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The effect of chaperone/co-chaperone overexpression on steroid receptor activity

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The effect of chaperone/co-chaperone overexpression on steroid receptor activity

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Content THEEFFECT OFCHAPERONE/CO-CHAPERONE OVEREXPRESSION ON
STEROIDRECEPTOR ACTIVITY
by
MaitreyeeKiranJathal
ADissertationPresented tothe
FACULTYOF THEGRADUATESCHOOL
UNIVERSITYOF SOUTHERNCALIFORNIA
InPartialFulfillmentofthe
RequirementsfortheDegree
MASTEROFSCIENCE
(MOLECULARPHARMACOLOGY)
August 2009
Copyright 2009 MaitreyeeKiranJathal
TableofContents
Abstract iv
Chapter1: Introduction 1
Chapter2: MolecularChaperones 3
2.1 HSP70,HSP90andHSF1: majorplayersinthestressresponse . . . . . 3
2.2 HSP70andHSP90: structures,functionsandco-chaperones . . . . . . . 4
Table2.1: ComponentsoftheHSP90/HSP70chaperonemachinery . . . 5
2.3 HSP90: Chaperoningsteroidhormoneaction . . . . . . . . . . . . . . 7
Chapter3: HSP90: thecancerchaperone 9
Chapter4: IntersectionofMolecularChaperonesandSteroidReceptors-I:
Stress activatesSRs 12
4.1 SteroidReceptors: a briefstructuraldescription . . . . . . . . . . . . . 12
4.2 IntersectionofMolecularChaperonesandSRs . . . . . . . . . . . . . . 14
4.3 StressactivatesSRs -experimentalproof . . . . . . . . . . . . . . . . . 14
Chapter5: IntersectionofMolecularChaperones andSteroidReceptors -II 20
5.1 Enhancedsteroidreceptorsignaling-hominginonHSP90 . . . . . . . 20
5.2 Chaperone levels influence steroid receptor activity - experimental evi-
dence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
5.3 HSP90co-chaperones: equalpartnersinalteringsteroidreceptoractivity 26
Chapter6: HSP90: ageneticcapacitor 28
Chapter7: Future perspectives andconcluding remarks 30
Bibliography 34
ii
ListofFigures
2.1 ThechaperoningcycleofHSP90 [WL05] . . . . . . . . . . . . . . . . 8
2.2 InhibitingtheHSP90 chaperone[WL05] . . . . . . . . . . . . . . . . . 8
4.1 Structural organization of steroid receptors; top - schematic 1D amino
acid sequence of a nuclear receptor, bottom- 3D structures of the DBD
(bound to DNA) and LBD (bound to hormone) regions of the nuclear
receptor. The structures shown are of the estrogen receptor. Structures
of N-terminal domain (A/B), hinge region (D), and C-terminal domain
(F)arerepresentedbyred,purple,andorangedashedlinesrespectively.
Source: http://en.wikipedia.org/wiki/Nuclear hormone receptor . . . . . 13
iii
Abstract
Cellular stress is present during tumour development. Stressed cells in most tissues
increasetheproductionof‘molecularchaperones’-proteinsthatmaintaintheconforma-
tionand functionof client substrates. Upregulatedchaperone levelsconstitutean adap-
tive response that enhances cell survival. Tumour cells display constitutively-elevated
levels of molecular chaperones, probably reflecting efforts at maintaining homeostasis
inahostileenvironment. ThemolecularchaperoneHSP90isunique-itsproteinclients
are primarily involved in cellular proliferation. Nuclear receptors (NR) are signaling
molecules and HSP90 clients whose aberrant activity is widely implicated in oncogen-
esis. It iscurrently unknownif stress-inducedchaperone overexpressioninfluences NR
activity and potentially cancer progression. It is also possible that stress alters NR lig-
and specificity. These agonists, antagonists or ‘selective nuclear receptor modulators’
modulate NR activity in cancer chemotherapy. Understanding chaperone influence on
NRsignalingshouldfacilitatethedesignofpatient-specifictreatmentstrategies.
iv
Chapter1
Introduction
Cellular stress is a prominent feature in various stages of tumour development. This
stress may be the result of pathophysiological features of the tumour environment e.g.
acidosis, glucose-deprivation and hypoxia. It is well-known that following exposure
to proteoxic stress (including heat, heavy metals, hypoxia and acidosis), cells in most
tissues dramatically increase the production of a small group of proteins collectively
knownas‘stressproteins’or‘heat-shockproteins’(HSPs)or‘molecularchaperones’.
Molecular chaperones are required to promote and maintain the conformation, and
therefore the function, of their client proteins. Their basal levels guard the proteome
from the dangers of misfoldingand aggregation. Increased chaperone levelsare part of
anadaptiveresponsethatenhancescellsurvival. Thereforeitisunsurprisingthattumour
cells display constitutively-elevated levels of molecular chaperones. This increased
chaperone expressionprobablyreflects theeffortsofmalignantcellstomaintainhome-
ostasis in a hostile environment. However, in addition to facilitating this survival, evi-
dencealsoindicatesthatchaperoneproteinsallowtumourcellstowithstandintracellular
alterations. Mutationsincritical signalingmoleculesthat wouldotherwisebe lethalare
1
notonlytoleratedbutservetoactivelypromoteoncogenesis. Inthisregard,themolecu-
larchaperoneHSP90isuniquebecauseitsclientsareprimarilysignalingmoleculesthat
are intimatelyinvolvedincellulargrowthandproliferationpathways.
One major class of signal transduction molecules is the family of nuclear receptors
(NR), whose aberrant activityis widely implicatedin oncogenesis. Some examplesare
the androgen, estrogen, glucocorticoid and progesterone receptors (AR, ER, GR and
PR respectively). It is currently unknown, however, whether stress-induced chaperone
(or co-chaperone) overexpression influences NR activity hence potentially cancer pro-
gression. Some data documenting GR, PR and AR activation by stress is available but
little such evidence for the ER or other NRs exists. Furthermore the molecular mech-
anism(s) by which chaperone overexpression influences NR behaviour remains largely
unknown. Inaddition,onemayspeculatethatstressaltersthespecificityofanNRforits
ligands. These molecules are tissue-specific agonists, antagonists or ‘selective nuclear
receptormodulators’. ManyoftheseligandsareusedtomodulateNRactivityincancer
chemotherapy. The AR and ER are of special concern considering the widespread use
of their agonists and antagonists in oncotherapy. For example, tamoxifen and ralox-
ifeneareusedagainsttheERinthetreatmentofbreastcancer. Dihydroxyflutamideand
finasteride are used to inhibit AR signaling in prostate cancer chemotherapy. Develop-
ing our understandingof the complexitiesof chaperone influence on NR signaling will
eventuallyfacilitatethedesignofpatient-specifictreatmentstrategies.
2
Chapter2
MolecularChaperones
Molecular chaperones facilitate the folding of proteins into a stable and functional
conformation. These chaperones are present in unstressed cells and play a criti-
cal role in normal protein homeostasis. They assist in assembly and disassembly of
protein complexes, inhibit improper protein aggregation (due to crowding or thermal
denaturation) and direct newly formed proteins to target organelles for final packag-
ing [Kre02]. The presence of stressful stimuli (e.g. hyperthermia [OKR02], hypoxia
[ICD
+
93], ATP depletion [MVB
+
99, NKB
+
01], free radicals [BM98, BRG
+
99],
hypothermia [FH99, PDP
+
07], desiccation [JS07], viruses [JM00, SP01], ethanol
[OKG
+
05,TB97,WSG
+
00]inducesanincreaseinchaperoneproductionwhichserves
toprotectthe cellsintheface ofadverseenvironmentalconditions. In thecase of acute
misfolding and aggregation caused by the stressors, irreversibly-harmed proteins may
betargetedtotheproteasomefordegradation[Hay96,MMM98,WL05].
2.1 HSP70,HSP90andHSF1: majorplayersinthestressresponse
The most prominent molecular chaperones are the ‘heat shock proteins’ (HSPs/hsps)
HSP70,HSP90andHSP27. Theseproteinsareabundantlyexpressedevenundernormal
3
cellularconditions[WL05]. Forexample,HSP70andHSP90isoformseachcomprise1-
2% of totalcellularprotein content[WL05]. The inducible,elevatedsynthesisof stress
proteinsisfacilitateddirectlybytrimerizationoflatentheatshockfactors(HSFs)ontar-
getedgenes. UnstressedcellshavealowbackgroundlevelofHSF1,whosetranscribing
activity is
˜
5% that of severely heat-stressed cells [Dil06]. Impositionof stress causes a
rapidreprogrammingofgeneexpressionmechanismssuchthatHSPsynthesisisgreatly
increased. Prior to stress, HSF1 is transcriptionally inactive because it is bound to an
HSP70/HSP90-containing ‘multichaperone’ complex (see below, also see [GGF
+
01]).
StresscausestheaccrualofdenaturedproteinsthatcompetewithHSF1forHSP90mul-
tichaperone complexes, resulting in unbindingand homotrimerizationof HSF1. Multi-
pleHSFs(denotedHSF1-4)provideredundancyoffunctionandspecializationofstress
control signals [Mor98]. HSF1 is the primary transcription factor required for expres-
sionofHSPsunderallconditions[BDV93,TMH
+
04]. HSF2AfunctionswithHSF1in
acoupledprocessundertheactionofexternally-appliedstress[HSG
+
03,SSM94].
2.2 HSP70andHSP90: structures,functions andco-chaperones
HSP70 and HSP90 do not function alone. Instead, they typically function as compo-
nentsof larger machines- ‘multichaperonecomplexes’- thatcontainother chaperones,
co-chaperones, modulators of ATPase activity and various accessory proteins (TABLE
1,[WL05]). Boththesechaperonescontainintramolecularinteractingdomainsthatcou-
ple ATP binding/hydrolysisto substrate binding/folding. HSP27 does not contain such
anATPaseregionbutinconcertwithHSP70can returnunfoldedproteinstotheirATP-
dependent refolding and re-activation conformation. The C-terminal ends of cytosolic
HSP70 and HSP90 isoforms possess the amino acid sequence EEVD, which serves as
4
the recognition motif for tetratricopeptide (TPR) domains found in a family of TPR-
containing co-chaperone proteins eg. HOP, FKBP51/52. These proteins serve as a
molecular‘bridge’linkingtheHSP70andHSP90multichaperonecomplexes.
HSP70 influences the productive folding of proteins to their native states and thus
preventstheirmisfoldingandaggregationbybindingtoexposedstretchesofhydropho-
bic amino acids in the substrate (or ‘client’) protein [MM04]. This folding machine
carries outcycles of substratebindingand release that are accelerated by ATP hydroly-
sisandmodulatedbyassociationwithregulatoryco-chaperones(HSP40,HIPandHOP,
seeTABLE1)thatinteractwithHSP70 andregulateitsATPaseactivity. Thesubstrate-
bindingdomainofHSP70 islocalizedtoa25kDaC-terminaldomain(CTD) withsub-
strate access controlled by a C-terminal lid that exposes the peptide-binding domain in
theATP-boundformandallowssubstratebindingtooccur whenHSP70isintheADP-
bound form. Opening and closing of the lid is governed by conformational changes
associated with ATP binding and hydrolysis that occur within the cleft of a 45 kDa N-
terminal bi-lobed region. Nucleotide exchange, which results in ATP binding in the
place of ADP, causes substrate release, thus allowing HSP70 to enter a new round of
substratebindingandrelease.
HSP90residesprimarilyinthecytoplasm,whereitexistspredominantlyasahomo-
dimer. There are two major cytoplasmic forms, HSP90α (the inducible isoforms)
and HSP90β (constitutive form) [SKC
+
04]. A third isoform, HSP90N was recently
discovered and found to be associated with cellular transformation [GVR
+
02]. This
75 kDa isoform lacks the 223-amino acid-long NTD, and is thereby bereft of the
nucleotide/inhibitorbindingsite. Instead,itpossessesashort,30-amino-acidhydropho-
bic sequence preceded by a putative myristylation signal. It has been identified as a
novel component of the RAF kinase signalosome, associates with that kinase and tar-
gets it to the plasma membrane for activation [GVR
+
02], thus substitutingRas kinase.
5
Table2.1: ComponentsoftheHSP90/HSP70chaperone machinery[WL05]
AdditionalHSP90analoguesincludeGRP94intheendoplasmicreticulumand TRAP1
inthemitochondrialmatrix[CSt
+
98,SKC
+
04]
HSP90isoformsconsistofthreemaindomainsthathaveimportantfunctionalinter-
actions. Heterodimers (αβ) and higher oligomers of both isoforms are also known to
exist [SKC
+
04]. HSP90 possess a 25 kDa N-terminal domain (NTD) characterized by
a‘Bergeratfold’[WL05]. ThisstructuralmotifbelongstotheGHKL(bacterialGyrase,
HSP90, histidine Kinase, MutL) superfamily (but has no similarity to the ATP-binding
domains found in other kinases or HSP70). The Bergerat fold also houses the binding
sitefortheadeninenucleotidesADPandATP.NucleotideexchangeandATPhydrolysis
(byHSP90itself,withtheassistanceofco-chaperones)driveHSP90tobind,chaperone
and release client proteins. The NTD is also the bindingsite for HSP90 inhibitorssuch
as geldanamycin (GA) and radicicol (RD) [SWN
+
95, WMC
+
94]. It has been found
that HSP90’s chaperoning function is critically dependent upon its ability to bind and
hydrolyse ATP [PPR
+
98]. The 12-kDa C-terminal domain (CTD) contains additional
6
ATP-binding sites (inhibitedby novobiocin),co-chaperone binding sites (characterized
by‘TPR’ domains)andisalsoinvolvedinHSP90 dimerization. Themiddlesegmentis
implicatedasamajorsiteforclientproteininteractionsandcontributesakeyconserved
arginineresidue(Arg380)thatisessentialforHSP90’sATPaseactivity[PP06].
2.3 HSP90: Chaperoning steroidhormoneaction
Chaperone‘cycling’isdrivenbymultipleroundsofATPhydrolysisresultinginiterative
rounds of client binding and release [MM04]. This process has been detailed by the
Smith laboratory [RCC
+
04, SWN
+
95] and will be described for the estrogen receptor
(ER),anHSP90 client.
NewlysynthesizedERassociateswithHSP70,HSP40andtheadapterHIP(HSP70-
interacting protein) to form an early complex (part a, below). The ER’s hydrophobic
hormone-binding domain is partially exposed in this complex and HSP90 binds to this
region in association with the co-chaperone HOP (HSP70/HSP90-organizing protein)
and displaces HSP40 to form an intermediate complex. In an ATP-dependent manner,
HSP90 fully exposes ER’s hormone-binding domain, and the co-chaperone p23 stabi-
lizes the ATP-bound HSP90. Cyclophilin 40 (CYP40) fills an open tetratricopeptide
repeat(TPR) acceptor siteonHSP90 tocompleteamaturecomplex.
In the absence of ligand, the ER is released from the mature complex to undergo
additionalcycles of chaperone interactions. Estrogen binding, however, leads to a con-
formational change in the ER, which releases chaperone componentsand leads to tight
binding of the receptor protein to estrogen response elements (EREs) and the recruit-
mentofco-activatorsneededtodriveER-mediatedtranscription[WL05].
The binding of an HSP90 inhibitor (eg. geldanamycin) ‘locks’ the chaperone in
an alternative conformation that prevents normal cycling and the formation of mature
chaperone complexes. The ER accumulates in an intermediate complex that recruits
7
Figure2.1: ThechaperoningcycleofHSP90 [WL05]
Figure2.2: InhibitingtheHSP90chaperone [WL05]
E3ubiquitinligaseanddrivesproteasome-mediateddegradationof theprotein,thereby
dramaticallyloweringcellularlevelsofthereceptoranddisruptingitsfunction.
8
Chapter3
HSP90: thecancerchaperone
HSP90 is a unique chaperone because the majority of its substrates are potential onco-
proteins. Chiefamongthemareproteinkinases(eg. ERBB2,C-RAF,B-RAF,AKTand
BCR-ABL) and nuclear receptors (NRs, eg. ER, AR, PR and GR) that are intimately
involved in cellular proliferation and survival pathways [MM04, ST08, WL05]. Inap-
propriateactivationofthesesignalingpathways-viaaberrantexpressionormalfunction
of particular components - is known to occur during acute or chronic stress, for exam-
ple, in the hostilehypoxic, acidic, nutrient-deprivedconditionsthat characterize malig-
nant tumour development. Therefore, it is unsurprisingthat many human cancers, both
solidtumoursand haematologicalmalignancies,displayconstitutivelyelevatedchaper-
onelevels[MM04,WL05].
At a physiological level, the increased abundance of chaperones/co-chaperones in
advanced cancers is one reflection of a cytoprotective stress response to the harsh
tumour microenvironment[Han97, Jaa99]. Unfortunately, in many types of cancer this
overexpression portends a poor prognosis in terms of response to therapy and rate of
survival [CC05]. For instance in breast cancer, HSP90α overabundance [JSC
+
92] is
detected in Stage IV tumours; these tumours represent the clinically most advanced
9
and aggressive grade of malignancies and are characterized by the presence of dis-
tant metastases [MKP
+
00]. A recently-conducted large scale study assessed quantita-
tivelythe relationshipof high HSP90 levelsin clinicalbreast cancer settingsand found
thatHSP90 overexpressionwasstronglyassociatedwithdecreased survival[PKG
+
07].
Elevated expression of HSPs 27, 70 and 90 (either individually or in combination)
has also been widely reported in uterine, renal and endometrial cancers, as well as
in osteosarcoma. Increased HSP90β transcription has been shown in patients suffer-
ing from systemic lupus erythematosus [TDM
+
93] while protein overexpression of
that isoform has been detected in chronic pancreatic tumours [ONT
+
00]. HSP90β
expression is also implicated in multidrug resistance (MDR) through its interaction
with P-glycoprotein, a key player in that phenomenon [BPH
+
96]. The overabun-
dance of HSP90 proteins in biopsy samples is considered a poor indicator of therapeu-
tic outcome [CCT
+
93, HZR00, Jaa99, JM00, NKM
+
98, TGH
+
00]. Elevated expres-
sion of HSP90N in acute and chronic pancreatic carcinoma has also been reported
[GVR
+
02, ZUB08, SGH
+
98]. There is also data suggesting that the overexpression
of the co-chaperone Cdc37 may promote inappropriate cell proliferation and constitute
animportantearlystepinthedevelopmentofhumanprostatecancer [SYD
+
00].
At the molecular level, increased chaperone/co-chaperone activities might allow
tumour cells to cope with imbalanced signaling and thereby escape the apoptotic death
that would otherwise ensue [WL05]. Supporting this notion is the direct experimental
observation that HSP90 obtained from tumour cells was found to possess much higher
ATPaseactivitythannormalcellderivedHSP90[KBB04,KTS
+
03]. Further,thisover-
active HSP90 was present entirely in multichaperone complexes [KTS
+
03]. It is thus
reasonable to assume that tumour progression may be linked to HSP90 ATPase activ-
ity. As the cancer progresses towards malignancy and demand for HSP90 chaperoning
10
functionexceedssupply,thecompleteusageoftumourHSP90mightprovideaselection
pressureleadingtotheupregulationofthechaperone.
As tumour cells gradually accumulate mutant and/or overexpressed signaling
molecules,HSP90mayengageitselfintheactivechaperoningandstabilizationofthese
oncoproteins. It adopts a high-activity form that is further characterized (and possi-
bly aided) by bound co-chaperone proteins. It is plausible that these mutant clients are
also conformationallylabile and possessgreater dependence on the chaperone machin-
ery for their structural stability. Consequent chaperone overexpression may therefore
represent an adaptive response that allows the tumour cells to configure signal trans-
duction pathways in order to permit unrestrained proliferation [MM04]. A vicious
cycle is thus set up. The enhanced dependence of mutant HSP90 kinase clients upon
the HSP90 chaperone complex (as compared to the same for wild-type clients) has
beenevidencedfortheproteinp53[BTB
+
96,SPD
+
96,WSP
+
98],tyrosinekinasev-src
[WMC
+
94,XL93,XSL99]andmorerecentlythereceptortyrosinekinaseErbB2/HER-
2[XYX
+
05].
The ability of HSP90 to stabilize such altered molecules, as one part of the cancer-
specific stress response, is a powerful and dangerous property that has been studied
extensively in the context of HSP90-client kinase behaviour. Less is known, how-
ever, about the nature of this stress response against the backdrop of HSP90’s interac-
tions with its other important client substrates - the nuclear steroid hormone receptors.
(Unlessspecificallymentioned,theterms‘nuclearreceptor’(NR)and‘steroidreceptor’
(SR) willbe used interchangeably). The remainder of thisarticle will describe our cur-
rent knowledgeof the interdependentrelationshipbetweenHSP90 and itsSRclientsin
anabnormalenvironment.
11
Chapter4
IntersectionofMolecularChaperonesandSteroid
Receptors-I:StressactivatesSRs
4.1 SteroidReceptors: abrief structuraldescription
The steroid receptors (SRs) share common structural features. All contain a separate
ligand-bindingdomain(LBD)and‘activationfunction2’(AF2)intheirCTDs. TheAF2
is a site for coactivator binding and is thus important for transcriptional activity. The
LBD is linked to the DNA-binding domain (DBD) by a hinge region (H) that contains
a nuclear localization signal (NLS). The DBDs are the most highly-conserved regions
of the receptors and each contain two zinc-finger binding motifs. The NTDs are the
least-conserved regions in both sequence and length but all contain at least one region
termed‘activationfunction1’(AF1)thatisrequiredforoptimaltranscriptionalactivity.
There are six SRs in higher vertebrates: androgen, estrogen, glucocorticoid, min-
eralocorticoid and progesterone receptors (AR, ERα, ERβ, GR, MR and PR, respec-
tively). All are associated with HSP90 in the absence of their cognate ligands. For
someSRs,itisthoughtthattheformationoftheSR-HSP90complexstabilizestheLBD
so that it remains in a high-affinity form, ready to bind ligand when required. For all
SRs this chaperone heterocomplex is thought to keep the SR transcriptionally inactive
12
Figure 4.1: Structural organization of steroid receptors; top - schematic 1D amino acid
sequenceofanuclearreceptor,bottom-3DstructuresoftheDBD(boundtoDNA)and
LBD (bound to hormone) regions of the nuclear receptor. The structures shown are of
theestrogenreceptor. Structuresof N-terminaldomain(A/B), hingeregion(D), andC-
terminaldomain(F)arerepresentedbyred,purple,andorangedashedlinesrespectively.
Source: http://en.wikipedia.org/wiki/Nuclear hormone receptor
in the absence of its ligand. As explained earlier, SR association with HSP90 occurs
inan ordered,step-wisefashionandisnecessary forthemaintenanceoftheunliganded
receptorinastatereadytobindandrespondtohormone[BHC
+
07]. Thesteroidrecep-
tors have themselves been described in great detail under normal cellular conditions.
The nature of their behaviour in an environment ravaged by stress or, in other words,
one that harbours an increased level of molecular chaperones is also an area worthy of
inquiry.
13
4.2 IntersectionofMolecularChaperonesandSRs
The earliest evidence for potential cross-talk involving stress proteins and NRs arose
a couple of decades ago when HSP90 was retrieved with GR within untransformed
(ie. hormone-free) GR complexes [CBJ
+
85, MBG
+
86, STS
+
85, SYB
+
85]. This dis-
covery quickly led to investigations into the possible convergence of steroid and stress
responses. Early attempts in answering this question were made by testing the effects
of steroid ligandson levels of hsps [HGS96]. There are reports of the induction of low
molecular-weighthspsinDrosophilacellsthat were treated withtheinsect steroidhor-
moneecdysterone[BW83,IB82]. Steroidsalsoinducetheproductionofhigh-molecular
weight hsps (HSP90) in rodent bladder [SHP01] and neuronal cells [LRC
+
02]. Estra-
diol and its metabolites upregulate the levels of chaperones in human breast cancer
cells [CBD
+
00, KLK
+
05]. The AR agonists dihydrotestosterone (DHT) and dehy-
droepiandrosterone (DHEA) have been shown to increase chaperone and co-chaperone
levels in human and murine prostate cancer cells [DMT
+
06, LBB
+
06]. In all cases,
it has been postulated that steroid-induced chaperone overexpression contributes to
tumorigenesis.
4.3 StressactivatesSRs-experimentalproof
Thereissomedatatoshowthatavarietyofstressconditions-heat,chemicals,mechan-
ical strain and hypoxia - is able to activate SRs [EEF
+
92, HGS96, JSC
+
01, KLK
+
09,
MFB
+
05,MSK
+
03,San92,SXL
+
93,WGT84,ZCJ
+
00]. Inmostcases,theapplication
of stress dramatically increased the transcriptional activity of a particular NR as mea-
sured by a reporter plasmid (the bacterial enzyme chloramphenicol acetyl transferase
(CAT) or firefly luciferase) under the hormonal control of a promoter containing the
appropriate hormone response element (HRE) sequence [EEF
+
92, HGS96, KLK
+
09,
14
MFB
+
05, MSK
+
03, SXL
+
93, ZCJ
+
00]. In two instances, the indicator of NR activ-
ity was transcription rate of a specific NR target gene [WGT84] or the phosphory-
lation status of a downstream effector kinase [JSC
+
01]. All experiments were per-
formedin-vitro,mostusingimmortalized,stably-transfectedmammaliancelllines. The
different NRs studied were the AR [KLK
+
09], ER [JSC
+
01, WGT84, ZCJ
+
00], GR
[HGS96, MFB
+
05, MSK
+
03, SXL
+
93] and PR [EEF
+
92]. The salient features and
certainlimitationsofeach studywillnowbedescribedinchronologicalorder.
Wolffe et al [WGT84] investigated the effect of heat-shock on ER in primary cul-
turesofmaleXenopuslaevishepatocytesasafunctionofestrogen-inducedvitellogenin
(VTG)geneexpression. Whilethephysiologicalrelevanceofthecellsystemisunclear
(anNRfoundprimarilyinfemaleswasstudiedinmaleamphibianlivercells),thestudies
by Wolffe and colleagues offered the first evidence that stress could be used to manip-
ulate intracellular levels of SR and the transcription and stability of an SR-induced,
developmentalgenemRNA.
Coincident with hsp production, different regimens of heat stress abolished the
estrogen-activated transcription and accumulation of VTG mRNA. Concomitant with
hsp production, heat-shock caused the destabilization of VTG mRNA accumulated by
prior treatment with estrogen. Estrogen treatment prior to heat-shock allowed for the
immediate resumption of VTG gene transcription on return to normal temperatures.
Heat-shock applied to hormonally-naive (ie untreated) male hepatocytes delayed VTG
expression after removal of stress. In other words, hormone pre-treated cells recovered
much faster from heat-shock (recovery being assessed by VTG mRNA transcription
rate) than did hormone un-treated (and heat-shocked) cells. The presence of hormone
during heat-shock failed to alleviate this paralysed response. Estrogen pre-treatment
alsoseemed toprotectERproteinfromheat-shockinactivationby allowing75% ofthe
15
receptorstoberetainedinthenucleus. Wolffeetalestablishedatight,quantitativecor-
relation between the accumulation of nuclear ER and the absolute rate of transcription
of VTG in estrogen-treated male Xenopus hepatocytes. While heat-shock resulted in a
total loss in ER hormone-binding activity, return of hormonal responsiveness in these
cells was found to be associated with recovery of nuclear ER protein levels. This cor-
roborates the concept that steroidal regulation of gene transcription may be dependent
upontheaccumulationofhormone-occupiedSRinthenucleus.
Edwards et al [EEF
+
92] studied the effect of stress on the transcriptional activity
of a CAT reporter gene regulated by a promoter that contained progesterone response
elements (PREs). They used heat-shock for most of their experiments but performed
one reporter assay using sodium arsenite as a form of chemical stress. Edwards and
co-investigators noted a direct, temporal relationship between stress-enhanced reporter
expression and hsp synthesis. They observed that heat-shock treatment substantially
enhanced hormone-dependent reporter activity but that sodium arsenite produced an
even greater reporter response. (Sodium arsenite also caused greater upregulation of
HSP levels than did heat-shock). Heat-shock treatment in the absence of hormone did
notstimulateCATgeneexpressionnordiditaffecttranscriptionfromacontrolreporter,
suggesting that enhanced receptor activity was due to an effect on PR-mediated pro-
cesses and not to a general effect on transcription. Stress-enhanced, PR-mediated tran-
scriptionwasalsounchangedatthePR-PRE(iereceptor-DNA)bindinglevel,regardless
ofthepresenceofhormone.
Sanchez and groups [San92, SXL
+
93] presented much information on the intracel-
lular movement and transcriptional activity of GR in stressed cells. It must be noted
though that some of their results [SXL
+
93] are confounded by the presence of high
(and therefore physiologically irrelevant) levels of GR expression in their model sys-
tem: 15-to30-foldgreaterthanwild-typeGRlevels. Followingheat-shock,unliganded
16
GR in GR-overexpressing cells traveled from the cytoplasm to the nucleus and consis-
tently displayed a small but reproducible activation of GR transcription in the absence
of hormone. (These same cells produced a dramatic increase in GR transcriptionwhen
hormone was added after heat-shock treatment). This hormone-free, stress-enhanced
GR-mediatedtranscriptionrequired thepresence of a functionalreceptor DNA-binding
domain (DBD) because cells containing DNA-binding deficient GR failed to perform
eitheraction. TheseexperimentssuggestedthatstressedNRsmightlocalizetothesame
high-affinity nuclear acceptor sites that are characteristic of the hormone-bound and
transcriptionally-active receptor. This would offer NRs a second, ligand-independent
route towards activation. Further studies [HGS96, San94] dealt extensively with the
kinetic relationship between heat stress and GR transcriptional activity. The behaviour
ofaGR-responsiveCATreporterwasanalysedinmurinefibroblastcellsthatweresub-
jected to elevated temperatures - a large increase in CAT expression was seen. They
termed the dramatic increase in stress-induced NR activity a ‘heat-shock potentiation
effect’. Hu suggested that the well-defined ‘peaks’ of potentiation seen during recov-
ery periodsmayhave beenduetothe synthesisor activationof aheat-inducibleprotein
factor. They did not explicitly say that a strong contender might be one or more of the
HSPs.
Zaman et al [ZCJ
+
00] showed that mechanical strain activated an ER-responsive
vector in transiently-transfected rodent fibroblasts. They used the classical ER antago-
nistICI184780toverifythatthisstress-increasedreporteractivitywasdueindeedtoER
signaling-additionoftheantagonistblockedthestrain-relatedenhancementofreporter
activity and cell proliferation. Jessop and co-workers [JSC
+
01] separately measured
ER(Ser122)andkinase(ERK-1)phosphorylationinresponsetomechanicalstressand
demonstrated that ER utilised a kinase-mediated signaling pathway to achieve target
geneexpression.
17
Recent studies by Mitsiou et al added to the findings of previous researchers
[MFB
+
05, MSK
+
03]. They too demonstrated enhanced GR-regulated reporter activ-
ity following heat-shock and hormone addition. They also observed a positive corre-
lation between the concentration of active GR protein (i.e. receptor able to bind hor-
mone) and the amount of HSP70 rendered insoluble by heat-shock. They noted that
stressed GR possessed fewer hormone-binding sites than the unstressed receptor did
but that the affinity of those sites remained unaffected by the stress treatment. Mit-
siou and co-workers did not attempt to define a direct relationship between hsp levels
andGR-drivenreporter activity. Later, theyreportedthefindingthatheat-stressapplied
to previouslyheat-conditionedcells resulted in evengreater GR-mediated transcription
enhancement. They suggested that CAT mRNA stabilisation as well as stimulation of
GR-mediated transcription accounted for this increase. They speculated that mRNA
stabilisationmighthave been due to the inhibition(in expressionor activity)of a labile
nuclease that was required for mRNA turnover. A different interpretation of ‘stimula-
tion of GR-mediated transcription’ is that a greater number of activated receptors bind
theirrespectiveDNApromoterelementsperunittime. Thismaybestudiedviaanelec-
trophoretic mobility shift assay (EMSA) where stressed (or control) immune-isolated
receptors are incubated with a short oligonucleotide containing the sequence of their
promoterelementbefore beinganalysedonanagarosegel.
Most recently, Khandrika and group [KLK
+
09] showed that the AR could be acti-
vated by stress in a ligand-independent manner. For three weeks, human prostate can-
cer cells were subjected to continuous cycles of hypoxic stress followed by recovery.
The activity of a luciferase reporter enzyme driven by an AR-target-gene-specific pro-
moter was increased 6 fold in stressed as compared to control cells while cellular AR
protein levels were unchanged. These cells also possessed increased lifespans, higher
clonogenicity and enhanced invasiveness when compared to control cells. Separately,
18
intensity of stress (ie 12h hypoxia vs. 4h hypoxia) was also positively correlated with
increased AR transcriptional activity. Khandrika and co-workers then measured the
phosphorylationstatusofakinase(MAPK)knowntobedownstreamofAR.Theyfound
that enzyme modification too was proportional to the intensity of stress. (It must be
remembered that MAPK is a stress-activated kinase and so serves as general marker
of internal stress rather than a phenomenon attributed solely to AR activation). Their
resultsclearlyshowedthatcontinuedstressactivelyselectsforanaggressive,androgen-
independentphenotype.
19
Chapter5
IntersectionofMolecularChaperonesandSteroid
Receptors-II
5.1 Enhancedsteroidreceptorsignaling -homing inonHSP90
All these data prove that stress and NR activation pathways intimately influence each
other. Each investigation considers, in some way, the interesting possibility that hsp
production is the driving force behind stress-enhanced reporter activity. Only Edwards
andMitsiouexperimentallyimplicateHSP70asthedeterminantofincreasedGRactiv-
ity. However, it is worthwhile considering the possibility - and strong likelihood - that
the other, large, stress-inducible chaperone, HSP90, is the agent of change. Some rea-
sonsforthiswillbepresentedbelow.
First, it is well-knownthat HSP90 is adept at preventingirreversible aggregationof
thermally-denaturedproteins[WBZ
+
92]. Equallywell-documentedistheessentialrole
of HSP90 in maintainingthe ligand-bindingabilityof itsSR clients. Prior to hormone-
binding, the AR, ER-α, GR, MR and PR are typically found bound to HSP90. (This
contrasts with other classes of NR, for example the retinoic acid receptor (RAR), that
displaynoapparentaffinityforthechaperone). HSP90interactionslocalizetotheLBD
20
of SRs. This ligand-binding ‘cleft’ appears to be collapsed in the unliganded, HSP90-
free SR such that the receptor itself must change its conformation to allow entry of the
ligand[GK01].
Second, the HSP90 chaperone machinery carries out an ATP-dependent ‘opening’
reaction of the LBD - the rate-limiting step in the overall process [KSP02]. Once the
cleft is open, it remains open only as long as the metastable complex with HSP90 is
maintained. This is evidenced in studieswhere the removalof HSP90 results in loss of
ligand-bindingabilityoftheGRandPR[MML
+
00]. Ligand-bindingisreadilyrestored
in the presence of HSP90 [MML
+
00, SDM
+
90, Smi93]. ERα is interesting in that its
ligand-binding ability is stable even in the absence of HSP90, yet the NR assembles
with HSP90 in a manner identical to that of the GR and PR [ALL
+
97, PKG
+
90]. The
liganded form of the NR is said to be ‘transformed’ or ‘activated’ because it can now
movetothenucleusandbetranscriptionallyactive[PT97].
Third, it has been shown that HSP90 is required both for NR movement into and
within the nucleus [PGH
+
04]. The rationale is that a hidden ‘nuclear localization sig-
nal’(NLS1)isrevealedwhenHSP90isremovedfromtheligand-boundNR-thebinding
ofligandfavoursclosureoftheligand-bindingcleft,promotingenoughofaconforma-
tional change in the NR to expose the NLS1 thereby initiating the process of nuclear-
directedNRmovement. [SPM
+
93].
Fourth, one may assume that concomitant with hormone-binding, HSP90 removal
andsubsequentNRstructuralalterationgivesrisetootherproteininteractionsitesonthe
NR.Thesebindingsitesmightbeutilisedbyco-chaperonesthatsomehowassistHSP90-
mediated regulation of NR signaling. We now know that specific HSP90 cochaperone
proteinstermed‘immunophilins’participateinNRsignalingbyenablingNRmovement
towards the nucleus. HSP90 facilitates linkage of NRs to the cytoskeletal motor sys-
tem and it does this via immunophilinproteins. For example, the HSP90 co-chaperone
21
FKBP52 localizes to microtubules and exists in cytosolic complexes with cytoplasmic
dynein [Cza94]. Subsequently it has been shown that other HSP90 co-chaperones, eg.
cyclophilin 40 (CyP40) and protein phosphatase 5 (PP5) also serve as linkers to the
dyneinmotorsystem[GHM
+
02].
Fifth, HSP90 is also involved in intra-nuclear NR trafficking. Kang and co-
workers [KDC
+
94] haveshownthat a GR-HSP90-immunophilinheterocomplexenters
the nucleus via the nuclear pores. Elbi et al [EWR
+
04] have shown that HSP90 is
requiredforgeneralintra-nuclearmobility. Theydevelopedapermeabilizedcellsystem
inwhichtranscriptionally-activenucleiweredepletedofsolublefactorsrequiredforGR
nuclear mobility- GRmovedunfettered when incubated withrabbit reticulocyte lysate
butlostthisabilitywhentreatedwithgeldanamycin,anHSP90-specific inhibitor. Yang
andcolleagues[YLD97]provedthatnuclearGRreleasedfromchromatincouldrecycle
to chromatin upon rebinding hormone without exiting the nucleus. Investigators then
showed that this recycling was inhibited by the HSP90 inhibitor geldanamycin added
eitherpriortoorfollowinghormonetreatment[CGS
+
97,LD99,Smi93,Whi96].
Finally,chromatinimmunoprecipitationexperimentsrevealedHSP90localizationto
nuclearglucocorticoidresponseelements(GREs)inahormone-dependentmanner,con-
firmingthe chaperone’s involvementinGR-transcriptionalactivation[FY02, MEL
+
07,
SMH
+
04]
Allthese examplessubstantiatetheclaim thatHSP90 isextensivelyinvolvedin NR
signaling. From them one may easily hypothesizethat HSP90 has the potentialto alter
NRbehaviourandactivityatanyormanystepsinthesignaltransductioncascade. One
of the simplest ways in which this might occur is via alteration of chaperone protein
levels. Modification of protein levels, via overexpression or by siRNA knock-down,
is a widely used method for studying the functional importance (or lack thereof) of
proteins. Oneoftheeasiestwaysinwhichtoalterchaperonelevelsinaphysiologically
22
relevant setting (bearing in mind that many overexpression experiments are performed
withabnormally-highamountsof theparticularproteinthuslendinga certain degreeof
suspicion to eventual results) is by challenging one’s experimental system with a form
ofstress(thermal,chemical,oxidativeetc).
As described earlier, the majority of groups employed elevated temperatures (or
‘heat-shock’) as their chosen stressor. Heat indiscriminately deposits its energy and
thus causes cell-wide damage. Heat affects the stability of proteins and causes their
denaturation. Given that the induced synthesis of hsps upon stress can be viewed as
an amplification of their basic chaperone function, heat-shock seems a valid model to
study chaperone function and regulation in mammalian cells [EdV91]. It is important
to remember that the application of most stressors to cells will result in a co-ordinated
inductionofallheat-induciblechaperones(eg. HSP27,HSP70,HSP110etcandnotjust
HSP90)forcell-wideheat-resistance. Controlled,selectiveoverexpressionofchaperone
proteins is therefore necessary in order to discern the specific effect(s) of individual
HSPs.
Recall that nearly all groups, from Wolffe et al to Khandrika and co-investigators,
suggested that chaperone upregulation may have been responsible for stress-increased
reporter activity in their respective experimental systems. None, however, pursued an
investigationintowhichspecificchaperonemightinfluenceNRactivity. Forthereasons
mentioned previously and some data discussed hereafter, HSP90 presents itself as a
viablecandidate.
23
5.2 Chaperone levels influence steroid receptor activity - experi-
mentalevidence
An early paper by Picard and co-workers [PKG
+
90] studied the effect of lowering
HSP90 levels in yeast cells. The strain of S. cerevisiae thus constructed possessed
a galactose-inducible HSP82 (yeast homologue of human HSP90) plasmid that was
expressed throughout the cells. The investigators also transfected expression and
reporter plasmids for the GR. The level of HSP82 was regulated such that it could be
lowered
˜
20-fold relative to wild-type expression levels of HSP82. Picard et al. found
that low levels of HSP82 expression rendered hormone-free receptors (or ‘aporecep-
tors’) largely unbound to the HSP82 chaperone. These aporeceptors were also tran-
scriptionally inactive. Addition of hormone activated the receptors but with markedly
reduced transcriptionefficiency. Dosesof varioussynthetichormonalligandsknownto
strongly activate receptor-mediated transcription also produced little or no effect when
HSP82 expression was repressed. Significantly, the results from this paper indicated
thataporeceptormoleculesthathadneverbeencomplexedwithachaperone(ieHSP82)
were qualitatively different from those whose previous chaperone interaction had been
reversedwithhigh-saltextractionorelevatedtemperatures.
Studies by Sabbah [SRR
+
96] used an in vitro, cell-free system to carry out EMSA
experimentsshowingthatthebindingofcalfuterineERtoaradiolabeledEREoligonu-
cleotide was inversely dependent upon the relative concentration of added purified
HSP90. Sabbah et al were correct in highlighting the imprecision of the methods they
used to quantify the concentrationsof expressed/overexpressedreceptor and chaperone
andstressedthattheirresultswerelargelyqualitative. Atlowconcentrationsofprepared
HSP90, ER was capable of forming complexes with its cognate ERE. High concentra-
tions of HSP90 (10-fold molar excess over receptor levels), inhibited specific ER-ERE
24
binding. (TheDNA-bindingabilityoftheubiquitoustranscriptionfactorsNF1andAP1
remained unaffected by these high HSP90 levels). Sabbah and group also determined
that the inhibitoryeffect of increased HSP90 levelscouldbe reversed by increasingthe
concentration of the ERE oligonucleotide. They also showed that HSP90 dissociated
ERE-boundreceptor andthatthisrecently-releasedreceptorwasabletorebindERE.
Caruso et al investigatedthe effect of HSP90 overexpressionon the activitiesof the
arylhydrocarbonreceptor(AhR)andER[CLB99]. HSP90overexpressioninhibitedthe
activity of an AhR responsive reporter. However HSP90 overexpression did not block
theinductionofanER-responsiveplasmid. Theirresultsareincontrasttothosereported
by Sabbah et al. One reason for this discrepancy might be the model systems - human
MCF-7 (twokinds- wild-typeand‘Adriamycin-resistant’)and T47Dbreast cancer cell
linesknowntopossessendogenousAhRandER[CLB99]versusanartificially-created
environment used by Sabbah - and their influences on chaperone-NR interactions. A
secondreasonmightbethemannerofHSP90overexpression-useofanuclear-targeted
HSP90 expression vector by Caruso and group rather than direct addition of purified,
full-length,‘pooled’HSP90fromvariousmammaliansourcesbySabbahetal.
Kang and co-workers [KMD
+
99] performed experiments to study the effect of
HSP90 overexpression on the activity of a glucocorticoid-dependent promoter. Like
Caruso et al, they used an expression plasmid that produced nuclear-targeted HSP90.
Kang et al. worked with yeast, murine and human breast carcinoma cells. They
increased the nuclear HSP90/GR ratio and discovered that glucocorticoid responses
wereattenuated. Thislevelofinhibitionwasmostpronouncedathighconcentrationsof
hormone (ie when receptor and chaperone are maximallydissociatedfrom each other).
Purified HSP90 physically interacted in vitro with previously in vivo-activated GR and
inhibited receptor binding to its cognate response element. This same in vivo, ligand-
activated GR was then able to reassociate with HSP90, suggesting that HSP90 played
25
a role in receptor recycling. Kang and group also pointed out that the inhibitory GR
response was directly proportional to the level of HSP90 overexpression and was not
seenincelllinesthatwereunabletoretainahighlevelofthechaperone protein. Given
however that their experimental results were procured in a system that registered a 30-
fold increase in nuclear HSP90 (relative to wild-type nuclear HSP90 levels) they may
nothaveobservedaphysiologicallyvalideffect.
5.3 HSP90co-chaperones: equalpartnersinalteringsteroidrecep-
toractivity
A similarbodyof research existsto showthat co-chaperone levelsdirectly alter steroid
receptor signaling - p23 and CHIP overexpression respectively enhance and dimin-
ish ER-mediated gene transcription [FPN05, ORB
+
06] while FKBP52 overexpression
potentiates GR and AR transcriptional activity [CPC
+
05, RRC
+
03]. Overexpression
of the HSP70 co-chaperone BAG-1L enhances AR transcriptional activity in human
prostateLnCaP cellswithoutalteringARproteinlevels[FTR98].
As in many of the HSP90 experiments, one should cautiously approach the results
observed by increasing the numbers of biomolecules to artificially high levels. Such
upregulationalterstheinternalbalanceofcellularfactors[FTR98]andsodataobtained
using such a system will additionally reflect the repercussion of events occurring in
an unnatural physiological setting. Such experimental influence can be minimized by
allowingbiomolecularoverexpressiononlyto a physiologicallyplausibleextent. Some
studies employ a second method of altering protein levels via RNA knock-down. This
hasthepartialadvantageofreducingextantcellularRNAlevelsthusavoiding‘molecu-
larover-crowding’causedbytheoverexpressiontechnique.
26
That said, the p23 and CHIP results are reasonable giventhe use of physiologically
relevantcellsystems(humanbreastcancercells)andtheexperimentalinvestigationinto
chaperone overexpression on specific ER-target genes. The FKBP52-AR studies are
somewhatincompleteinthatARbehaviourisanalysedonlyinan‘ARnull-background’
HeLacellsystemandnotinprostatecancercells(amorenaturalphysiologicalsetting).
Also, both FKBP52 studies would benefit from experiments that determined the effect
ofco-chaperoneoverexpressiononAR-target genes.
The evidence presented above argues in favour of the important role of HSP90 (or,
perhaps more appropriately, the HSP90 chaperone machinery) in supporting steroid
hormone action. As specific roles for chaperone and co-chaperones in NR complexes
are coming into focus, it would be worthwhile considering the potential for regulating
steroid hormone responsiveness in tissues through expression of relevant chaperones
andco-chaperones. Indeed,thesameevidencecorroboratesjustthat-directoverexpres-
sion of chaperones (or co-chaperones) does alter NR signaling by either increasing or
decreasingNRactivity. Patternsofchaperone/co-chaperoneexpressioncouldalsoinflu-
ence NR activity. Cheung-Flynn et al [CPC
+
05] studied adult mouse tissues, focusing
on four androgen target organs (testes, epididymis, anterior prostate and seminal vesi-
cles). They discovered that all nine proteins of interest (HSP90, HSP70, HSP27, HOP,
HIP, FKBP51/52, PP5, CyP40 and p23) were detected in each organ, but that the pat-
tern of protein expression levels was different in each. It is reasonable to assume that
ARactivity(andthatofotherHSP90clientproteins)differsbasedonthechaperone/co-
chaperone cohort in that particular organ. Cheung-Flynn and co-workers didnot inves-
tigatethis. Additionally,alteringthe relativeconcentrationsof co-chaperones wouldbe
expected to shift the balance of HSP90 complexes,which could then go on to affect its
clientsandprocessesthataresensitivetothosecomplexes.
27
Chapter6
HSP90: ageneticcapacitor
The studies described so far relied on increasing or decreasing the amounts of
chaperone/co-chaperone protein while observing the resultant effects on NR activity.
HSP90 is essential for the survival of all eukaryotes tested, whereas HSP90-knock-out
results in only a mildly-thermosensitive phenotype in E. coli [WL05]. In Drosophila
cells, compromising the function of HSP90 induced epigenetic alterations in gene
expression as well as heritable alterations in chromatin state [SQL03, SLX
+
02]. This
impliedthat HSP90 could conceal inherent genetic variation withinpopulationsof cer-
tain organisms [QSL02, RL98]. As a consequence of its protein chaperoning function,
HSP90 allows polymorphicvariants of crucial signalingpathways to accumulate while
the pathway as a whole retains sufficient function to maintain wild-type phenotypes.
This‘buffering’attheproteinlevelbyHSP90funnelscomplexdevelopmentalprocesses
into discrete, well-defined outcomes (a process termed ‘canalization’ [Wad42]) despite
underlying genotypic variation, and it seems to be essential for the robust expression
of uniform phenotypes under basal conditions [RXG
+
05]. Under stressful conditions,
however,someoftheunstableclientproteinsofHSP90mightbecomeevenmoreunsta-
ble, creating an increased demand for HSP90 in facilitating the refolding of its usual
client proteins as well as the new, stress-destabilized ones. The buffering capacity of
28
HSP90isthusexceededandpreviouslyhiddengeneticvariationsnowbecomeavailable
for natural selection and enhance the survivalof distinctgenotypes withina population
[RL98].
The same reasoning has been applied to tumour biology where it has been pro-
posed that, at the protein level, HSP90 might function as a biochemical buffer of the
extensive genetic heterogeneity that is characteristic of most cancers [SQL03]. During
cancerprogression,themalignantphenotypemightbreakloosewhenthebufferingabil-
ity of HSP90 is overloaded in tumour cells as a result of normal aging, the increased
load of mutant/misfolded oncoproteins, the hostile tumour microenvironment - or all
these factors acting in concert. As a result, diversity within the tumour cell population
would increase and accelerate the selection pressure in favour of invasive, metastatic
and drug-resistant phenotypes [GV03]. It must be cautioned that the possibility of a
‘Dr. Jekyll-Mr. Hyde’ scenario also exists with respect to the use of HSP90 inhibitors.
The ability of HSP90 inhibitors to affect multiple oncogenic pathways simultaneously
(as a consequence of the large number of signal transduction molecules that are clients
of the chaperone) is a therapeutically attractive feature of these compounds. However,
purposely repressing HSP90’s buffering activity (by inhibiting HSP90’s chaperoning
function) raises the chance of revealing mutationsthat enhance the survival and malig-
nant progression of some tumour cells within the entire population. This evolutionary
viewofmalignantprogressionsuggeststhatdefinitivecontrolof acancer willprobably
be achieved most effectively by first understanding and then altering the mechanisms
of function of the key determinants that shape its ability to adapt and evolve - a prime
exampleofwhichmightbeHSP90.
29
Chapter7
Futureperspectivesandconcludingremarks
HSP90 protein expression is known to be high in several cancers, including breast
[JSC
+
92, YNN92, YNT
+
96, NKM
+
98, CSF
+
98, CBJ
+
01, MM04] and prostate
[WMC
+
94, CEF
+
02, SSR03, GTA
+
03]. The AR and ER are critical signaling fac-
tors in breast and prostate tumour progression. Both SRs are both bona fide clients of
HSP90 and several small molecule inhibitors of HSP90 effectively block the ligand-
binding ability of NRs and promote their degradation. Frontline therapies in both can-
cers involve the extensive use of small molecules that interfere with the activation of
eitherNR,eitherdirectlybyinhibitinginteractionoftheNRwithitsligandorindirectly
by reducing synthesis of the mitogenic ligand. For example, tamoxifen, raloxifene and
anastrozole are used against the ER in the treatment of breast cancer. Bicalutamide,
dihydroxyflutamide and finasteride are used to inhibit AR signaling in prostate cancer
chemotherapy. Given the direct, extensively researched link between AR/ER activity
and disease progression, it is unusual that less work has been done on studying the
influenceofastressfulenvironmentonthesignalingbehaviourofeitheroftheseNRs.
As the first step in contributingto the ever-growingbody of informationdocument-
ing theintersection of molecularchaperones and nuclear receptors, thisauthor believes
that a thorough investigation into the effect of stress on the transcriptional activity of
30
the human ER in normal and tumour cells should be undertaken. Studies may begin
by measuring whether ER transcriptional activity is affected by heat-shock. The use
of heat as a cellular stressor is a well-characterized in the literature. One may then
attempttocorrelatechangesinERtranscriptionalactivitywiththekineticsofupregula-
tion of HSP90 protein levels. A potential cause-and-effect relationship may be studied
bytransfection-mediatedanalyses.
GiventheimportanceofHSP90inmalignantprogressionandtheapplicationofNR-
targetedpharmaceuticals,aninterestingextensiontothephenomenonofstress-activated
NR activitywould be an investigationinto altered NR ligand specificity. To the best of
this author’s knowledge, no such work exists. One study has assessed HSP90 expres-
sion in aggressive breast tumours [KLK
+
05] and recent clinical work correlated inten-
sity of HSP90 expression with decreased patient survival in breast cancer [PKG
+
07].
Thatgrouphoweverwasunabletodirectlyexploretheintermediatequestionofwhether
HSP90 expression intensity correlated with these patients’ responses to chemotherapy,
whichthenultimatelyaffectedtheirsurvivalchances. Thistopicisofspecialinterestfor
itiswell-knownthatHSPs(HSP27andHSP70)areimplicatedinbotheffectivenessand
tolerance to chemotherapy [PVM
+
06, TMD02, VGT
+
98]. Similar studies for HSP90
howeverremainfewandfarbetween.
The stress response in solid tumours - characterized by the massive overexpression
of HSPs - leads to the induction of resistance to drugs that act primarily on rapidly
dividing cells. This resistance is reversible or decays upon removal of the stress con-
ditions [TT99, TNT
+
03]. The phenomenon of drug resistance has been studied most
frequentlyonlywithHSPs27and70withrespecttoDNA-interactingagentseg. anthra-
cyclines (doxorubicin), antimetabolites (methorexate, fluorouracil), epipodophyllotox-
ins (etoposide), the taxanes (paclitaxel) and vinca alkaloids (vincristine, vinblastine)
31
[CC05, HW00, TMD02, VGT
+
98]. There seems to be virtually no information avail-
able on HSP expression - in particular HSP90 - and association with effectiveness of
steroid-receptor-targetedchemotherapy.
Further, experimentalevidence supportsthedual capabilityof anticancer drugs act-
ing not only as lethal agents for tumour cells but also themselves inducing an adaptive
stress response in the neoplastic environment. Also, each tumour cell (and tissue) type
mayshowadistinctresponseanddrug-specificgeneexpressionpattern,dependingupon
thearrayandconcentrationofthevariouschaperonesandco-chaperonesitharbours.
Inaddition,theactivityofenzymaticchaperonesmayalsoplayaroleindetermining
response to a therapeutic ligand. It has been shown that tumour-derived HSP90 pos-
sesses a much higher ATPase activity than does normal-tissue-derived HSP90 and that
thisoveractivechaperoneexistsexclusivelyinachaperonecomplex[KTS
+
03]. Further,
tumour HSP90 has over 100-fold greater sensitivity for HSP inhibitors (eg. 17-AAG)
as compared to non-tumour HSP90 [KBB04, KTS
+
03]. Reports suggest that mutant
oncoproteinclientsare somehowresponsiblefor HSP90’s enhanced bindingaffinity. A
secondattractivehypothesisisthatthemulti-chaperone-boundHSP90,orothercompo-
nents of the multi-chaperone complex, are responsible for catalyzing a conformational
change in geldanamycin which enable it to bind with exceptional selectivity to HSP90
[Nec03]. Finally, complexation of HSP90 might lead to a conformational change in
its own ATP-binding pocket such that this new, altered shape facilitates binding to gel-
danamycin).
It would be interesting to undertake studies that compared the drug responses of
humantumourcelllinesandsolidtumourtissuesamplesthateitherdidordidnotover-
express HSP90. Would these cells exhibit altered morphology? What would be the
ATPaseactivityofHSP90ineachcase? WouldHSP90overexpressioncorrelatewithits
ATPase activity? Would this ATPase activity subsequently affect drug response? One
32
maypredictthatproteotoxicstresswouldconvertinactive(orweakly-active)HSP90into
complexedHSP90withhigherenzymaticactivity. Wouldtherebedifferentresponsesto
chemotherapy in tumours that overexpressed normally-active HSP90 vs. tumours that
harboured normal protein levels of overactive HSP90? Are there polymorphic differ-
ences within HSP90 obtained from various types of cancers and do these genetic vari-
ations contribute to altered therapeutic responses? At the time of this writing, there do
not seem to be answers to these questions. Further work is needed to elucidate the bio-
logical significance of chaperone overexpression and the potential influence on patient
chemotherapeuticresponses.
Much progress has been made toward characterizing molecular chaperones and
delineating their roles in steroid receptor function. An equal amount of effort has been
madeinunderstandingthemechanismsbywhichNRsactivateorinhibitgenetranscrip-
tion. Theevidencepresentedinthismanuscriptprovesbeyonddoubtthatthetwofields
engageinamplecross-talk. Furtherinvestigationintheareawillexpandourunderstand-
ing of chaperone expression as determinants of cellular responses to steroid hormones
indifferentphysiologicalcontexts. It willalsopositivelyimpactoureffortsinselecting
appropriatetherapeuticregimensinthetreatmentofbreastandprostatecancers.
33
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46

Abstract (if available)

Abstract Cellular stress is present during tumour development. Stressed cells in most tissues increase the production of ‘molecular chaperones’ - proteins that maintain the conformation and function of client substrates. Upregulated chaperone levels constitute an adaptive response that enhances cell survival. Tumour cells display constitutively-elevated levels of molecular chaperones, probably reflecting efforts at maintaining homeostasis in a hostile environment. The molecular chaperone HSP90 is unique - its protein clients are primarily involved in cellular proliferation. Nuclear receptors (NR) are signaling molecules and HSP90 clients whose aberrant activity is widely implicated in oncogenesis. It is currently unknown if stress-induced chaperone overexpression influences NR activity and potentially cancer progression. It is also possible that stress alters NR ligand specificity. These agonists, antagonists or ‘selective nuclear receptor modulators’ modulate NR activity in cancer chemotherapy. Understanding chaperone influence onNR signaling should facilitate the design of patient-specific treatment strategies.

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Asset Metadata

Creator Jathal, Maitreyee Kiran (author)

Core Title The effect of chaperone/co-chaperone overexpression on steroid receptor activity

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology

Publication Date 07/31/2009

Defense Date 07/01/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag activation,cancer,chaperone,OAI-PMH Harvest,proliferation,signaling,steroid receptor,Stress

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Duncan, Roger F. (committee chair), Hamm-Alvarez, Sarah F. (committee member), Tahara, Stanley M. (committee member)

Creator Email maitreyee.jathal@gmail.com,maitreyt@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2441

Unique identifier UC1459607

Identifier etd-Jathal-2889 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-171767 (legacy record id),usctheses-m2441 (legacy record id)

Legacy Identifier etd-Jathal-2889.pdf

Dmrecord 171767

Document Type Thesis

Rights Jathal, Maitreyee Kiran

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu