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Unit of Metabolic Medicine, Imperial College School of Medicine at St. Mary’s, London W2 1PG, United Kingdom
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Abstract |
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 Top Abstract I. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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I. Introduction |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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II. Peripheral Estrogen Synthesis and Blood and Tissue Estrogens in Breast Cancer |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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B. Aromatase
Soon after the discovery of the extraglandular route of estrone
formation from androstenedione, it became apparent that the extent of
this conversion was related to body weight (3, 22). In normal weight
subjects about 1% of androstenedione is converted to estrone whereas
in obese subjects this can increase up to 10%. The increase in the
peripheral conversion of androstenedione to estrone associated with
obesity most likely accounts for the increased risk that such subjects
have for the development of endocrine-dependent cancers (23, 24).
In weight-matched subjects an increase in peripheral estrogen synthesis also occurs with aging (25). The increase in aromatase activity that occurs with aging, as detected from in vivo studies, was confirmed in one investigation in which adipose tissue aromatase activity was measured in vitro (26). In contrast, in another study an increase in in vitro aromatase activity was detected in adipose tissue taken from perimenopausal women (19.5 pg/mg/3 h) as compared with tissues from younger women (3.2 pg/mg/3 h) while that from menopausal women had conversion rates below 11 pg/mg/3 h (27). In a recent study, however, in which competitive RT-PCR analysis was used, levels of P450 aromatase transcripts in adipose tissue from buttocks, thighs, and abdomen of women were found to increase with advancing age (28).
An important finding to emerge from the first studies into the regulation of aromatase activity in vitro was the observation by Simpson and colleagues (29, 30, 31) that the synthetic glucocorticoid, dexamethasone, in the presence of FCS, could markedly stimulate aromatase activity. It was subsequently shown that the endogenous glucocorticoid, cortisol, could also stimulate in vitro aromatase activity (32). While glucocorticoids can stimulate aromatase activity in vitro, attempts to obtain evidence for such a role for glucocorticoids in vivo were not successful. The peripheral conversion of androstenedione to estrone in women given dexamethasone or Synacthen injections did not increase during therapy (33), with similar results being obtained in investigations carried out in monkeys (34). The reason for the failure of glucocorticoids to enhance in vivo aromatase activity remains puzzling but will be discussed later in the review in the light of current knowledge of the control of aromatase activity.
C. Estrone sulfatase
Blood and breast tissue concentrations of estrone sulfate are much
higher than for the unconjugated estrogens (35, 36, 37) and, furthermore,
the half-life of estrone sulfate (10–12 h) in blood is much longer
than for unconjugated steroids (20–30 min) (38). Estrone sulfate may
therefore act as a reservoir for the formation of estrone after
hydrolysis by estrone sulfatase (11, 12). The activity of estrone
sulfatase is much higher than the aromatase in normal and malignant
breast tissues and, in contrast with the aromatase, is present in most
breast tumors (5). Using physiological substrate concentrations,
formation of estrone via the sulfatase pathway was found to account for
a 10-fold greater amount of breast tumor estrone than that formed via
the aromatase route (39). In rat nitrosomethylurea-induced mammary
tumors, a model used to investigate hormone-dependent tumors, up to
50% of the estrone was found to originate in situ from the
hydrolysis of estrone sulfate (40).
D. E2DH
Estrone, formed from either androstenedione or estrone sulfate, is
converted to estradiol by E2DH. It was originally thought that E2DH, as
such, was responsible for the interconversion of estrone and estradiol
and that it could act in either an oxidative or reductive direction
dependent upon cofactor availability. However, infusions of either
[3H]estrone or [3H]estradiol in women with
breast tumors revealed that within tumors little metabolism of
[3H]estradiol occurred whereas [3H]estrone
was readily converted to [3H]estradiol (41). For benign
and malignant breast lesions a positive correlation was found between
E2DH activity in adipose tissue surrounding the breast lesion and the
degree of obesity of the individual (42). It is now apparent that E2DH
(Type I) is present in breast tumors and that this dehydrogenase is
responsible for the reduction of estrone to estradiol (13, 14).
E. Origin of breast tumor estrogens
In contrast to the low levels of estrogens found in the plasma of
postmenopausal women, preliminary measurements of breast tumor estrogen
concentrations, using a mass fragmentography technique, initially
indicated that breast tumors may contain high estradiol concentrations
(43). Several investigations, using fully validated RIA techniques,
have now compared plasma estrogen concentrations with those in normal
and malignant breast tissues. A consensus has emerged from these
investigations indicating that the concentrations of estrone and
estradiol in both normal and malignant breast tissues are significantly
higher than the levels in plasma. Furthermore, estrogen concentrations,
and in particular estradiol, are higher in malignant than in normal
breast tissue (43, 44, 45, 46, 47). A consistent finding from these investigations
was that the tumor-plasma estradiol ratio (up to 20-fold) was much
higher than that for estrone (44, 45). One intriguing finding to emerge
from measurements of breast tissue estrogens was the observation that
while tissue estrone concentrations in postmenopausal women reflected
the decrease in estrogen production that occurs at menopause, tumor
estradiol concentrations were independent of menopausal status with
similar levels being detected in tumors from pre- and postmenopausal
women (48, 49, 50).
As estrogens in breast tumors could originate via uptake from the circulation, the estradiol content of estrogen receptor positive (ER+) or ER negative (ER-) tumors were compared. While some evidence was obtained for higher estradiol concentrations in ER+ than in ER- tumors (51, 52, 53), no consistent correlation was detected between ER and estradiol content as might be expected if uptake and retention was the major source of tumor estradiol (45, 47, 52).
An alternative and more likely explanation is that since the enzymes necessary for estradiol synthesis are present in most breast tumors, in situ formation makes a major contribution to the high estradiol content of these tumors. In an attempt to obtain information on the origin of estrogen found in breast tumors, a double isotopic infusion technique was developed to allow uptake of estrogen from the circulation to be differentiated from in situ estrone synthesis in normal and malignant breast tissues (54). In some breast tumors there was little evidence of in situ estrone synthesis, but in others up to 90% of the estrone content of tumors was identified as resulting from in situ synthesis. Furthermore, significant in situ estrone formation in normal breast tissue was detected only in tissue adjacent to tumors actively synthesizing estrogen (54).
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III. Influence of Breast Tumor Location on Enzyme Activities in Adjacent Tissues |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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Support for this concept was provided by results from a study that found a highly significant correlation between the size of malignant, but not benign, breast tumors and E2DH activity in adipose tissue adjacent to the tumor (56). E2DH activity within breast tumors was also found to correlate with activity in normal breast tissue taken some 2–5 cm from the tumor (57).
Similar investigations revealed that the location of a tumor within the breast could also influence aromatase activity in the breast quadrant in which the tumor was located. Miller and his colleagues (58, 59) made the important observation that aromatase activity in the tumor-bearing breast quadrant was always higher than in non-tumor-bearing quadrants. This finding has now been confirmed in two further studies of mRNA expression (60) and tissue activity level (61), and only one other study produced conflicting results (62). Therefore, results from three independent investigations have confirmed that the location of a tumor within the breast is able to influence aromatase expression and activity in adjacent tissues.
Some evidence that estrone sulfatase activity may also be influenced by tumor location has also been obtained, but further investigations are required to confirm the significance of this observation (63).
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IV. Proposed Model for the Regulation of Estrogen Synthesis in Breast Tumors |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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, will first be described
with the supporting evidence discussed in detail later in the review.
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As previously noted, while only 40–50% of breast tumors possess aromatase activity (9), most tumors have highly active estrone sulfatase and E2DH (Type I) enzyme systems (5). Immunocytochemical studies to examine the location of aromatase in breast tumors have provided evidence for a stromal (65, 66) and epithelial (67, 68) location for this enzyme complex. Measurements of aromatase activity in stromal tumor-derived fibroblasts or epithelial MCF-7 breast cancer cells have revealed that a much higher level of aromatase activity is present in stromal cells (69). Immunocytochemical analysis of the location of E2DH has revealed that the enzyme is located in the cytoplasm of malignant epithelial cells (70).
Cytokines, and in particular IL-6 and tumor necrosis factor-
(TNF), have emerged as having crucial roles in regulating estrogen
synthesis in breast cancer cells. IL-6 and TNF both stimulate
aromatase, E2DH, and estrone sulfatase activities and furthermore can
also act synergistically to enhance the activities of these enzymes.
While IL-6 can be secreted by breast tumor-derived fibroblasts and
macrophages, a major source of IL-6 produced within breast tumors is
thought to be the infiltrating lymphocytes.
The ability of IL-6 to stimulate aromatase activity can be
markedly potentiated by the IL-6 soluble receptor (IL-6 sR). IL-6 sR is
produced by malignant epithelial cells and fibroblasts derived from
malignant, but not normal, stromal cells, but again, a major source of
IL-6 sR within breast tumors is from the TAMs and TILs. Shedding of
IL-6 sR from malignant epithelial cells can be increased by estradiol
with the antiestrogen, tamoxifen, being able to block the
estradiol-stimulated increase in shedding. The IL-6 receptor (IL-6R)
and/or IL-6 sR can interact with the gp130 component of the IL-6R
system, and this is required for the induction of signal transduction
that results from IL-6 binding to its receptor. As the gp130 protein
appears to be expressed ubiquitously, it is possible that IL-6, in
association with its soluble receptor, could stimulate aromatase
activity in other cells within the body not expressing the IL-6R. It is
also likely, but remains to be shown, that IL-6 in combination with the
IL-6 sR, may also enhance E2DH activity in breast cancer cells,
although there is indirect evidence for such an interaction. TNF,
which is secreted by adipocytes and cells of the immune system, can
increase expression of the gp130 protein component of the
IL-6R-signaling system, and this probably accounts for the synergy that
occurs between IL-6 and TNF in their ability to stimulate estrogen
synthesis. Overall, it has emerged that there is a complex system of
regulating estrogen synthesis within breast tumors that appears to be
mainly coordinated by cytokines. The result of the coordinated
stimulation of estrogen synthesis may account for the high
concentrations of estradiol found in breast tumors.
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V. Evidence in Support of Proposed Model of Cytokine Regulation of Estrogen Synthesis |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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Analysis of breast tumor cytosol and BCF led to the identification of
several factors that could stimulate estrogen synthesis, including the
insulin-like growth factors, Types I and II (81). An albumin-like
factor was also identified in breast tumor cytosol (82). Some types of
human serum albumin were subsequently shown not only to stimulate E2DH
and aromatase activities but also to potentiate the ability of growth
factors, such as IGF-I, or cytokines, such as IL-1, to stimulate the
activities of these enzymes (73, 83) (Fig. 2). The ability of IL-1 to interact with
human serum albumin was examined as it had previously been reported
that IL-1-like activity was associated with BSA (84). In view of the
proposed model of the regulation of estrogen synthesis by cytokines, it
is of interest that some types of albumin have recently been reported
to increase production of IL-6 and PGE2 by macrophages
(85). It is possible that a modified form of albumin in breast tumors
may have a similar immunostimulatory role.
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IL-6 was also identified in conditioned medium (CM) from cultured
breast tumor-derived fibroblasts as one of the factors responsible for
the ability of this medium to stimulate E2DH reductive activity in
MCF-7 breast cancer cells (87, 88, 89). Recombinant IL-6 (rIL-6) also
stimulated E2DH activity in these cells although its ability to
increase enzyme activity was not confirmed in another investigation
(90). The degree to which rIL-6-stimulated E2DH activity was lower than
that achieved by CM containing a comparable concentration of IL-6,
suggesting that other factors may also be present in the CM and able to
potentiate the stimulatory effect of IL-6 on E2DH activity. Since
TNF is produced by adipocytes (91, 92), and with adipose tissue
forming a substantial proportion of tissue in the breasts of
postmenopausal women, a combination of TNF and IL-6 was tested for
its ability to stimulate E2DH activity. TNF markedly potentiated the
ability of IL-6 to stimulate E2DH activity although in vitro
this combination of cytokines inhibited cell proliferation (90). TNF
also stimulates estrone sulfatase activity in MCF-7 cells and aromatase
activity in fibroblasts derived from normal and malignant breast
tissues, with TNF and IL-6 also acting synergistically to enhance
the activities of these enzymes (93, 94) (Fig. 3). Other cytokines that are members of
the IL-6 superfamily, including IL-11, oncostatin M, and leukemia
inhibitory factor, can also increase aromatase activity in cultured
adipose tissue stromal cells (95).
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Several different factors that can stimulate estrogen synthesis
in cultured breast cancer cells or adipose tissue stromal cells have
now been identified in breast tumor cytosol, BCF, and CM from breast
tumor-derived fibroblasts. However, it is apparent that cytokines, and
in particular IL-6 and TNF, are emerging as the primary factors
regulating aromatase (93), E2DH (89, 90), and estrone sulfatase (94)
activities.
B. Potentiation of cytokine stimulation of estrogen synthesis
While cytokines can stimulate aromatase, E2DH, and estrone
sulfatase activities in breast cancer cells and fibroblasts, the extent
of stimulation by a single cytokine is usually relatively modest. The
finding that some human serum albumin preparations markedly potentiated
the ability of cytokines and growth factors to stimulate in
vitro aromatase activity (73) suggested that such a mechanism may
also be effective in breast tumors.
Further evidence in support of the potentiation of cytokine stimulation of enzyme activity was provided by a comparison of the effects of recombinant cytokines, such as IL-6, and CM from tumor-derived fibroblasts in which the concentration of IL-6 was measured. While there is no doubt that such CM does contain IL-6, the initial report that rIL-6 stimulates E2DH activity was difficult to confirm (90). In the initial study it was apparent that whereas CM containing IL-6 at a concentration of 2 ng/ml resulted in a 250% stimulation of E2DH reductive activity, rIL-6 at the much higher concentration of 80 ng/ml stimulated activity by only 150% (89). In another study CM containing IL-6 at a concentration of 5 ng/ml stimulated E2DH reductive activity almost 1000-fold, whereas rIL-6 from several different sources had only a modest effect on the activity of this enzyme (90). It was concluded from this investigation that in addition to IL-6, other factors, such as other cytokines and/or proteins, must also be present in CM from tumor-derived fibroblasts. Such factors must be able to greatly potentiate the ability of cytokines, such as IL-6, to stimulate enzyme activity.
Similar differences in the ability of cytokines to stimulate aromatase activity have also been noted. The initial finding that IL-6 could stimulate aromatase activity was detected using tumor-derived fibroblasts (79). In contrast, using stromal cells derived from subcutaneous adipose tissue, Simpson and his colleagues were initially unable to confirm an effect of IL-6 on aromatase activity (personal communication). It seemed possible, therefore, that the difference in the ability of fibroblasts derived from breast tumors and stromal cells derived from adipose tissue may depend upon the secretion of a coregulatory protein by the former cells.
Simpson and his colleagues (95) recently showed that the combination of
IL-6 with its soluble receptor (IL-6 sR) resulted in a marked
stimulation of aromatase activity in stromal cells derived from
subcutaneous adipose tissue. The ability of IL-6 sR to potentiate the
stimulatory effect of IL-6 on aromatase activity in fibroblasts derived
from subcutaneous adipose tissue was recently confirmed (98).
Significant aromatase activity was detectable in IL-6-stimulated
fibroblasts, but the combination of IL-6 sR plus IL-6 resulted in a
21-fold greater stimulation of activity than with IL-6 alone (Fig. 4).
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VI. The Role of Soluble Cytokine Receptors in Cytokine Action |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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The combination of IL-6 plus IL-6 sR has been shown to enhance the secretion of acute phase proteins by Hep G2 cells (108) and the inhibition of growth of T47D breast cancer cells (109), compared with the effects of IL-6 alone. It is likely, therefore, that by the shedding of IL-6 sR by one cell type, and after ligand binding, the complex could act on cells that only express gp130 at their surface (99). Such cells would not normally react to IL-6, but it is known that gp130 is present on all cell types whereas the IL-6R is not expressed ubiquitously. Using a cytokine gene transfer-based tumor rejection model, IL-6 sR was also shown to be active in vivo (110).
B. Regulation of gp80 and gp130
Recent studies have provided important insight into the regulation
of gp80 and gp130. IL-6 at high concentrations down-regulates IL-6R
(gp80), resulting in cells becoming desensitized (108). Glucocorticoids
regulate IL-6R (gp80) expression, but their effects differ for
different cell types. Treatment of monocytes with dexamethasone results
in down-regulation of IL-6R (gp80) mRNA expression (111), whereas in
hepatocytes and epithelial cells glucocorticoids have a positive effect
on gp80 mRNA expression (112). IL-6 can increase the expression of
gp130 mRNA to a small extent, whereas expression is markedly
up-regulated by a combination of IL-6 and dexamethasone in Hep G2 cells
(113). In human UAC (U Amnion Cell) and Hep 3B (hepatoma) epithelial
cells, in addition to IL-6, IL-1 and TNF also increase expression of
gp130 mRNA (114). The increase in gp130 expression caused by TNF is
the most likely explanation for the marked synergy seen between IL-6
and TNF in their ability to stimulate estrogen synthesis in breast
cancer cells.
C. Regulation of IL-6 sR shedding
In a recent investigation, the effects of steroids, cytokines, or
12-O-tetradecanoyl phorbol-13-acetate on the release of IL-6
sR from MCF-7 breast cancer cells was examined (98). Treatment of cells
with estradiol resulted in a marked increase in the release of IL-6 sR,
and this estradiol-stimulated release was almost completely abolished
by the antiestrogen, 4-hydroxytamoxifen. Both IL-6 and TNF increased
IL-6 sR release, as did dexamethasone and 12-O-tetradecanoyl
phorbol-13-acetate. IL-6 sR was also detected in CM from another ER+
breast cancer cell line, T47D, but not in the ER- MDA-MB-231 cell
line, a result that was recently confirmed (97). IL-6 sR was also
detected in CM collected from malignant tumor-derived fibroblasts but
not from benign or normal breast tissue-derived fibroblasts (98). CM
from lipopolysaccharide-stimulated macrophages and lymphocytes also
contained high concentrations of IL-6 sR. Concentrations of IL-6 sR
were higher in cytosol prepared from malignant breast tissue than in
cytosol from normal breast tissue. The finding of significant
concentrations of IL-6 sR in fibroblasts derived from malignant but not
normal (mainly adipose) breast tissue offers a ready explanation for
the discrepancy in the ability of IL-6 alone to stimulate aromatase
activity in cells derived from these different tissues (73, 95). The
ability of estradiol to increase the release of IL-6 sR, and inhibition
of this stimulation by 4-hydroxytamoxifen, could also account for the
effects that these compounds have on estrogen synthesis in breast
cancer cells. Estradiol markedly potentiates the ability of IL-6 to
stimulate E2DH reductive activity in MCF-7 cells (115), while tamoxifen
inhibits the ability of IL-6 to stimulate enzyme activity (116).
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VII. Origin of Estrogen-Stimulating Factors in Breast Tumors |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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activity
was also measured in these biopsy samples, as a marker of cell
proliferation, and in the two specimens showing an increase in
aromatase activity there was a corresponding increase in DNA polymerase
activity. This was one of the first findings to suggest that breast
tumor aromatase might be capable of producing a biological effect,
i.e., produce sufficient estrogen to induce cell
proliferation. Further support for this concept was provided by other
observations arising from this study (54). A marked decrease in
in situ tumor estrone synthesis was associated with a
decrease in tumor estrone concentrations and an increase in cell
nuclear condensation, a marker of apoptosis (122, 123). A similar
result showing an increase in breast tumor aromatase activity, when
measured in vitro after 4-hydroxyandrostenedione therapy,
was also obtained in another study (124). At the time it was difficult to comprehend what factors might be capable of stimulating tumor aromatase activity in the presence of 4-hydroxyandrostenedione that effectively blocked peripheral aromatase activity. The wound healing that occurs after obtaining a biopsy sample would be associated with macrophage and lymphocyte invasion. It is possible that infiltration by these cells of the immune system may have provided sufficient cytokines to stimulate aromatase activity in these two samples, even in the presence of 4-hydroxyandrostenedione.
A further indication that macrophages and lymphocytes may have an
important role in regulating tumor aromatase activity came from a study
of aromatase activity in breast tissue obtained from a woman who had
previously undergone breast augmentation by silicone injection, which
was not contained within a capsule (61). In normal breast adipose
tissue, aromatase activity is relatively low (
10 fmol/mg protein/3
h) (5). For the subject who had had breast augmentation, aromatase
activity of up to 400 fmol/mg protein/3 h was detected (61).
Histological examination of this tissue revealed the presence of
inflamed tissue with extensive macrophage and lymphocyte invasion.
Furthermore, a significant correlation was found between aromatase
activity in samples of this breast tissue and the ability of tissue
explants to produce IL-6, although the cells in the tissue responsible
for IL-6 production were not identified.
B. Macrophages and lymphocytes in breast tumors
There is evidence that as much as 50% of the volume of breast
tumors is comprised of macrophages and lymphocytes (125), and it is
therefore likely that such cells are the major source of cytokines that
are present in tumors and available to stimulate estrogen synthesis.
The finding of a strong association between oncogene amplification and
dense lymphocytic infiltration of tumors lends additional support for
an important role for these cells in regulating tumor growth (126).
Evidence to support such a role for macrophages and lymphocytes was
obtained by collecting CM from lipopolysaccharide-stimulated peripheral
blood monocytes and lymphocytes. In the presence of dexamethasone, CM
from macrophages and lymphocytes stimulate aromatase activity to a
greater degree than ever previously detected (61). CM from these cells
also stimulate E2DH reductive and estrone sulfatase activities in MCF-7
breast cancer cells (127).
Breast tumor cells are known to secrete a number of chemokines, such as IL-8 and MCP-1, which attract both macrophages and lymphocytes (128, 129, 130, 131). Epithelial cells derived from breast tumors have recently been shown to secrete large amounts of the chemokine IL-8 (132).
Thus, in addition to the possibilities of breast tumors producing factors that might stimulate estrogen synthesis in adjacent tissues, or that enhanced estrogen synthesis in normal breast tissue may favor tumor development, it is also possible that increased estrogen synthesis in tumors and adjacent tissues results from macrophage and lymphocyte invasion. Further support for a three-component model (i.e., epithelial, stromal, and immune cells) as being important in regulating intratumoral estrogen synthesis has recently been put forward (133).
The attraction of macrophages and lymphocytes to the margins of a tumor
could account for the original observation of a significant correlation
between tumor size and E2DH activity in tissue adjacent to the tumor.
As shown in Fig. 5, the correlation
between E2DH activity in tissue adjacent to the tumor and tumor size
(56) (Fig. 5A) is very similar to the correlation relating
CD4+ invasion and tumor size (134) (Fig. 5B). Similarly,
the presence of a greater number of macrophages and lymphocytes in the
breast quadrant bearing a tumor, with the subsequent release of
aromatase-stimulatory cytokines, is the most likely explanation for the
consistent finding of an association between tumor location and breast
adipose tissue aromatase activity.
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VIII. Regulation of Lymphocyte Cytokine Production |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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). It is now evident
that T helper (Th) cells, a type of lymphocyte, can exist as two
distinct subsets (135, 136). Th cells can mature to either a Th1 or Th2
phenotype, each of which secretes a characteristic set of cytokines.
For example, Th1 cells secrete IFN
, IL-2, and TNFß, while Th2
cells secrete IL-4, IL-5, IL-6, and IL-10. Cytokines produced by Th2
cells therefore include IL-6 which, as previously discussed, is
emerging as having an important role in regulating breast tumor
estrogen synthesis. Furthermore the responses of Th1 and Th2 cells are
mutually inhibitory. IFN, which is secreted by Th1 cells, inhibits
secretion of cytokines by Th2 cells, while IL-10, which is secreted by
Th2 cells, inhibits the Th1 cytokine response.
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IX. Clinical Observations Explained by Proposed Model of Cytokine Regulation of Estrogen Synthesis |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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). It is now known that plasma IL-6 and
TNF concentrations are elevated in patients with septic shock, and
the observation by Nunez therefore provides important evidence in
support of a role for these cytokines in regulating peripheral estrogen
synthesis (147, 148).
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C. The effect of weight on peripheral aromatase activity
TNF, which stimulates aromatase, E2DH, and estrone sulfatase
can act, as previously discussed, synergistically with IL-6 to increase
the activities of these enzymes. In addition to being produced by cells
of the immune system, TNF is also secreted by adipocytes (91, 92).
However, a greater amount of TNF is secreted by adipocytes from
obese subjects than by those of lean individuals (149). Thus, although
the increased mass of adipose tissue, in which aromatization takes
place, of obese subjects is likely to contribute to their increased
production of estrone (3), it is likely to be potentiated by their
increased production of TNF from adipocytes.
D. The discriminant function test
Some years ago Bulbrook and Hayward developed the discriminant
function test as a marker for women who subsequently developed breast
cancer (150). The excretion of low levels of androgen metabolites of
DHA indicated women at risk of developing breast cancer and were also
associated with an unfavorable prognosis in women with breast cancer.
These studies were extended to show that the discriminant function
test, i.e., the ratio of 11-deoxy-17-oxosteroids (mainly
etiocholanolone and androsterone, which are derived from adrenal DHA)
to that of 17-hydroxyglucocorticoids (largely derived from cortisol)
improved the predictive power of such hormone measurements. While it
was not readily apparent 30 yr ago why the test had such predictive
value, it was recently postulated (151) that the discriminant function
test may, in fact, be a marker of Th1/Th2 cytokine production. Adrenal
androgen production is reduced in women with breast cancer, and thus an
imbalance in the DHA-glucocorticoid ratio would favor the production of
Th2 cytokines including IL-6. Increased production of IL-6 would result
in an increase in estrogen synthesis in peripheral and breast tissues.
There is also some evidence that in addition to T lymphocytes,
malignant cells themselves might also secrete Th2-type cytokines (152).
E. Stress and breast cancer
The role of stress in increasing the risk of breast cancer is
controversial, but a recent study showed convincing evidence for a
higher number of life-stress events in women with breast cancer but not
benign breast disease (153). Although the way in which stress
influences the development of breast cancer is very complex (154),
stress, as previously discussed, would be expected to increase
glucocorticoid production. As breast cancer occurs most commonly in
postmenopausal women, a time when DHA/DHA-S production is in decline,
then any increase in stress would move the T-helper cell response in
the direction of Th2 cytokine production and could result in increased
estrogen synthesis. BALB/c mice, subjected to water stress after Meth A
tumor transplantation, have an increase in their tumor size and tumor
growth rates compared with unstressed animals (155).
F. Immunosuppression and breast cancer risk
The incidence of breast cancer in kidney transplant recipients
receiving immunosuppressive therapy is 25% lower than expected in a
normal population (156). Immunosuppressive drugs, such as cyclosporin
A, act to inhibit white blood cell (wbc) production and the secretion
of cytokines by these cells. Therefore in immunosuppressed women
reduced amounts of cytokines would be available to stimulate estrogen
synthesis in breast and other peripheral tissues and may contribute to
the reduced incidence of breast cancer in these patients.
Support for the use of immunosuppressive drugs leading to a reduction in wbc cytokine production and estrogen synthesis has been obtained (69). Collection of CM from wbcs of an immunosuppressed subject revealed a marked reduction in its cytokine content and its ability to stimulate aromatase activity compared with CM collected from a woman with breast cancer.
G. Failure of glucocorticoids to stimulate aromatase activity in
vivo
If IL-6 is an important factor regulating in vivo
aromatase activity, then this might offer some explanation for the
discrepancy, as previously noted, in the ability of glucocorticoids to
stimulate in vitro but not in vivo aromatase
activity (30, 33, 34). Dexamethasone can only induce aromatase activity
in vitro in the presence of FCS, but the reason for this and
the factors facilitating the ability of dexamethasone to stimulate
aromatase activity are not known. A factor with a molecular mass in the
region of 150–300 kDa has been isolated in FCS as being responsible
for stimulating aromatase activity, but its identity is not yet known
(157). As dexamethasone induces IL-6R expression, it is possible that
it acts in vitro to increase the number of IL-6Rs on cells
(158), thereby increasing the ability of IL-6, which may be present in
FCS in some complexed form, to stimulate aromatase activity. However,
while glucocorticoids act to increase IL-6R expression, they act
in vivo to inhibit IL-6 gene expression, possibly as part of
a feedback mechanism to limit the response to stress and infection
(159). Inhibition of IL-6 gene expression by glucocorticoids in
vivo could therefore account for their inability to stimulate
in vivo aromatase activity.
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X. Summary and Future Perspectives |
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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, have important roles in stimulating
estrogen synthesis in breast cancer cells although it is likely that
other stimulatory factors remain to be identified. Cells of the immune
system, which are attracted to infiltrate tumors by chemokines, are
probably the major source of cytokines and cytokine-soluble receptors
that have been found to stimulate estrogen synthesis. However, IL-6
derived from stromal tissue and TNF from adipocytes within the
breast are also likely to contribute to the production of regulatory
cytokines. A major paradox that has emerged from much of the research carried out during the last few years to isolate factors that stimulate estrogen synthesis is the realization that most factors that stimulate enzyme activity in vitro also inhibit cell growth (160). The only possible exception to these findings was for IGF-I/II, which while stimulating E2DH activity, also tended to increase cell growth (81). A possible explanation for this paradox may lie with the fact that macrophages and lymphocytes, which probably produce most of the cytokines that stimulate estrogen synthesis, are trying to act to inhibit the growth of tumor cells, i.e., carrying out their immunosurveillance role. However, in vivo with the availability of the appropriate substrates, i.e., androstenedione, estrone, and estrone sulfate, the stimulation of the activities of the enzymes that utilize these substrates produces sufficient estradiol to overcome the normal inhibitory effect that cytokines have on cell growth. Also, it is possible that the production of IL-6 sR by tumor fibroblasts and/or invading macrophages and lymphocytes overcomes the normal desensitization that would be expected to occur if these cells were producing large amounts of IL-6. Thus, in addition to oncogene activation, which can result in overexpression or aberrant functioning of growth factors and their receptors, it is possible that IL-6 sR production by cells that are in close proximity to malignant cells is yet another mechanism whereby normal cell growth mechanisms are subverted, thus enabling tumor cells to proliferate.
The concept that increased production of estrogens enables tumor cells
to overcome the inhibitory effects of the immune system is in keeping
with the hypothesis first put forward some years ago by Prehn and
Lappé (161). They postulated that the immune response was
unlikely to have evolved for the purpose of promoting tumor growth and
speculated "that the effects of the immune system on tumor growth
must be the inadvertent consequences of some attributes of the response
that are more advantageous to the individual." It also remains a
possibility, however, that in addition to IL-6 and TNF, which
inhibit in vitro cell growth, other cytokines may not only
stimulate estrogen synthesis but also cell growth. Leukemia-inhibitory
factor, for example, which stimulates aromatase activity (95), is known
to stimulate the proliferation of MCF-7 breast cancer cells (162) and
to be produced by ER- but not ER+ breast cancer cells (163). It is
also possible that another cytokine, IL-3, which has been shown to be
secreted by fibroblast derived from male breast tissue but not female
and which can inhibit E2DH reductive activity (164), may also have a
role in the complex regulation of breast tumor estrogen synthesis.
The last 10 yr has been an exciting period for research into the regulation of breast tumor estrogen synthesis. Not only have the enzymes involved in estrogen synthesis been isolated and their genes cloned (13, 14, 15, 16, 17, 18, 19) but specific inhibitors for some of the enzymes have also been developed (165, 166, 167, 168). However, it is becoming evident that the use of enzyme inhibitors alone may not be sufficient to produce the anticipated clinical benefits of total estrogen synthesis blockage (169, 170). At some stage in the near future it will therefore be necessary to develop novel strategies for inhibiting the growth of hormone-dependent breast tumors. Investigations to reveal the complex signaling pathway (95) whereby cytokines control enzyme activity may lead to the development of specific inhibitors of the signal transduction pathways that lead to increased estrogen synthesis in tumors. Understanding the interaction of cytokines with their receptors (171), and the factors that regulate their production and expression, could lead to the development of cytokine receptor antagonists that could have a therapeutic role in the treatment of breast cancer.
If, as discussed in this review, DHA or a related metabolite, such as androstenediol or androstenetriol (172, 173), does have an important antiglucocorticoid role in regulating the Th1/Th1 balance, it should be possible to obtain further evidence of an imbalance in cytokine production and thus a role for this steroid in the etiology of breast cancer. Also, the possibility of using DHA or DHA-S for the chemoprevention of breast cancer would appear to warrant further investigation.
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TopAbstractI. IntroductionII. Peripheral Estrogen...III. Influence of Breast...IV. Proposed Model for...V. Evidence in Support...VI. The Role of...VII. Origin of Estrogen...VIII. Regulation of Lymphocyte...IX. Clinical Observations...X. Summary and Future...References |
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