Cli. Biochem, Vol. 25, pp. 407-414, 1992 Printed in the USA. All rights reserved.

0009-9120/92 $5.00 + .00 Copyright © 1992 The Canadian Society of Clinical Chemists.

Role of Estrogen Receptor Variants in the Development of Hormone Resistance in Breast Cancer MELS SLUYSER Division

of Tumor

Biology, The Netherlands Cancer Institute, Plesmanlaan 1066 CX Amsterdam, The Netherlands

Recent evidence suggests that the progression to hormone resistance in some breast tumors is due to mutations in the estrogen receptor (ER). Various types of ER variants have been found in breast cancer biopsies and breast cancer cell lines. The ER variants include dominant-positive receptors that are transcriptionally active in the absence of estrogen, and dominantnegative receptors that are themselves transcriptionally inactive but prevent the action of the normal receptor. The mechanisms by which these variants cause loss of hormonal control is becoming clear. ER variants may be prognostic factors for breast cancer. By modifying the action of ER variants, it should be possible to develop new strategies for treatment of malignant breast disease.

121,

Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; EIA, enzyme immuno-assay; HPLC, high performance liquid chromatography; ERE, estrogen responsive element; wt, wild-type; RFLP, restriction fragment length polymorphism; GR mice, Grunder strain mice; HD, hormone-dependent; HI, hormone-independent. Manuscript received November 30, 1990; revised May 20, 1992; accepted May 22, 1992.

endocrine response because about 12% of E R + / PgR+ breast tumors do not show a hormonal response (2). Some tumors are E R - / P g R + (2); this is surprising, because normally one might expect that tumors that are negative for ER would also be negative for PgR. Why are there these discrepancies between hormone receptor levels and hormone response, and why do tumors that initially respond to endocrine therapy develop resistance to this treatment? In 1985 we proposed that these phenomena are caused by mutated or truncated estrogen receptors that act constitutively, i.e., enhance transcriptional activity even in the absence of hormone. The action of these deviant receptors would resemble that of oncogenes and we therefore suggested that when steroid hormone receptors would be sequenced, they would turn out to have structural homology with certain oncogenes (3). This hypothesis proved to be correct when later it was discovered that steroid receptors are members of a superfamily of DNA-binding proteins with a "zinc-finger" motif (4). This superfamily also includes receptors for thyroid hormone (T3), retinoic acid, vitamin D3, dioxin, and several "orphan receptors" whose ligands are not yet known. The viral oncogene v-erb-A is a member of this class of proteins; v-erb-A is the malignant counterpart to the cellular thyroid hormone receptor c-erb-A (5). Not only v-erb-A, but also various other members of the nuclear hormone receptor superfamily exhibit oncogenic potential (6). The structure of the estrogen receptor (consisting of regions A - F ) is depicted in Figure 1. In the absence of hormone, the receptor is believed to be associated with a 90 kDa heat-shock protein (hsp90) via domain E. Upon binding of estradiol, the ER dissociates from hsp90 and binds as a homodimer to the estrogen-responsive element (ERE) on the nuclear DNA. Regions E/F of the receptor serve as the dimerization site. The receptors bind to the ERE via their C region (DNA-binding domain). Transcription activation of the estrogen-responsive gene then takes place via domains A/B, C, and E of the receptor.

CLINICALBIOCHEMISTRY,VOLUME25, DECEMBER1992

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KEY WORDS: estrogen receptor variants; hormone dependency; breast cancer. Introduction

strogen receptor protein is an important progE nostic factor for patients with breast cancer. The presence of estrogen receptor (ER) correlates with response to endocrine therapy in patients with metastatic disease, and is associated with prolonged disease-free and overall survival in patients with primary disease. Tumors lacking ER and progesterone receptor (PgR) generally grow faster than tumors containing ER and PgR. This may explain why these receptor-negative tumors behave more aggressively. However, the correlation between ER positivity and endocrine dependence is not perfect. Approximately 40% of ER-positive tumors fail to regress with endocrine therapy. It has been hypothesized that PgR may be a more effective marker of endocrine responsiveness, since PgR is the end product of estrogen action (1), But even PgR, or PgR in combination with ER, does not give a perfect correlation with

SLUYSER

.2N A I

B

Icl°l

I t- oo.

Function

binding of hsp i

nuclear translocation

I I l I

dimerization DNA binding transcription activation

Figure 1 -- Structure of the estrogen receptor protein and functional domains. This article discusses data (summarized in Table 1) indicating that mutations take place in the ER of m a m m a r y tumors, and that the resulting ER variants contribute to the development of hormone resistance in breast cancer. TABLE 1

Estrogen Receptor Variants in Breast Cancer

Conclusions Exon 5-deleted ER trans-activates constitutively Frame-shift mutations in domains C and E Exon 7-deleted ER inhibits binding of wtER to ERE Exon 3-deleted ER inhibits binding of wtER to ERE Two point mutations in B-domain. No effect on ER status Nonfunctional exon 3-deletion ER mutant 50 and 3 5 - 3 7 kDa ER variants in mouse mammary tumors 47 kDa ER variant in human breast cancer Rapid assay of ER mutants with HPLC ER gene amplification in 6/14 cases No ER gene amplification or rearrangement Differences in ER gene methylation in ER+ and E R - tumors PvuII RFLP in ER gene in 14/20 males PvuII RFLP correlates with age, not with ER expression PvuII RFLP in intron 1. No correl, with age or ER expr. HindIII RFLP correlates with PgR expression No change in ERmRNA Nucleotide mismatch in ER causes low estrogen binding Small-size ER mRNA encodes truncated 37 kDa ER protein Exon 2 intron boundary change in ER ER detected immunologically in HI tumors Aberrant interaction of ER with cell nucleus Weak or no binding of ER variants (50 kDa) to ERE Difference in ER phosphorylation between hormone-dependent and -independent tumors

408

Reference 7 8 9,10 11 47 9 23,24 27 25 12 13 19 14 16 17 18 48 20,49 21,22 22 26 28,30 29 34

Estrogen receptor variants ER MUTANTS

IN B R E A S T C A N C E R

About 2% of all breast tumors are E R - / P g R + . The ER - / P g R + phenotype could result from a variant ER that is unable to bind hormone and thus appears negative in a ligand-binding assay, but is still f u n c t i o n a l in s t i m u l a t i n g t h e e s t r o g e n responsive PgR. This transcriptionally active ER variant might be a potential mechanism for a tumor's escape from hormone control. Experimental data, in which ER mRNA was reverse transcribed and the resulting ER cDNA was sequenced, support this notion. Studies by F u q u a et al. (7) demonstrated the presence of an ER lacking exon 5 (which encodes part of the hormone-binding domain). This E R I E 5 variant was the predominant ER m R N A expressed in three E R - / P g R + tumors. The ERSE5 constitutively activated transcription of a normally estrogen-dependent gene construct in yeast cells. Studies by Graham et al. (8) with T47D h u m a n breast cancer cell lines showed the presence of ER m u t a n t s in hormone independent E R - / P g R + sublines. Their experiments revealed three ER complementary DNA mutants, including two frame-shift/termination mutants that would encode ERs truncated in the DNAbinding domain and in the hormone-binding domain, and a third m u t a n t with a large in-frame deletion spanning the hinge region and a part of the hormone-binding domain. If expressed, these mutant ERs would lack hormone-binding capacity and would be undetected by the anti-ER antibodies currently in clinical use. Some ER variants are not themselves transcriptionally active, but prevent the action of the normal receptor. This is the case for an ER variant lacking exon 7 that has been detected in ER + / P g R - breast tumors (9,10). Since exon 7 encompasses part of the hormone-binding domain, the variant itself presumably does not bind hormone. Besides the ERSE7, the ER + / P g R - tumors therefore presumably also contain an estrogen-binding receptor that is not able to induce PgR. The exon 7-deleted ER variant inhibited the binding of wild-type (wt) ER to its cognate response element (10). Studies by Wang and Miksicek (11) with T47D cells showed a number of variant ERs, one of which lacked exon 3 encoding the CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992

ESTROGEN RECEPTOR VARIANTS

second zinc finger of the DNA-binding domain. This variant inhibited transcription activation in a dominant negative fashion when cotransfected with the wt ER and reporter plasmid, and also inhibited DNA binding of w t E R in a gel mobility shift assay in vitro. Since this ERSE3 is not able to bind to its response element, the observed inhibitory effect probably occurs through p r o t e i n - p r o t e i n interactions. This could involve the formation of a heterodimer between mutant and wt receptors, competition for a limiting transcription factor, or both. Certain mutations in the estrogen receptor gene may cause nonfunctional ER. Cases of nonfunctional mutants have been reported, e.g., an exon 3 deletion variant that is inactive in the yeast expression vector assay (9), and two deletion mutants, ERSE2 and ERSE7 that had little or no effect on trans-activation by the full-length estrogen receptor (11).

change. Using an RNase protection assay, Garcia et al. (20) found a nucleotide mismatch in the B-coding region that correlated with low ligand binding, in 8 of 66 ER-positive tumors. The mismatch corresponded to a C to T transition at nucleotide 257, resulting in an alanine to valine substitution. Smaller size ER mRNA variants resulting from deletions of the hormone-binding domain were reported by Murphy and Dotzlaw (21) due to the introduction of 84 unique amino acids at the exon 3 boundary (amino acid 253). This sequence was followed by a stop codon resulting in a truncated 37 kDa protein (21,22). The same group found another ER variant with an insertion of six unique amino acids at the exon 2 intron boundary (amino acid 214), followed by a stop codon for a total of 220 amino acids (22). E R PROTEIN V A R I A N T S

ER

G E N E A M P L I F I C A T I O N AND RESTRICTION F R A G M E N T L E N G T H POLYMORPHISM

W h e r e a s some a u t h o r s r e p o r t e d s t r u c t u r a l changes in the ER transcripts, others did not find a

Variant ER proteins have been detected in mamm a r y t u m o r cytosols. In our l a b o r a t o r y , 3Htamoxifen aziridine affinity-labeling studies of mammary carcinomas of the Grunder (GR) inbred mouse strain have revealed that the 65 kDa fulllength (wt) receptor is replaced by 50 kDa and 35 kDa ER proteins when the tumors progress from hormone dependence to hormone independence during serial transplantations (23). In some GR transplant series, the 65 kDa ER is completely replaced by 50 kDa ER in the hormone-independent transplant passages, whereas in other tumor lines there is a partial replacement and both the 65 kDa and low molecular forms are observed. ~-Chymotrypsinlike protease activity generally is lower in the hormone-independent tumors than in the hormonedependent tumors, indicating that the lower molecular size of ER in the hormone-independent tumors cannot be attributed to the increased level of this protease activity (23). Studies on MXT mouse mammary tumors also provide evidence for low molecular weight ER variants. Experiments involving labelling of ER with 3H-tamoxifen aziridine and antiER monoclonal antibodies revealed the presence of 65, 50, and 37 kDa estrogen receptors in MXT tumor cytosols (24). These ERs disappeared during transplantation, and were replaced by a 35 kDa band in late hormone-independent passages. We have used GR mouse mammary carcinomas as a model for evaluating the effect of ER variants, present in the tumor cytosols, on the prognostic reliability of the receptor assay. With high performance liquid chromatography (HPLC), it is possible to separate the w t E R from low-molecular weight forms of ER that still bind hormone. HPLC is a rapid method for determining the relative levels of the hormone-binding ER forms. Our data indicate that whereas both wt and low molecular weight ER bind estradiol and react with antibody directed to the hormone-binding domain in the Abbott enzyme immunoassay (EIA) test, only the full-length receptor causes estrogen-responsive growth of the tumors.

CLINICALBIOCHEMISTRY,VOLUME25, DECEMBER1992

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Amplification of the estrogen receptor gene has been reported in breast cancer by some investigators, whereas others did not find ER gene amplification. Nembrot et al. (12) reported a 1.6- to 3-fold amplification in 6 of 14 cases, whereas Koh et al. (13) did not find ER amplification or rearrangement in 34 breast cancer patients. A P v u I I restriction fragment length polymorphism (RFLP) was reported in the ER gene of 14 of 20 males by Castagnoli et al. (14), and according to Hill et al. (15) this RFLP correlated with ER expression in 188 breast cancer patients. Parl et al. (16) however found the P v u I I RFLP to be correlated with age, but not ER expression. In a follow-up study by the latter group (17), the P v u I I RFLP was located within intron 1, and this time no correlation with either age or ER expression was seen in 260 breast cancer patients. A H i n d I I RFLP of the ER gene was reported by Wanless et al. (18) in a small percentage of breast cancer patients, and this correlated with PgR expression. ER GENE METHYLATION

Falette et al. (19) has reported that normal breast and adjacent tumor tissue differ in methylation pattern of the ER gene. A difference in ER gene methylation was also found between ER-positive and ERnegative tumors. However, there was no difference in receptor activity as a function of methylation. We have detected differences in methylation patterns of genomic ER between different passages of mammary tumors in GR mice (Sluyser et al. unpublished). E R MESSAGE

SLUYSER

This suggests that prognostic clinical tests, currently in use for estimating ER in mammary tumors, may be impaired by the presence of low molecular ER variants in the tumor samples (25). Immunocytochemical studies have revealed the presence of estrogen receptors in hormone nonresponsive mammary tumors of C3H mice (26). Variant estrogen receptor protein forms have also been found in human breast cancer cytosols. Whereas 36% of the tumors contained only the full-length 65 kDa receptor, 49% contained both 65 kDa and 47 kDa ER in variable amounts. The remaining 15% of breast tumors contained only the 47 kDa ER. The ability to induce PgR in response to estrogen was correlated with the presence of the 65 kDa ER (27). Immunocytochemical studies of human breast tumors show two types of defective ER: those that are unable to bind to the nucleus in a hormone-filled state and those that bind to the nucleus as naked ER (28). Gel retardation assays in vitro showed that the ER of some ER-positive tumors did not bind (or bound only weakly) to a synthetic ERE. This decrease of E R - E R E interaction was associated with a 50 kDa variant dimer or a 50/67 kDa heterodimer of wt plus variant (29). Anti-receptor antibodies have been used to characterize the nuclear binding of ERs in vitro. Studies on ER-positive breast tumors, revealed that tumors in which the ER exhibited abnormalities in nuclear binding behavior (ligand-independent nuclear binding or no nuclear binding) failed to respond to hormone therapy (30). See Table 1 for a summary of reports on ER variants.

low estradiol binding in some mammary tumor cytosols may be due to the low phosphorylation of the ER present in these cytosols (34).

Discussion

A possible role of phosphorylation in steroid hormone receptor function has long been recognized. Based on the effects of ATP on hormone binding and activation of steroid receptors, it has been suggested that the steroid binding as well as transformation of steroid receptors is influenced by phosphorylationdephosphorylation processes (31,32). Whereas most phosphorylations in steroid hormone receptors take place at serine residues, the estrogen receptor is phosphorylated at tyrosine; tyrosine residue(s) crucial for hormone binding have been localized in the hormone-binding domain of ER (33). The effect of phosphorylation on the hormonebinding capacity of ER has been investigated by us in hormone-dependent and hormone-independent mammary carcinomas of GR mice. Our data suggest that the ER of hormone-independent tumors generally is less phosphorylated in vivo than the receptor of hormone-dependent tumors, so that the receptor of the former tumors is more susceptible to ATP phosphorylation and ATP-induced estradiol binding. Western blots of ER with antiphosphotyrosine antibody showed that in the hormone-independent tumors, the large ATP-induced increase of hormone binding to ER was associated with phosphorylation of tyrosine on the ER. These data indicate that the

The estrogen receptor mutants found in mammary tumors probably reflect changes in genomic ER sequence, since they do not correspond to normal splicing pattern of ER mRNA. The ER variant lacking exon 5 described by Fuqua et al. (7) lacks part of the hormone-binding domain. Although this ERIE3 variant was the major component of ER mRNA in some of the tumor samples, w t E R mRNA was also found in these samples indicating that the variant was due to an error-prone splicing event. Since tumor cells show heteroploidy and chromosomal rearrangements, some of the ER variants may result from exon skipping or chromosomal deletions causing the loss of a single exon in individual ER alleles. Wang and Miksicek (11) have argued that exon loss may be a naturally occurring event in primary breast tumors. Why do some ER m u t a n t s act constitutively, whereas others act as dominant repressors or do not appear to function at all? The estrogen receptor stimulates transcription by means of distinct functions, performed by intra-receptor sequences, including a transcription-activation function in the DNA-binding region and two nonacidic transcription activation functions, TAF-1 in the A/B region and TAF-2 in the E/F region (35) as shown in Figure 1. Both TAF-1 and TAF-2 are exhibited by the w t E R (36-38). TAF-1, in the N-terminal domain, is constitutively active while the activity of TAF-2, in the hormone-binding domain, requires the binding of estradiol. Studies on mouse ER have shown that the TAF-2 activity requires a region in the C-terminus of the hormone-binding domain between residues 538 and 552; this region is conserved among many nuclear hormone receptors and is also essential for transcriptional activation by other steroid receptors, e.g., the glucocorticoid receptor (39). Similar to the glucocorticoid receptor, the estrogen receptor contains acid transactivation functions; the latter are distinct from the nonacidic functions TAF-1 and TAF-2 (40). The ER also contains a constitutive DNA binding function that enables the ER to bind and thereby repress other activators of the ERE in the absence of estradiol (38). Estrogen receptor variants with inactivated TAF2, apparently use their intact TAF-1 to activate estrogen-responsive genes in an estrogen independent manner. An example of this would be the ERIE5 variant (7) that activates constitutively in yeast expression studies. The detection of this ERIE5 in E R - / P g R + breast tumors could explain the apparent paradox of the E R - / P g R + breast cancer phenotype in some instances. The ERSE7 v a r i a n t detected in E R + / P g R breast tumors (9) did not have transcriptional activity by itself, but inhibited the wtER. ERIE7 is a

410

CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992

PHOSPHORYLATION OF ER

ESTROGEN RECEPTORVARIANTS naturally occurring, alternatively spliced ER variant that would potentially encode an ER protein with a molecular weight of about 52 kDa (9). Since this E R I E 7 lacks a major part of the hormonebinding domain, it presumably does not bind estradiol and therefore cannot be responsible for the low molecular weight ER proteins found in tumor cytosols that are detected by ligand binding (23,24,27). The cause of the latter protein variants remains uncertain. It is not clear whether these 50 and 35 kDa ERs are due to mutations at the genomic DNA and mRNA level, or to proteolysis. It is also possible that mutations in the ER gene encode protein variants that are more susceptible to proteolysis than the wt receptor. When ERIE7 cDNA is expressed in yeast cells, the variant interferes with the normal transcriptional activation by w t E R in a dominant negative manner (9). Since ERSE7 still has the DNA-binding

domain, it might compete with w t E R for binding to the ERE. However, ERE interference cannot be the mechanism by which the E R I E 3 described by Wang and Miksicek (11) inhibits wtER, since their m u t a n t contains an inactivating deletion within the DNAbinding domain and as a consequence does not bind to ERE. It has been proposed that the E R I E 3 m a y exert its d o m i n a n t n e g a t i v e effect b y p r o t e i n protein interactions via a direct association between ERSE3 and wtER, but studies to detect this association have been unsuccessful (11). A mechanism for the repression by ERSE3 could be by formation of a nonproductive complex with a limiting component of the transcription machinery ("squelching"). TAF-1 and TAF-2 of h u m a n ER have been shown to interact with factors indispensable for mediating their activation function. The nature of these factors is unclear but it has been suggested that they are intermediary factors interposed between the enhancer

VARIANT NUCLEAR HORMONE RECEPTORS AS CONSTITUTIVE ACTIVATORS OF GENE TRANSCRIPTION

GENE FOR GROWTH REGULATORY FACTOR

NUCLEAR H O R M O N E RECEPTOR GENE

I

t

I hormone controlled cell growth

uncontrolled cell growth

Figure 2 - - The nuclear hormone receptor gene encodes a receptor that, upon binding of hormone, activates a gene for a growth regulatory factor (above). The receptor variant, however, activates the gene constitutively that results in uncontrolled growth (below). CLINICAL BIOCHEMISTRY,VOLUME 25, DECEMBER 1992

411

SLUYSER

factors (close to the ERE) and the basic factors (close to the hormone-regulated gene). TAF-1 and TAF-2 regions of ER might contact strings of these intermediary factors (40). Structural and functional analysis of estrogenregulated genes have revealed a common palindromic ERE, with the consensus sequence of 5'G G T C A N N N T G A C C - 3 ' . However, most EREs are imperfectly palindromic, although in this case they are less efficient in mediating transcriptional regulation by the receptor and they weakly bind the receptor. To explain the mitogenic effect of estrogen, it is assumed that the hormone regulates the expression of genes whose products control the cell cycle. In line with this hypothesis, estradiol has been found to induce the expression of c-fos, c-jun, and c-myc proto-oncogenes (41). Of interest is the finding that an ER m u t a n t lacking the hormone-binding domain activates the c-fos gene promoter in the absence of estrogen (42). In this way some ER mutants might influence the cell cycle. Some estrogen-responsive genes are activated by ER by a mechanism involving the F o s - J u n complex, a constituent of activator protein 1 (AP-1). An example is the proximal region of the chicken ovalbumin gene that contains a half-palindromic ERE that binds a nucleoprotein complex (AP-1) containing oncoproteins c-Fos and c-Jun. The latter proteins, together with ER, coactivate the ovalbumin promoter. ER variants lacking their DNA-binding domain can still function in the co-activation with

c-Fos and c-Jun, indicating that binding of ER to the ERE is not required. This suggests that ER directly interacts with the c-Fos/c-Jun (AP-1) complex (43). Overexpression of c-Jun or c-Fos proteins (but not of J u n D, also part of AP-1) inhibits ER activity in MCF-7 h u m a n breast cancer cells (44). This mechanism may also contribute to the loss of hormone response in some breast cancer cases. Conclusion There is more than one way in which ER can be involved in the loss of hormone control. Some ER mutants act in a dominant positive m a n n e r by causing cell proliferation without the requirement of estradiol (Figure 2). Others act as dominant negative oncogenes ("dononcs") by competing with the normal wt receptor for interaction with the ERE, or interfering with transcription factors that are necessary for normal ER action. That impairments in ER cause an increase in malignancy suggests that normally the ER is involved in growth control by activating a gene that is not activated by the ER variant, so that in the latter case the tumor growth is not suppressed (Figure 3). M a m m a r y cancer is a very heterogeneous disease (45,46), and perhaps there are also other ways, not directly involving ER, that hormone resistance m a y develop, e.g., because of the activation of oncogenes such as jun, fos, ras, or myc, or to mutations in the control regions of hormone-regulated genes that im-

VARIANT NUCLEAR HORMONE RECEPTORS AS DOMINANT NEGATIVE ONCOGENES (Dononcs)

NUCLEAR HORMONE RECEPTOR G E N E

I

TUMOR SUPPRESSOR GENE

ONCOGENE

II activated

t ~

hormone

Donor,: (dominant negative mutant)

,

j

J

activated

Figure 3 - - Activation of a hypothetical tumor suppressor gene by wild-type nuclear receptor (above). The tumor suppressor gene is not activated by the nuclear receptor mutant so malignant growth is not suppressed (below). 412

CLINICAL BIOCHEMISTRY,VOLUME 25, DECEMBER 1992

ESTROGEN RECEPTORVARIANTS pair growth control. F u t u r e research will have to determine the relative importance of these putative cellular defects in causing hor m one resistance in m a l i g n a n t breast disease.

altered expression of the progesterone receptor. Anticancer Res 1991; 11: 139-42.

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CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992