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Endocrinology |
Department of Medicine, College of Medicine, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß, mediate genomic
effects in target cells. The aim of this study was to determine the
relative mRNA expression levels for ER subtypes and ERß isoforms in
colon tumors, normal colonic mucosa, and colon cancer cell lines. ER
and ERß isoform mRNA levels were investigated in paired samples of
colon tumors and normal mucosa from 26 patients using comparative
reverse transcription-PCR and then Southern analyses.
Constitutive steroid hormone receptor mRNA levels were determined for
five colon adenocarcinoma cell lines using reverse transcription-PCR,
and ERß levels were further studied in Caco-2 cells using Northern
and Western analyses. ERß mRNA steady-state levels (relative to
glyceraldehyde-3-phosphate dehydrogenase mRNA) were significantly
decreased in colon tumors compared with normal mucosa in female
patients. ERß1 and ERß2 isoform mRNA levels were significantly
decreased in tumors from female patients, and ERß1 mRNA levels were
also significantly lower in tumors from female patients compared with
tumors from males. ER mRNA levels were much lower than ERß levels
and were similar between normal mucosa and tumor samples in both
genders. ERß mRNA was detected in Caco-2, T84, and SW1116 cell lines
and all lines were essentially negative for ER mRNA. Caco-2 cells
coexpressed ERß1, ERß2, and ERß5 mRNA, though a single protein
transcript was observed. ERß protein was detected in normal colonic
superficial epithelium, vascular smooth muscle and endothelium, and
enteric neurons by immunohistochemistry. These data show that ERß is
the predominant ER subtype in the human colon and that decreased levels
of ERß1 and ERß2 mRNA are associated with colonic tumorigenesis in
females. This information suggests that activation of ERß-mediated
processes in the superficial colonic epithelium may have a role in the
preventive effects observed for female gender and ERT usage. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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130,000 new cases of colon cancer will be diagnosed and about
50,000 people will die each year from colon cancer. Mortality rates
for colon cancer have fallen during the past 20 years because of early
detection from increased screening. Colon cancers include nonhereditary
and hereditary types. Sporadic colon cancers comprise the vast majority
of cases, and incidence rates increase logarithmically after age 40.
Hereditary colon cancers include familial adenomatous
polyposis and
HNPCC.3
Human colon cancers undergo a multistage carcinogenesis pathway from
adenomatous polyps to carcinoma (reviewed in Refs. 2
and
3
). A number of genetic events have been characterized and
include alterations in "tumor suppressor" and susceptibility genes
that normally encode for proteins regulating cell cycle progression and
programmed cell death (4)
. The adenomatous polyposis
coli gene and mismatch repair genes are altered
early in the neoplastic process, either as inherited or somatic
mutations. Additional somatic mutations in the transforming growth
factor ß receptor, the K-ras oncogene and the
deleted in colon cancer and p53 tumor suppressor
genes may occur during further progression. The probability that
a normal colonic epithelial cell will acquire a somatic gene alteration
is low, but 50% of the population at 70 years of age will develop a
polyp of which 5% are expected to develop into adenocarcinomas
(4)
. Patients with HNPCC develop polyps that often
progress to cancers because of defective DNA mismatch repair
genes that result in increased mutation rates.
Given the high incidence of colon cancer in the aging population and high mortality rates for advanced disease, new prevention strategies are needed. A possible protective effect for estrogens on colon cancer risk has been suggested by numerous epidemiological and experimental studies. At all ages, women are less likely than men to develop colon cancer (5, 6, 7) . Male rodents have higher aberrant crypt or tumor formation rates compared with females in several colon cancer models (8, 9, 10) . The protective effects of female hormones are also evident in families with HNPCC, because the lifetime risk of developing colon cancer is significantly lower in females than in males (30% versus 74%, respectively; Ref. 11 ). Preliminary data have been reported as well in the mouse colon cancer model for familial adenomatous polyposis that show reduced tumor numbers in intact females compared with ovariectomized females (12) .
ERT (alone or in combination with progestins) is estimated to reduce colon cancer risk by 30–40% in postmenopausal women (13, 14, 15, 16, 17) . In a recent review of 30 studies including case-controls or cohorts with 3 meta-analyses, 23 studies reported a protective effect, whereas only 1 study reported an adverse effect of ERT (18) . The majority of studies from 1995 or later showed protective effects, and these studies tended to control for more confounding variables such as aspirin use or smoking. The risk reductions were generally similar among recent ERT versus those who were on ERT for >5 years. The protective effects of ERT on incidence and size of polyps have also been reported (19, 20, 21) . Some investigators attribute the greater decline in colon cancer mortality rates in aged women compared with men to the increased use of ERT since 1990 (16 , 17) .
Estrogens modulate sexual development and reproductive functions in
addition to effecting the cardiovascular and central nervous systems
and bone (reviewed in Ref. 22
). Genomic effects of
estrogens are mediated by at least two related members of the steroid
receptor superfamily, ER and ERß. ERs act as ligand-activated
transcription factors and modulate gene expression by interactions with
promoter response elements or other transcription factors
(23)
. Studies are uncovering a diversity of functions for
each ER subtype. With either ER subtype, transactivation at an estrogen
response element is similar between 17ß-estradiol and the
antiestrogens tamoxifen and raloxifene. At an activating protein 1
response element, 17ß-estradiol increases reporter activity with
ER but inhibits it with ERß. However, with the antiestrogens,
transactivation via the activating protein 1 element is decreased with
ER and increased with ERß. These interactions are further
compounded by the ability of ER subtypes to form homodimers (/,
ß/ß) or heterodimers (/ß) and by cell-type specific expression
of ER coactivators and corepressors (24, 25, 26, 27, 28, 29)
.
To date, few studies have examined expression of ERß in the GI tract.
Studies from this laboratory using Northern analysis showed that ERß
mRNA was expressed as multiple transcripts and in greater abundance
than ER in the rat upper GI tract (30)
. Both ER and
ERß mRNA were detected in the epithelium of the stomach and upper
intestine by RT-PCR. Enmark et al. (31)
reported that ERß mRNA was detected by RT-PCR throughout the human GI
tract including colon. By in situ hybridization, ERß mRNA
was localized in the GI epithelium, whereas the muscle layers were
devoid of staining. In the midgestational human fetus, ER and ERß
mRNAs were coexpressed in stomach and colon with lower levels in small
intestine, as determined using RT-PCR (32)
. Moore et
al. (33)
described five ERß isoforms with different
COOH-terminal domains attributable to differential splicing at the exon
7–8 junction. Some nucleotide sequences were homologous between
ERß2, ERß4, and ERß5 isoforms in the 3' region of exon 8. In
normal human colon, ERß1, ERß2, and ER5 mRNA were detected
(33)
. In contrast, three colon cancer cell lines expressed
only ERß2 and ERß5 mRNA. ERß3 and ERß4 mRNA were detected only
in testis. Fiorelli et al. (34)
reported
expression of ERß1 mRNA in colon cancer cell lines and ERß2–5 mRNA
in HCT8 and HCT116 lines. Arai et al. (35)
also
reported that colon cancer cell lines express ERß mRNA but did not
study isoform levels.
The potential coexistence of ER subtypes and ERß isoforms increases the degree of complexity for determining ER-mediated functions. Analyses for ER subtypes in normal colon would be important for understanding mechanisms for potential protective effects of estrogens on colon cancer risk. Given the scarcity of information regarding ER mRNA expression levels in human colon, this study was performed to determine the relative mRNA levels of ER subtypes and ERß isoforms between paired samples of normal human colonic mucosa and colon tumors. ERß mRNA and protein levels were also analyzed in several colon cancer cell lines to determine whether ERß expression patterns were similar to normal colon. Immunohistochemistry was used to localize cell-specific distribution for ERß protein in normal colon.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Tumor samples included 3 polyps and 23 adenocarcinomas (14 moderately
differentiated). The normal mucosa was harvested adjacent to the tumor
or from distal resection margins. All patients gave informed consent in
accordance with the University of Florida Institutional Review Board or
other institutions using Declaration of Helsinki guidelines. Samples
were provided by the University of Florida Cancer Tissue Bank (UF,
n = 14) or purchased from the National Cancer
Institute Cooperative Human Tissue Network (NCI, n = 12). Patients were randomly selected from each source by gender
without exclusion for race. Medical histories were not obtainable,
however; only one female patient was 34 years of age (potentially
premenopausal), whereas the remainder were >52 years of age. One male
patient was 38 years of age and the remainder were >48 years of age.
After dissection by the pathologist, portions of tumor and the normal
mucosa were frozen promptly and stored in liquid nitrogen. Ten samples
from UF were stored for 2–3 years before assay, whereas other samples
were stored for 2–8 months before assay. Histopathology confirmed that
the tumor samples were comprised of 90% tumor cells, and that the
normal mucosa samples did not contain pathology.
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Oligonucleotide Primers.
Oligonucleotide primer pairs were designed for ER, ERß, PR, VDR,
and GAPDH using published literature or sequence information contained
in the National Center for Biotechnology Information GenBank database
with OLIGO 4.0 software (National Biosciences, Plymouth, MN; Table 2
). Oligonucleotide primers were tested using BLAST software to confirm
gene specificity and to determine exon locations (36)
.
ERß primer sets were designed to detect a region of the
NH2 terminus that is shared by all isoforms or to
detect specific exon 8 sequences to differentiate ERß1 from ERß2,
ERß4, and ERß5 isoforms. To compare mRNA levels on a
semi-quantitative level, the ER primer pair efficiencies were first
tested using full-length human cDNA as templates. PCR amplifications
were performed in parallel using 10-fold dilutions of each respective
template. Linearity of the integrated density signal for patient sample
ER and GAPDH was tested using a range of cDNA template amounts. PCR
primer pairs were selected that showed linear amplification rates using
30 (GAPDH) or 45 (ER) amplification cycles at a 55°C annealing
temperature. PCR products from two to three tissue samples were cloned
to verify sequence identity. Nucleotide sequences were determined by
automated sequencing at the University of Florida Interdisciplinary
Center for Biotechnology Research.
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0.5 g) were pulverized in a mortar and pestle
cooled with liquid nitrogen and homogenized in guanidine thiocyanate.
Cancer cells were washed with PBS and guanidine thiocyanate was added
to the culture dish. Poly(A)+-select mRNA was
prepared from Caco-2, HT29, and MCF-7 cells and normal colon samples
using poly-dT conjugated magnetic beads (PolyAT-tract kit, Promega) as
described (30)
. RNA concentration and quality were
assessed by spectrophotometric readings at 260 and 280 nm. Samples of
total RNA or poly(A)+mRNA were fractionated on a
6% formaldehyde-1.2% agarose gel and photographed after ethidium
bromide staining. The RNA samples were transferred to nylon membranes
using overnight capillary blotting in 20x SSC and were covalently
cross-linked to the membrane with UV light. Membranes were prehybridized for 15 min at 62°C in a solution containing 62 mM Na PPi (pH 7.2), 1 mM EDTA, 7% SDS, and 1% BSA in a rotating oven (38) . Human cDNA fragments were isolated by restriction enzyme digestion and gel electroelution before labeling with 32P-dATP by random primer extension (Decaprime, Ambion). The probes were denatured by boiling for 10 min and then added to the prehybridization solution. The membranes were hybridized overnight at 62°C and washed twice in 40 mM Na PPi (pH 7.2), 1 mM EDTA, and 5% SDS for 15 min at 65°C. Autoradiography was performed at -75°C with an intensifying screen for 0.5–5 days. Membranes were stripped with 0.5x SSC-0.5% SDS at 95°C between hybridizations.
Comparative RT-PCR and Southern Analysis.
To compare the relative abundance of specific mRNAs, equal amounts of
total RNA from groups of 5–9 patients or from cell lines were
transcribed into cDNA in parallel to insure similar conditions. Random
hexamer-primed cDNA synthesis was performed using 2 µg total RNA in a
final volume of 20 µl as described (30)
. Reverse
transcription reactions were carried out at 42°C for 1 h and
inactivated at 99°C for 5 min.
Comparative PCR was performed as described previously, with modification (30 , 39) . GAPDH mRNA was first amplified at a low cycle number as an internal standard before gel electrophoresis and photography. If needed, cDNA dilutions were adjusted and GAPDH levels reamplified with the aim to produce similar intensities for GAPDH signals between samples. A master PCR solution was made consisting of diluted Taq polymerase, buffer, and deoxynucleotide triphosphates (Idaho Technology, Idaho Falls, ID). Aliquots of the master PCR solution were added to microfuge tubes, on ice, containing sufficient volumes of sample cDNA and water to create a master sample solution for amplification of four to five genes. After mixing, 8-µl aliquots from each master sample solution were placed in cold tubes and 2 µl of primers were added (final, 5–10 pM). The cDNAs were amplified by PCR in a DNA air thermo-cycler programmed to heat to 94°C for 30 s, and then 45 cycles (94°C for 0 s, 55°C for 0 s, 72°C for 30 s). The reaction tubes for GAPDH were removed at 30 cycles and stored at -20°C until analysis. Aliquots (8.5 µl) of the PCR reactions were analyzed by electrophoresis in a 3% agarose gel and photographed. PCR products were transferred to a membrane, as described, for Northern analysis. Membranes were hybridized with 32P-labeled human cDNAs before autoradiography. Control amplification reactions were run concurrently. Positive controls were performed using reverse transcription reactions from cell lines or plasmid-derived cDNA inserts as templates. Negative control reactions for each primer pair contained the master PCR solution with water substituted for cDNA template.
Western Analysis.
Cells were grown in dishes and washed with PBS. Cell lysates
were harvested in radioimmunoprecipitation assay buffer [50
mM Tris-HCl (pH 7.4), 1% NP-40; 150 mM NaCl; 1
mM EGTA; 1 mM phenylmethylsulfonyl fluoride;
and 1 µg/ml aprotinin]. Human colon and rat ovaries were pulverized
and homogenized as described (40)
. Protein concentration
was determined by bicinchoninic acid analysis (Pierce) using BSA
as a standard. Protein samples were boiled in Laemmli sample buffer
containing ß-mercaptoethanol (Bio-Rad, Hercules, CA) for 5 min before
electrophoresis by 10% SDS-PAGE. Proteins were transferred to a
nitrocellulose membrane. After staining with Ponceau S dye, the
membrane was blocked with 5% nonfat dry milk in TBS [10
mM Tris-HCl (pH 7.4), 150 mM NaCl] for 2–4 h
at room temperature and incubated with affinity-purified rabbit
polyclonal antihuman ERß antisera (PA1–311, 1 µg/ml; Affinity
Bioreagents, Golden, CO; 06-629-MN, 2 µg/ml; Upstate Biotechnology,
Lake Placid, NY) diluted in TBS overnight at 4°C. The antisera were
produced with synthetic peptides representing a similar region of the
NH2 terminus that is conserved between human and
rat. After washing in TBS, the membrane was incubated with
peroxidase-conjugated donkey antirabbit antisera (1:10,000 in TBS-2%
nonfat dry milk; Amersham, Piscataway, NJ) and binding was detected by
autoradiography using enhanced chemiluminescence (Amersham). Duplicate
membranes were probed by substitution of the primary antibody solution
with either normal rabbit serum (1:2500) or with TBS-1%BSA
buffer to detect nonspecific staining.
Immunohistochemistry.
Formalin-fixed paraffin blocks for paired normal mucosa and moderately
differentiated colon tumors for three female patients were obtained
from the National Cancer Institute. Sections were processed and
analyzed by immunohistochemistry using the PA1.311 antisera (10
µg/ml) and the microwave antigen retrieval method, as described
(41)
. Controls included substitution of the primary
antibody with normal rabbit serum and preabsorption of the antisera
with peptide antigen as described by the supplier (PEP-011, Affinity
Bioreagents).
Data and Statistical Analyses.
Autoradiograms or photographs were scanned at 150-dpi resolution
using Adobe Photoshop 3.0, and integrated densities were determined
using NIH Image 1.61 software (PC version, Scion Corporation, MD). To
confirm that GAPDH steady-state mRNA levels were similar between tumors
and normal mucosa, relative densities for GAPDH mRNA transcripts
(Northern analysis) were expressed as ratios to the sample 18S rRNA
densities (ethidium bromide staining). Steroid hormone receptor levels
were expressed as ratios of integrated density for steroid hormone
receptor to GAPDH products. Statistical analyses for ANOVA and
correlations were performed using Crunch statistical package 3.0
(Crunch Software, Oakland CA). Repeated-measures ANOVA was used,
treating gender as a between-subject factor and sample type (tumor and
normal mucosa) as a within-subject factor to test whether there were
statistically significant differences in ER mRNA levels between tumors
and normal mucosa (post hoc t tests;
P < 0.05).
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RESULTS |
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Preliminary studies determined the optimal primer pairs and PCR cycle
conditions resulting in a linear relationship between ER and GAPDH mRNA
levels. GAPDH PCR products showed concentration-dependent amplification
at 30 cycles (Fig. 1)
, and GAPDH mRNA steady-state levels determined by RT-PCR were similar
between sample types [integrated density: tumor, 0.053 ± 0.001 (n = 26); mucosa,
0.047 ± 0.004 (n = 24)].
Several primer pairs for ERß cDNA were tested as shown in Table 2
.
The PCR primer pair for ERß exons 1–4 reliably produced a PCR
product that was visible by ethidium bromide staining and thus was
chosen for initial sample screening for overall ERß mRNA levels. To
compare the mRNA levels of ER and ERß on a semi-quantitative
level, the amplification efficiencies of the respective primer pairs
were tested using full-length cDNAs as templates. Similar signal
densities of the resulting PCR products were detected for each primer
pair with detection over a 2-log scale (Fig. 1)
. Differences in primer
efficiency for the ERß1 or ERß2 exon 7–8 primer pairs were also
tested using full-length ERß1 or ERß2 cDNA as templates.
Amplification efficiency was similar between the isoform pairs over a
2-log detection scale, although positive reaction products by Southern
analysis required 100-fold greater template concentrations. To
confirm the data for ERß2 and to detect coexpression of Eß5,
another primer was designed for a 3' terminal region of exon 8 that is
100% homologous between ERß2, ERß4, and ERß5 isoforms (Table 2)
.
This second primer set reliably produced PCR products for ERß2 and
ERß5 from patient samples that were visible by ethidium bromide
staining.
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55% of that in normal mucosa in female patients
(P < 0.02; Fig. 2
). In male patients, ERß levels in tumors were 80% of that in
normal mucosa (Fig. 2)
. ERß levels in normal mucosa from females were
generally higher than in normal mucosa from males [2.0 ± 0.4 (females) versus 1.2 ± 0.21
(males); P = 0.07].
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mRNA steady-state levels were much lower than
ERß mRNA levels because PCR products were usually not visible by
ethidium bromide staining, and autoradiography exposure times for
Southern analysis were longer. ER mRNA steady state levels were not
significantly different between tumor and mucosa in either males or
females, although tumor levels were generally lower than in normal
mucosa (Fig. 2)
. In an adenocarcinoma from a male patient, an ER
splicing variant was cloned that contained an exon-5 deletion and a
single conservative nucleotide substitution. Correlations between ER
subtypes and age were not observed for either gender.
To determine mRNA expression patterns of ERß isoforms and whether the
decrease in ERß mRNA levels in female tumors was specific for a given
ERß isoform, ERß1 and ERß2/ERß4/ERß5 mRNA levels were
analyzed. In female patients, ERß1 and ERß2 mRNA steady-state
levels were both significantly decreased in tumors compared with normal
mucosa (Fig. 3)
. In 1 of 10 females, ERß1 and ERß2 mRNA levels in a tumor were
higher than in normal mucosa (Fig. 3)
. In male patients, ERß1 and
ERß2 mRNA levels were not different between tumor and normal mucosa
(Fig. 3)
. ERß2 RT-PCR results were similar using either exon-8
primer. Between genders, ERß1 mRNA steady-state levels were
significantly lower in tumors, and ERß2 levels were significantly
higher in the normal mucosa of females compared with the corresponding
samples in males (Fig. 3)
. ERß5 mRNA levels were less abundant than
for ERß2, with no significant differences between samples (Fig. 3)
.
Several PCR products with higher molecular weights were detected using
ERß2 antisense primers in patient samples. Additional ERß1 PCR
products using the exon 1–4 primer set were also detected at smaller
molecular sizes, indicating possible detection of exon-deletion
splicing variants. However, all these products were detectable at much
lower intensity than the expected PCR product.
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. Moderately differentiated tumors comprised the majority of
samples for both females and males, and the ERß mRNA levels were
significantly decreased in female tumors (P < 0.05; ANOVA). Three polyps from female patients (63, 68, and 81
years of age) were analyzed, and overall ERß mRNA levels were
slightly lower, with greater variability, when compared with normal
mucosa. ERß1 mRNA levels were also lower [0.77 ± 0.034 (polyp) versus 1.57 ± 0.60 (mucosa)],
yet ERß2 and ERß5 mRNA levels were not different in polyps.
Human Colon Adenocarcinoma Cells Caco-2 Differentially Express
Steroid Hormone Receptor mRNA Levels.
ERß mRNA was detected in Caco-2, T84, and SW1116 colon cancer cell
lines (Fig. 4)
. A fainter signal for ERß mRNA was detected in HT-29 and SW48 cells.
As expected, ER mRNA was readily detected in the human breast
carcinoma line MCF-7 (Fig. 4)
. Four colon cell lines were negative for
ER mRNA expression, with extremely low levels detected in SW1116
cells compared with MCF-7 cells. PR mRNA was detected in MCF-7 cells,
with low levels detected in Caco-2 cells (Fig. 4)
. VDR mRNA levels were
comparable among cell lines except for lower levels in Caco-2 cells
(Fig. 4)
. Coexpression of ERß isoforms in Caco-2 cells was studied
using RT-PCR, and ERß1, ERß2, and ERß5 mRNA were detected (Fig. 5)
. Several smaller-sized ERß2 PCR products were also detectable using
the sense exon 2 and antisense exon 8 primers, but levels were
extremely low.
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1.7 kb was detected in Caco-2 and MCF-7 cells and in
normal human colon (Fig. 6)
. A faint signal at 7.2 kb was also detected in MCF-7 cells after
longer exposure times. On the basis of relative amounts of mRNA
analyzed by Northern analysis, ERß mRNA transcripts were most
abundant in Caco-2 cells, and ER mRNA transcripts were detected only
in MCF-7 cells (Fig. 5)
.
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60,000 in
Caco-2 using the PA1.311 antisera (Fig. 7)
. A tissue sample from rat ovary was included as a positive control
(42)
, and a doublet was detected at
Mr 58,000 with a fainter band at
Mr 73,000 (Fig. 6)
. ERß protein
in archival normal female samples was not detected by immunoblot
analysis using either primary antibody source. Replicate membranes for
each immunoblot were incubated without primary antibody or with normal
rabbit serum, and immunoreactive bands were not observed.
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. Enteric neurons in submucosa and myenteric plexi were also
immunopositive for ERß (Fig. 8)
. In the submucosa, ERß
immunoreactivity was detected in the nuclei of smooth muscle and in the
endothelial cells of large-sized arterioles (Fig. 8)
. Slight ERß
immunoreactivity was detected in superficial epithelium in one tumor,
but the surface epithelium was difficult to discern in two tumors
because of sample orientations. Formalin-fixed paraffin sections from
rat uterus, processed in parallel with the colon specimens, showed the
expected cellular distribution for ERß protein in glandular and
luminal epithelium (Fig. 8)
. Substitution of the primary antisera with
normal rabbit serum resulted in trace positive-staining in the
superficial epithelium and occasional mononuclear cells in the lamina
propria (Fig. 8)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß mRNA was observed, with ERß mRNA levels in greater
abundance than ER, as previously reported (31
, 33)
.
ERß mRNA steady-state levels were significantly decreased in colonic
tumors compared with normal mucosa in female patients, whereas in male
patients, ERß mRNA levels were not different between samples.
Although medical histories for ERT usage were not obtainable, ERß
mRNA levels in normal mucosa were similar regardless of age in females.
Nothing is known regarding in vivo hormonal regulation of
ERß expression in humans, although up-regulation of ERß mRNA levels
by 17ß-estradiol or down-regulation by progestin treatments have been
observed in human breast cancer cell lines (43
, 44)
.
Differences in overall ERß mRNA levels between tumors and normal
mucosa were paralleled by alterations in ERß1 and ERß2 mRNA levels.
Interestingly, ERß1 mRNA levels in female tumors were significantly
lower than in male tumors, emphasizing a possible gender-specific role
for the ERß1 isoform in colon tumorigenesis. ERß2 mRNA levels were
more abundant in normal mucosa of females compared with males with
decreased ERß2 mRNA levels in tumors indicating that this isoform
could also contribute to estrogen-mediated functions in colon. To date,
one group has reported functional studies for ERß2 in human pituitary
cells (45)
. In comparison to ERß1, ERß2 is truncated
at the COOH-terminus but has 26 unique amino acids because of
alternative splicing. Transfection experiments with ERß2 showed a
lack of ligand- and estrogen response element-binding and preferential
dimerization with ER. A dominant negative activity was demonstrated
only against ER transactivation. Important physiological differences
could result in cells that coexpress ERß isoforms depending on
constitutive ER levels. In tissue such as the colon, where ERß
isoforms predominate, each ERß isoform needs to be evaluated for
ligand-dependent -independent effects on cell growth, development, or
death. Very low levels for ERß1 and ERß2 splicing variants were
also detected by RT-PCR-Southern analysis. Other ERß1 splicing
variants, including exons 2, 3, 5, 6, and 5+6 deletions, have been
reported in breast and pituitary cancers (46, 47, 48)
. The
overall functional effect of these ERß variants would be expected to
be minimal because of low expression levels and presence in both normal
mucosa and colon tumors.
A trend between adenoma-carcinoma progression and ERß mRNA levels was
shown only for moderately differentiated carcinomas, because sample
sizes were too low in the other categories. Alterations in ERß mRNA
expression may occur after the initiation phase of colonic
carcinogenesis secondary to somatic mutations, hypermethylation of
promoter regions, or decreases in cell types that express ERß.
Successive alterations for several genes have been well-characterized
in the adenoma-carcinoma sequence. Hypermethylation of CpG islands in
the promoter region of genes, including ER, has been
observed during aging and tumor progression in the human colon and
results in decreased gene expression (49)
. This study
shows that terminally differentiated colonocytes express ERß protein,
whereas other studies indicate that ER protein may be localized in
the submucosa (31
, 50
, 51)
. Progressive loss of
differentiated cell types during cancer progression would be expected
in both genders, so alternative mechanisms need to be considered for
the gender differences.
Foley et al. (52) recently reported that ERß protein levels were decreased in colon cancer patients. Paired samples from 11 patients (5 males and 6 females) were studied using RT-PCR and immunoblot analysis. Although decreases in ERß protein levels were detected in both genders, alterations in ERß mRNA levels were not observed. Our studies show that decreased levels of ERß mRNA, including ERß1 and ERß2 isoforms, can be detected in female cancer patients. Assay conditions, such as total amount of RNA transcribed, enzyme sources, primer sensitivities, or sample populations, could account for the differences. The signal intensities for ERß protein levels were remarkably high given that ERß mRNA transcripts have not been detectable using standard amounts of poly(A)+ select mRNA by Northern analysis (31 , 45 , 53) . By immunoblot analysis, we were unable to detect specific bands for ERß protein in the cell lines using the antisera (Upstate Biotechnology) and protein isolation methods cited in the study by Foley et al. (52) , nor was ERß protein detected in colon samples using two antibody sources. This study does show that ERß protein was detected in the superficial epithelium rather than in crypt regions, so sample differences could account for the discrepancies.
In our study, ER mRNA was expressed in lower abundance than ERß
mRNA, with no difference in ER mRNA levels between tumors and normal
mucosa in both genders. Previous studies report conflicting results
regarding human colonic ER expression depending on the detection
method. 17ß-Estradiol ligand binding studies could detect either ER
subtype and cDNA probes, PCR primers, or antibodies directed against
the ligand and hormone-binding domains may cross-react between ER
subtypes because of sequence homology in these regions. One study
suggested that survival of patients with ER-positive normal mucosa
was longer than patients with ER-negative normal mucosa, whereas the
ER status of tumors had no prognostic value (54)
. In
another study using ligand-binding assays, ER-was detected in
similar amounts in normal mucosa and in colon tumors, and levels did
not vary by the sex or the age of patients or by the histopathological
grade of the tumor (55)
. Several other studies showed no
correlation in ER levels between tumor and normal mucosa by various
methods (50
, 51
, 56, 57, 58, 59)
.
Issa et al. (60)
showed that ER-promoter
hypermethylation increased as a direct function of age in human colon
regardless of gender and suggested that ER is a tumor suppressor
gene in human colon. ER mRNA expression was detected in normal
mucosa but not in tumors and cell lines (RT-PCR), and ER
over-expression suppressed growth in the RKO colon cancer cell line.
Our results show that ER mRNA can be detected by RT-PCR, albeit in
very low levels, in human colon tumors. Differences in PCR primer
efficiency or other technical considerations may account for the
difference in results. Paradoxically, overexpression of wild-type ER
in ER-negative cell lines and treatment with estrogens can lead to
antiproliferative effects and increased differentiation (reviewed in
Ref. 61
). It has recently been proposed that ERß
functions as a negative regulator for ER (22
, 62)
. It
would seem unlikely that ERß has such a role in the colon, inasmuch
as ER mRNA levels are much less abundant than ERß. For these
reasons, we propose that ERß may mediate estrogenic effects on colon
cancer susceptibility.
Given the difficulty in identifying wild-type ER in human colon, it
is not surprising that ER variants have not been reported. In this
study, an ER exon-5 deletion variant was cloned from a colon
adenocarcinoma of a male patient. ER variants have been reported in
several normal and neoplastic tissues (63
, 64)
. These
variants include nucleotide insertions, exon duplications, point
mutations, and alternative splicing resulting in exon-deleted
transcripts. The ER exon-5 deletion variant has a truncated
ligand-binding domain and is coexpressed in the majority of
ER-positive tissues, however the role of this ER variant in colon
cancer is expected to be minor.
Virtually nothing is known about ERß function in colonic epithelium.
Normal human colon cell lines are not generally available, because they
usually do not maintain a normal phenotype with passage. Human colon
cancer cell lines that are often used as models for aggressive (HT-29)
or absorptive (Caco-2) phenotypes were studied. Our data show that
Caco-2 cells express mRNA for ERß1, ERß2, and ERß5 isoforms.
Expression of ERß2 and ERß5 mRNA has been reported in other colon
cancer cell lines, whereas ERß1 and ERß4 mRNA were absent
(33)
. Fiorelli et al. (34)
reported expression of all five ERß isoforms in several other colon
cancer cell lines. The colon cell lines in the current study were
essentially negative for ER mRNA by RT-PCR in agreement with others,
although one study has reported detection of ER mRNA in Caco-2 cells
(34
, 60 , 65
, 66)
. The selection of various sublines
because of culture conditions could account for differences.
Multiple ERß mRNA transcripts have been observed in several human
tissues by Northern analysis, with ERß1 and ERß2 mRNA transcripts
reportedly expressed at 7.2–7.5-kb and 1.7-kb in testis
(31
, 45
, 53)
. ERß mRNA transcripts were detected at
1.7-kb in Caco-2 and MCF-7 cell lines and in normal human colon,
with a 7.2-kb mRNA transcript detected only in MCF-7 cells. Because
ERß protein was detected in Caco-2 cells at the expected molecular
weight, the low molecular-sized ERß mRNA transcript may result from
colon-specific regulatory factors on ERß isoform expression levels or
transcription start sites (67)
. Expression of ERß
protein in a cell line with an absorptive phenotype also supports our
data showing immunolocalization of ERß protein in superficial
epithelial colonocytes.
Two other steroid hormone nuclear receptors were analyzed in this
study. In particular, expression of PR was determined, because
interactions with ER are well known in some reproductive tissues.
Very low levels of PR mRNA were detected in these colon cancer cell
lines, suggesting that studies on the regulation of PR expression as a
marker of ERß function might be difficult. VDR mRNA was
expressed with an inverse relationship between ERß and VDR mRNA
levels observed between HT-29 and Caco-2 cells. Estrogenic
up-regulation of rat intestinal VDR has been observed in
vivo, but studies using intestinal cell lines have not been
reported (68)
. Caco-2 cells express functional aromatase,
an enzyme that converts steroids such as testosterone to estradiol and
estrone, so that ligand-activation of ERß protein could result from
exogenous or endogenous sources of 17ß-estradiol (69)
.
Females have a lower lifetime risk for developing colon tumors, even in families with HNPCC, and the use of ERT reduces colon cancer risk, implying that female hormones, namely estrogens, decrease susceptibility for colon cancer. This study suggests that ERß-mediated functions, in part, could be a potential mechanism by which estrogens alter susceptibility for colon cancers. Coexpression of ERß isoforms increases the degree of complexity in understanding mechanisms mediated by ER ligands, whether endogenous or exogenous. Given the increased usage of ERT and selective ER modulators in women for the prevention of various diseases, investigations on ligand activation of ERß-mediated functions in human colon are needed. Inasmuch as the numbers of elderly women are increasing and the survival rates for patients with advanced colon tumors have only modestly improved, consideration of new preventive strategies for colon cancer is also needed. The potential clinical significance of this study is that ERß may mediate chemopreventive effects for estrogens in the colon and selective ERß ligands might be a colon cancer prevention strategy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the American Cancer
Society, Florida Division (to M. C-T.). Preliminary results were
presented at annual meetings for the American Gastroenterological
Association (see Gastroenterology, 116: A595, 1999) and
the American Association for Cancer Research (see Proc. Am. Assoc.
Cancer Res., 41: 139, 2000). 
2 To whom requests for reprints should be
addressed, at Box 100214 JHMHC, Department of Medicine, College
of Medicine, University of Florida, Gainesville, FL 32610. Phone:
(352) 392-9621; Fax: (352) 392-3618; E-mail: thompmc{at}medicine.ufl.edu
3 The abbreviations used are: HNPCC, hereditary
nonpolyposis colorectal cancer; ER, estrogen receptor; ERT, estrogen
replacement therapy; GI, gastrointestinal; UF, University of Florida;
NCI, National Cancer Institute; RT-PCR, reverse
transcription-PCR; GAPDH, glyceraldehyde phosphate dehydrogenase; TBS,
Tris-buffered saline.
Received 3/24/00. Accepted 11/14/00.
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