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Endocrinological Oncology |
Departments of Internal Medicine (S.J.S., R.J.P., J.K.) and Pathology (L.T.), Wayne State University, Detroit, Michigan 48201; Breast Cancer Program, Barbara Ann Karmanos Cancer Institute (S.J.S., R.J.P., L.T.), Detroit, Michigan 48201; and the Department of Internal Medicine, University of Virginia Health Sciences Center (R.J.S.), Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Dr. Steven J. Santner, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201.
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Abstract |
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The present study sought to provide more direct evidence of the predominant role for stromal cell aromatase in breast tumor tissue. Accordingly, breast tumor stromal and epithelial cells were examined for aromatase enzyme activity and messenger ribonucleic acid (mRNA) expression. Stromal and epithelial cells from benign tissue served as a means of comparing activity and regulation in benign and tumor tissue. Enzyme activity in stromal cells from breast tumor tissue was low basally, but increased by 30- to 1200-fold when induced by dexamethasone. Combining dexamethasone with phorbol esters and cAMP produced an additional 1.2- to 4.1-fold stimulation. Analyses of exons III/V and exons IX/X demonstrated that aromatase mRNA expression was also substantially increased by these treatments.
Increases in enzyme activity and mRNA expression in cells from benign breast stroma paralleled those observed in tumor stroma, although the increases in enzyme activity were generally lower. In contrast to the responses observed in stromal cells, epithelial cells from breast tumor or nonmalignant breast tissue were unresponsive to dexamethasone, either added alone or in combination with phorbol esters and cAMP. This study provides direct biochemical evidence that aromatase is present in stroma within breast tumors, as in surrounding tissues, and suggests that estrogen synthesis within the tumor may modulate tumor growth via a paracrine mechanism.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine cellular localization, we recently compared aromatase activity measured biochemically with the level of immunohistochemical staining in the stromal and epithelial components of human tumors (14). Although staining occurred in both components, biochemical activity correlated only with staining in the stromal component. Stromal cells in neoplasms are a manifestation of the desmoplastic reaction, resulting in the appearance of fibrous tissue within the tumor. Recent studies have characterized these cells as myofibroblasts and demonstrated that their origin is mainly preexisting fibroblasts and smooth muscle, with some contribution from pericytic cells (15). These cells comprise a substantial proportion of the cells present in breast tumors, averaging 45% in one study (14).
Additional data from nonmalignant tissues also suggest that the stromal cell compartment may be an important source of aromatase (16, 17, 18, 19, 20, 21). In abdominal or benign breast adipose tissue, the stromal compartment contributes substantially to aromatase activity and is capable of dynamic responses to regulatory stimuli. On the other hand, studies of aromatase regulation in breast tumor epithelial cell lines in culture have revealed minimal stimulation by these regulators (22, 23, 24).
To provide direct biochemical evidence that the stroma is a significant source of aromatase in breast tumors, we have cultured stromal cells, measured their aromatase activity, and examined the regulation of this enzyme. In addition, we examined whether stromal cells from benign tissues from patients with tumors could also provide a substantial source of aromatase. Our findings indicated that myofibroblasts isolated from either breast tumor or benign stromal cells synthesize estrogens in vitro and that stimulators of aromatase transcription can increase enzyme activity by 3–4 orders of magnitude. In contrast, aromatase in epithelial breast cells was unresponsive to these agents. We conclude that the stromal compartment from breast tumors can serve as a site for sufficient estrogen production to stimulate tumor growth and that aromatase in stromal cells is capable of dynamic regulation by a variety of activators of transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stromal cell cultures were prepared from benign and breast
cancer tissues from each of three patients at either the Geraldine
Brush Cancer Research Institute (San Francisco, CA) or the Karmanos
Cancer Institute (Detroit, MI). Benign tissue was taken from the same
breast as the tumor at a site as distant as possible from the tumor and
was macroscopically nonmalignant. Patients were all postmenopausal,
estrogen receptor positive, and ranged in age from 57–72 yr. The
stromal cell isolation procedure is similar to that previously
described (25, 26) and is outlined here. Tissue pieces were trimmed of
fat and minced into fine (1- to 2-mm) pieces with scissors. The pieces
were incubated overnight in either Hams’s F-12 medium or Waymouth’s
MB 752/1 medium containing 15% calf serum, hyaluronidase (100 U/mL),
and collagenase (150 U/mL) and filtered the next day to remove
remaining undigested pieces. Cells were cultured routinely in
Waymouth’s MB 752/1 medium with 10–15% calf serum and used during
passages 4–13, except where noted. Cells were characterized using
monoclonal antibodies to 
-smooth muscle actin (Dako Corp.,
Carpenteria, CA), vimentin (Dako), desmin (Shandon Lipshaw, Pittsburgh,
PA), and cytokeratins 14 (Novocastra Laboratories, Newcastle on Tyne,
UK) and 18 (Dako). The procedure used was similar to that previously
described (27), except that methacarn fixation was used. All stromal
cell preparations, whether from tumors or benign tissues, were negative
for cytokeratins 14 and 18, negative for desmin, and positive for
-smooth muscle actin and vimentin. Based on these results, all
preparations, whether from benign or tumor tissue, appeared to be
mostly, if not completely, myofibroblasts (15, 28, 29, 30, 31).
Nonmalignant breast epithelial cells were obtained from three sources. One culture of normal epithelial cells was obtained from a reduction mammoplasty (Clonetics Corp., San Diego, CA) and was obtained at passage 8. These cells were passaged once into HMEC growth media (Clonetics Corp.). They were again passaged into this medium, allowed to grow to approximately 50% confluence, and stepped down into similar medium without hydrocortisone. After 1 day, experiments were set up similarly to those using stromal cell preparations. A nonmalignant breast cell line, MCF-10F, immortalized epithelial cells obtained from breast tissue of a patient undergoing a sc mastectomy for fibrocystic disease (32), were routinely grown in DMEM-Ham’s F-12 medium to allow the cells to grow as attached cells. They were passaged into IMEM containing 5% FCS and grown in this medium for 1 week before aromatase assay. The final culture tested was made up of epithelial cells isolated from benign breast tissue obtained from a patient with breast cancer. The original tissue was trimmed of fat and treated with collagenase and hyaluronidase similarly to tissues from which stromal cells were isolated, except that the resulting cells were grown in DMEM-Ham’s F-12 (1:1) containing 5% horse serum, insulin, epidermal growth factor, hydrocortisone, and cholera enterotoxin (32) until approximately 50% confluence was reached. At this point, the cells were stepped down into IMEM containing 5% FCS, but without hydrocortisone or other additives, for 1 day before experiments were initiated. The MCF-7 cell line, a breast tumor cell line derived from epithelial cells, was also tested. These cells were grown routinely in IMEM containing 5% FCS (33).
Experimental protocol and aromatase enzyme assays
Experiments were initiated by adding the test compounds in Waymouth’s medium (stromal cell cultures), HMEC medium without hydrocortisone (normal epithelial cells), or IMEM (MCF-10F, MCF-7, and benign breast epithelial cells). Cells were refed 24 h later (except where noted) with the same medium containing the test compounds. After an additional 24 h, flasks were again refed for 6 h with medium containing the test compounds and 1.8 µCi 1ß-[3H]androstenedione (DuPont-New England Nuclear, Boston, MA; final concentration, 20–25 nmol/L) to measure aromatase activity by tritiated water release (7). All test compounds were added as 1000- to 2500-fold concentrated solutions in 100% ethanol, except dibutyryl cAMP (dbcAMP), which was added as a 10-fold concentrated solution in medium. The final alcohol concentration was 0.2%, except during the aromatase assay when it was 0.3%. The source of the media and test compounds was Life Technologies, except for dbcAMP and phorbol 12,13-diacetate (PDA), which were obtained from Sigma Chemical (St. Louis, MO).
After the 6-h incubation for the aromatase assay, the medium was poured off and stored at -20 C until purification. The cells were washed with PBS and scraped off into additional PBS and frozen until assayed for protein by the Bradford method (34). In experiments involving analysis of messenger ribonucleic acid (mRNA), parallel cultures were washed with PBS and total RNA isolated. The purification method used for isolating the tritiated water produced is similar to that previously described (7), except that 150 µL 40% trichloroacetic acid containing 20 mg charcoal were added to 450 µL medium. After centrifugation, the supernatant was added to a column containing 20 mg activated charcoal in AG50-X4 cation exchange resin (Bio-Rad, Melville, NY) for support. The recoveries of tritiated water in samples were assessed by adding 2 µCi [3H]water in phosphate-buffered saline to tubes containing trichloroacetic acid-charcoal. These were then processed in the same way as the samples. Three different assays with five tubes each produced recoveries of 79.0 ± 0.6%, 79.8 ± 0.6%, and 81.0 ± 2.5% of the added tritiated water. The final values for aromatase enzyme activity via tritiated water release were not corrected for recovery, except for the comparison of aromatase by tritiated water release and direct product isolation. The direct product isolation method used in this comparison is similar to that previously reported (35). Flasks were run in parallel with the tritiated water flasks and used 1.8 µCi [7-3H]androstenedione in each flask. Estrone and estradiol were purified using previously established thin layer chromatography methods (35), and [14C]estrone (500 dpm) and [14C]estradiol (250 dpm) were added as recovery markers.
RNA analysis
The molecular methods used were those described by Sambrook et al. (36), unless otherwise specified. After removal of medium for aromatase assay, flasks were rinsed twice with phosphate-buffered saline, and RNA was extracted via a guanidium isothiocyanate method (37) using RNA isolator (Genosys Biotech, The Woodlands, TX). The RNA concentration was determined spectrophotometrically. This concentration was verified, and mRNA integrity was evaluated by denaturing formamide-formaldehyde 1.0% agarose gel electrophoresis followed by hybridization with a glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe. Oligonucleotides for PCR and hybridization probes were obtained from Genosys, except G3PDH amplimers and probe, which were purchased from Clontech (Palo Alto, CA). Aromatase exon III and exon IV/V PCR amplimers (38) were used to produce a 196-bp product from aromatase mRNA vs. a predicted more than 10-kilobase product from genomic DNA (39, 40). Also used were aromatase exon IX and exon X amplimers (41) to produce a 293-bp product vs. a predicted more than 1.5-kilobase product from genomic DNA (39, 40). Aromatase oligonucleotide hybridization probes were exon IV (sense, 5'-ATTACAGCTCTCGATTCGGCAGCAA-3') (42) and exon IX (sense, 5'-CCCCAAACCCAATGAATTTACTC-3') (39). G3PDH PCR amplimers and probe produce and detect a 452-bp product from G3PDH mRNA.
Reverse transcription (RT) of 0.5 µg total RNA was performed using the RNA PCR kit (Perkin-Elmer Corp, Foster City, CA) containing 10 mmol/L Tris-HCl (pH 8.3); 50 mmol/L KCl; 5 mmol/L MgCl2; 1.0 mmol/L each of deoxy (d)-ATP, dCTP, dGTP, and dTTP; 4 µmol/L random hexamer primers; 1 U/µL ribonuclease inhibitor; and 2.5 U/µL murine leukemia virus reverse transcriptase. After a 10-min incubation at room temperature, the reaction was carried out for 30 min at 42 C, 5 min at 99 C, and 5 min at 4 C in a Perkin-Elmer 9600 thermal cycler. RT controls lacked reverse transcriptase. An aliquot of the RT product from each sample, containing the equivalent of 0.1 µg RNA originally, was added to a PCR reaction mixture containing 0.4 µmol/L of each amplimer; 10 mmol/L Tris-HCl (pH 8.3); 50 mmol/L KCl; 2 mmol/L MgCl2; 0.4 mmol/L each of dATP, dCTP, dGTP, and dTTP; and 2.5 U AmpliTaq DNA polymerase. After a 1-min incubation at 94 C, there were 35 (aromatase) or 30 (G3PDH) PCR cycles of 94 C for 0.5 min, 58 C (aromatase exon III and IV/V amplimers) or 60 C (aromatase exons IX and X and G3PDH amplimers) for 0.5 min, and 72 C for 1.5 min. The reaction was terminated by an additional incubation of 5 min at 72 C.
Aliquots of each PCR product, containing the equivalent of 20 ng RNA originally, were electrophoresed through NuSieve (3:1) agarose (FMC Bioproducts, Rockland, ME) in 1 x Tris borate EDTA (TBE) buffer, using a 100-bp ladder (GenSura Laboratories, Del Mar, CA) as a molecular size reference. Gels were stained with ethidium bromide, photographed, and blotted to GeneScreen Plus membranes (New England Nuclear Research Products, Boston, MA) with 10 x SSC (standard saline citrate) buffer. To evaluate the quantity of PCR product, serial 1:2 dilutions were made and denatured in 0.4 N NaOH and 25 mmol/L Na2 ethylenediamine tetraacetate, followed by neutralization in 20 x standard saline phosphate EDTA (SSPE) buffer. The solutions were then applied to membranes under gentle vacuum using a 96-well dot blot apparatus (Schleicher and Schuell Corp., Keene, NH). Oligonucleotide probes were 5'-end labeled with To polynucleotide kinase to a specific activity of approximately 8 x 106 cpm/mmol. Hybridizations were carried out in 3 mol/L tetramethylammonium chloride, 50 mmol/L Tris-HCl (pH 7.0), 2 mmol/L Na2 ethylenediamine tetraacetate, 0.1% SDS, 5 x Denhardt’s solution, and 100 µg/mL sheared heat-denatured salmon testis DNA overnight at 56 C. Washing was under stringent conditions, up to 10 min at 56 C in 5 x SSPE and 0.1% SDS. Autoradiography was performed at -76 C to optimize analysis in the linear range for evaluation by computerized scanning laser densitometry (model 300A, Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin and
vimentin, and negative staining for cytokeratins 14 and 18. Cells from
either source contained very low unstimulated aromatase activity (Fig. 1
). However, 100 nmol/L dexamethasone (DEX), a regulator
of aromatase transcription, increased enzyme activity by 30- to
1200-fold. Combining dbcAMP and PDA with DEX produced a 1.2- to
4.1-fold stimulation over that achieved with DEX added alone. dbcAMP
and PDA added together caused little or no increase in aromatase
activity over that in unstimulated controls. When dbcAMP and PDA were
added with DEX, cooperative effects were observed. These effects were
variable, but greater than additive stimulation occurred (1.1- to
almost 4-fold) over that provided by the sum of activities with DEX
alone and with PDA and dbcAMP without DEX. Although breast tumor
myofibroblasts generally exhibited higher levels of stimulated activity
than those of the contiguous benign tissue, these differences were not
significant. A larger number of samples will need to be studied to
confirm this trend. Letrozole (CGS 20267), a proven aromatase inhibitor
(43), completely blocked enzyme activity, providing evidence that this
assay was specifically measuring aromatase activity.
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). Activity measured by direct product isolation was
slightly higher than that seen using the tritiated water assay,
indicating that the tritiated water assay was not overestimating
activity, as has been observed in several other tissues (44, 45, 46, 47). The
final estrogen concentration (estrone plus estradiol) achieved in the
medium was 4.6 nmol/L, a level sufficient to stimulate tumor growth
in vivo (48) in hormonally responsive tumors.
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). DEX
(10-7 mol/L) caused nearly maximal stimulation of activity
(either with or without PDA and dbcAMP) in benign breast stromal cells,
whereas in stromal cells from breast tumors 10-5 mol/L DEX
provided an additional stimulation of enzyme activity, at least when
combined with PDA and dbcAMP.
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. Enzyme activity was only slightly higher after 6
h of treatment with either DEX alone or DEX in combination with PDA and
dbcAMP. Activity increased to maximal levels by 30 h and was
maintained at 54 h.
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), a finding consistent with enzyme activity results.
However, unlike enzyme activity, essentially peak mRNA levels occurred
within 6 h and were maintained at 30 and 54 h (Fig. 5A). As
expected, Letrozole, which inhibits enzyme activity directly, had no
effect on aromatase mRNA levels. As defectively spliced transcripts
producing nonfunctional enzyme (missing exon X) have been observed in
rat ovary and Leydig tumor cells (51), analysis was repeated using exon
IX and X amplimers and an exon IX probe. The results were very similar
to the experiments in which the exon III and IV/V amplimers were used
(Fig. 5B). This suggests that the entire sequence is transcribed and
that defectively spliced aromatase transcripts are not responsible for
the discordant enzyme activity and mRNA levels observed at 6 h.
Differences in relative mRNA levels were not due to sample variations
in RNA quantitation or RT efficiency, as essentially equal amounts of
G3PDH PCR product were detected in all samples after hybridization with
a G3PDH probe (Fig. 5C).
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). These results demonstrated a 16- to
64-fold relative increase in aromatase mRNA accumulation in treatment
groups (DEX alone or DEX, PDA, and dbcAMP) over control levels at all
time points studied. Comparison of the aromatase enzyme activity and
mRNA levels after treatment clearly showed the increase in mRNA levels
after 6 h relative to enzyme activity (Fig. 7). The
stimulation of aromatase activity and mRNA levels and the degree of
stimulation observed after treatment were similar in the stromal cells
prepared from benign breast tissue of this patient (data not
shown).
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).
Additionally, these treatments caused little or no change in aromatase
mRNA levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor stromal cells are a component of the desmoplastic reaction that occurs commonly in several types of neoplasm and nearly uniformly in breast cancer. It involves recruitment of simple fibroblasts and vascular smooth muscle cells and results in the interspersing of stromal and neoplastic cells. We previously estimated the prevalence and magnitude of the desmoplastic reaction in human breast tumors and found stromal cells in all tumors (comprising a minimum of 15% of all cells), on the average representing 45% of the total cell population in the tumor (25–75th percentile, 39–50%) (14).
Several observations suggest that the desmoplastic stromal cells contribute predominantly to estrogenic stimulation of tumor epithelial cells. First, aromatase activity measured biochemically in total tumor homogenates correlated significantly with histological aromatase scores in tumor stromal, but not tumor epithelial, cells (14). Second, the activity of aromatase in cultured stromal cells from tumors is substantial when studied under optimal conditions (Ref. 52 and this report), whereas that in epithelial cells of diverse origin is minimal (Refs. 22–24 and this report). Finally, tumors contain a large proportion of stromal cells that are in close proximity to the tumor epithelial cells (14, 15). Similar levels of aromatase were found in the surrounding benign cells. This source may be significant during carcinogenesis or the early stages of tumor growth, but would be of decreasing importance as the tumor enlarges. Although no conditions resulting in high levels of aromatase enzyme activity have yet been discovered in breast tumor epithelial cells, further work is necessary to understand the potential for autocrine regulation of estrogen levels in these cells.
These studies demonstrated that DEX substantially increased aromatase activity. Although not necessarily reflecting a physiological effect of glucocorticoids in vivo, these observations indicate the potential for regulation by endogenous factors. The observations of Utsumi et al. (11) provide additional support for this concept. They found that aromatase mRNA was alternatively spliced in human breast tumors using exon 1b or 1c, suggesting preferential stimulation of enhancer elements by endogenous factors. Interestingly, they found that exon 1c expression in lymph node metastasis correlated with the presence of scirrhous carcinoma, which contains a large stromal component. In our studies, regulation of aromatase was achieved at least in part by aromatase mRNA accumulation, as previously observed in adipose tissue and skin fibroblasts (21, 49, 50). Treatment with DEX alone substantially increased both enzyme activity and mRNA level. However, the combination of DEX, PDA, and dbcAMP further stimulated enzyme activity severalfold without having an apparent effect on mRNA level. These results suggest that mechanisms other than transcriptional regulation may also be operating to regulate estrogen synthesis via aromatase in these cells. Effects on message stabilization, rate of translation, posttranslational processing, or alterations in aromatase protein half-life are all possibilities that could be tested.
Although myofibroblast cultures produced from the stromal component of both benign breast and breast tumor tissues were responsive to glucocorticoid stimulation of aromatase, none of the epithelial cells, whether cultured from nonmalignant or tumor tissue, demonstrated significant responses to DEX, either alone or in combination with phorbol esters and cAMP. However, the cellular localization of aromatase is controversial (14, 53, 54, 55), and it remains possible that additional factors might be capable of stimulating aromatase activity in epithelial cells. A series of factors, including steroids, growth factors, phorbol esters, and cAMP, either alone or in some combinations, have been used to attempt to stimulate enzyme activity in MCF-7 or T47D breast cancer cells. Although statistically significant increases in activity were reported, the total stimulation produced by any factor was never greater than a doubling (22, 23), whereas increases of 3–4 orders of magnitude were achieved in stromal cells. Also, the maximal aromatase activity in epithelial cells was much lower than that in stromal cell preparations. These data support the concept that stromal aromatase may be a major contributor to estrogen production in breast tumors, although contributions from other enzymatic sources, such as estrone sulfatase (35, 56), are not ruled out. These data further suggest that if in situ estrogen production via aromatase significantly stimulates the growth of hormonally responsive breast tumors, it would require paracrine interactions between the stromal and epithelial components because the stromal compartment appears to contain most of the estrogen synthetic machinery, whereas epithelial cells contain the estrogen receptors (57, 58).
Support for the existence of parenchymal influences on the stromal
component is provided by a study indicating that the stromal cells
closest to the tumor can convert to a myofibroblastic pattern of
immunohistochemical staining (15). Normal breast fibroblasts growing in
three-dimensional collagen gels convert to myofibroblasts in the
presence of breast tumor epithelial cells, but not in their absence.
The degree of conversion to myofibroblasts was inversely related to the
distance from the tumor cell nests. In addition to providing evidence
for paracrine interactions between stromal and epithelial cells, this
study suggests that myofibroblasts would be close to and within the
tumor in vivo and, thus, could provide mitogenic support of
the tumor. Our cells, both malignant and benign, were characterized
immunohistochemically as myofibroblasts, a finding consistent with a
number of studies in vivo and in cultured cells (15, 28, 29, 30, 31), whereas breast tissue from individuals without cancer appears
to contain few myofibroblasts (15, 28, 29, 31). Conditions of tissue
culture can alter the phenotypic properties of stromal cells. Serial
passage of fibroblasts can cause the expression of -smooth muscle
actin, and fibroblasts can apparently convert to myofibroblasts in
culture (15). Nevertheless, the demonstration of myofibroblasts in the
stromal component of virtually all breast tumors studied suggests that
study of cultured myofibroblasts can yield useful information
concerning the in vivo interactions between stromal and
parenchymal components of breast tumors.
In summary, the aromatase enzyme is highly regulated and appears to be localized to the stroma of breast tumor and benign breast tissues. Under some circumstances, this enzyme in breast tumor appears capable of producing sufficient quantities of estrogens in vitro to stimulate tumor growth in vivo in hormonally responsive individuals. However, definitive conclusions regarding the relative contributions of the epithelial and stromal components awaits the development of cell-specific enzyme assays for aromatase.
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Acknowledgments |
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Footnotes |
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Received February 26, 1996.
Revised August 21, 1996.
Accepted August 28, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4-androstenedione to estrone in normal males and
nonpregnant normal, castrate and adrenalectomized females. J Clin
Endocrinol Metab. 27:1103–1111.[Medline]
. Mol Cell Endocrinol. 106:17–21.[CrossRef][Medline]
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