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Protein in Serous Adenocarcinomas of Ovarian, Tubal, and Peritoneal Origin
From the Departments of Microbiology and Immunology*
and
Biochemistry,§
Temple University School of
Medicine, Philadelphia, Pennsylvania; Department of
Pathology,
University of Virginia Health
Sciences Center, Charlottesville, Virginia; and Department of
Pathology,
Fox Chase Cancer Center,
Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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protein in 16 serous adenocarcinomas originating
from the ovaries, fallopian tubes, and the peritoneum.
Using an affinity-purified antiserum specific for RAR and a
monoclonal antibody recognizing the full-length estrogen receptor
molecule (clone 6F11), we performed immunohistochemistry on
frozen tissue sections and examined the relationship between RAR and
estrogen receptor protein expression by comparing the percentage of
immunostained tumor cells for either receptor. Our findings indicate a
strong linear relationship between the percentages of RAR- and
estrogen receptor-labeled tumor cells as determined by linear
regression analysis (P < 0.005,
r = 0.825). A modest inverse relationship was found
between the percentage of RAR-positive tumor cells and histological
grade, attesting to a differentiation-dependent trend
(P < 0.04). No significant relationship was found
between RAR-labeled cells and clinical stage
(P = 0.139), site of tumor origin (ovaries
versus fallopian tubes versus peritoneum)
(P = 0.170), and primary
versus metastatic lesion (P =
0.561). Thus, serous adenocarcinomas are capable of expressing
RAR and estrogen receptor despite high histological grade and
advanced stage of neoplastic disease. Compared with the heterogeneous
localization of RAR in cancer cells, there was widespread
RAR immunoreactivity in tumor-infiltrating lymphocytes,
vascular endothelial cells, and stromal fibroblasts,
underscoring the value of immunohistochemistry in the accurate
determination of RAR/(RXR) content in tumor specimens.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retinoids are metabolites of retinol (vitamin A) and are considered to
be important signaling molecules in the modulation of growth and
differentiation of normal and neoplastic cells.10-13
They
have been shown to prevent mammary carcinogenesis in
rodents,13
inhibit the growth of human cancer cells
in vitro,10-12
and be effective chemopreventive
and chemotherapeutic agents in a variety of human epithelial and
hematopoietic neoplasms.14,15
Retinoic acid (RA) has been
shown to be an effective growth modulator of human ovarian carcinoma
cell lines, imparting an inhibitory effect on ovarian tumor cell growth
using RA either alone or in combination with other
differentiation-inducing agents.16-19
The effects of RA on
ovarian cancer cells are thought to be mediated by nuclear RA receptors
(RARs) and retinoid X receptors (RXRs).20-22
These nuclear
receptors are members of the steroid/thyroid receptor superfamily and
can modulate gene transcription through a variety of
mechanisms.23-25
RARs include RAR, RARß, and RAR
,
each of which exhibit high affinity for both all-trans-RA
and 9-cis-RA, whereas RXRs include RXR, RXRß, and
RXR and are activated by 9-cis-RA.26,27
Each
RAR subtype comprises various isoforms as a result of different
promoters and alternative splicing: the RAR gene contains two
promoters transcribing two distinct isoforms, identified as RAR1 and
RAR2 in humans.28
Concomitant with ligand binding, the receptor-ligand complexes bind to their respective response elements (RARE and RXRE), located in the regulatory regions of a number of retinoid target genes.11,21,29 Receptor binding to specific response elements and the resulting gene activation or inhibition occurs through the formation of heterodimers between RARs and RXRs; however, gene activation can also occur through RAR or RXR homodimers.12 It has been suggested that although RXR ligands mediate transactivation through RXR homodimers, they are largely inactive in mediating transactivation through RAR/RXR heterodimers.30-32 This raises the possibility that RARs, and not RXRs, are active in the various retinoid-mediated processes.33
Despite several previous studies looking at the RAR/RXR signaling
pathways in RA-sensitive and RA-resistant human ovarian cancer cell
lines, until now, the cellular distribution of RAR proteins in human
ovarian tumor specimens is unknown. In the present study, we have
evaluated by immunohistochemistry the cellular localization of RAR
protein and have examined whether there is a relationship between
RAR and estrogen receptor (ER) labeling of tumor cells in surgical
specimens of serous adenocarcinoma originating in the ovaries and the
secondary müllerian system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patient data are summarized in Table 1
. All 16 patients had undergone
exploratory laparotomy, total abdominal hysterectomy, bilateral
salpingo-oophorectomy, and debulking surgery. Patient ages ranged from
36 to 80 (median age, 64.5). Fifteen of 16 patients harbored
intermediate (grade II) to high grade (grade III) serous papillary
adenocarcinomas, and in one patient the tumor contained mixed serous
papillary and endometrioid adenocarcinomas (Table 1
, case 4). In 11 of
16 patients there was a clearly defined ovarian primary lesion, in 3
patients the tumors were of peritoneal origin (Table 1
, cases 5, 15,
and 16), and in one instance the origin of the serous adenocarcinoma
was traced to the fallopian tube (Table 1
, case 11). The majority of
patients had advanced Fédération Internationale des
Gynaecologistes et Obstetristes (FIGO) stage III/IV disease at
presentation (13 of 16 stage III, 1 of 16 stage IV). Two patients with
ovarian and tubal serous papillary adenocarcinomas, respectively, were
considered to be stage IC (Table 1
, cases 3 and 11). In 6 of 16
lesions, the specimens evaluated in this study were derived from
metastatic deposits to the contralateral fallopian tube (Table 1
, case
9), pelvis (Table 1
, case 12), omentum (Table 1
, cases 5 and 7), and
transverse mesentery (Table 1
, case 2).
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Ovarian tumor specimens were obtained from the Department
of Pathology, Fox Chase Cancer Center, under institutional Internal
Review Board approval. All specimens were collected prospectively
during a 2-year period (1995 to 1997). The tumor tissue samples
procured for this investigational study were microdissected from, and
were representative of, the surgical pathology specimens. The presence
of viable (nonnecrotic) tumor tissue was determined grossly by the
prosector. Surgically resected tumor samples were promptly embedded in
ornithine carbamoyltransferase and were kept frozen in -70°C until
further processing. Patient characteristics, source of tumor specimens,
histopathological diagnosis, tumor grade, and FIGO stage at the time of
surgery are summarized in Table 1
(also, see above).
Antibodies
A rabbit polyclonal antibody to RAR and a mouse monoclonal
antibody to ER were used. RAR1 (C-20; Santa Cruz Biotechnology,
Inc., Santa Cruz, CA), is an affinity-purified polyclonal antibody
raised against a peptide corresponding to amino acids 443 to 462
mapping at the COOH terminus of the RAR1 of human origin, which is
identical in sequence to the corresponding region of RAR2. C-20 does
not cross-react with RARß or RAR isoforms. The immunogen of the
immunoglobulin (Ig) G1 mouse monoclonal antibody (clone 6F11) to human
ER (Novocastra Laboratories Ltd., Newcastle on Tyne, UK) is prokaryotic
recombinant protein corresponding to the full-length ER molecule.
Determination of Specificity of the RAR Antibody by Western Blot
Recombinant RAR and recombinant RARß proteins were
prepared as S-Tag fusion proteins as previously
described.34
Briefly, Escherichia coli K12
strain BL21(DE3) cells were transformed with the expression of
plasmid pET-29a (Novagen, Madison, WI) containing full-length
cDNA of mouse RAR or mouse RARß. Because the cDNAs are cloned in
frame with an S-Tag marker, the proteins are expressed as fusion
proteins that can be monitored by probing for the presence of the
S-Tag. Bacterial extracts containing either recombinant RAR or
recombinant RARß were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and then transferred to
polyvinylidene difluoride paper by electroblotting. The blots were
probed with either S-Tag antibody or RAR antibody (C-20), and
protein bands were detected by alkaline phosphatase as described
previously.35
The anti-RAR antibody only recognizes the
RAR protein and not the RARß protein (Figure 1)
. This confirms the specificity of the
RAR antibody used in this study.
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Cryostat sections (5 µm thick) were air-dried for 1 hour and
then fixed in cold acetone at -20°C for 1 hour. Immunohistochemical
staining was then performed according to the avidin biotin complex
peroxidase method as previously described,36
using
commercially available kits, rabbit IgG and mouse IgG ABC Elite
Vectastain (Vector Laboratories, Burlingame, CA) for polyclonal and
monoclonal antibodies respectively. Briefly, sections were incubated
with goat (rabbit IgG kit) or horse (mouse IgG kit) sera for 1 hour to
reduce nonspecific binding. Sections were incubated with either
anti-RAR (dilution 1:50) or anti-ER (dilution 1: 50) antibodies
according to the manufacturers' recommendations for 1 hour at
room temperature. Antigen-antibody complexes were detected with
anti-rabbit (or anti-mouse)-biotinylated avidin-horseradish peroxidase
complex. The sections were then developed with 3,3'-diaminobenzidine as
the peroxidase substrate. All experiments with the RAR antibody were
performed in duplicate. Two complete immunostained sets of slides were
generated: one without counterstaining to ensure unambiguous nuclear
localization or lack thereof, and the other with light counterstaining
with Mayer's hematoxylin (Sigma Chemical Co., St. Louis, MO) to
facilitate histological evaluation. The RA-sensitive human ovarian
carcinoma cells (CAOV) grown in coverslips served as the positive
control for RAR (Figure 2)
. The CAOV
cell line has been shown previously to constitutively express RAR
mRNA.19
Negative controls included normal rabbit IgG (1.25
µg/ml) and nonspecific mouse ascites fluid (Becton Dickinson,
Mountain View, CA), which were used instead of specific rabbit
anti-RAR1 or mouse monoclonal antibody 6F11 to ER, respectively.
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Morphological assessment of immunostained tissue preparations and
manual cell counting of immunolabeled tumor cells were performed.
Initially, representative areas of the histological tumor specimen were
selected under low-power magnification. Between 633 and 1271 epithelial
tumor cell nuclei were evaluated per case, in 20 representative
high-power fields (40x). Only tumor cells with unequivocal nuclear
localization, irrespective of intensity of staining, were counted as
positive. Staining of the nuclei of tumor-infiltrating lymphocytes
(TILs) and the nuclei of nonneoplastic mesenchymal cells, such as
fibroblasts and vascular endothelial cells, was excluded from the cell
counts. Immunostained preparations were evaluated by two observers
(CDK, IS) independently. The number of positive tumor cells (the
numerator) in relation to the total number of tumor cells (the
denominator) was recorded as the labeling count for each individual
case. The percentage of immunolabeled tumor cells for a given tumor
specimen was expressed as a mean labeling percentage based on the
number of high-power fields examined. Mean labeling percentages for
histological grades II and III were calculated from the total number
of representative specimens examined. Interobserver agreement was
within 15% (
= 0.82).
Statistical Methods
RAR- and ER-labeled cell fractions were expressed in
percentages. Overall variations in the percentages of RAR-labeled
cells were evaluated by analysis of covariance using a main effects
model for tumor origin, histological tumor grade, clinical (FIGO)
stage, and metastasis, with ER percentages as the covariate.
Relationships between the percentages of RAR-labeled cells and
ER-labeled cells were evaluated by linear regression analysis, the
Pearson correlation coefficient, and data plotting; the individual
relationships between the percentages of RAR-labeled cells with
metastatic source, tumor origin, histological grade, and FIGO stage
were evaluated by one-way analysis of variance. A P value
less than 0.05 was considered to be significant. The general linear
model with LSMEANS posthoc testing and the plot procedures of
the SAS package (SAS Institute, Cary, NC) were used for these
statistical analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ER Proteins
RAR immunohistochemical staining was detected in all specimens,
although the degree and cell-type distribution of immunolabeled cells
varied widely among tissue specimens. Overall, RAR-positive tumor
cells were present in more well differentiated portions of tumor
growing as papillary fronts (Figures 3
,
A, B, and C, and Figure 4
, A and C) and much
less prominent in poorly differentiated areas of tumor typified by
dense cellularity and a predominantly solid pattern of growth (Figures 3E and 4
, E and F). The percentage of RAR-labeled tumor cell nuclei
was somewhat increased in the moderately differentiated,
intermediate-grade (grade II) tumors containing variably prominent
tubulopapillary structures, as compared with the less differentiated,
high-grade (grade III) serous adenocarcinomas: the mean RAR labeling
percentages for grades II and III were 67% (range 58 to 84%) and 30%
(range 2 to 66%), respectively. A certain degree of RAR
intratumoral staining heterogeneity was present in high-grade tumors,
and when present it was usually associated with better differentiated
papillary foci (Figure 3D)
. However, RAR staining was also detected
in poorly differentiated areas of tumor, either in scattered,
individual cells, or in clusters of tumor cells (Figure 3E)
.
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staining was detected in benign, mononuclear cells
infiltrating haphazardly the tumor stroma, known as TILs (Figures 3F and 4
, E and F). RAR-positive TILs occurred either as prominent
nodular aggregates (Figure 3F)
or as scanty stromal mononuclear cell
infiltrates (Figure 4
, E and F). In one of our cases representing a
high-grade, FIGO stage IV serous adenocarcinoma of peritoneal origin
(Table 1
, case 16), RAR staining was present in TILs but was largely
absent in tumor cells (Figure 4
, E and F). Also, RAR staining was
present in nonneoplastic mesenchymal cells, consistent with fibroblasts
of the desmoplastic tumor stroma, in endothelial cells of tumor blood
vessels (Figure 3B
, arrowheads), and in resident ovarian stromal cells
(not shown).
A distinctive ER nuclear staining was detected variously in all
specimens. Immunoreactivity among tumor cells was particularly
widespread in papillary areas (Figure 4
, B and D) but was also present
in poorly differentiated areas of tumor (not shown). The number of
ER-labeled cells in any given tissue specimen was consistently higher
as compared with RAR (the mean ER labeling percentages for grades II
and III were 80% (range 71 to 94%) and 53% (range 36 to 74%),
respectively), but followed a differentiation-dependent trend similar
to that of RAR. A strong linear relationship was found between
percentages of RAR- and ER-immunolabeled tumor cells (Table 2
, Figure 5
) (also, see below).
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-positive tumor cells (Table 1
, cases 7 and 16)
(Figure 5)
: The first, case 7, was derived from an omental metastasis
of a grade III/FIGO stage IIIB serous adenocarcinoma of ovarian origin.
The second specimen, case 16, was from a grade III/FIGO stage IV serous
adenocarcinoma of peritoneal origin (Figure 4
, D and F). TILs and stromal fibroblasts of the tumor were ER negative; however, ovarian stromal cells were ER positive (not shown).
Statistical Analysis
The percentages of ER-labeled cells and the tumor origin were
found to be jointly related to the percentages of RAR-labeled cells
using analysis of covariance (P < 0.005), and
on posthoc testing, the percentages of RAR-labeled cells when
corrected for the covariate effects of ER labeling in the analysis of
covariance model were found to be significantly higher in serous
adenocarcinomas of ovarian origin than in homologous tumors of
peritoneal origin (P < 0.01). Interestingly,
when the relationship of RAR and tumor origin was examined by
analysis of variance (without the information supplied by the ER
labeling), no statistical significance was found (Table 2)
. Results of
statistical analysis of the individual relationships between RAR
immunohistochemical labeling percentages of tumor cells and ER
percentages, metastatic source of the specimen, tumor grade, and FIGO
stage are shown in Table 2
. A strong linear relationship was found
between the percentages of RAR- and ER-immunostained tumor cells
(r = 0.825) by linear regression analysis and
data plotting (Table 2
, Figure 5
). A modest relationship
(P < 0.04) was found between the percentages of
RAR-positive tumor cells and histological grade with slightly higher
labeling counts noted in grade II as compared with grade III serous
tumors. No statistically significant relationship was found,
respectively, between RAR immunoreactivity and FIGO stage or
specimen sampling from metastatic versus primary tumor
sites.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein in Serous
Adenocarcinomas
This study provides new data with regard to the cellular
distribution of RAR protein in 16 frozen, surgically resected serous
adenocarcinoma specimens originating in the ovaries, fallopian tubes
and the pelvic peritoneum, ie, the so-called secondary müllerian
system. Also, it examines the relationship between RAR and ER
protein expression by comparing the percentage of immunostained tumor
cells for either receptor in surgical specimens. The relatively small
number of cases evaluated notwithstanding, this study indicates a
strong linear relationship between the percentages of RAR- and
ER-immunostained tumor cells as determined by linear regression
analysis (Table 2
, Figure 5
). RAR and ER immunoreactivities are
present in both intermediate- (grade II) and high-grade (grade III)
lesions, corresponding, for the most part, to advanced FIGO stage
serous ovarian adenocarcinomas. A modest inverse relationship is found
between the percentage of RAR-positive tumor cells and histological
grade, attesting to a differentiation-dependent trend (Table 2)
.
Because histological grade in ovarian adenocarcinomas is largely a
function of differentiation, there is a higher percentage of
RAR-positive tumor cells in grade II tumors with papillary areas as
compared with the grade III tumors. However, grade III tumors may also
contain papillary foci, as well as a relatively small number of poorly
differentiated RAR-positive cells. Conversely, no significant
relationship is found between RAR-labeled cells and such categorical
variables as FIGO stage; site of origin of tumor, ie, from ovary,
fallopian tubes, or pelvic peritoneum; and source of specimen from an
intra-abdominal metastasis (as opposed to the primary site) (Table 2)
.
Thus, RAR tumor cell labeling is present in specimens from primary
ovarian, as well as metastatic tumor implants in the omentum,
peritoneum, and parametria, indicating that serous carcinomas are
capable of expressing RAR, and also ER, despite high histological
grade and advanced clinical stage.
Previous in vitro studies have shown that RAR plays a
major role in the growth inhibition of mammary cancer
cells37,38
and ovarian cancer cells39,40
by
retinoids in a dose-dependent manner. Still, the presence of RAR in
intermediate- to high-grade, advanced-stage serous tumors demonstrated
in this study is similar to that described previously in breast
carcinomas.41
van der Leede and collaborators41
have proposed an apparent uncoupling of RAR expression and
proliferation inhibition, offering a threefold explanation for this
phenomenon: 1) perturbed transcriptional regulation as part of tumor
progression; 2) loss of mechanisms of RAR down-regulation; and/or 3)
insufficient retinoid levels to achieve down-regulation, hence
culminating in overexpression of RAR.41
It remains to be
determined whether there are alterations in the levels of RAR
isoforms in cancer cells because of alterations in the factors
regulating RAR gene transcription at the promoter level. In this
regard, there is evidence of estrogen-induced expression of RAR1
mRNA, but lack of RAR2 transcripts, in ER-positive breast carcinoma
cell lines.38
Interestingly, serum retinol has been found
to be significantly lower in ovarian cancer patients,42
although the actual content of retinoids in ovarian adenocarcinoma
specimens is unknown. Collectively, malignant tumors in vivo
may exhibit dysregulation of cellular differentiation signaling
pathways, which may also involve RARs/RXRs.
To date, there has been only a small number of immunohistochemical
studies aimed at the localization of RARs in normal and neoplastic
tissues. This may be attributed to several confounding factors. The low
concentration of individual RAR epitopes may hinder their detection by
immunohistochemistry,43,44
even in neoplastic cells, such
as leukemia cells.45
At this time, there is only a limited
number of commercially available subtype-specific antibodies. Their
immunohistochemical performance in chemically fixed tissues is not well
defined and may be punctuated by unexpected (or even spurious)
localizations. Also, the presence of cellular retinoid binding proteins
may hinder the localization of RARs, a problem that has been addressed
previously in the context of autoradiography.46
Thus far,
immunohistochemical studies on surgical tumor specimens are limited to
the evaluation of RAR in breast carcinomas.41,47
The
latter two studies have used archival, formalin-fixed,
paraffin-embedded tissue sections and different antibodies. In one of
them, unexpected RAR cytoplasmic staining of tumor cells has been
detected in addition to nuclear staining.41
We have also
observed a similar cytoplasmic localization for RAR in a large
number of formalin-fixed, paraffin-embedded ovarian tumor specimens,
which led us to withdraw these data from the present study. Clearly, an
abnormal processing of RAR in tumor cells deserves consideration in
this respect;41
however, in our view, this appears less
likely, because cytoplasmic staining has also been detected, by us, in
benign epithelial and mesenchymal cells in paraffin, but not in frozen,
acetone-fixed sections of ovarian, adnexal, and other unrelated tissues
(CDK, Y. Yu, IS, and KJS, unpublished observations).
To minimize potential artifacts introduced either by the use of
cross-linking fixatives or by conventional tissue processing and
paraffin embedding, we elected to perform immunohistochemical staining
only on frozen sections fixed briefly in cold acetone in -20°C. This
is a reliable method that renders unambiguous nuclear localizations, at
least as evidenced with the RAR antibody used in this study. That
the anti-RAR antibody recognizes RAR in bacteria is also
supported by the expected nuclear immunolocalizations in human cells
in situ, thus collectively providing strong evidence for
specificity. Additional issues regarding accurate determination of
steroid receptors in general and RARs in particular in surgical
specimens include tumor cell heterogeneity and receptor expression by
nonneoplastic cell types within the tumor, such as TILs, vascular
endothelial cells, and stromal fibroblasts. Thus, immunohistochemistry
is the single most appropriate and accurate method of determining the
cellular source of steroid receptor protein in ovarian tumor
specimens.48
RAR Localization in TILs
The widespread presence and intensity of RAR staining in TILs
is noteworthy. It is detected in >80% of mononuclear inflammatory
cells randomly dispersed either in the periphery or within
sheets of cancer cells. Although all 16 specimens contain
RAR-positive mononuclear cells in varying proportions, 6 of 16
tumors exhibit prominent TILs. A similar RAR staining pattern of the
TILs has been described, in passing, in a series of breast
carcinomas.41
Although the immunological significance of
TILs in epithelial tumors in general and in ovarian carcinomas in
particular is unclear, it has been suggested that they may represent
tumor cytolytic oligoclonal T-cell responses.49
Retinoids are multicellular immunomodulators both in vivo
and in vitro, including but not limited to various tumor
cell types, human and murine thymocytes, fibroblasts, Langerhans'
cells, natural killer cells, and T lymphocytes.50
Both
retinol and RA induce vigorous proliferative responses on human
peripheral blood mononuclear cells after stimulation with anti-CD3
antibodies, which is specifically mediated through the clonotypic
T-cell receptor-CD3 complex and correlates with the up-regulation of
surface T-lymphocyte adhesion/activation markers, as well as an
augmentation of interleukin-2 and interferon-
transcripts.50
It has been shown that RA promotes
proliferation and induces RAR gene expression in murine T
lymphocytes and that RA and RAR might function in T cells as
ligand-inducible transcriptional enhancer factors.51
It
remains to be determined whether the expression of RAR in ovarian
carcinoma TILs is related to tumor antigen-specific or oligoclonal
T-cell response(s).
Relationship of RAR and ER Immunoreactivity Profiles
Despite the exclusion of RARA, the gene for RAR, as a candidate
gene for brca-1 (the susceptibility gene for hereditary
breast-ovarian cancer),52
there is nonetheless evidence to
suggest that ovarian and breast cancer cells may exploit similar
signaling pathways for growth and differentiation, insofar as they are
both steroid hormone-dependent neoplasms. Compared with
mammary53
and endometrial cancers54
in which
the ER status is prognostically significant, a linear relationship
between ER level and survival has not been established in ovarian
carcinomas.55-57
By biochemical radioligand assays,
ovarian carcinomas have been shown to contain ER in the 60% tumor
range.9
Others have reported that this percentage declines
to 38% when immunohistochemistry is used as the method of
detection.48
Using solely cryostat sections and monoclonal
antibody 6F11, we have found ER mean labeling percentages of 80% for
grade II and 53% for grade III serous tumors. Interestingly, Slotman
and coworkers58
have found no correlation between tumor
ploidy and histological grade, stage of disease, and ER content in
ovarian adenocarcinomas.
There are several lines of evidence suggesting a positive relationship
between RAR and ER gene59
and protein57
expression in breast carcinomas and human mammary carcinoma cell lines.
RAR appears to be required for inhibition of anchorage-independent
growth either by natural (all-trans-RA) or by
conformationally restricted retinoids with agonist activity in the
MCF-7 ER-positive breast carcinoma cell line.60
Moreover,
although a statistically significant correlation has been established
between RAR and ER mRNA in primary breast tumors, no such
correlation has been found to exist between ER levels and expression of
either RARß, RAR, RXR, RXRß, or RXR mRNA
levels.61,62
It is believed that the relationship between
RAR and ER gene expression is partly caused by estradiol enhancement
of RAR gene expression,59,63
which is mediated through
an imperfect half-palindromic estrogen response element and Sp1
motifs.63
In contrast to RAR, the mechanism responsible
for the retinoid sensitivity of breast cancer cells does not involve
transcriptional modulation of the RXRs by RA.64
Most
ER-negative breast carcinomas express lower levels of RAR and are
largely resistant to the growth-inhibition effects of RA compounds
in vitro.65
However, estradiol-independent
enhancement of RAR gene expression has been demonstrated in certain
ER-negative breast cancer cell lines, such as SKBR-31 and
MDA-MB-435.65
RA-mediated growth inhibition in these lines
is accomplished via a 72-bp fragment of RAR promoter that
contains unique cis elements.65
To date, there
are conflicting reports with regard to a relationship between RAR
and ER in breast tumors at the protein level. Recently, Han and
co-workers47
have reported that RAR expression is
significantly increased in ER-positive breast tumors as determined by
immunohistochemistry and image cytometry. This is in contrast to a
previous immunohistochemical study claiming that no such relationship
exists between RAR and ER status.41
It has been previously shown that 17-ß-estradiol may regulate growth
in human ovarian carcinoma lines.66
17-ß-Estradiol can
stimulate the growth of populations of ER-positive ovarian carcinoma
cells that may in turn be associated with changes in the cellular
levels of steroid hormone receptors.66
Our findings in
serous ovarian carcinomas are consistent with those by Han and
collaborators47
in breast carcinomas, insofar as they
support a relationship trend between RAR and ER expression in
certain steroid hormone-dependent epithelial neoplasms. However, as
alluded to above, a definitive determination in this regard awaits the
evaluation of a larger sample of tumors.
Concluding Remarks and Future Directions
In the past, encouraging results have been obtained with RA-based
treatment of patients with locally advanced squamous cell carcinoma of
the uterine cervix67
and cisplatin-resistant metastatic
endometrial adenocarcinoma.68
Because RAR plays a major
role in retinoid-mediated growth inhibition of breast and ovarian
cancer cells in vitro, it is also possible that patients
with breast41
and ovarian carcinomas could be responsive to
retinoids independently of their responsiveness to antiestrogen
regimens. Based on their specific RAR/RXR profile and method of
delivery in vivo, ovarian carcinomas may be amenable to a
variety of therapeutic interventions using conformationally restricted
retinoid ligands.40
This underscores the importance of
future studies aiming to elucidate the full RAR/RXR profile in serous
adenocarcinomas of the ovary and secondary müllerian system.
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Acknowledgements |
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Footnotes |
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Supported by National Cancer Institute grant RO1 CA64945 (to KJS) and by Research Supplement for Individuals with Disabilities grant 3 RO1-CA64945-02S1 from the National Cancer Institute's Comprehensive Minority Biomedical Program (to CDK).
This work is dedicated to Dr. Demetrios Katsetos on the occasion of his 70th birthday.
Accepted for publication May 18, 1998.
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References |
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