Download Steroid/Thyroid Receptor-like Proteins with

(CANCER RESEARCH 50. 451-458. February I. 1990]
Review
Steroid/Thyroid Receptor-like Proteins with Oncogenic Potential: A Review
Mels Sluyser
Division of Tumor Biology; The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Abstract
Mutated or truncated forms of certain members of the steroid/thyroid
receptor superfamily have oncogenic potential. The aberrant forms com
pete with the normal receptor for binding to the responsive element on
the DNA and thus interfere negatively with the normal transcription
control mechanism. Oncogenes that arise from dominant negative muta
tions may therefore be called "domines." to distinguish them from reces
sive types such as that causing retinoblastoma ("renoncs"). It is possible
that dononcs are also responsible for the loss of hormonal responsiveness
of some tumors during progression.
Research during the past few years has revealed the existence
of a class of genes that encode ligand-dependent DNA-binding
proteins with a zinc-binding "finger" motif. Because the steroid
have this general structure, and the superfamily also includes
genes for retinoic acid receptors, vitamin D3 receptors, and
dioxin receptors. More members of this superfamily are likely
to exist (3). The genomic organization of these genes probably
follows the pattern found for the progesterone and estrogen
receptor genes. These genes are split into 8 exons. The A/B
region is almost entirely encoded within a single exon, and each
of the putative "zinc fingers" of region C is encoded separately.
Region E is assembled from 5 exons (5, 6). Some members of
the steroid/thyroid hormone receptor superfamily are shown in
Fig. 1.
Viral erb-A and Thyroid Hormone Receptors
Viral erb-A was originally discovered when it was found that
the ability of avian erythroblastosis virus to transform erythroblasts is determined by two oncogenes, v-erb-A and v-erb-B (7this article to give a complete review of this gene superfamily,
inasmuch as excellent reviews have already been published by 9). Both erb-A and erb-B oncogenes are homologous to avian
and mammalian chromosomal DNA sequences c-erb-A and cother authors (cf. Refs. 1-3), but rather to look at certain
erb-B (10). Cellular erb-B is a truncated form of the epidermal
members that may be involved in malignant transformation
growth factor receptor (11). The erb-A oncogene potentiates
and/or tumor progression.
the transforming activity of erb-B and appears to be responsible
for the early blockage of cell differentiation within the erythroid
General Structure of Steroid/Thyroid Receptors
lineage (12). In transfection experiments, the v-erb-A oncogene
product
by itself is sufficient to transform erythrocyte cells (13).
In 1985 Sluyser and Mester (4) proposed that the products
The
human
(14) and chicken (15) c-erb-A genes, the cellular
of certain oncogenes may bear structural resemblance to steroid
counterparts of the viral oncogene v-erb-A, represent thyroid
hormone receptors. This idea was based on the hypothesis that
the loss of hormonal dependency of certain tumors might be hormone (T3) receptors.
The erb-A genes encode a cysteine-rich domain that shows
due to the appearance of mutated or truncated steroid receptorhigh
homology with the putative DNA-binding domain of ste
like proteins that act constitutively, i.e., enhance transcriptional
roid hormone receptors (13-20). The amino acid sequence of
activity even in the absence of hormone. These aberrant proteins
this domain in \-erb-A is almost identical to that of the glucowould be either produced due to mutations in steroid receptor
corticoid
receptors (21, 22) and estrogen receptors of various
genes or encoded by oncogenes that have homology with steroid
species
(23,
24). A thyroid hormone receptor called erb-A-T
receptor genes (4).
has been described for the human testis which is closely related
Subsequently, investigations in several laboratories show that
to the chicken erb-A (25).
steroid receptors have structural homology with the avian erythInterestingly, several c-er¿>-A-relatedgenes exist in the human
roblastosis virus oncogene v-erb-A.
genome
(26). Of the multiple human homologues to v-erb-A
Steroid hormone receptor proteins contain different domains
the most closely related homologue is the \\c-erb-\\ gene on
which have been designated A-F (1, 2). The assignment of these
human chromosome 17 (27) that probably encodes erb-A-T.
domains is based on degrees of homology between steroid
Another
homologue, the hc-erb-AB gene, is located on human
receptors; i.e., regions A, C, and E are highly conserved between
chromosome
3(13).
these receptors, whereas this is less the case for regions B, D,
The existence of multiple thyroid hormone receptors suggests
and F. Subsequent studies revealed that these regions serve
different functions. Domain C contains two zinc-binding "fin
that these receptors play different roles from one tissue to the
gers" that interact with DNA. Domain E is the region to which other. This is confirmed by studies in the rat where of 3 thyroid
the hormone molecule binds. The D region separating C and E hormone receptors described, one (called r-erb-Aß-2)is ex
has been called "hinge region" because it is thought to act as a pressed only in the pituitary gland (28) whereas other homo
logues of the v-erb-A oncogene are expressed in other tissues.
hinge between the DNA-binding and hormone-binding do
Of interest is that alternative splicing generates messages en
mains. The A/B domain may serve to modulate the receptorcoding rat c-erb-A proteins that do not bind thyroid hormone
DNA interactions and also may cause hormone-independent
trans-activation (see below). Thyroid hormone receptors also (29) (Table 1).
The thyroid hormone receptor, in the absence of its ligand,
Received 9/7/89; accepted 10/24/89.
blocks the activity of a responsive promoter. This suppression
The costs of publication of this article were defrayed in part by the payment
is abolished when thyroid hormone is added to the system,
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
which stimulates expression. The oncogenic analogue of the
451
and thyroid receptors belong to this class, it has been called the
"steroid/thyroid receptor superfamily." It is not the purpose of
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STEROID/THYROID
410
315
456.
639
232
169 —¿
120
53—1 102
263
RECEPTOR-LIKE
462|—l 4*
198 —¿
153
gag
ER
GR
PR
MR
VDR
T3Ru
T3R|i v-erbA
RAR
HAP
Fig. 1. Schematic amino acid comparison of members of the steroid/thyroid
hormone receptor superfamily. Primary amino acid sequences have been aligned
on basis of regions of maximum amino acid similarity. Amino acid numbers are
those for the human receptors with the exception of \-erb-\. GR, glucocorticoid
receptor; MR, mineralocorticoid receptor; VDR, vitamin D3 receptor; T¡R,thyroid
hormone receptor; RAR. retinoic acid receptor; HAP, hepatoma-associated pro
tein (epithelial type retinoic acid receptor. RAR£).The DNA-binding regions
(region C) are shaded (1-3).
Table 1 Cellular homologue to the v-erb-A oncogene in the rat
Expression+
HomologueT-erb-Aa-\ binding
Skeletal muscle, brown fat
r-erb-Aa-2"
Brain, hypothalamus
-tKidney, liver
r-erb-A0-\
r-erb-A.ß-2T3
+
Pituitary gland
" r-ero-An-2 is a truncated form of the thyroid hormone receptor that lacks
the thyroid hormone (T5) binding domain. Data taken from Ref. 29.
thyroid hormone receptor, \-erb-A, acts as a constitutive re
presser in this system and, when coexpressed with the receptor,
blocks activation (30).
The \-erb-\ product has a lower affinity than the normal
receptor for binding to a thyroid hormone-responsive element
in the long terminal repeat of Moloney murine leukemia virus,
which binds c-erb-Aa protein. Overexpressed v-erb-A protein
may therefore interfere negatively with normal transcriptional
control mechanisms (31), suggesting that in fact v-erb-A is a
transcriptional regulator run amok.
PROTEINS
corresponding to a predicted polypeptide with a relative molec
ular mass of 51 kDa (35).
Subsequent studies by Benbrook et al. (36) revealed that the
hap gene product is a retinoic acid receptor. This was demon
strated by using the "finger-swap"1 approach in which the DNAbinding ("finger") domain of the unknown receptor is ex
changed with the DNA-binding domain of a receptor for which
specific HREs have been identified. The hybrid receptor is used
to transfect susceptible tissue culture cells together with a
reporter gene containing the relevant HRE. In the transient
cotransfection experiment by Benbrook et al. (36) the chloramphenicol acetyltransferase reporter gene was linked to a
promoter containing the estrogen-responsive element. It was
then found that only retinoic acid induced chloramphenicol
acetyltransferase gene expression strongly at a physiological
concentration (2.5 x IO"8 M).
This finding is at variance with the report by de-Théet al.
(35) who stated that the hap protein did not specifically bind
retinoic acid. The apparent discrepancy is resolved by the fact
that specific binding of retinoic acids to their receptors cannot
be carried out with protein in vitro, as done by de-Théet al.,
but can be demonstrated only indirectly by using the fingerswap approach.
In the rat, this novel retinoic acid receptor gene is strongly
expressed in various organs but not liver. The size of the
transcript ranges from 2.4 to 9.5 kilobases in these organs. This
retinoic acid receptor gene shows a considerable specificity for
epithelial-type tissue including skin and has therefore been
named RARE (34). The discovery of the RARK gene apparently
contributing to a hepatocarcinoma is surprising inasmuch as
vitamin A and other retinoids are generally considered to have
antitumor activity. The RARK gene might, however, contribute
to tumor development when expressed erroneously in liver
tissue, where it is normally silent. Retinoids are known to
maintain the proliferative state of epithelial cells and this type
of cell proliferation, when induced in other types of tissues by
erroneous RARK expression in the presence of retinoic acid,
may lead to tumor development (36).
Glucocorticoid Receptor Mutants
Retinoic Acid Receptors
The discovery that the DNA-binding domain of the steroid
and thyroid hormone receptors is highly conserved has led to
the use of the DNA sequences encoding these domains as
hybridization probes to scan the genome for related, but novel,
ligand receptors. In this way a cDNA1 was cloned encoding a
polypeptide which turned out to be a receptor for the morphogen retinoic acid (32, 33).
Dejean et al. (34) found an integration of hepatitis B virus
next to a liver cell DNA sequence with a striking resemblance
to that of v-erb-A and steroid receptors. They proposed that
this gene (called hap) is usually silent or transcribed at a very
low level in normal hepatocytes but becomes inappropriately
expressed as a consequence of the hepatitis B viral integration,
thus contributing to the cell transformation.
Cloning and sequence analysis of the corresponding comple
mentary DNA from a human cDNA library revealed that the
cellular hap gene has an open reading frame of 448 amino acids,
The glucocorticoid receptor has been cloned (20), and the
functions of various domains have been analyzed (37-39). The
amino acid residues involved in attachment of the ligand have
been identified (40). Two separate sequences within the gluco
corticoid receptor, NL1 and NL2, act as signals for the hor
mone-dependent nuclear localization of the receptor (41). The
carboxyl terminus of the human receptor contains a 30-amino
acid sequence (named "r2") that functions as an activation
domain. A similar and independent activity has also been iden
tified in the amino-terminal region of the receptor. These two
sequences in the molecule are both acidic but are structurally
unrelated (42).
Removal of 29 amino acids from the carboxy terminus of the
rat glucocorticoid receptor leads to only very little (1%) loss of
hormone-inducible activity, but further deletions cause loss of
hormone-inducible activity (39, 43). While deletions up to 180
carboxy-terminal residues fail to produce a biological response
in either the presence or the absence of steroid, longer carboxyterminal deletions or truncation of the amino-terminal part of
the domain induce transcriptional activation even in the absence
of hormone. Truncation at both ends of the molecule resulted
in a polypeptide of only 150 amino acids which is still effective
in constitutive activation (43).
1The abbreviations used are: cDNA. complementary DNA; HRE, hormoneresponsive DNA element; MMTV-LTR, mouse mammary tumor virus long
terminal repeat; PR, progesterone receptor; hER, human estrogen receptor; ER,
estrogen receptor; RFLP, restriction fragment length polymorphism; SCLC,
small cell lung carcinoma.
452
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STEROID/THYROID
RECEPTOR-LIKE
Regions in the carboxy and amino termini of the glucocorticoid receptor that increase transcription but are not involved in
DNA binding may be moved to other parts of the receptor or
attached to heterologous binding domains and still maintain
function by increasing transcription (42). Such studies can also
be done using systems in which glucocorticoid receptors exert
negative effects. The results suggest that the negative effects on
transcription that glucocorticoid receptors exert in some sys
tems are generated via steric hindrance. The amino terminus is
not critical for this repression but both the DNA- and hormonebinding domains are required for efficient repression to occur
(44). These data on receptor structure and function are of
interest because certain cells (e.g., mouse T-cell lymphomas
and human lymphoblastic leukemia cells) are known to contain
mutant glucocorticoid receptors with structural defects which
causes an inability to mediate in glucocorticoid-induced cell
lysis. Because growth of the normal ("wild-type") cells is inhib
ited by adding glucocorticoids, this makes isolation of glucocorticoid-resistant cell variants quite easy. The majority of these
resistant cells possess glucocorticoid receptors with structural
and functional defects. Four major abnormalities have been
identified: r~ (receptor deficient), nt~ (nuclear transfer defi
cient), nt' (nuclear transfer increased), and act1 (activation la
bile). The r~ phenotype occurs most often and is characterized
by low or undetectable hormone binding. Some r~ cells contain
a polypeptide that cross-reacts with anti-receptor antibodies
and has the same molecular weight as the wild-type receptor
(M, 94,000). This suggests a mutation in the hormone-binding
domain (45-47). The r~ phenotype may in some cells also arise
from lack of expression of receptor with no gene product or
specific mRNA detectable (48). Whatever the cause of the r"
phenotype may be, this phenomenon is not due to gross DNA
rearrangements or deletions and therefore perhaps may be due
to point mutations (49).
The glucocorticoid receptor of the nt1 phenotype is a M,
40,000 polypeptide that represents the amino-terminally trun
cated form of the wild-type receptor. These nt' receptors are
synthesized as shorter polypeptides rather than being processed
from larger molecules. The mRNA of these truncated receptors
is about 1.5 kilobases shorter than the wild-type mRNA from
which the 5' sequences are missing. However, Southern blot
analysis did not reveal any genomic deletions or rearrangements
(47, 49). Therefore it seems likely that transcription is initiated
correctly and that aberrant splicing is responsible for the trun
cated nt' mRNA (38).
Receptors of the act' phenotype are relatively labile in the
sense that the hormone is dissociated easily when these recep
tors are activated. The act' receptors, however, behave in a
manner similar to that of wild-type receptors if the hormone is
attached covalently by cross-linking labeling and thus unable to
dissociate upon activation of the complex (50).
Glucocorticoid receptors have also been studied at the protein
level. Mild treatment with proteases generates fragments that
structurally resemble the nt' receptors of glucocorticoid-resistant cells and that resemble these nt' receptors in exhibiting
increased nonspecific affinity for DNA in vitro. However, this
does not mean that these molecules can activate transcription
since other studies show that an intact amino terminus (A/B
region) is required for the glucocorticoid receptor to be able to
activate efficiently the HRE of the MMTV-LTR (37, 51).
Analysis of the normal rat liver glucocorticoid receptor pro
tein indicates that the amino terminus is blocked (52). Gluco
corticoid receptors are phosphoproteins (53-55), and serine
(53) or threonine (55) residues are phosphorylated depending
PROTEINS
on the conditions. Whether these phosphorylations serve a role
in receptor functioning, and if so in what way, is unclear at
present (56).
Steroid receptors that are isolated from cytosols often are
found to be complexed with M, 90,000 heat shock protein (57).
There is a debate whether these complexes serve a role in
steroid hormone action or are just artifacts of the isolation
procedure. Whatever the answer may be, phosphorylation of
the receptor does not appear to play a role in the association
or dissociation of glucocorticoid receptor from M, 90,000 heat
shock protein (58). When rat thymus cells are depleted of ATP
by anaerobiosis, the specific glucocorticoid-binding capacity of
these cells disappears, and it rapidly reappears when ATP levels
are restored. In cells deprived of ATP the glucocorticoid recep
tor is present in a "null receptor" form that cannot bind
hormone and that is bound in the nuclei of the ATP-depleted
cells. It is possible that the null receptor is present as the
dominant form in r~ mutant cells such as described above (59).
Progesterone Receptor A and B Forms
PRs in chicken and human tissues are represented by two
molecular forms, A and B, with molecular weights of 79,000
and 109,000, respectively. Equimolar ratios of these A and B
forms have been found in the cytosols of chicken oviduct tubular
gland cells and in human breast cancer T-47D cells (60, 61).
Immunoanalysis and peptide mapping of the photoaffinitylabeled proteins indicate that the A and B forms are structurally
related (60, 62, 63). Furthermore, both forms bind to DNA
(62). This finding has been confirmed by DNasel footprinting
experiments using the hormone-responsive element of the
chicken lysozyme gene (64). Expression of the cloned cDNA
produced a protein that resembles the natural B form (M,
109,000).
A protein corresponding in size to form A (M, 79,000) was
produced by expressing an amino-terminally truncated receptor
starting at Met-128 or by internal initiation during in vitro
translation (65, 66).
Evidence has been presented that the A and B proteins are
derived by alternate initiation of translation from a single
mRNA transcript (67). Comparison of the sequences surround
ing the AUG triplets in rabbit (68), human (65), and chicken
(69) PRs indicates only partial conservation and does not
provide an explanation for only a single form of PR (the B
form) in rabbit, in contrast to the two protein forms (A and B)
found in humans and chickens. It has therefore been proposed
that the secondary structural context of these sequences on the
mRNA may play a role in determining whether alternate initi
ation of translation will take place (67).
The A/B region of the chicken progesterone receptor plays a
crucial role in the differential activation by progesterone of the
ovalbumin gene and the MMTV-HRE. This is shown by the
fact that whereas progesterone receptor form B preferentially
activates the MMTV-LTR promoter, its naturally occurring
amino-terminally truncated form (form A) can activate the
promoter of both genes (70).
The chicken progesterone receptor gene appears to be present
as a single copy and to consist of a 38-kilobase transcription
unit (6). An alternative polyadenylation signal is present near
the 5' end of the second intron; this results in a truncated
mRNA. The putative protein product of this variant (1.8 kilobases) mRNA would contain only the amino-terminal region
and one-half of the DNA-binding region. If actually present in
cells, such truncated receptors might compete with normal
453
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STEROID/THYROID
RECEPTOR-LIKE
receptors for available steroid-regulatory elements on target
genes. Alternatively, such molecules may exist as dangerous
cellular variants if they retain any biological activity, since the
repressive hormone-binding regulatory domain is absent (6).
obtained from MCF-7 cells contains a glycine 400 (GGG) to
valine 400 (GTG) mutation compared to the ER of a human
genomic library (5). In addition, two silent mutations (Ser-10,
TCC -»TCT; Thr-594, ACG -^ ACA) are present in the MCF7 ER cDNA. Interestingly, a glycine is found at position 400
in the chicken ER (74), mouse ER (24), rat ER (73), and
Xenopus ER (75) sequences suggesting that it is the MCF-7 ER
which differs from the wild-type sequence. The Gly-400^VaI400 mutation is in the hormone-binding domain of the cloned
hER and destabilizes the ER structure, thereby decreasing its
apparent affinity for estradiol at 25°C,but not at 4°C.This
Estrogen Receptor Variants
The hER gene has been mapped to human chromosome 6
(3, 71), and the murine ER gene to mouse chromosome 10
(72). Comparison of the ERs of various species reveals complete
(100%) homology between the DNA-binding domains of these
species (16, 24, 73, 74). The ER of the cold-blooded organism
Xenopus laevis exhibits high similarity in amino acid sequence
with the human and avian ERs. In the putative DNA-binding
region, its amino acid sequence differs at only 1 of 83 amino
acids (75).
The DNA-binding region (region C) plays a role in specific
recognition and binding to the estrogen-responsive elements of
target genes, whereas region E is indispensable for hormone
binding. Receptors that lack the hormone-binding domain,
however, still recognize specific responsive DNA elements,
while the isolated hormone-binding domain binds estradiol with
wild-type affinity (23, 76). The length of the hinge region D,
which joins the DNA- and hormone-binding domains, can be
substantially altered without affecting ER function (76).
In order to elucidate the role of the hormone-binding domains
of hER and human glucocorticoid receptor, these carboxyterminally located sequences were joined to the DNA-binding
domain of the yeast transcription factor GAL-4 (77). Stimula
tion of transcription by these chimeric receptors from GAL-4responsive reporter genes was hormone dependent. The chi
meric receptor only bound tightly to nuclei when hormone or
antihormone was present. However, the antihormone did not
cause transcription activation, indicating that the hormonebinding region of receptors has a dual function by both causing
hormone (or anti-hormone)-dependent
binding to nuclei, and
hormone (but not antihormone)-dependent
activation of tran
scription.
Of interest also was that GAL-ER [147-251] binds tightly to
nuclei even in the absence of hormone and that it competes for
GAL-4-activated transcription. Apparently, the ER sequence
147-251 does not efficiently mask the DNA-binding domain
in GAL-ER [147-251]; hence this chimeric receptor can by its
own accord attach to the GAL-4 binding sites in the absence of
hormone (77).
The transcriptional activation function located in the hor
mone-binding domain of hER is encoded by separate codons.
The results suggest that the protein surface responsible for the
activation function is generated by the three-dimensional fold
ing of the hormone-binding domain and is most likely created
from dispersed elements (78). This finding is of interest because
the location of transcriptional activation domains within steroid
receptors have been controversial. Whereas receptors that are
deleted for the hormone-binding domain show constitutive
activation, the magnitude of this activation ranges from only
5% to full wild-type activity depending on the receptor and the
experimental conditions. This indicates that the NH2-terminal
A/B region is important for activation, an idea that is also
supported by the fact that NH2-terminal deletions of the hER
(70, 76) and chicken PR (69, 79) can reduce the ability of the
receptor to activate transcription, but only on certain pro
moters, suggesting that the A/B region may be interacting with
promoter-specific factors.
It has been reported that the human ER cDNA clone pOR8
PROTEINS
point mutation may be a cloning artifact, because the valine
codon GTG was not found in the sequence of three other hER
cDNA clones derived from MCF-7 cell RNA. However, it is
also possible that a minor population of MCF-7 cells had this
mutation or that a minor fraction of hER mRNA is mutated
posttranscriptionally
(80). RFLP has been reported in ER
genes. With restriction enzyme PvuU, absence of a 0.6-kilobase
restriction fragment was found to be associated with ER-negative human breast cancer cells; instead the ER-negative cells
had a restriction fragment of 1.6 kilobases. The RFLP is
probably located within sequences encoding the DNA- or hor
mone-binding regions (81). The finding that ER-positive and
ER-negative breast tumors differ significantly in RFLP is of
interest since it suggests that ER activation or lack of activation
is associated with different alÃ-eles(designated PI and P2). The
molecular basis of this phenomenon is unknown, but it might
affect the proper splicing of ER mRNA. There is the possibility
of point mutations being responsible for the effect. Perhaps
sequence analysis of ER mRNA from breast cancer patients
may elucidate this matter (81).
Pssl restriction enzyme analysis has identified a single twoallele polymorphism in which the 1.4-kilobase alÃ-eleoccurs in
91% of North American Caucasians and the 1.7-kilobase alÃ-ele
in 9% (82).
Multiple ER mRNA variants have been reported for X. laevis.
The Xenopus ER encodes 4 mRNAs approximately 9, 6.5, 2.8,
and 2.5 kilobases long. It is possible that these derive from
different polyadenylation signals (75). Transcription initiation
sites at 10 major sites have been reported close together for the
mouse ER gene; however, in the mouse uterus only a single ER
mRNA transcript is observed (24). Of interest is that when the
mouse ER cDNA is tested for functional activity by transfection, it shows not only hormonally dependent activity but also
some constitutive activity. The latter may possibly be due to
the presence of mutated or truncated receptors (24, 83).
The transcripts required to encode a protein the size of the
human ER protein (M, 65,000) is about 2 kilobases. In human
uterus the size of the ER mRNA is, however, found to be about
4.2 kilobases, and therefore about one-half of the mRNA ap
pears to be untranslated (82). This is also the case with the ER
mRNA of MCF-7 human breast cancer cells which show a short
5' and a very long 3' untranslated region (16, 17).
The degree of transcriptional stimulation by truncated mu
tants of human ER is dependent on cell type and promoter
context (84).
ER Proteins
Studies at the protein level have revealed ER variants in
various tissues. Such studies generally involve covalent labeling
of cytosolic ER with ['HJtamoxifen aziridine in the presence of
protease inhibitors and subsequent identification of the labeled
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STEROID/THYROID
RECEPTOR-LIKE
receptors by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Investigations of this type carried out with mouse uterus
demonstrated a major ER component (M, 65,000) with minor
fragments with molecular weights of 54,000 and 37,000. Per
haps the low molecular weight forms originate from the A/r
65,000 holoreceptor by proteolytic degradation, but the possi
bility that the M, 65,000, 54,000, and 37,000 species are differ
ent gene products cannot be ruled out (85). Similar experiments
in our laboratory using ['H]tamoxifen aziridine tagging of es
trogen-dependent and -independent GR mouse mammary tu
mors show essentially similar results. In these tumors we de
tected M, 65,000, 50,000, and 35,000 receptors. Of interest was
that there was a shift towards the low molecular weight forms
when these mouse tumors became hormonally independent
during serial transplantations in syngeneic mice.2
A protease with a-chymotrypsin activity that increased the
affinity of ER for binding to DNA has been described in
mammary tumors of C3H mice (86). However, there is also
evidence for a specific hormonally regulated modification of
ER. The response, as detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, is the formation of a closely
spaced ER doublet (M, 65,000 and 66,500) in nuclei of the
mouse uterus (87). A possible explanation for the doublet might
be a phosphorylative mechanism, inasmuch as it has been
reported that ER from calf uteri is phosphorylated and dephosphorylated by a nuclear kinase and a cytosolic phosphatase
(88). It should be pointed out that even if some of the low
molecular weight forms of ER observed in some tissues turn
out to be proteolytic cleavage products of the holoreceptor, this
proteolysis may have functional significance. The cleavage is at
the border regions of the functional domains and may serve to
separate these regions. Furthermore, it has been reported that
the ER molecule itself has proteolytic activity that is responsible
for its own transformation to the active state (89).
PROTEINS
The loss of hormonal dependence in breast cancer is due to
the emergence of autonomous cell clones that progressively
achieve dominance in the tumor mass (90, 91). This apparent
heterogeneity of the tumors suggests that combined chemohormonal therapy would be a more effective treatment for breast
cancer than either single modality alone. Animal studies indi
cate this to be the case; however, these studies also show that
even the combined treatment does not cure but only extends
the latency period of the tumor (92, 93). This is also observed
in human studies where the combined approach causes higher
response rates and longer relapse-free intervals than either
modality used singly. Even with chemoendocrine combinations,
however, complete remissions remain relatively uncommon and
cure of metastatic diseases remains impossible (94). The reason
for this disappointing result apparently is that the cytotoxic
drugs used in studies thus far are not sufficiently effective in
destroying the highly malignant subclones that emerge during
the tumor progression. It seems likely that breast cancer pro
gression is a highly complex affair, resulting in the emergence
of increasingly malignant cells by natural selection. The ques
tion therefore is, "Where do these cells come from and what is
the mechanism by which they are able to achieve dominance?"
outgrowth of the tumor and the progression steps involves
different oncogenes. Several oncogenes have been implicated in
human mammary cancer including myc, ras, src, neu, and
perhaps others (95). Many of the chromosomes frequently
altered in human breast cancer (i.e., chromosomes 1, 3, 6, 7,
and 11) contain sequences which encode human cellular onco
genes. Examples of this are c-blim, N-ras, and c-ski on chro
mosome 1; c-ra/on chromosome 3; c-myb on chromosome 6;
c-erbB and c-met on chromosome 7; and H-ras and c-ets on
chromosome 11 (96). It has been reported that c-myb expression
shows a strong association with human breast cancers that have
high estrogen receptor levels (97). The myb gene, like the ER
gene, is on human chromosome 6 (98) and mouse chromosome
10 (72). Whether these genes are within the same region on
these chromosomes is not known. If this turns out to be the
case, it raises the interesting possibility that the myb oncogene
is involved in the mechanism by which levels of ER are en
hanced in hormone-dependent breast cancers. Kasid et al. (99)
have reported that when human breast cancer MCF-7 cells were
transfected with v-Ha-ras, these cells no longer responded to
exogenous estrogens in culture and were fully tumorigenic
without estrogen supplementation when tested in nude mice.
These transformed cells still had high levels of ER. The authors
concluded that MCF-7 cells that acquire an activated ras gene
can bypass the hormonal regulatory signals that trigger the
neoplastic growth of the breast cancer cell line. Interestingly,
transfection of the v-Ha-ras oncogene into MCF-7 cells causes
these cells to secrete increased levels of several growth factor
activities constitutively, suggesting that growth of hormonally
independent breast cancer cells might be stimulated by these
growth factors (100). Cultured ER-negative cell lines constitu
tively secrete relatively high concentrations of several growth
factors such as transforming growth factor a, insulin-like
growth factor I, platelet-derived growth factor, an epithelial cell
colony-stimulating factor, mammary-derived growth factor, and
autocrine motility factor (95). Secretion of several of these
growth factor activities in ER-positive lines is regulated by
estrogen: estrogen deprivation or antiestrogen treatment re
duces the growth factor secretion; whereas estrogen administra
tion increases it (95). Thus, ER-negative cells might have a
growth advantage due to constitutive growth factor secretion.
However, results by Osborne et al. (101) are at variance with
this hypothesis. These authors inoculated ER-negative MDA231 human breast cancer cells in castrated female athymic nude
mice and found that the resulting tumors did not support
growth of MCF-7 cells inoculated in the opposite flank, which
required estrogen supplementation for growth. In this model
system, growth factors therefore were not capable of replacing
estrogen for growth stimulation of MCF-7 breast cancer cells.
These data make it seem unlikely that the loss of estrogen
dependence in breast cancer can be explained simply by the
constitutive production of growth factors that replace estrogen
in stimulation of cell growth. If therefore growth factors are
not responsible for sustaining hormone-independent
growth,
how is this growth sustained? We have proposed that mutated
and/or truncated steroid receptor-like proteins act as constitu
tive activators (4). Of interest is the fact that estrogen receptor
variants have been reported in human mammary cancers and
that the relative amounts of these proteins increase with loss of
hormonal dependency (102-104).
Assuming that a breast tumor originates from a single trans
formed breast tissue cell, it seems likely that the subsequent
Conclusions
Loss of Hormonal Dependence
' B. Moncharmont.
Duijvcnbode,
C. C. J. De Goeij, G. Ramp,
and M. Sluyser, manuscript
A. W. M. Rijkers.
S. van
In recent
in preparation.
field
>ears
of Steroid
we
have
hormone
seen
action.
a tremendous
The
exciting
revolution
in the
discovery
of the
455
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STEROID/THYROID RECEPTOR-LIKE PROTEINS
relationship between the structures of steroid and thyroid re
ceptors with that of the viral oncogene v-erb-A makes it tempt
ing to speculate that activation of some of these receptor genes
is implicated in oncogenesis.
That \-erb-A can exert its specific effects on erythroblasts
even though it cannot bind thyroid hormones apparently is due
to changes in hormone binding domain E of c-erb-A having
occurred. It seems possible that these mutations prevent the E
region from masking the DNA-binding region (domain C),
thereby permitting constitutive binding of the receptor to DNA.
In the progesterone receptor gene there is an alternative
polyadenylation signal near the 5' end of the second intron,
and this could result in a truncated mRNA. The putative protein
product of this variant (1.8 kilobases) mRNA would contain
only the NH2-terminal domain and one-half of the DNAbinding domain of the complete receptor and would lack hor
mone-binding activity. If actually present in cells, such trun
cated receptors might compete with normal receptor forms for
available steroid-regulatory elements on target genes and thus
have oncogenic potential (86). Of interest also is the difference
between the complete progesterone receptor (form B) and its
truncated form (form A) in its ability to activate specific genes
(70).
The question can be raised whether the low molecular weight
forms of steroid receptors that have been found in various
tissues serve a physiological function. It is important to estab
lish which of these forms are due to changes at the genomic or
mRNA level and which are caused by secondary events, e.g.,
phosphorylation or proteolysis. Proteases have been reported
that can degrade estrogen and progesterone receptors selectively
(105). Steroid receptors are particularly susceptible to proteases
at sites in the molecule linking the various domains, and it is
possible that proteolysis serves a physiological function by
causing these regions to be released in the cell. It is also possible
that changes in steroid receptor genes cause increased suscep
tibility of the encoded proteins to endogenous proteases. In
addition to the estrogen-dependent
rra/w-activation domain
located at the COOH-terminal region of ER, the ER molecule
also contains a /rani-activation domain located in the NH2terminal region which is active even if no hormone is present
(104). One can conjecture that when COOH-terminally mu
tated ER molecules are released in cells, this may lead to
constitutive activation of transcription. Of interest is the report
that gestodene, a synthetic progestin, binds to the ER of malig
nant breast tumors but not to the ER of normal breast, liver,
pancreas, or endometrium. This might be indicative of a malig
nant change in the ER of breast neoplasms (106).
Of interest also is the report that restriction fragment length
polymorphism in the ER gene is associated with the presence
or absence of a functional ER protein in human breast cancer
cells (81).
The presence of abnormal ER variants in breast cancer (102,
103, 107) suggests that a subclassification of tumors based on
functional abnormalities of ER may predict refractoriness to
hormone therapy. Abnormal methylation of the ER gene re
duces ER mRNA levels in human endometrial carcinomas ( 108)
and this may be the case in breast cancers.
It has been reported that malignant human prostatic tissue
contains a 4-5S androgen receptor form that is not present in
normal prostate (104). Besides being involved in liver carcinogenesis (hap gene) and induction of avian leukemia (\-erb-A),
certain members of the steroid/thyroid receptor family may
play a role in other specific types of cancer. Changes in the
coding region of the DNA-binding domain of glucocorticoid
receptors have been reported between mouse lymphoma cells
and an androgen-dependent
mouse mammary tumor (109).
Therefore, changes in steroid receptors at the genomic level
may be associated with certain malignant states.
Deletions of chromosome 3 have been reported in SCLC and
many non-small cell tumors of the lung (110). In the case of
SCLC a consensus deletion (3p21—>25)has been defined which
contains a putative suppressor gene (111). The thyroid hormone
receptor T^Rß(c-erb-Aß)locus maps to this region on chro
mosome 3, and the erb-Aßhybridization to DNA extracted
from these tumors is reduced, suggesting that either T-tRß
or
another erô-A-related gene found at this locus may be the
suppressor gene. It is also possible that another suppressor gene
is located at this locus, perhaps the RARßgene which maps to
chromosome 3p24 (112). It has been proposed that in SCLC,
both alÃ-elesof the c-erb-A gene are inactivated, one by a
chromosomal deletion and the other by a more subtle mutation.
Dononcs
Herkskowitz (113) has proposed that some oncogenes may
be examples of naturally occurring dominant negative muta
tions. Both dominant and recessive mutations are known to
cause cellular transformation. Recessive mutations, such as that
causing retinoblastoma, involve loss of wild-type functions. By
contrast, \-erb-A may be an example of a dominant negative
mutation, inasmuch as this oncogene acts as a constitutive
repressor and, when coexpressed with the thyroid-hormone
receptor, blocks activation of the thyroid hormone-regulated
gene (30). Thyroid hormone action is also inhibited by a nonhormone-binding form of the c-erb-A protein that is generated
by alternative mRNA splicing (29, 114).
Oncogenes that arise from dominant negative mutations may
therefore be called "dononcs," to distinguish them from reces
sive types such as that causing retinoblastoma ("renoncs"). In
conclusion, steroid receptors have moved from the role of
prognostic guides and guides to therapy into a new role, that of
potential pathogenic factors in oncogenesis. In the next few
years we can expect to be able to define the structures and
function of the abnormal transcripts of these and other DNAbinding "finger" proteins and to establish their roles in onco
genesis and tumor progression in specific types of cancer.
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Steroid/Thyroid Receptor-like Proteins with Oncogenic
Potential: A Review
Mels Sluyser
Cancer Res 1990;50:451-458.
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