP90 inhibitor STA-1474 exhibits biologicactivity against osteosarcoma cell lines, Int. J. Cancer 125 (2009) 2792–2801.

[62] W. Ying, Z. Du, L. Sun, K.P. Foley, D.A. Proia, R.K. Blackman, D. Zhou, T. Inoue, N.Tatsuta, J. Sang, S. Ye, J. Acquaviva, L.S. Ogawa, Y. Wada, J. Barsoum, K. Koya,Ganetespib, a unique triazolone-containing Hsp90 inhibitor, exhibits potentantitumor activity and a superior safety profile for cancer therapy, Mol. CancerTher. 11 (2012) 475–484.

[63] D.A. Proia, K.P. Foley, T. Korbut, J. Sang, D. Smith, R.C. Bates, Y. Liu, A.F.Rosenberg, D. Zhou, K. Koya, J. Barsoum, R.K. Blackman, Multifaceted interven-tion by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activatedJAK/STAT signaling, PLoS One 6 (2011) e18552.

[64] A.J. Woodhead, H. Angove, M.G. Carr, G. Chessari, M. Congreve, J.E. Coyle, J.Cosme, B. Graham, P.J. Day, R. Downham, L. Fazal, R. Feltell, E. Figueroa, M.Frederickson, J. Lewis, R. McMenamin, C.W. Murray, M.A. O'Brien, L. Parra, S.Patel, T. Phillips, D.C. Rees, S. Rich, D.M. Smith, G. Trewartha, M. Vinkovic, B.Williams, A.J. Woolford, Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydrois oindol-2-yl]methanone (AT13387), anovel inhibitor of the molecular chaperone Hsp90 by fragment based drug design,J. Med. Chem. 53 (2010) 5956–5969.

[65] B. Graham, J. Curry, T. Smyth, L. Fazal, R. Feltell, I. Harada, J. Coyle, B. Williams, M.Reule, H. Angove, D.M. Cross, J. Lyons, N.G. Wallis, N.T. Thompson, The heatshock protein 90 inhibitor, AT13387, displays a long duration of action in vitroand in vivo in non-small cell lung cancer, Cancer Sci. 103 (2012) 522–527.

[66] D. Mahadevan, D.M. Rensvold, S.E. Kurtin, J.M. Cleary, L. Gandhi, J.F. Lyons, V. Lock,S. Lewis, G. Shapiro, First-in-human phase I study: results of a second-generationnon-ansamycin heat shock protein 90 (HSP90) inhibitor AT13387 in refractorysolid tumors, ASCO Meet. Abstr. 30 (2012) 3028.

[67] M.G.Marcu, A. Chadli, I. Bouhouche,M. Catelli, L.M.Neckers, Theheat shockprotein 90antagonist novobiocin interacts with a previously unrecognized ATP-binding domainin the carboxyl terminus of the chaperone, J. Biol. Chem. 275 (2000) 37181–37186.

[68] S.B. Matthews, G.A. Vielhauer, C.A. Manthe, V.K. Chaguturu, K. Szabla, R.L. Matts,A.C. Donnelly, B.S. Blagg, J.M. Holzbeierlein, Characterization of a novel novobi-ocin analogue as a putative C-terminal inhibitor of heat shock protein 90 inprostate cancer cells, Prostate 70 (2010) 27–36.

[69] J.D. Eskew, T. Sadikot, P. Morales, A. Duren, I. Dunwiddie, M. Swink, X. Zhang, S.Hembruff, A. Donnelly, R.A. Rajewski, B.S. Blagg, J.R. Manjarrez, R.L. Matts, J.M.Holzbeierlein, G.A. Vielhauer, Development and characterization of a novelC-terminal inhibitor of Hsp90 in androgen dependent and independent prostatecancer cells, BMC Cancer 11 (2011) 468.

[70] B.H. Kang, J. Plescia, T. Dohi, J. Rosa, S.J. Doxsey, D.C. Altieri, Regulation of tumorcell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone net-work, Cell 131 (2007) 257–270.

[71] B.H. Kang, J. Plescia, H.Y. Song, M. Meli, G. Colombo, K. Beebe, B. Scroggins, L.Neckers, D.C. Altieri, Combinatorial drug design targetingmultiple cancer signalingnetworks controlled by mitochondrial Hsp90, J. Clin. Invest. 119 (2009) 454–464.

[72] B.H. Kang, M.D. Siegelin, J. Plescia, C.M. Raskett, D.S. Garlick, T. Dohi, J.B. Lian, G.S.Stein, L.R. Languino, D.C. Altieri, Preclinical characterization of mitochondria-targeted small molecule hsp90 inhibitors, gamitrinibs, in advanced prostatecancer, Clin. Cancer Res. 16 (2010) 4779–4788.

[73] B.H. Kang, M. Tavecchio, H.L. Goel, C.C. Hsieh, D.S. Garlick, C.M. Raskett, J.B. Lian,G.S. Stein, L.R. Languino, D.C. Altieri, Targeted inhibition of mitochondrial Hsp90suppresses localised and metastatic prostate cancer growth in a genetic mousemodel of disease, Br. J. Cancer 104 (2011) 629–634.

[74] S.J. Arlander, A.K. Eapen, B.T. Vroman, R.J. McDonald, D.O. Toft, L.M. Karnitz,Hsp90 inhibition depletes Chk1 and sensitizes tumor cells to replication stress,J. Biol. Chem. 278 (2003) 52572–52577.

[75] J. Hubbard, C. Erlichman, D.O. Toft, R. Qin, B.A. Stensgard, S. Felten, C. TenEyck, G. Batzel, S.P. Ivy, P. Haluska, Phase I study of 17-allylamino-17demethoxygeldanamycin, gemcitabine and/or cisplatin in patients with re-fractory solid tumors, Invest. New Drugs 29 (2011) 473–480.

[76] G. Iyer, M.J. Morris, D. Rathkopf, S.F. Slovin, M. Steers, S.M. Larson, L.H. Schwartz,T. Curley, A. DeLaCruz, Q. Ye, G. Heller, M.J. Egorin, S.P. Ivy, N. Rosen, H.I. Scher,D.B. Solit, A phase I trial of docetaxel and pulse-dose 17-allylamino-17-demethoxygeldanamycin in adult patients with solid tumors, Cancer Chemother.Pharmacol. 69 (2012) 1089–1097.

[77] G.J. Riely, R. Stoller, M. Egorin, D. Solit, J. Dunbar, A. Savage, J.Walker, D. Grayzel, R. Ross,G.J. Weiss, A phase Ib trial of IPI-504 (retaspimycin hydrochloride), a novel Hsp90 in-hibitor, in combination with docetaxel, ASCO Meet. Abstr. 27 (2009) 3547.

[78] J.S. Kauh, R.D. Harvey, T.K. Owonikoko, B.F. El-Rayes, D.M. Shin, S. Murali, C.M. Lewis,M.D. Karol, F. Teofilovici, Y. Du, H. Fu, F.R. Khuri, S.S. Ramalingam, A phase I and phar-macokinetic study of multiple schedules of ganetespib (STA-9090), a heat shock pro-tein 90 inhibitor, in combination with docetaxel for subjects with advanced solidtumor malignancies, ASCO Meet. Abstr. 30 (2012) 3094.

[79] K. Camphausen, P.J. Tofilon, Inhibition of Hsp90: a multitarget approach toradiosensitization, Clin. Cancer Res. 13 (2007) 4326–4330.

[80] E.E. Bull, H. Dote, K.J. Brady, W.E. Burgan, D.J. Carter, M.A. Cerra, K.A. Oswald,M.G. Hollingshead, K. Camphausen, P.J. Tofilon, Enhanced tumor cell radiosen-sitivity and abrogation of G2 and S phase arrest by the Hsp90 inhibitor17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, Clin. Cancer Res.10 (2004) 8077–8084.

[81] R. Enmon, W.H. Yang, A.M. Ballangrud, D.B. Solit, G. Heller, N. Rosen, H.I. Scher,G. Sgouros, Combination treatment with 17-N-allylamino-17-demethoxygeldanamycin and acute irradiation produces supra-additive growth suppressionin human prostate carcinoma spheroids, Cancer Res. 63 (2003) 8393–8399.

[82] H.J. Ochel, G. Gademann, In vitro combined modality treatment of prostatecarcinoma cells with 17-(allylamino)-17-demethoxygeldanamycin and ionizingradiation, Anticancer Res. 26 (2006) 2085–2091.

[83] R. Yu, S. Mandlekar, S. Ruben, J. Ni, A.N. Kong, Tumor necrosis factor-relatedapoptosis-inducing ligand-mediated apoptosis in androgen-independent pros-tate cancer cells, Cancer Res. 60 (2000) 2384–2389.

[84] Y. Ma, V. Lakshmikanthan, R.W. Lewis, M.V. Kumar, Sensitization of TRAIL-resistant cells by inhibition of heat shock protein 90 with low-dose geldanamycin,Mol. Cancer Ther. 5 (2006) 170–178.

[85] V. Georget, B. Terouanne, J.C. Nicolas, C. Sultan, Mechanism of antiandrogenaction: key role of hsp90 in conformational change and transcriptional activityof the androgen receptor, Biochemistry 41 (2002) 11824–11831.

[86] C. Tran, S. Ouk, N.J. Clegg, Y. Chen, P.A. Watson, V. Arora, J. Wongvipat, P.M.Smith-Jones, D. Yoo, A. Kwon, T. Wasielewska, D. Welsbie, C.D. Chen, C.S.Higano, T.M. Beer, D.T. Hung, H.I. Scher, M.E. Jung, C.L. Sawyers, Developmentof a second-generation antiandrogen for treatment of advanced prostate cancer,Science 324 (2009) 787–790.

[87] J. Zou, Y. Guo, T. Guettouche, D.F. Smith, R. Voellmy, Repression of heat shocktranscription factor HSF1 activation by HSP90 (HSP90 complex) that forms astress-sensitive complex with HSF1, Cell 94 (1998) 471–480.

[88] N. Zaarur, V.L. Gabai, J.A. Porco Jr., S. Calderwood, M.Y. Sherman, Targeting heatshock response to sensitize cancer cells to proteasome and Hsp90 inhibitors,Cancer Res. 66 (2006) 1783–1791.

[89] N. Dakappagari, L. Neely, S. Tangri, K. Lundgren, L. Hipolito, A. Estrellado, F.Burrows, H. Zhang, An investigation into the potential use of serum Hsp70 as anovel tumour biomarker for Hsp90 inhibitors, Biomarkers 15 (2010) 31–38.

[90] S. Kummar, M.E. Gutierrez, E.R. Gardner, X. Chen, W.D. Figg, M. Zajac-Kaye, M.Chen, S.M. Steinberg, C.A. Muir, M.A. Yancey, Y.R. Horneffer, L. Juwara, G.Melillo, S.P. Ivy, M. Merino, L. Neckers, P.S. Steeg, B.A. Conley, G. Giaccone,J.H. Doroshow, A.J. Murgo, Phase I trial of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein inhibitor,administered twice weekly in patients with advanced malignancies, Eur.J. Cancer 46 (2010) 340–347.

[91] R.K. Ramanathan, M.J. Egorin, C. Erlichman, S.C. Remick, S.S. Ramalingam, C. Naret,J.L. Holleran, C.J. TenEyck, S.P. Ivy, C.P. Belani, Phase I pharmacokinetic and pharma-codynamic study of 17-dimethylaminoethylamino-17-demethoxygeldanamycin,

218 M.M. Centenera et al. / Biochimica et Biophysica Acta 1835 (2013) 211–218

an inhibitor of heat-shock protein 90, in patients with advanced solid tumors,J. Clin. Oncol. 28 (2010) 1520–1526.

[92] R. Bagatell, G.D. Paine-Murrieta, C.W. Taylor, E.J. Pulcini, S. Akinaga, I.J. Benjamin, L.Whitesell, Induction of a heat shock factor 1-dependent stress response alters thecytotoxic activity of hsp90-binding agents, Clin. Cancer Res. 6 (2000) 3312–3318.

[93] F. Guo, K. Rocha, P. Bali, M. Pranpat, W. Fiskus, S. Boyapalle, S. Kumaraswamy, M.Balasis, B. Greedy, E.S. Armitage, N. Lawrence, K. Bhalla, Abrogation of heat shockprotein 70 induction as a strategy to increase antileukemia activity of heat shockprotein 90 inhibitor 17-allylamino-demethoxy geldanamycin, Cancer Res. 65(2005) 10536–10544.

[94] A.K.McCollum, C.J. Teneyck, B.M. Sauer, D.O. Toft, C. Erlichman,Up-regulation of heatshock protein 27 induces resistance to 17-allylamino-demethoxygeldanamycinthrough a glutathione-mediated mechanism, Cancer Res. 66 (2006) 10967–10975.

[95] M.V. Powers, P.A. Clarke, P. Workman, Dual targeting of HSC70 and HSP72 inhibitsHSP90 function and induces tumor-specific apoptosis, Cancer Cell 14 (2008)250–262.

[96] F. Lamoureux, C. Thomas, M.J. Yin, H. Kuruma, E. Beraldi, L. Fazli, A. Zoubeidi, M.E.Gleave, Clusterin inhibition usingOGX-011 synergistically enhances Hsp90 inhibitoractivity by suppressing the heat shock response in castrate-resistant prostate cancer,Cancer Res. 71 (2011) 5838–5849.

[97] K.L. Jhaveri, N.M. Iyengar, A. Corben, S. Patil, M. Akram, R. Towers, R. Sakr, C.Hudis, T.A. King, N. Rosen, S. Chandarlapaty, S. Modi, Biomarkers that predictsensitivity to heat shock protein 90 inhibitors (HSP90i), ASCO Meet. Abstr. 30(2012) 10618.

[98] S. Modi, A. Stopeck, H. Linden, D. Solit, S. Chandarlapaty, N. Rosen, G. D'Andrea,M. Dickler, M.E. Moynahan, S. Sugarman, W. Ma, S. Patil, L. Norton, A.L. Hannah,C. Hudis, HSP90 inhibition is effective in breast cancer: a phase II trial oftanespimycin (17-AAG) plus trastuzumab in patients with HER2-positive meta-static breast cancer progressing on trastuzumab, Clin. Cancer Res. 17 (2011)5132–5139.

[99] L.V. Sequist, S. Gettinger, N.N. Senzer, R.G. Martins, P.A. Janne, R. Lilenbaum, J.E.Gray, A.J. Iafrate, R. Katayama, N. Hafeez, J. Sweeney, J.R. Walker, C. Fritz, R.W.Ross, D. Grayzel, J.A. Engelman, D.R. Borger, G. Paez, R. Natale, Activity ofIPI-504, a novel heat-shock protein 90 inhibitor, in patients with molecularlydefined non-small-cell lung cancer, J. Clin. Oncol. 28 (2010) 4953–4960.

[100] K. Wong, M. Koczywas, J.W. Goldman, E.H. Paschold, L. Horn, J.M. Lufkin, R.K.Blackman, F. Teofilovici, G. Shapiro, M.A. Socinski, An open-label phase II studyof the Hsp90 inhibitor ganetespib (STA-9090) as monotherapy in patientswith advanced non-small cell lung cancer (NSCLC), ASCO Meet. Abstr. 29(2011) 7500.