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Endocrine-Related Cancer(1998) 5 1-14

Endocrine-Related Cancer(1998) 5 1-14 1998 Journal of Endocrinology Ltd Printed in Great Britain1351-0088/98/005-001 $08.00/0

Introduction

The ability of oestrogens and androgens to stimulate thegrowth of a number of endocrine cancers is well

established and several endocrine therapies are widelyused to reduce the availability of the hormones or to blocktheir action. These include non-steroidal hormoneantagonists such as tamoxifen and flutamide, steroidalcompounds which include ICI 182780 and cyproteroneacetate, and both steroidal and non-steroidal aromataseinhibitors. Although we have learned a great deal about themolecular mechanism of steroid hormone action, it is stillunclear how hormones stimulate the proliferation oftumour cells and how hormone antagonists function. Inpart this is a result of the ability of oestradiol andtestosterone to regulate the expression of many proteinsimplicated in the control of cell proliferation making it

difficult to identify the crucial targets. Some of thesetargets are growth factors and/or their receptors whichsuggests that the mitogenic effects of steroids may bemediated by indirect autocrine or paracrine mechanisms(Clarke et al. 1991, Roberts & Sporn 1992). Alternativelysince steroids regulate the expression of certain cyclins orkinase inhibitors (Musgrove & Sutherland 1994, Altucciet al. 1996) they may control cell cycle progressiondirectly. Recent work suggests that as well as the cyclinD1 gene, cyclin D1 itself may be a crucial target (Zwijsenet al. 1997) but additional proteins could also be importantin different subsets of tumours.

The identification of target genes for steroid hormones

is further complicated by the observation that receptorsignalling is cross-coupled with that of other signallingpathways. It used to be thought that signalling by receptorswas relatively straightforward compared with most othersignalling pathways, since the receptor itself is atranscription factor. It is now clear, however, that growthfactors, neurotransmitters and other hormones are able tomodulate the activity of steroid hormone receptors (Poweret al. 1991, Aronica & Katzenellenbogen 1993, Ignar-Trowbridge et al. 1993). This means that alterations in theactivity of receptors and the expression of individual targetgenes involved in cell proliferation is determined not only

by hormonal signals but also by changes in othersignalling pathways, which undoubtedly take place duringbreast and prostate cancer progression. Receptors are alsocapable of regulating the activity of a number of other

transcription factors, either directly or indirectly, andthereby modulate the ability of other signalling pathwaysto control gene transcription (Shemshedini et al. 1991,Philips et al. 1993, Stein & Yang 1995, Webb et al. 1995).

Although the precise role of steroid hormones in cellproliferation is still ill-defined, tremendous progress hasbeen made in elucidating the role of hormones in receptoractivation and the mechanism by which hormoneantagonists block this activity. In particular two recentadvances have been made which provide new insights intosteroid hormone action and the function of nuclearreceptors in general. First, models for the three-dimensional structure of the ligand binding domain of

several receptors have been determined (Bourguet et al.1995, Renaud et al. 1995, Wagner et al. 1995, Brzozowskiet al. 1997) and secondly, novel proteins have beenidentified (Cavaills et al. 1994, Halachmi et al. 1994)which interact with receptors in a ligand dependentmanner and may play a role in gene transcription. Thesedevelopments are helping to provide a betterunderstanding of the mechanism of action of bothhormones agonists and antagonists and the links betweennuclear receptors and other signalling pathways. In thisreview we will outline these advances, with reference tothe oestrogen receptor (ER), and discuss their relevance toantioestrogen therapy and tamoxifen resistance.

Steroid hormone receptors are membersof the nuclear receptor family

Steroid hormone receptors are members of the nuclearreceptor family, a large group currently totallingapproximately 150 different proteins, which function astranscription factors in many different species includingboth invertebrates and vertebrates. In addition to steroidhormone receptors, the nuclear receptor family consists ofreceptors for retinoids, thyroid hormone, fatty acids andprostaglandins and a number of so-called orphan

Molecular mechanisms of steroid

hormone actionR White and M G Parker

Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, 44 Lincolns Inn Fields,London WC2A 3PX, UK

(Requests for offprints should be addressed to M G Parker)


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White and Parker: Molecular mechanisms of steroid hormone action

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Figure 1 (A) Organisation of functional domains in the nuclear receptors. DBD and LBD refer to the

positions of the DNA binding domain and the ligand binding domain respectively. The relative

positions of the two transactivation domains AF1 and AF2 are also shown. (B) Schematic diagram

of steroid hormone receptor binding to DNA. The receptors bind as homodimers to palindromic

sequences. The specific sequences of simple response elements for the ER and other steroid

hormone receptors are shown.


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receptors, the ligands of which have yet to be identified(Parker 1993, Mangelsdorfet al. 1995). They are highlyrelated in both primary amino acid sequence and the

organisation of functional domains suggesting that manyaspects of their mechanism of action are conserved. Thus,progress in our understanding of steroid hormone actionhas been facilitated by studies of many nuclear receptorfamily members.

The sex steroid receptors are not only expressed in sexaccessory tissues but in many other types of cellsincluding liver, bone, pituitary and cardiovascular cells.Androgen receptors are encoded by a single gene whereasthere are two genes for ERs, ER and ER (Kuiper et al.1996, Mosselman et al. 1996). Our current knowledge ofthe distribution of ER (Kuiper et al. 1997) is based onmRNA analysis either by RT-PCR or by in situ

hybridisation and the relative levels of ER and ERprotein levels have yet to be determined. Analysis of micemade devoid of ER by targeted gene disruption(Bocchinfuso & Korach 1977) indicates that this receptoris essential for uterine growth and mammary glanddevelopment but not for mediating the inhibitory effects ofoestrogens in vascular injury (Jafrati et al. 1997). Thus, itis possible that ER and ER have distinct functions insome tissues but not in others. Undoubtedly the role ofthese individual receptors will be confirmed in the futureby gene knock-out experiments to generate mice lackingeither both receptors or one isoform only and analysing theresulting phenotypes.

A simplified diagram of the organisation of functionaldomains in nuclear receptors is shown in Fig. 1A. Thereceptors are characterised by a highly conserved DNAbinding domain and a moderately conserved ligandbinding domain which also functions in dimer formationand transcriptional activation (Parker 1993, Mangelsdorfet al. 1995). A second dimerisation domain is locatedwithin the DNA binding domain itself and the receptorscontain two transcriptional activation functions by whichthey are able to regulate the expression of target genes;AF1, which is located in the N-terminal or A/B domain,and AF2 in the ligand binding domain. When activated byhormone binding, the receptors may act directly as

transcription factors by binding to specific DNAsequences, termed hormone response elements, found inthe vicinity of target genes (Evans 1988, Beato 1989). Thesequences of simple binding sites for the steroid receptorsare shown in Fig. 1B. Composite response elements havealso been identified which bind receptors in addition toother transcription factors such that the binding of oneinfluences, either positively or negatively, the activity ofthe other (Diamond et al. 1990). In addition, it is nowevident that nuclear receptors are also capable ofregulating the transcription of genes that lack hormoneresponse elements by modulating the activity of other

transcription factors such as AP-1 (Philips et al. 1993,Webb et al. 1995) and NF-B (Stein & Yang 1995). Todate most of the analysis which has been carried out has

been to determine the mechanism by which receptorsfunction as DNA dependent transcription factors but it islikely that the transcription of many genes is regulatedindirectly by non-classical mechanisms.

Nuclear receptors function as liganddependent transcription factors

In the absence of hormone, steroid hormone receptorsexist as inactive oligomeric complexes with a number ofother proteins including chaperon proteins, namely theheat shock proteins Hsp90 and Hsp70 and cyclophilin-40and p23 (Smith & Toft 1993, Pratt & Toft 1997). The role

of Hsp90 and other chaperons may be to maintain thereceptors folded in an appropriate conformation torespond rapidly to hormonal signals. Whether differenttypes of receptor complexes occur in cells, eachcontaining a subset of chaperons and able to respond todifferent types of signal, or whether receptors are foldedinto a mature complex containing chaperons in anassembly line type of process is as yet unclear (Bohen etal. 1995, Pennisi 1996). Following hormone binding, theoligomeric complex dissociates allowing the receptors tofunction either directly as transcription factors by bindingto DNA in the vicinity of target genes or indirectly bymodulating the activity of other transcription factors.

Nuclear receptors may be classified into four typesbased on their dimerisation and DNA binding properties.Steroid hormone receptors bind to DNA as homodimerseither to simple response elements comprising shortpalindromic sequences or to composite response elementswith other transcription factors. Oestrogen responseelements consist of inverted repeats of the sequence A/GGGTCA separated by three nucleotides, whereas thereceptors for androgens, glucocorticoids and progestinsbind to inverted repeats of the sequence AGA/GACA, alsoseparated by three nucleotides. In contrast, a second groupof nuclear receptors, including the retinoic acid receptor(RAR), thyroid hormone receptor (TR) and vitamin D

receptor, bind to DNA as heterodimers in combinationwith the retinoid X receptor (RXR) (Mangelsdorf et al.1995). The binding sites for these receptors consist ofdirect repeats of the sequence A/GGTCA separated by upto five nucleotides with the DNA binding specificity ofdifferent receptors being determined to some extent by thespacing of the repeats. In addition RXR itself and someorphan receptors bind to direct repeats as homodimerswhile a final group of orphan receptors are also able tobind as monomers to single copies of the extended bindingsite sequence TCAAGGTCA. The major part of thedimerisation interface is formed from sequences within


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the ligand binding domain of the receptors while a second

dimerisation function is located within the DNA bindingdomain itself (Kumar & Chambon 1988, Fawell et al.1990a).

Transcriptional activation by steroid receptors ismediated by at least two distinct activation functions; one,referred to as AF1, is located in the N-terminal domain anda second, AF2, is in the hormone binding domain (Lees etal. 1989, Tora et al. 1989). In the ER, and probably in otherreceptors as well, the absolute and relative activities ofthese two domains vary depending on the target promoterand the cell type. However, while the two activationdomains have the potential to act independently, they

appear to interact with each other in the context of the

intact receptor in a way which has yet to be determined.The N-terminal activation function, AF1, variesconsiderably in both size and primary amino acidsequence in different steroid hormone and nuclearreceptors. A number of studies have shown that thisdomain is a target for phosphorylation by other signallingpathways (Ali et al. 1993, Kato et al. 1995, Bunone et al.1996). The second activation function, AF2, is induced byhormone binding and a short sequence encoding anamphipathic -helix in the C-terminal part of the ligandbinding domain, which is conserved in all trans-criptionally active nuclear receptors, has been shown to be

Figure 2 Diagrams of models of the structure of the ligand binding domains of nuclear

receptors to demonstrate the effect of agonist binding. Panel A shows the arrangement of

sequence elements for three different receptors, human RXR in the absence of ligand and

human RARand rat TR in the presence of their respective ligands. The numbers 1 to 12

correspond to separate -helices and s1 to s4 to -strands. The position of amino acids which

are in close contact with ligand are marked and are found in helices 3, 5, s1 and s3, the loop

between helices 6 and 7 and helices 11 and 12. Panels B and C show diagrams of the folded

structure in the absence and presence of ligand and the relative positions of helices 1 to 12

(H1-H12). This shows the realignment of helices 10 and 11 and the alteration in position of

helix 12 to form a lid over the ligand binding pocket. This repositioning results in the formation

of a new surface on the ligand binding domain, indicated by the white circles, which is believed

to be required for the interaction with coactivator proteins.


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essential in receptor function (Danielian et al. 1992,

Saatcioglu et al. 1993, Barettino et al. 1994, Durand et al.1994). However, it is doubtful whether this short sequencefunctions autonomously since mutation of conservedresidues in other parts of the ligand binding domain, whichdo not affect ligand binding or dimerisation, also generatetranscriptionally defective receptors suggesting that AF2is derived from a number of different elements (ODonnell& Koenig 1990, Henttu et al. 1997).

Structure of the ligand binding domain ofnuclear receptors

A major advance in our understanding of how nuclearreceptors function has been the determination of modelsfor the ligand binding domain based on crystal structuresof three different receptors either in the absence orpresence of their cognate ligands. The secondary structureof all three nuclear receptors is shown in Fig. 2A andschematic diagrams of models of the structures obtainedin the absence and presence of ligand in Fig. 2B and Crespectively. The structure of the ligand binding domain ofRXR crystallised in the absence of ligand, consists of 12-helices and two -strands arranged in three layers toform an antiparallel -helical sandwich (Bourguet et al.1995). This receptor was crystallised in the form of adimer with the dimer interface formed mainly from helices9 and 10. Subsequently, crystals of the ligand binding

domains of RAR(Renaud et al. 1995) and TR (Wagneret al. 1995) generated in the presence of their respectiveligands were shown to have a similar overall structure, but

with one major exception. The C-terminal helix, helix 12,

which protrudes beyond the core of the ligand bindingdomain in the unliganded RXR, is folded back across thecore of the domain in both RARand TR. It has thereforebeen proposed that in the case of RAR, ligand bindingresults in a realignment of helices 10 and 11 which re-arrange to form a continuous helix. This creates ashortened helix 12, corresponding to the amphipathic -helix described earlier, which is released from the core ofthe domain to fold back over the top of the ligand bindingpocket where it forms both contacts with the ligand and asalt bridge in helix 4. Helix 12 is similarly aligned in thecase of the TR although a salt bridge is not formed.Protein sequence comparisons between different receptorssuggest that their overall helical structure is conserved(Wurtz et al. 1996) and unpublished results indicate thatthe structures of peroxisome proliferator activatedreceptor (PPAR) and ER are consistent with thisprediction. Thus it appears that a major role for ligandbinding is an alteration in the conformation of the ligandbinding domain to form a novel surface. This may thenallow the binding of coactivators and other regulatoryproteins.

Recruitment of receptor interactingproteins

Gene transcription depends on the formation of a pre-

initiation transcription complex that includes basaltranscription factors and one of the roles of transcriptionalactivators, including steroid hormone receptors, is thought

Table 1 Receptor interacting proteins.

Predicted size

Group Name(s) Protein (kDa) Receptor binding Function(s)

I CBP/p300 CBP 265 ER, RAR, RXR, TR Histone acetylase

p300 300 ER, RAR, RXR, TR Bind transcription factors

p160 SRC-1a 156.7 ER, RAR, RXR, TR, PR

RIP160 SRC-1e/NCoA-1 152.3

ERAP160 TIF2 159.6 ER, RAR, RXR, TR, PR Form complexes with CBP/p300GRIP1

NCoA-2

pCIP 152 ER, RAR, TR, PR

II ARA70 ARA70 70 AR Androgen receptor coactivator

RIP140 RIP140 128 ER, RAR, RXR, TR, PR Multiple nuclear receptor

binding sites

TIF1 TIF1 112 ER, RAR, TR, PR Chromatin remodelling?

SUG1 SUG1 46 RAR, RXR, ER, TR, PR, VDR Associated with proteosome

TRIP1

AR, androgen receptor; PR, progesterone receptor; VDR, vitamin D receptor.


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to be the stabilisation of this complex. Although receptorshave been shown to bind directly to basal transcriptionfactors in vitro the significance of these interactions is

unclear since they are unaffected by mutations in thereceptor that destroy transcriptional activity (Sadovsky etal. 1995). The involvement of additional targets is sug-gested by the observation that AF2 activity is inhibitedwhen receptors are overexpressed suggesting that limitingdownstream target proteins, required for gene trans-cription, are being sequestered (Tasset et al. 1990). Anumber of candidate target proteins have been detected inextracts from mammalian cells which bind to the ligandbinding domain of receptors in a ligand dependent manner(Cavaills et al. 1994, Halachmi et al. 1994). Since theseproteins fail to interact with mutant receptors which aretranscriptionally inactive it has been proposed that they

may have a role in ligand dependent transcriptionalactivation by AF2.

Receptor interacting proteins may be classified intotwo groups (Table 1). The first group consists of twofamilies of proteins which appear to stimulate trans-cription and represent bona-fide coactivators. One family,originally identified as proteins of 160 kDa, consists of anumber of related proteins; the steroid receptor coactivatorproteins (SRC-1a and SRC-1e) (Onate et al. 1995, Kameiet al. 1996), transcription intermediary factor TIF2(Voegel et al. 1996) (independently identified as GRIP-1(Hong et al. 1996)) and CREB binding protein (CBP)interacting protein (p/CIP) (Torchia et al. 1997). The

second family comprises CBP and p300, which wereoriginally shown to function as coactivators for cAMPresponse element binding protein (CREB), the trans-cription factor that mediates responses to protein kinase Astimulation. Subsequently, however, CBP/p300 wereshown to function as coactivators for many othertranscription factors and may play a central role in manysignalling pathways (Janknecht & Hunter 1996, Shikamaet al. 1997).

The second group contains a number of unrelatedproteins which also interact with the ligand bindingdomain but their role in receptor function is still unclear.This group includes a 140 kDa protein termed RIP 140

(Cavaills et al. 1995), TIF1 (Le Douarin et al. 1995),ARA70 (Yeh & Chang 1996), which has been shown tostimulate transactivation mediated by the androgenreceptor, and TRIP1/SUG1 (Lee et al. 1995, vom Baur etal. 1996). RIP 140 stimulates receptor mediated trans-cription in yeast (Joyeux et al. 1996) and contains anactivation domain which is functional in mammaliancells (LHorset et al. 1996) but it is still unclear whether itis a bona-fide coactivator. TIF1 has been shown to interactwith proteins associated with heterochromatin and mayplay a role in chromatin remodelling (Le Douarin et al.1996). All these proteins have been shown to contain one

of more copies of a short sequence motif LXXLL which isnecessary and sufficient to mediate the binding of ligandbound nuclear receptors. This motif has been described as

a signature sequence for the interaction of proteins withnuclear receptors (Heery et al. 1997, Torchia et al. 1997).

Other proteins have also been isolated which interactwith other regions of the ligand binding domain. Forexample two related proteins, N-CoR and SMRT, whichfunction as repressors for RXR and TR in the absence ofligand (Horlein et al. 1995, Heinzel et al. 1997, Nagy etal. 1997), have been reported to bind to oestradiol andprogesterone receptors in the presence of antagonists(Jackson et al. 1997, Smith et al. 1997). A detaileddescription of all these proteins, both activators andrepressors, is beyond the scope of this article; they arereviewed in detail elsewhere (Horwitz et al. 1996, Heery

& Parker 1997) and therefore we will focus on theimportance of coactivators in steroid hormone actiontogether with potential interactions between receptors andother transcriptional regulators.

Structure and function of coactivators fornuclear receptors

To date, it appears that the p160 proteins and CBP/p300are the most important of the receptor interacting proteinsin terms of their ability to potentiate the transcriptionalactivity of receptors. The p160 proteins SRC1, TIF2 andp/CIP share significant sequence homology, most notably

in a central region found to be required for interaction withthe ligand binding domain of nuclear receptors. Theyshow ligand dependent binding to nuclear receptors invitro but fail to bind to transcriptionally inactive receptorsin which mutations have been introduced in the conservedhydrophobic residues in helix 12 (Kamei et al. 1996).Similarly mutations in conserved residues in helix 3 whichalso impair AF2 also prevent binding of these coactivators(Henttu et al. 1997). This suggests that, in the presence ofligand, helices 3 and 12 form a composite surfacerecognised by the p160 proteins, a hypothesis which isconsistent with the models of the holo ligand bindingdomains. Similarly, CBP/p300 have been reported to bind

to the ligand binding domains of nuclear receptors in aligand dependent manner and increase AF2 activity intransfected cells (Chakravarti et al. 1996, Hanstein et al.1996, Kamei et al. 1996, Yao et al. 1996). This binding isalso dependent on the integrity of helix 12, implying thatthese proteins recognise a similar if not identical bindingsite (Heery et al. 1997). The p160 and CBP/p300 familymembers also interact directly with one another (Hansteinet al. 1996, Kamei et al. 1996, Yaoet al. 1996) but we havelittle information about the stoichiometry of binding ofany of these proteins to receptors. For example, it isunclear whether the proteins bind to receptors


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independently or as complexes, whether they bindsequentially or simultaneously, which may pose stericproblems given their relative sizes, and whether there is

redundancy or receptor selectivity. These questions arebeing tackled by many laboratories at the present time.

When coactivators are recruited to receptors they mayserve at least two distinct roles. First, they may stabilisethe formation of a pre-initiation transcription complex ofDNA polymerase and basal transcription factors, andsecondly they may remodel chromatin by destabilising thebinding of nucleosomes. Regions required for trans-criptional activation have been found in both p160 andCBP/p300 family members. These have been identified byfusing fragments of these proteins to a DNA bindingdomain and testing their ability to stimulate transcriptionof reporter genes in transfected cells. In SRC1, one

corresponds to the region involved in CBP binding andmay reflect the recruitment of CBP and/or p300 to thepromoter. In CBP, an activation domain corresponds to aregion that binds to TATA-box binding protein (TBP) andso may directly interact with the basal transcriptionmachinery. Both families of proteins also encode histoneacetyl transferases that acetylate lysine residues onspecific histones which may destabilise nucleosomes topromote gene transcription (Ogryzko et al. 1996). Inaddition, they are also reported to be capable of recruitingP/CAF, another histone acetyl transferase (X-J Yang et al.1996). What emerges from these studies is that co-activators are multidomain proteins that presumably

function either independently or as complexes and thegoal is now to determine the precise roles and relativeimportance of each of these domains when they arerecruited by different receptors to target genes. A modelshowing some of the functions of coactivator proteins isshown in Fig. 3.

Role of hormonal agonists andantagonists in coactivator recruitment

Although crystal structures have yet to be solved for theligand binding domain of any one receptor in the ligandedand unliganded state, the overall similarity of the

structures for apo-RXR and holo-RAR and the high

level of sequence conservation in the nuclear receptorfamily, allows a model to be proposed for the mechanismof action of hormone agonists and antagonists. The

realignment of helix 12 as a lid over the ligand bindingpocket provides a potential molecular explanation for thedisparate activities of different ligands. Ligands whichfunction as agonists would be expected to allow theformation of an interacting surface made up from residuesin helices 3 and 12. In contrast, ligands that fail to allowthe correct realignment of helix 12 are likely to functionas antagonists. Interestingly, although the structure of theligand appears to determine its affinity for binding to thereceptor it does not seem to be as important in determiningAF2 activity, implying that the role of agonists may besimply to release helix 12 from an inactive conformationrather than to promote its correct realignment. This may

be true in the case of the ER since many environmentaloestrogens that bind relatively poorly still function asagonists (Soto et al. 1991, White et al. 1994, Jobling et al.1995).

Hormone antagonists may be classified into two basictypes, either mixed agonists/antagonists or pure ant-agonists, exemplified by the antioestrogens tamoxifen andICI 182780 respectively. The binding site in the receptorfor both types of compound overlaps with that of oestra-diol and so they act as competitive inhibitors of oestrogenaction (Dauvois & Parker 1993). In vitro assays andtransient transfection studies suggest that tamoxifenbinding allows the receptor to dimerise and bind to DNAwith high affinity but blocks transcriptional activitymediated by AF2. The agonist activity is thought to bederived primarily from AF1, which is active even whentamoxifen is bound to the receptor (Berry et al. 1990,Danielian et al. 1993, Tzukerman et al. 1994). Thereforetamoxifen has the potential to act as an antagonist whenthe transcriptional activity of the receptor is mediated byAF2 but as an agonist on promoters or in cell types whereAF1 is active. The pure antioestrogens, however, seem toblock the activity of both AF1 and AF2. Evidence isemerging that part of the mechanism of antagonism maybe a result of the recruitment of corepressors to thereceptor (Horwitz et al. 1996, Jackson et al. 1997, Smithet al. 1997). The lack of AF2 activity in the presence of

Figure 3 Interactions between receptors and

coactivator proteins. Activated receptors bind p160

proteins and proteins of the CBP/p300 family in a ligand

dependent manner. This DNA bound complex may

then stimulate the expression of hormone responsive

genes by a number of possible mechanisms including

the remodelling of chromatin, interaction with other

transcription factors and the stabilisation of the pre-

initiation complex which consists of DNA polymerase

and basal transcription factors.


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either type of antioestrogen is likely to reflect the incorrectrealignment of helix 12. This has recently been confirmedby the determination of models for the structure of theligand binding domain of the ER both in the presence ofoestradiol and in the presence of the antagonist raloxifene(Fig. 4). The major difference in the two structural modelsis an alteration in the positioning of helix 12 which is aconsequence of the need to accommodate the long, bulkyside chain of the antioestrogen in the ligand binding cavity(Brzozowski et al. 1997). The failure to correctly re-position helix 12 and the inability to form an appropriatesurface for the binding of coactivator proteins in thepresence of hormone antagonists may be analysed in vitro.Addition of either tamoxifen or ICI 182780 preventsbinding in assay systems in which coactivator proteins areanalysed for their ability to associate with the ligandbinding domain of the ER (Henttu et al. 1997). Similarlythese antagonists inhibit the stimulatory effects ofcoactivators such as SRC1 on receptor mediated trans-criptional activation in mammalian cells. Finally it may besignificant that many steroid antagonists, e.g. tamoxifen,ICI 182780 and RU486, are of greater size than the naturalligand suggesting that interference with the realignment ofhelix 12 may be a common mechanism of hormoneantagonism.

In addition to their effects on the realignment of helix12 pure antioestrogens have been found to suppress the

action of ERs in other ways. They clearly reduce theintracellular concentration of ERs by reducing their halflife (Gibson et al. 1991, Dauvois et al. 1992). In thepresence of ICI 182780, receptors accumulate in thecytoplasm as a consequence of a block in nuclear uptakeof the protein (Dauvois et al. 1993) and it is conceivablethat this aberrant accumulation and/or conformationtriggers degradation. It has also been proposed that ICI182780 blocks receptor dimerisation and, as aconsequence, inhibits DNA binding (Fawell et al. 1990b).These observations, however, were based on in vitroexperiments and have been questioned by a number ofworkers analysing the effects of antioestrogens in intactcells (Reese & Katzenellenbogen 1991, Metzger et al.1995).

Although it may be possible to assign all anti-oestrogens as either mixed agonists/antagonists or pureantagonists it is unlikely that their mechanism of actionwill be precisely the same as either tamoxifen or ICI182780. Partial agonists are particularly interestingbecause their agonist activity is cell specific. Thus, bothraloxifene and tamoxifen act as antagonists in breastcancer and agonists in bone while their action differs in theendometrium where only raloxifene is an antagonist(Draper et al. 1996, N N Yang et al. 1996). This differencemust reflect distinct conformations induced in the receptorby the two antioestrogens which might, for example,

Figure 4 A model for the structure of the ligand binding domain in the presence of hormone agonists and

antagonists. Although antagonists may bind with high affinity the structure of the domain may be unable to

fold to generate the surface required for the binding of other proteins and realignment of helix 12 with helices

3 and 5 may be prevented. The helix numbers are based on those descr ibed by Brzozowski et al. (1997).

These differ from those described by Wurtz et al. (1996) and presented in Fig. 2 due to the presence of an

additional helix in ER, helix 4.


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differentially affect the sensitivity of the N-terminalactivation domain to phosphorylation by other signallingpathways or alter interactions between AF1 and AF2.

Ligand independent stimulation of ERs

The transcriptional activity of the ER has been reported tobe modulated by a number of other signalling pathwaysinvolving different protein kinase cascades. The bestcharacterised of these is the ability of epidermal growthfactor (EGF) to modulate the activity of the ER. It wasinitially observed that the effects of oestrogen on themouse uterus can be mimicked by the administration ofEGF to animals and that this is a result of the activation ofthe ER in an oestrogen independent manner (Ignar-Trowbridge et al. 1992). These results have been

confirmed by analysing the uterotrophic response in ERknock-out mice, where the oestrogen-like effects of EGFare absent, demonstrating that these responses aredependent on the expression of the ER (Curtis et al. 1996).The activation of ERs by EGF has been analysed in cell

lines (Ignar-Trowbridge et al. 1993, 1996) and shown tobe mediated by the MAP kinase pathway whichphosphorylates a serine residue at position 118 in AF1 (Aliet al. 1993, Kato et al. 1995, Bunone et al. 1996).Phosphorylation of this residue seems to be required forAF1 activity but little is known about its role intranscriptional activation. Since AF1 is functional in thepresence of tamoxifen it is likely to be important in theagonist effects of this antioestrogen. Cell specificvariations in AF1 activity probably reflect differences inthe stimulation of the MAP kinase pathway in response togrowth factors and this may account for the cell-specificdifferences observed for the agonist activity of tamoxifen.Agents that increase cyclic AMP levels and stimulateprotein kinase A activity also increase transactivation bythe ER, an effect which was first demonstrated by treating

cells with the neurotransmitter dopamine (Power et al.1991). However, it is not known whether this is achievedby phosphorylation of the receptor itself or other proteinsinvolved in transcriptional activation.

Figure 5 Activation of the oestrogen receptor. The receptor may be activated by ligand binding and in addition by

stimulation with growth factors acting via MAP kinase (MAPK) to phosphorylate AF1. The receptor may also be a

target for tyrosine kinase(s) resulting in activation of AF2. Partial antagonists such as tamoxifen block ligand

dependent activity but not growth factor stimulation of AF1. Pure antagonists prevent ligand dependent activation

and may inhibit other pathways by completely blocking the action of the receptor protein.


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The ER is also modified by phosphorylation ontyrosine residues, in particular at Y537 in the humanprotein (Arnold et al. 1995), which is located at the N

terminus of helix 12 in the hormone binding domain.Phosphorylation of this tyrosine residue is not absolutelyrequired for AF2 activity since it can be replaced withphenylalanine without affecting oestrogen dependentactivation (White et al. 1997). However, analysis of othermutations introduced at this position suggests thatphosphorylation may represent an alternative mechanismfor ligand independent activation of the ER for thefollowing reason. Replacement of the tyrosine residuewith charged residues, either aspartic acid or glutamicacid, which might mimic tyrosine phosphorylation,generates constitutively active receptors (Weis et al. 1996,White et al. 1997). We think that this is likely to occur as

a consequence of the realignment of helix 12 to form theinteracting surface required to recruit coactivators.Surprisingly an alanine replacement or a serine at thisposition has a similar effect. One interpretation of theseresults is that the tyrosine residue takes part inhydrophobic interactions which maintain the receptor inan inactive state and that activation is achieved bydisruption of these interactions allowing the release ofhelix 12, analogous to the conformational change thatoccurs as a result of ligand binding. The importance of thisresidue, and the possibility that phosphorylation mayrepresent a means for activating the receptor, is suggestedfrom the sequence conservation between both ER and

ER, which have been analysed from a number ofdifferent species all of which have a tyrosine residue inthis position. However, the identification of a growthfactor/kinase pathway which both phosphorylates andactivates the receptor at this position remains elusive. Asummary of the various pathways which may be involvedin the activation of the receptor is shown in Fig. 5.

Interactions between nuclear receptorsand other transcription factors

Interactions may also occur within the cell nucleusbetween receptors and other transcription factors, for

example AP-1. This transcription factor is implicated as acritical target for many signalling pathways that regulatecell differentiation, proliferation and transformation(Angel & Karin 1991). It is important as a target forgrowth factors which stimulate the phosphorylation ofFos/Jun family members and may also play a role ingrowth inhibition by retinoids which have been shown toblock AP-1 activity (Pfahl 1993, Chen et al. 1995).Oestrogens, which stimulate the proliferation of MCF-7breast cancer cells, increase growth factor induced AP-1activity whereas antioestrogens, which inhibit growth ofthese cells, inhibit the activity of AP-1 (Philips et al. 1993,

Webb et al. 1995). A number of alternative mechanismshave been described to explain how nuclear receptors maymodulate the activity of other transcription factors. These

include the binding of receptors to DNA at compositeresponse elements in association with other factors, e.g.the control of proliferin gene expression by the gluco-corticoid receptor and AP-1 (Diamond et al. 1990), andthe oestrogen regulation of ovalbumin gene expression(Gaub et al. 1990). The recent discovery of the ability ofreceptors to associate with proteins such as CBP/p300,which themselves have the potential to make multiple con-tacts with other transcription factors, suggests a possiblemechanism for the integration of different signallingpathways at target gene promoters. For example, it hasbeen reported that the ability of receptors to repress AP-1activity results from the sequestering of CBP (Kamei et al.

1996) although other factors, as yet unidentified, have alsobeen proposed (Saatcioglu et al. 1994, 1997).

The problem of tamoxifen insensitivity andtamoxifen resistance

There is no a priori reason why ER positive breasttumours should all be sensitive to tamoxifen treatmentsince their proliferation may be completely independent ofthe activity of the ER. Similarly, tumours may becometamoxifen resistant as a consequence of further geneticchanges that allow them to proliferate in the absence offunctional ER. However, a relatively high proportion of

tamoxifen resistant tumours are still sensitive to alternateendocrine therapies suggesting that the proliferation of asubset of tumours is receptor dependent.

The molecular basis for the tamoxifen resistance ofthis type of tumour is unknown but several possibilitieshave been proposed. A point mutation has been identifiedat amino acid 537 in the human ER from a breast tumoursample in which a tyrosine residue, previously shown tobe a site of protein modification by phosphorylation, asdescribed earlier, is altered to asparagine. This generates aconstitutively active protein which appears to beinsensitive to the affects of antioestrogens (Zhang et al.1997). However, mutation of this residue to a number of

other amino acids, including alanine and serine, results inreceptor proteins which although constitutively active areinhibited by treatment with either tamoxifen or ICI182780 (Weis et al. 1996, White et al. 1997). Receptormutations (Jordan et al. 1995) and variants (Fuqua 1994,Fuqua et al. 1995) have also been proposed to account fortamoxifen insensitivity. In particular, a variant lacking theoestrogen binding domain has been reported to exhibitconstitutive activity and stimulate the proliferation ofbreast cancer cells in vitro in the presence of tamoxifen.We have been unable to confirm this observation when theexpression of this variant was induced in cell lines and we


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conclude that changes in other signalling pathways mustalso be necessary (Rea et al. 1996). One candidate is theMAP kinase pathway, which potentiates the activity of the

N-terminal activation domain, AF1, even in the presenceof tamoxifen. Tamoxifen, like oestrogen, seems topromote the binding of the receptor to stimulate theexpression of target genes and it is conceivable that AF1mediated transcription may therefore be increased in cellswith enhanced MAP kinase activity. These target genes,however, may remain sensitive to alternative endocrinetreatments. For example, ER dependent transcriptionwould remain sensitive to aromatase inhibitors becausethere would be insufficient available oestrogen to activatethe receptor. Similarly, ICI 182780 treatment would notonly reduce ER levels but may inhibit both activationdomains. Thus, the ability of alternative endocrine

therapies to block tumour growth in tamoxifen resistantpatients might reflect the failure of the receptor to activatea subset of target genes even when the N-terminal domainAF1 is stimulated by other signalling pathways.

Future prospects

An understanding of the molecular mechanisms of actionof the ER is not only beginning to provide an explanationfor the function of clinically useful antioestrogens but isalso suggesting novel therapeutic targets. These include,for example, the interactions between coactivators andreceptors which can be disrupted by specific peptides invitro (Heery et al. 1997, Torchia et al. 1997). It maytherefore be possible to prevent these interactions in vivoand block the agonistic effects of oestrogens. The twoareas in which progress has been relatively slow in thepast, namely the identification of the crucial oestrogenregulated target genes involved in cell proliferation andthe mechanism of tamoxifen resistance, remain veryactive areas of research. Hopefully, with a betterunderstanding of both the mammalian cell cycle andalternate intracellular signalling mechanisms, moreprogress will continue to be made in the future.

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