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Endocrinological Oncology |
Neuroendocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. Sushela S Chaidarun, Neuroendocrine Unit, Bulfinch 457, Massachusetts General Hospital, Boston, Massachusetts 02114.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2ER to 7ER, have been identified in breast cancer and
have been postulated to have important pathogenetic and clinical
implications in tumorigenesis and/or development of hormone resistance.
Because pituitary tumors, particularly prolactinomas, are known to be
E2-dependent, we investigated alternatively spliced ER
variant messenger ribonucleic acid expression in 40 human pituitary
tumors of various phenotypes and normal pituitary tissues, using
reverse transcription-PCR and Southern blot analyses. Nine of 11
prolactinomas readily expressed multiple ER variants (2ER, 4ER,
5ER, and 7ER), whereas 6 of 11 tumors showed faint expression of
3ER. Four of 7 glycoprotein hormone-producing tumors that
synthesized FSHß expressed 2ER, 5ER, and 7ER. In 9 GH- and
10 ACTH-secreting tumors examined, the expression of normal and variant
ER was restricted to tumors that also exhibited scattered PRL
immunoreactivity. Variant and normal ER were not found in three null
cell tumors (oncocytomas) that showed negative immunoreactivity for all
pituitary hormones or their subunits. In contrast, only 4ER and
7ER were uniformly detected in normal pituitaries. 6ER was not
detected in any normal or neoplastic pituitary specimen studied. We
conclude that multiple alternatively spliced ER variants are
coexpressed with normal ER in a tumor phenotype-specific manner. In
addition, ER variants 2ER and 5ER were found to be tumor
specific. Future functional studies will be required to determine
whether coexpression of multiple ER variants along with normal ER
confers a pathophysiological role in pituitary hormone regulation
and/or tumor cell proliferation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mitogenic and regulatory effects of E2 are mediated through its nuclear receptor (ER), a ligand-activated transcriptional factor of the steroid receptor superfamily. ER is encoded by eight exons and is composed of several functional domains important for transcriptional activation function as well as DNA and ligand binding (7, 8). Sequence analysis of the ER gene reveals strong homology to the viral v-erbA oncogene, suggesting that ER is a cellular homolog of this oncogene (9). In addition to its well characterized role as an E2-activated transcription factor, ER has recently been shown to interact directly with peptide growth factors, independent of E2 (10, 11). Therefore, ER may mediate cellular proliferation through a number of intracellular mitogenic signaling pathways.
In other hormone-dependent tumors such as breast cancer, several
altered ER isoforms generated by alternative messenger ribonucleic acid
(mRNA) splicing (termed ER variants) have recently been identified
(12, 13, 14). Some of these ER variants have been shown to have
differential effects on E2-responsive gene expression. For
example, an exon 5 ER spliced variant (5ER) that lacks most of the
hormone-binding domain of the receptor has been shown to constitutively
activate E2-responsive genes (15). Coexpression of this
variant with the wild-type ER in MCF-7 human breast cancer cells
confers resistance to the estrogen antagonist tamoxifen (12, 13).
Furthermore, an ER isoform lacking most of the hormone-binding domain
that is structurally similar to 5ER has been shown to activate
c-fos promoter independent of E2 administration
(16). Therefore, this variant may stimulate cell proliferation
independent of E2 and may also contribute to clinical
antiestrogen resistance. In contrast, an exon 3 ER spliced variant
(3ER) that lacks a portion of the DNA-binding domain has been shown
to have a dominant negative effect on wild-type receptor
transcriptional activation when cotransfected with wild-type receptor
in HeLa cells (17). This variant may, therefore, be able to modulate
E2 sensitivity in the tumor cells. Differential exon
alternative splicing of the ER gene, therefore, can give rise to a
variety of variant receptor isoforms that may potentiate the diverse
actions of E2 through a single receptor gene.
In the pituitary, wild-type ER mRNA has been recently demonstrated in
normal and adenomatous lactotrophs and gonadotrophs (18, 19). However,
the expression of ER variants in normal and neoplastic pituitary cells
has never been studied. Altered ER gene expression in
E2-sensitive pituitary adenomas may modulate normal ER
function affecting both neoplastic cell proliferation and hormone
secretion. Therefore, we investigated the expression of all potential
exon alternate splice variant mRNAs, designated 2ER to 7ER, in 40
human pituitary adenomas of different phenotypes and normal pituitary
tissues, using reverse transcription-PCR (RT-PCR), dideoxy sequencing,
and Southern blot analyses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pituitary adenomas were obtained from 40 patients who underwent
transsphenoidal surgery. Patients with clinically nonfunctioning
adenomas (n = 10) ranged in age from 37–79 yr (median, 54 yr),
and all had normal serum glycoprotein hormone (GPH) and free
-subunit levels with serum PRL levels of less than 100 µg/L.
Immunocytochemical staining using specific antibodies against GPH
subunits (common , FSHß, LHß, and TSHß) was positive in 7 of
10 tumors (i.e. GPH tumors), whereas antibodies against GH,
PRL, and ACTH were negative. The other three tumors were negative for
all specific pituitary hormone antibodies (i.e. null cell
tumors). Patients with macroprolactinomas (n = 11) ranged in age
from 17–60 (median, 27 yr), and all had elevated PRL levels (210 to
>8000 µg/L) and immunostaining consistent with the diagnosis.
Patients (n = 9) with GH-secreting adenomas ranged in age from
27–78 yr (median, 38 yr), and all had clinical and biochemical
evidence of acromegaly, with elevated serum levels of GH (9–90 µg/L)
and insulin-like growth factor I (699–1233 µg/L). All somatotroph
adenomas showed strong immunostaining for GH, and 4 tumors also showed
rare to scattered PRL staining (GH/PRL tumors). Patients with
corticotroph tumors [n = 10; 5 microadenomas (diameter, <1 cm)
and 5 macroadenomas] ranged in age from 20–59 yr (median, 48 yr), and
all microadenomas had dexamethasone suppression testing and petrosal
catheterization results consistent with Cushing’s disease. All
corticotroph adenomas exhibited positive immunostaining for ACTH,
whereas only one tumor also showed scattered staining for PRL (ACTH/PRL
tumor).
To ensure that surgical specimens were not contaminated with a significant number of normal pituitary cells, all PRL-, GH-, and ACTH-secreting as well as null cell tumors studied were negative for LHß mRNA expression in RT-PCR assay (data not shown). All GPH-producing tumors were negative for Pit-1 mRNA, a member of the POU domain of transcription factors that is specifically expressed in GH, PRL, and TSH cells.
The control normal pituitary tissues used in these studies were obtained within 3–6 h postmortem and snap-frozen in liquid nitrogen (National Disease Research Interchange, Philadelphia, PA). The normal human pituitary complementary DNA (cDNA) library (Clontech, San Diego CA) represents pooled mRNA from nine men and women, aged 15–83 yr, from tissue obtained 1–3 h postmortem.
RNA extraction and RT-PCR analysis of mRNA
Pituitary adenomas were obtained in phosphate-buffered saline after transsphenoidal surgery and frozen in liquid nitrogen. Total RNA was extracted using a single step, acid guanidinium isothiocyanate phenol/chloroform technique (20), followed by enzymatic digestion of genomic DNA with 1 U RQ1 deoxyribonuclease/µg total nucleic acid at 37 C for 1 h (Promega, Madison, WI) and quantitated by UV spectrophotometry. To prepare cDNA, 1 µg total RNA was reversed transcribed in 50 mmol/L Tris-HCl (pH 8.3), 5 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 0.25 mmol/L spermidine, 200 µmol/L deoxy-NTPs (dNTPs), and 12 U AMV reverse transcriptase (Promega), with random hexamers (1 µg) as first strand cDNA primers. RT reactions were carried out at 25C for 10 min, followed by a 10-min elongation step at 42 C and heat inactivation at 99 C for 5 min.
To screen the entire ER gene for all possible single exon-spliced
variants, we used six sets of specific primers covering each exon
region that would detect both normal and variant ER (Fig. 1
and Table 1). Oligonucleotide primers
were designed using Oligo software (National Biosciences, Minneapolis,
MN) and compared to GenBank sequence libraries to assure specific
amplification of human ER cDNA. Reactions without AMV reverse
transcriptase were also carried out with each RNA sample to exclude
genomic DNA contamination as a source of amplified signal. All tumors
were negative for receptor signal in minus RT reactions (data not
shown). To control for potential nonspecific RNA degradation in
pituitary tumor RNA preparations, samples were tested for the presence
of GAPDH mRNA by PCR, and all were positive. Oligonucleotide primers
for control PCR of human glyceraldehyde phosphate dehydrogenase
(GAPDH), Pit-1, and LHß were (5'-3'): GAPDH-U, gag cca gat cgc tga
gac; GAPDH-L, ttc tcc atg gtg gtg aag; Pit-1U, cat tta ctt cgg ctg ata;
Pit-1L, agg ttg atg gct ggt ttc; LHß-U, gct cca ggg gct gct gct; and
LHß-L, cga cag ctg aga gcc aca ggg.
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-32P]dCTP (100 nCi/reaction) in the
PCR reactions. PCR primers (12.5 pmol) were used for each reaction, and
amplifications were carried out in an MJ thermocycler (MJ Research,
Watertown, MA). All reactions were amplified for 35 cycles (1 min at 94
C, 1 min at the optimal annealing temperature, and 1 min and 15 s
at 72 C). All amplified products were fractionated by 6% nondenaturing
Tris/borate/EDTA PAGE (Protogel, National Diagnostics, Atlanta, GA) and
exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) for 1–12
h. Sequence analysis of ER variants
To confirm the identity of each ER splice variants, specific PCR products of the appropriate sizes were isolated from a 1% agarose gel using GlasPac/GS purification kit (National Scientific, San Rafael, CA) and cloned into pGEM-T plasmid vectors (Promega). Nucleotide dideoxy sequencing was performed according to the manufacturer’s instructions using either a universal 40 primer or specific ER primers and Sequenase version 2.0 (U.S. Biochemical Corp., Cleveland, OH).
Southern blot analysis
To determine whether pituitary tumors express ER isoforms with
multiple exon deletions, we performed Southern blot analysis of ER PCR
products encompassing exons 1–6 and exons 4–8 using specific ER
primers U1n/L6c and U4n/L8c, respectively (Table 1
). PCR reaction was
performed without radioactive labeled nucleotides, and the products
were fractionated by TAE-1.5% agarose gel electrophoresis and
transferred to a nylon membrane. Southern blot hybridization was
performed as previously described (21). ER cDNA probes appropriate for
each ER fragment were prepared by random hexamer labeling with
[-32P]dCTP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR analysis of normal and alternatively spliced ER variant
mRNAs in 40 human pituitary tumors is summarized in Table 2. Full-length ER mRNA was detected in all lactotroph
adenomas and 57% of the GPH-producing adenomas. Normal ER mRNA was
also found in four mammosomatotroph adenomas with mixed GH/PRL
immuno-staining and in a single corticotroph tumor with scattered
PRL immunoreactivity, but was not detected in pure somatotroph tumors
or corticotroph tumors, which exhibited positive immunostaining for
only GH or ACTH, respectively.
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shows RT-PCR data for 2ER mRNA expression in
lactotroph and GPH-producing pituitary tumors. 2ER and the expected
wild-type ER mRNA were detected in the majority of lactotroph and
GPH-producing tumors (82% and 57%, respectively). Of the 7
GPH-producing tumors, only adenomas that immunostained for FSHß
(tumors 1, 2, 5, and 7) expressed both normal and 2ER mRNA (Fig. 2B). In contrast to the high prevalence of 2ER expression found in
these pituitary subtypes, 2ER was not detected in somatotroph,
corticotroph, or null cell tumors (Table 2). 3ER was barely detected
in only 6 of 11 prolactinomas and a single mammosomatotroph adenoma.
Control RT-PCR of GAPDH mRNA was positive in all tumors (data not
shown).
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shows RT-PCR data for normal ER as well as
4ER through 7ER mRNA expression in 11 lactotroph tumors. 4ER
mRNA (Fig. 3A) was detected in the majority of lactotroph adenomas
(82%). 4ER mRNA was also detected in 50% of mammosomatotroph
adenomas with mixed GH/PRL immunostaining, 1 GPH-producing tumor, and 1
corticotroph adenoma with mixed ACTH/PRL immunostaining. It was not
detected in any of the pure somatotroph tumors or corticotroph tumors,
which exhibited positive immunostaining for only GH or ACTH,
respectively. 4ER was not detected in any null cell tumor studied
(Table 2). 5ER mRNA (Fig. 3B) was abundantly expressed in 82% of
the prolactinomas studied and was also found in 57% and 50% of
GPH-producing and mixed GH/PRL tumors, respectively. However, 5ER
mRNA was not observed in any of the other pituitary tumor subtypes
examined. RT-PCR analysis using specific primers to detect 6ER mRNA
expression in lactotroph adenomas exhibited only a single band
representing normal ER mRNA (Fig. 3C). Furthermore, no 6ER mRNA was
detected in any pituitary tumor sample studied (Table 2). 7ER mRNA
expression was detected in all lactotroph adenomas (Fig. 3D) as well as
in 57% of GPH-producing and all mixed GH/PRL adenomas (Table 2).
However, 7ER mRNA could not be amplified from any other human
pituitary tumor subtype.
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ER spliced variant expression in normal human pituitaries
The expression of alternatively spliced ER variant mRNAs in four
normal human pituitary specimens and a normal human pituitary cDNA
library pooled from nine normal men and women is shown in Fig. 4. In contrast to the observed expression of 2ER in
lactotroph and GPH-producing tumors, 2ER mRNA was undetectable in
all normal pituitaries studied (Fig. 4A). All normal pituitaries except
normal sample 1 expressed 4ER mRNA at levels comparable to those
found in lactotroph and GPH-producing tumors (Fig. 4B). However, 5ER
mRNA was not detected in the pooled normal human pituitary cDNA library
and was only barely detected in two normal pituitary samples (Fig. 4C).
This low level of expression in normal pituitaries was in contrast to
the readily detectable expression of 5ER variant mRNA in lactotroph
and GPH-producing tumors. 7ER mRNA was expressed in all normal
pituitary specimens studied at levels comparable to those of the tumors
(Fig. 4D). 3ER and 6ER mRNA were not detected in any normal
pituitary specimens studied (data not shown).
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Representative sequence data of the truncated ER variants, 2ER
and 5ER, are shown in Fig. 5. These data confirm that
exons 2 and 5 are excluded from the ER mRNA. Both exon 1–3 and exon
4–6 alternative splice events introduce novel nonsense mutations,
interrupting the open reading frame. This observed alternate splicing
of the ER mRNA results in truncated ER protein isoforms due to a
premature stop codon introduced by frame-shifting of the wild-type open
reading frame. 2ER encodes a severely truncated ER protein that
retains only the A/B domain of the hormone-independent transcription
activation region, whereas 5ER protein possesses the A/B domain and
the DNA-binding region, but lacks most of the hormone-binding domain.
In contrast, 3ER and 4ER sequence were joined in-frame and
encoded for full-length ER proteins that lack the second zinc finger
DNA-binding and hinge regions, respectively, whereas 7ER lacks only
the carboxyl-terminal portion of the hormone-binding domain (data not
shown).
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To determine whether pituitary tumors express unique ER isoforms
with multiple exon deletions, we performed Southern blot analysis of ER
PCR products encompassing exons 1–6 and exons 4–8 using specific ER
cDNA probes appropriate for each fragment, as shown in Fig. 6A. Hybridization of a specific ER cDNA probe to
Southern blots containing ER exon 1–6 PCR products from 11
prolactinomas (Fig. 6B) revealed the variant bands of the expected size
for each single exon splicing (2ER, 4ER, and 5ER) along with
the wild-type ER mRNA. No larger deletion bands compatible with
specific ER isoforms with multiple exon deletions were identified by
either the product size or sequence analysis. Southern blot analysis of
ER exon 4–8 PCR products (Fig. 6C) also confirmed the presence of only
single exon-spliced variants (5ER and 7ER). These data confirm
the presence of multiple ER isoforms generated by a single exon
splicing mechanism in human pituitary adenomas and suggest that an
alternative mRNA-splicing event resulting in ER isoforms with multiple
exon deletions is rare in pituitary tumors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6ER, were coexpressed with
wild-type ER mRNA in a tumor phenotype-specific manner. Prolactinomas
displayed the greatest diversity of variant ER mRNA expression,
including 2, 3, 4, 5, and 7ER, although 3ER mRNA was
expressed at much lower levels. GPH-producing tumors that synthesized
FSH also expressed multiple ER spliced variants (2ER, 5ER, and
7ER), particularly 2ER, which expressed at levels comparable to
those of the wild-type ER mRNA. Variant and normal ER were not found in
any other tumor phenotype, except for mixed GH/PRL and ACTH/PRL tumors,
in which low levels of both normal and variant ER were also detected.
Interestingly, only 4ER and 7ER were uniformly expressed in
normal pituitaries at levels comparable to those in the tumors. 2ER
and 5ER, which were abundantly expressed in lactotroph and
gonadotroph tumors, were nearly undetectable in normal tissues. Using
Southern blot analysis, no multiple exon deletion of more than a single
exon was identified. These data demonstrate that the expression of
certain alternatively spliced ER variants in human pituitary tumors is
both tumor specific and tumor phenotype specific, and that a wide array
of ER isoforms can be detected in the majority of PRL- and
GPH-producing tumors. Although multiple isoforms of ER variants are coexpressed with wild-type ER in human pituitary tumors of lactotroph and gonadotroph origins, their functional consequences are unknown. Because full-length ER is comprised of several functional domains important for DNA binding, hormone binding, and maximal transcription activation, deletion of a specific domain due to alternative mRNA splicing may give rise to a variant receptor with altered receptor activity. ER variants may have differential biological effects at several levels 1) by competing the ability of wild-type ER to bind with high affinity to E2, 2) by affecting the formation of stable ER homodimers after E2 binding, or 3) by altering the trans-activation of ER at E2-responsive gene promoters. Therefore, differential exon alternative splicing of the ER gene can give rise to a variety of variant receptor isoforms that may potentiate the diverse actions of E2 through a single receptor gene.
Little is known about the 2ER variant that encodes only the A/B
domain of hormone-independent transcriptional activation function.
Sequence analysis indicates that the protein product encoded by this
variant is severely truncated and lacks domains critical for binding
estrogen response elements or E2. However, the A/B domain
of the receptor has been shown to be important for stimulating
transcription from certain E2-responsive genes such as pS2
(7), c-fos (22), and C3 (23) and is also critical for growth
factor interactions (cross-talk) with ER signaling pathways (10).
Moreover, functional analysis of an ER mutant containing only the A/B
domain similar to 2ER has been shown to be highly effective in
repressing Fos-mediated transcription in HeLa cells. Fos protein is
known to antagonize transcription of the c-fos gene promoter
induced by ER (22). Therefore, the 2ER truncated receptor may retain
selective transcriptional activity and act as a coactivator or
repressor of wild-type ER and may also play a role in cross-coupling
between the nuclear receptor and other growth signal transduction
pathways.
5ER was also found to be expressed in PRL- and GPH-producing tumors
and was not readily detected in most normal pituitary tissues studied,
including a normal pituitary cDNA library obtained from nine men and
women. This variant has been shown to have constitutive transcriptional
activity on E2 response elements and confer
tamoxifen-resistant growth in human breast cancer MCF-7 cells (13, 15).
A mutagenesis-generated ER isoform lacking most of the hormone-binding
domain structurally similar to the 5ER-encoded protein has also been
shown to activate c-fos promoter independent of
E2 administration (16). Therefore, 5ER might also have
constitutive activity and function to up-regulate the growth
factor-induced transcriptional response in human pituitary tumors.
However, whether this ER variant possesses similar transcriptional
activity in pituitary cells remained to be determined.
Two ER variants (3ER and 6ER) were not readily detectable in any
pituitary specimens. 3ER, which lacks a portion of the DNA-binding
domain, has been shown to have a dominant negative effect on wild-type
receptor transactivation function in HeLa cells (17). However, the
absence of 3ER in human pituitary suggests that this dominant
negative isoform may not have a significant pathophysiological role in
human pituitary tumorigenesis. 6ER, which lacks a portion of the
hormone-binding domain, has never been observed in any normal or
neoplastic human tissue type (14, 24, 25). Our results, which failed to
detect 6ER mRNA in normal and adenomatous human pituitary specimens,
are consistent with these studies.
Two ER variants (4ER and 7ER) were found in both normal and
neoplastic pituitary, similar to those in other tissues. A 4ER
variant has been shown to be expressed in both normal and tumor cells
of breast, uterus, and brain (25). This ER variant lacking the hinge
region and a small fraction of the hormone-binding domain of the
receptor was unable to bind E2 or a synthetic
E2-responsive element and was transcriptionally inactive in
transfection assays using human embryonic carcinoma P19EC and chorionic
carcinoma JEG3 cell lines (25). In contrast, 7ER has been shown to
have a dominant negative effect on wild-type ER in a yeast expression
vector system (26). However, 7ER was detected in both normal and
adenomatous pituitary specimens in our studies and has also been found
in both normal and neoplastic breast tissues (14) as well as human
meningioma (25).
Our data demonstrate the following. 1) Multiple, alternatively spliced
ER variant mRNAs lacking a single exon were coexpressed with wild-type
ER in a tumor phenotype-specific manner. 2) The majority of
prolactinomas as well as a subset of GPH-producing adenomas that
synthesized FSH expressed 2ER, 4ER, 5ER, and 7ER. No ER
variants were found in somatotroph, corticotroph, or null cell tumors,
and 6ER was not detected in any tumor studied. 3) Normal pituitaries
uniformly expressed only 4ER and 7ER in addition to normal ER,
whereas 2ER and 5ER mRNA appeared to be pituitary tumor specific.
Coexpression of variant and wild-type receptors is compatible with
potential interactions between ER and its variant isoforms in
estrogen-sensitive pituitary cell types. Specific ER variants may have
a role in modulating E2 sensitivity in normal and
adenomatous cells, whereas others may be involved in promoting aberrant
cell growth and abnormal hormone production in the tumors. Therefore,
the identification of ER variant mRNAs in human pituitary tumors is a
first step in investigating their functional significance in the
pituitary as well as their potential role in modulating pituitary
neoplastic cell growth and hormone biosynthesis.
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Footnotes |
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2 Recipient of the Massachusetts General Hospital Medical Discovery
Award.
Received November 7, 1996.
Revised December 13, 1996.
Accepted December 26, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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