Regulation of aromatase activity within the breast

J. SteroidBiochem.Molec.Biol.Vol. 61, No. 3-6, pp. 193-202, 1997

Pergamon

PII: S0960-0760(96)00202-6

© 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0960-0760/97 $17.00+ 0.00

Regulation of A r o m a t a s e Activity Within the Breast W . R . M i l l e r , ~* P . M u l l e n , ~ P . S o u r d a i n e , t C . W a t s o n , 2 J. M . D i x o n 3 a n d J. T e l f o r d 1 ~ICRF Medical Oncology Unit, University Dept. of Clinical Oncology, Western General Hospital, Edinburgh EH4 2XU, U.K.; 2Roslin Institute, Roslin, Midlothian EH25 9PS, U.K. and 3Breast Unit, Western General Hospital, Edinburgh EH4 2XU, U.K.

L o c a l o e s t r o g e n b i o s y n t h e s i s w i t h i n t h e b r e a s t c a n b e h i g h l y v a r i a b l e , in v i t r o a r o m a t a s e a c t i v i t y b o t h in b r e a s t c a n c e r s a n d m a m m a r y a d i p o s e t i s s u e d i s p l a y i n g o v e r a 40-fold r a n g e b e t w e e n t h e highest a n d l o w e s t levels. E v i d e n c e is p r e s e n t e d to s h o w t h a t : (i) t r a n s c r i p t i o n a l a c t i v i t y m a y i n f l u e n c e o e s t r o g e n b i o s y n t h e s i s w i t h i n b r e a s t c a n c e r s in t h a t b o t h a r o m a t a s e m R N A a n d S T A T n u c l e a r b i n d i n g a r e c o r r e l a t e d p o s i t i v e l y to in vit•o a r o m a t a s e a c t i v i t y ; (ii) t h e l o c a l p r e s e n c e o f c a n c e r m a y e n h a n c e a r o m a t a s e a c t i v i t y in p a r t i c u l a t e f r a c t i o n s a n d p r i m a r y f i b r o b l a s t c u l t u r e s f r o m m a m m a r y a d i p o s e t i s s u e ; (iii) t u m o u r e x t r a c t s a n d b r e a s t c y s t fluids m a y i n ä u c e a r o m a t a s e in c u l t u r e d f i b r o b l a s t s , t h e a c t i v e p r i n c i p l e s r e s p o n s i b l e f o r t h e s e effects b e i n g i n c o m p l e t e l y d e f i n e d ( a l t h o u g h t h e c o m b i n a t i o n o f i n t e r l e u k i n ( I L ) - 6 a n d its s o l u b l e r e c e p t o r d r a m a t i c a l l y e n h a n c e s a r o m a t a s e a c t i v i t y , it is u n c l e a r w h e t h e r this p a r t i c u l a r c y t o k i n e s y s t e m c a n a c c o u n t f o r t h e s t i m u l a t o r y effects o f b r e a s t e x t r a c t s a n d fluids); (iv) t h e a r o m a t a s e a c t i v i t i e s in b o t h b r e a s t c a n c e r a n d a d i p o s e t i s s u e s a r e s u s c e p t i b l e to c l a s s i c a l a r o m a t a s e i n h i b i t o r s s u c h as a m i n o g l u t e t h i m i d e a n d 4 - h y d r o x y a n d r o s t e n e d i o n e ( a n d to n e w e r i n h i b i t o r s s u c h as C G S 1 6 9 4 9 a n d C G S 2 0 2 6 7 a t low n a n o m o l a r c o n c e n t r a t i o n s ) b u t r e d u c e d s e n s i t i v i t y to 4 - h y d r o x y a n d r o s t e n e d l o n e m a y b e o b s e r v e d in c e r t a i n b r e a s t c a n c e r s . T h e s e findings m a y have i m p o r t a n t implications for the d e v e l o p m e n t a n d progression of h o r m o n e - d e p e n d e n t c a n c e r s w i t h i n t h e b r e a s t . © 1997 E l s e v i e r S c i e n c e L t d

fr. Steroid Biochem. Molec. BioL, Vol. 61, No. 3-6, pp. 193-202, 1997

INTRODUCTION T h e development of the breast and the growth of cerrain breast cancers appear to be dependent upon oestrogen [1]. Although in premenopausal w o m e n this h o r m o n e may be supplied by the ovary, in postmenopausal w o m e n major sites of oestrogen synthesis appear to be peripheral [2-5]. T h e breast itself has the potential for such biosynthesis, both m a m m a r y adipose tissue and breast cancers displaying the aromatase activity necessary to convert androgen precursor into oestrogen [6, 7]. Although levels of aromatase activity in the breast are relatively low, they are comparable with those in other peripheral sites [8, 9], and such oestrogen is being produced on site within a potentially sensitive organ. It is therefore of import-

ance to determine the factors which might influence and regulate oestrogen biosynthesis within the breast; this is the subject of the present paper.

MATERIALS AND M E T H O D S

Breast tissues

Breast cancers were obtained either by biopsy or at mastectomy from patients with histologically confirmed malignancy of the breast. Adipose tissue was derived at operation from the breasts of w o m e n presenting with either benign or malignant conditions of the breast. Breast cyst fluids were obtained by needle puncture of palpable cysts. Specimens from which cells were to be cultured were immediately transferred aseptically into sterile phosphate-buffered saline Proceedings of the IV International Aromatase Conference, Tahoe (PBS). All remaining tissues were put on ice in the City, CA, U.S.A., 7-11 June 1996. *Correspondence to Prof. W. R. Miller. operating theatre and transported to the laboratory. 193

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Tissue homogenates T u m o u r s to be assayed for aromatase activity were finely sliced with scissors and sonicated in Krebs Ringer phosphate buffer twice for 1 min using an MSE sonicator on the maximum setting. H o m o g e n a t e s of t u m o u r to be included in cell culture were prepared by disruption in « M E M m e d i u m using a Silverson homogenizer ( m a x i m u m speed for 10 s x 2) and centrifuged at 1000g for 10 min. T h e resultant supernatant was sterilized through filters and added to cultures at a fmal concentration of 0.25 and 2.5%. Particulate fractions Adipose tissue was carefully dissected from breast parenchyma, homogenized by hand in a glass to glass homogenizer in phosphate buffer and centrifuged at 800g for 5 min. T h e resultant supernatant was separated from the upper layer of fat and lower pellet of cell debris using a Pasteur pipette and was centrifuged at 100,000g for 1 h. T h e pellet was resuspended in buffer and used as a particulate fraction in the aromatase assay. Fibroblast cell cultures Gross fat was teased from breast adipose tissue and the stromal-enriched fraction was chopped into small pieces. Aliquots (2 g) were then placed into universal containers and minced. Collagenase (10 ml; 2 mg/tal) was added to each vial and incubated for 30 min at 37°C in a shaking water bath. T h e contents were allowed to settle for 10 min and the liquid phase was aspirated from below the surface lipid layer and centrifuged at 3000 r p m for 10 min. T h e resulting pellet was washed twice in PBS and resuspended in ~ - M E M supplemented with penicillin, streptomycin and heatinactivated fetal calf serum (15%). T h e cell suspension was then aliquoted into 60 m m Petri dishes (4 ml per dish) and grown as monolayer cultures in an atmosphere of 5% CO2:95% air. After 48 h cultures were washed twice with PBS and fresh media added. Cells were then allowed to grow to confluence. Before assaying for aromatase activity, primary cell lines were incubated with dibutyryl cyclic A M P (1 m M ) in the absence of F C S for 4 days, whereas passaged cells were incubated with dexamethasone (1 #M) in the presence of F C S (15%) for 18 la. Aromatase assays Aromatase activity in minces of breast cancer was determined by isolation of [3H] oestradiol after incubation with 7«[3H] testosterone as described previously [10]. In brief, t u m o u r minces were incubated for 2 h at 37°C in Krebs Ringer phosphate buffer ( p H 7 . 4 ) containing [7c~3H] testosterone (22.5/~Ci) and an N A D P H generating system. T h e reaction was stopped by the addition of methanol, and radioinert

oestradiol (500 #g) was added to monitor procedural losses. Metabolites were extracted into ethyl acetate and purified by extensive thin layer chromatography. Oestrogen fractions were characterized by chemical derivative formation (diacetates and methyl ethers), purity being based on the criteria that: (a) chromatographic mobility of oestradiol and derivatives was similar to that of authentic standards; and (b) constant specific radioactivity between patent steroid and derivatives. T h e formal level of detection for oestradiol was 0.02% of the original precursor; values in excess of this figure were considered to be positive evidence of aromatization. T h e determination of aromatase activity in adipose tissue was based on the m e a s u r e m e n t of [3H] water after incubation with [lfl3H] A4-androstenedione. Particulate fractions were incubated for 5 h at 37°C in phosphate buffer with [lfl3H] A4-androstenedione (1/tCi, 100 nM) and an NADPI-I generating system. Blank incubations were performed with bovine serum albumin (1.5 mg/ml) in place of the particulate fraction. Aliquots of each incubated system were dispensed into ice-cold chloroform, shaken and centrifuged. T h e aqueous phases were then mixed with 5% charcoal in phosphate buffer and centrifuged at 2000g for 15 min. T h e resultant supernatant was decanted into a counting vial containing N E 2 6 0 scintillant (10 tal) and counted. Aromatase activity was also determined in cultured fibroblasts using a similar technology, except that incubation was performed on monolayers growing in Petri dishes. This involved removing " s p e n t " tissue culture media, and washing dishes with PBS. T o each dish, [lfl-3H]-androstene dione (2 ~tCi; 100 nM), in « - M E M was added and incubation was carried out at 37°C for 5 h under an atmosphere of 5% CO2:95% air. Blank incubations consisted of dishes containing media but no cells. After incubation all dishes were placed on ice for 15 min, the m e d i u m aspirated, transferred to ice-cold chloroform (5 ml), shaken vigorously and centrifuged for 5 min at 3000 rpm. T h e radioactivity in the aqueous phase was measured after charcoal extraction as described above. P450 Aromatase transcripts Total R N A was extracted using a modification of the m e t h o d of Auffrey and Rougeon [11]. In brief, frozen tumours (0.3-0.4 g) were pulverized in liquid nitrogen, suspended in 3 M lithium chloride - ó M urea, sonicated and left overnight at 4°C. Total R N A was recovered by centrifugation (10,000g), and resuspended in 1 0 m M Tris p H 7.5, 0.5% SDS, 5 m M E D T A containing proteinase K (50/~g/ml) and incubated at 37°C for 2 0 m i n . Samples were extracted twice with phenol/chloroform, once with chloroform and precipitated with NaCl/ethanol. R N A was resuspended in water at the concentration of 1/~g//~l and stored at - 8 0 ° C . Quantitation of aromatase cyto-

Regulation of Aromatase Activity within the Breast chrome P450 m R N A was measured by a differentialsize polymerase chain reaction (PCR)-aided transcript titration assay [12, 13]. A 32 bp deleted P 4 5 0 a r o m c D N A was constructed as an intemal control [14]. One microlitre (1/ag) of total R N A from breast tumours (1 #g/1/tl) and 1/tl of different amounts of internal control (cRNA) were added to a mix (18/tl) with the following composition: 17.2/tl of water, 2/tl of 10 × T a q buffer, 1.5/tl of MgCI2 (25 m M ) , 0.6/tl of d N T P (10 m M ) , 0.6/tl of each 5'- and 3'-oligonucleotides (20/aM). T h e mixture was heated at 95°C for 1 min using a heating block, and was quicldy chilled on ice. T a q buffer containing 50 units M M u L V reverse transcriptase (Rnase H minus; Promega, S o u t h a m p t o n , U.K.), 10 units RNasin ribonuclease inhibitor (Promega) and 2.5 units T a q D N A polymerase were added to each sample. Reverse transcription was performed at 37°C for 60 min, and then stopped by raising the temperature to 95°C for 2 min. T h e amplification was then carried out through 30 P C R cycles. Because of the low levels of P 4 5 0 a r o m transcripts in breast tumours, a second P C R was performed using a nested primer of the 3'-oligonucleotide. T h e product of the first reverse transcriptase ( R T ) - P C R (I/tl) was amplified in a final volume of 20/tl of P C R solution through 26 cycles. Resulting products (452 bp and 420 bp) were separated on a 5% polyacrylamide gel. Autoradiographs were quantitated using a computerized laser densitometer. T h e initial a m o u n t of the target P 4 5 0 a r o m was determined at the crossing point of the curves which correspond to a ratio of unity. T h e double-strand oligonucleotide used for the deletion of the P 4 5 0 a r o m c D N A was m a d e by annealing of the 23 m e r 5'-oligonucleotide 5 ' - G A T C G C T A T C GTGGTTAAATGCA-3' with the 15 m e r 3'-oligonucleotide 5'-TTTAAC CACGATAGC-3'. The sequences of the 19 m e r 5'-oligonucleotide and of the 20 m e r 3'-oligonucleotide used for the R T - P C R were 5'-GTCGTGTCATGCTGGACAC-3' (exon V) and 5'-AAGGCTTTGCGCATGACCAA-3' (exon IX), respectively. T h e sequence of the nested 20 m e r 3'oligonucleotide used for the second P C R was 5 ' TCACCAATAACAGTCTGGAT-3' (exons VIII and IX).

Nuclear binding activity by electrophoretic mobility-shifi assay (EMSA) Nuclear extracts were prepared by a modification of the m e t h o d of D i g n a m [15], as previously described [16]. Briefly, breast samples (100 mg) were ground to a fine powder under liquid nitrogen and then dispersed in buffer. T h e powder was then homogenized at 4°C and filtered through two layers of Miracloth. T h e filtrate was centrifuged at 2000 r p m for 10 min; the resultant nuclear pellet was washed in buffer, recentrifuged and the nuclei were lysed. C h r o m o s o m a l D N A and debris were removed by cen-

195

trifugation at 35,000 r p m for 30 min and the supernatant dialysed for 4 h against 100 volumes of buffer containing glycerol. Insoluble material was removed by brief centrifugation and the cleared nuclear extract aliquoted and flash frozen in liquid nitrogen. E M S A was carried out using a 17 bp double-stranded oligonucleotide which contained the highest affmity binding sequence for Stat5 (GATTCCGGGAACC G C G T ) and 4 #g of nuclear extract. Complexes were resolved on native 6% polyacrylamide gels which were fixed and dried before quantitation and autoradiography.

RESULTS

Variation in aromatase activity within the breast Aromatase activity was measured in 236 breast cancers by product isolation. Activity was detected (>_0.02% conversion of 7~3H testosterone to oestradiol) in 166 (70%), with levels ranging from 0.02 to 0.83% (Fig. 1). Using 3H release from 1/j3H androstenedione, aromatase activity was observed in the particulate fractions from all 100 specimens of breast adipose tissue, with levels varying from 3 to 120 fmol/ m g protein/h (Fig. 1). Therefore using different assay methods and sources of tissue, a 40-fold range of activity was evident for aromatase within the breast.

Correlation between aromatase mRNA

activity and level of

Measurements of aromatase activity (by »H release) and P 4 5 0 a r o m transcripts were m a d e in 20 breast cancers. Activity and aromatase m R N A were detected in all samples. T h e level of transcript ranged from 50 mol/#g total R N A (corresponding to 0.8 x 1 0 - 4 amol) to 2450 mol//tg total R N A (corresponding to 40.6 × 10 -4 amol). T h e m e a n + S E M of aromatase transcript levels was 695 _ 169 mol. T h e relationship between aromatase activity and m R N A transcripts is shown in Fig. 2. T h e correlation by Spearman Rank test between the two parameters was not significant (P = 0.22). However, when plotted as a scattergram the distribution of the points reveals that whereas the majority of tumours (15/20) showed a relatively close correlation between activity and m R N A , five tumours showed disparate values between activity and m R N A (two tumours showed a low aromatase activity despite high transcript levels, and conversely three displayed high aromatase activity with low transcript levels). Sufficient material from one of the tumours which demonstrated low aromatase activity, despite having high transcript levels, was available for further analysis. T o determine if the low activity could be explained by the presence of an inhibitor in the particulate fraction, a dilution experiment was performed by mixing equal volumes of particulate fraction from this t u m o u r with another

196

W.R. Miller et al. Adinose Tissue (100}

Breast Cancer (236} Activity (%c°nversi°n)

Activity (fmol/mg protein/hr)

0.85

0.30 0.17

50

0.10

33

0.05

20

0.02
i 0

10 3 10

20

30 40 Number

50

60

70

0

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20 Number

30

Fig. 1. Level o f in vitro a r o m a t a s e activity in c o n s e c u t i v e s e r i e s o f b r e a s t c a n c e r s a n d m a m m a r y a d i p o s e tissues.

which had high aromatase activity and correspondingly high transcript levels. T h e results of this experiment indicate that the aromatase activity of the mixed particulate fractions was lower than the expected average value. This would be consistent with the presence of an endogenous inhibitor within the t u m o u r with low aromatase activity. A control experiment in which dilution was performed with particulate fractions from tumours having concordant low and high aromatase activity/mRNA transcripts produced the expected effect of average activity. Relationship with the S T A T family of transcription factors

Nine breast cancers were assayed for S T A T binding activity and aromatase activity by 3H release. S T A T complexes were evident in nuclear extracts of all specimens, but the a m o u n t of retarded probe varied

~" Z

between individual cancers. After quantitation the tumours were classified according to S T A T binding levels, using a stratification based on the larger series which have been reported previously [17]. This showed that tumours with S T A T levels greater than the median value possessed significantly higher levels of aromatase activity than those with S T A T levels lower than the median (Fig. 3). Association between breast cancer and aromatase in m a m mary adipose tissue

T o extend our previous studies in which aromatase activity in adipose tissue was shown to be elevated in quadrants of the breast in which cancer was located, a further eight m a s t e c t o m y specimens were investigated. Aromatase activity, as detected in particulate fractions derived from adipose tissue from each quadrant of

220 Activity (fmol/mg protein. hr) 60 ~

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Fig. 2. R e l a t i o n s h i p b e t w e e n t u m o u r a r o m a t a s e activity a n d P450arom transcrlpts.

< median

> median

STAT Transcription Fig. 3. T u m o u r a r o m a t a s e a c d v i t y in b r e a s t c a n c e r s w i t h h i g h a n d low S T A T n u c l e a r b i n d i n g .

Regulation of Aromatase Activity within the Breast

(a)

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.p~ Palpable ~~" tumor

(b)

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(c)

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197

shown in Fig. 4(b). Once again quadrants involved with t u m o u r tended to display higher aromatase activity than cultures derived from non-tumour-bearing quadrants. In four out of six cases the highest activity was thus in fibroblasts derived from quadrants with t u m o u r and the lowest activity was only seen in culrufes from a single tumour-bearing quadrant. T h e mean level of aromatase was higher in fibroblasts from tumour-bearing quadrants (compared with noninvolved quadrants) in five of the six mastectomy specimens. T h e level of aromatase activity in primary cultured fibroblasts also correlated with that found in the subcellular fractions from the tissue assayed at biopsy. T h e ordering of activity throughout the breast was thus identical in one breast and only showed a single transposition of activity between quadrants in the remaining breasts. In contrast, fibroblasts passaged on and incubated in the presence of dexamethasone failed to demonstrate a clear correlation between levels of aromatase and the quadrants from which the cultures were derived (Fig. 4(c)). For example, the fibroblasts with either the highest or lowest aromatase activity were derived from involved quadrants in three breasts in each case. Similarly, the level of aromatase activity in passaged cultures did not reflect the activity in particular fractions from the original excised material.

The effects of adding tumour extracts and breast cyst fluids to cultured fibroblasts

=, Highest

~

Palpable tumor

Fig. 4. Relationship b e t w e e n t u m o u r location within the breast and a r o m a t a s e activity in: (a) particulate fractions of m a m m a r y a d i p o s e tissue; (b) p r i m a r y fibroblast cultures of m a m r n a r y a d i p o s e tissue; (c) p a s s a g e d fibroblast cultures of

these mastectomy specimens, is shown diagrammatically in Fig. 4(a). T h e quadrants with the highest and next highest aromatase activity were each involved with t u m o u r in five of the breasts, whereas those with the lowest and next to the lowest were each involved in only orte case. Average aromatase activity was higher in tumour-bearing quadrants compared with tumour-free quadrants in six of the eight mastectomies. Although this positive association between t u m o u r involvement and aromatase activity showed the same trend as that observed previously, it failed to reach statistical significance. Fibroblast cultures were derived from each quadrant of all mastectomy quadrants. However, two sets became infected during the pre-incubation period with dibutyryl cyclic AMP. Results relating to aromatase activity of the remaining primary cultures are

T u m o u r homogenates were capable of a dose-related induction of aromatase activity in dexamethasone-treated cultures (data not shown). Effects were evident with both malignant and benign tumours, but the magnitude of effect could differ markedly between different homogenates. However, there was a tendency for the effects of extracts from malignant tumours to be quantitatively greater than those from benign tumours (data not shown). U n d e r the same conditions the addition of breast cyst fluids had a similar action, although the degree of effect varied greatly between different cyst fluids (Fig. 5). T h e levels of a variety of hormones, growth factors, cytokines and proteins were measured in the cyst fluids. Positive relationships were evident between aromatase stimulation and amounts of certain components within the cysts. As can be seen in Fig. 5, cyst fluids which contained substantial levels of interleukin (IL)6 produced a greater enhancement of aromatase activity than fluids with low or undetectable amounts of IL-6. However, within the group of fluids containing substantial amounts of IL-6 the quantitative relationship with the degree of stimulation and a m o u n t of IL-6 was relatively poor. Furthermore, the addition of IL-6 in amounts corresponding to levels in cyst fluids failed to produce stimulation of aromatase activity. Indeed as is shown in Fig. 6, IL-6 at a concentration of 50 ng/ml (which is greater than amounts

198

W.R. Miller et al. 20' IL6 0

0

0

0

0.5

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CF5

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22.3

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-FCS

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CF10

Fig. 5. T h e e f f e c t s o f b r e a s t c y s t f l u i d s o n a r o m a t a s e a c t i v i t y i n c u l t u r e d f i b r o b l a s t s o f m a m m a r y s u e . F i g u r e s r e f e r to c o n c e n t r a t i o n s ( n g l m l ) o f I L - 6 i n t h e c y s t f l u i d s .

detected in any cyst fluid) induced only a slight stimulation of activity. However, if this concentration was added in the presence of a soluble IL-6 receptor, a marked stimulation of aromatase activity was observed. T o explore the possibility that the stimulatory effects of cyst fluids were caused by the simultaneous presence of IL-6 and soluble IL-6 receptors and that the difference between cyst fluids might reside more in levels of soluble receptors, studies were performed in which IL-6 was added to cyst fluids which had a differing capacity to induce aromatase. These results are shown in Fig. 7 and indicate that IL-6, added in combination to cyst fluids, produced no greater effect in cyst fluid alone.

adipose tis-

related inhibition at micromolar and nanomolar concentrations, respectively; newer drugs such as C G S 1 6 9 4 9 and C G S 2 0 2 6 7 were even more potent. However, studies in which in vitro aromatase activity was investigated in patients before and during treatment with 4-hydroxyandrostenedione indicated that occasionally values paradoxically rose on treatment (Fig. 9). Similarly, aromatase in approximately 15% of breast tumours appeared to be resistant to 4-hydroxyandrostenedione when the drug was added to in vitro incubates (data not shown). This resistance appears at least specific to 4-hydroxyandrostenedione as other aromatase inhibitors such as aminoglutethimide and C G S 1 6 9 4 9 retain their efficacy (Table 1).

Sensitivity of breast aromatase to inhibitors

Aromatase in both breast cancer and m a m m a r y adipose tissue may be blocked by classical aromatase inhibitors. As is shown in Fig. 8, aminoglutethimide and 4-hydroxyandrostenedione thus produced a dose-

DISCUSSION

Despite the comparatively low levels, aromatase activity in both breast cancer and adipose tissue can vary markedly with over a 40-fold difference between the lowest and highest values. Given that local pro-

g

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adipose tissue.

Control

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CF1 + IL6

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Fig. 7. T h e e f f e c t s o f I L - 6 a n d c y s t f l u i d s a l o n e a n d in c o m b i nation on aromatase activity in cultured fibroblasts of mammary adipose tissue.

Regulation of Aromatase Activity within the Breast

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Fig. 8. T h e effects o f a r o m a t a s e i n h i b i t o r s o n a r o m a t a s e a c t i v i t y in e i t h e r c u l t u r e d f i b r o b l a s t s o f m a m m a r y a d i p o s e t i s s u e o r m i c r o s o m a l f r a c t i o n s o f a b r e a s t cancer. AMG, a m i n o g l u t e t h i m i d e ; 4OHA, 4 - h y d r o x y a n d r o s t e n e d i o n e ; 16949, CGS16949; 20267, CGS20267.

d u c t i o n o f oestrogen m i g h t have clinical significance in terms o f maintaining the g r o w t h o f h o r m o n e d e p e n d e n t cancer, it was o f interest to determine factors which m i g h t influence aromatase within the breast. In the present study we have presented two sets o f data which suggest that m a j o r regulation occurs at the level o f gene transcription. T h e r e was thus a clear positive quantitative relationship b e t w e e n aromatase activity and P 4 5 0 a r o m m R N A . A l t h o u g h the correlation did n o t reach statistical significance, the n u m ber o f t u m o u r s studied was small and the association was c o n f o u n d e d by a m i n o r i t y of t u m o u r s which potentially represent different subsets o f cancers. W e were also able to detect a significant positive relationship b e t w e e n J A K - S T A T transcriptional activity and that of aromatase. T h e s e observations w o u l d be con-

(A)

(B)

aromatase activity pmol oestradiol/g tissue

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aromatase activity % change

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sistent with the finding of a S T A r r binding site in the p r o m o t e r region o f the aromatase gene [18]. A l t h o u g h there is a generally positive association between transcriptional and aromatase activity, it was noticeable that again a single t u m o u r a p p e a r e d as an outlier. It is therefore w o r t h considering reasons for this discord a n c e b e t w e e n aromatase and transcriptional activities. First, for logistical reasons aromatase transcripts, S T A T binding and e n z y m e activity m e a s u r e m e n t s were m a d e on separate portions o f individual t u m o u r s which, because o f the k n o w n heterogeneity o f breast cancers m a y give rise to inherent variations. H o w e v e r , t u m o u r s with discordant values were o f no special type and were no m o r e h e t e r o g e n e o u s t h a n the other cancers. Second, it is conceivable that the 3H release assay, which does not definitively identify an oestrogen p r o d u c t , does not reflect aromatase activity.

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Table 1. Examples of differential sensitivi~y of aromatase in breast cancers to inhibitors Sensitive phenotype A

Insensitive phenotype B

55% 6% 5% C 64% 29% 36% 9%

93% 7% 23% D 165% 111% 29% 7%

+40HA (10 -s M) +40HA (10 -7 M) + A M G (10 -4 M) +40HA +40HA +16949 +16949

(10 -s M) (10 -7 M) (10 -s M) (10 7 M)

T u m o u r minces were incubated with 7ct3H testosterone in the absence and presence of inhibitor and oestradiol fractions characterized. Values a r e a percentage of aromatase observed in control incubations (without inhibitor). T u m o u r s A and C are examples of a phenotype sensitive to 4-hydroxyandrostenedione whereas tumours B and D are relatively insensitive to the drug.

Indeed, it has been suggested that this type of assay can produce false-positive results when used to detect low levels of aromatase activity [19]. Our unpublished experience is also that it is possible to encounter tumours which release 3H from ]]~3H androstenedione into an aqueous phase without being able to detect radioactively labelled oestrogen by product formation. However, we have utilized the product isolation assay for aromatase activity in a small series of different cancers, and observed that tumours could display the phenotype with low/no aromatase activity despite the presence of relatively high amounts of m R N A transcripts. Third, in those tumours with high m R N A and low aromatase it is possible that m R N A was not transcribed into protein, and conversely in those cancers with low m R N A and high aromatase that the protein may be stabilized. Finally, it is possible that endogenous activators/inhibitors of the enzyme activity might have an overriding influence. It was to explore this possibility that we mixed a particu-

late fraction from a t u m o u r displaying low aromatase activity and high m R N A with that from a t u m o u r having concordant high levels of m R N A and activity. That the resultant enzyme activity was disproportionately low would be consistent with the presence of an endogenous inhibitor. At this stage the nature of the agent(s) remains a matter of speculation. In terms of m a m m a r y adipose tissue, the present study confirmed that areas of the breast bearing cancer tend to have higher aromatase activity than noninvolved quadrants. There are several potential reasons for this, including: (i) tumours grow preferentially in areas which possess locally high aromatase activity; (ii) fat adjacent to tumours is inherently different or may disproportionately contain populations of cells which possess high aromatase activity; (iii) tumours secrete factors into their local environment which induce aromatase activity. In order to understand bettet the basic observation we have performed more detailed studies and shown that primary fibroblasts derived from breast quadrants which are involved with malignancy also tend to display higher aromatase activity in culture. The n u m b e r of fibroblasts in such cuhures do not vary according to the quadrants from which they were derived, and therefore the differences in aromatase are unlikely to be due to the original biopsies having variable proportions of fibroblasts. It seems more likely therefore that either the fibroblasts are inherently different when derived from cancer-bearing quadrants, or are contaminated with cells which have high aromataseinducing factors or have been influenced by the close proximity of tumour. In the case of the latter scenario it might be expected that any t u m o u r influence would disappear on passage, which was the case in the present investigation. However, a complicating factor in the studies was that, in order to demonstrate aromatase activity in passaged fibroblasts, it was

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Regulation of Aromatase Activity within the Breast n e c e s s a r y to c u l t u r e in the p r e s e n c e o f g l u c o c o r t i coids. W h e t h e r t h e results a r e a f e a t u r e o f p a s s a g e or t h e c h a n g e in i n d u c i n g a g e n t f r o m d i b u t r y l cyclic A M P to d e x a m e t h a s o n e is still to b e resolved. T h e d e x a m e t h a s o n e - t r e a t e d c u l t u r e s were also u s e d as a m o d e l s y s t e m in w h i c h to test t h e ability o f b r e a s t - d e r i v e d extracts a n d fluids to i n d u c e a r o m a t a s e activity. T h e s e studies s h o w the clear p o t e n t i a l o f s u c h extracts a n d fluids to p o t e n t i a t e a r o m a t a s e activity. T h e t e n d e n c y for h o m o g e n a t e s o f m a l i g n a n t t u m o u r s to p r o d u c e q u a n t i t a t i v e l y g r e a t e r effects would be consistent with cancers having inherently h i g h e r S T A T t r a n s c r i p t i o n a l activity [17], a n d b e i n g able to p r o d u c e factors w h i c h c a n i n d u c e a r o m a t a s e activity in t h e s u r r o u n d i n g s t r o m a l fibroblasts. (In this r e s p e c t it is r e l e v a n t t h a t s o m e b u t n o t all studies have shown that aromatase within breast cancers t h e m s e l v e s m a y i m m u n o l o c a l i z e to the s t r o m a l c o m p o n e n t [20, 21].) T h e i d e n t i t y o f i n d u c i n g factors r e m a i n s to b e definitively d e t e r m i n e d . G r o w t h factors a n d c y t o k i n e s , such as I G F - I , I L - 6 a n d I L - 8 m a y b e s t i m u l a t o r y [7, 2 2 - 2 4 ] . H o w e v e r , o u r u n p u b l i s h e d results i n d i c a t e t h a t b l o c k i n g a n t i b o d i e s d o n o t n e c e s s a r i l y a t t e n u a t e effects in t u m o u r extracts a n d b r e a s t fluids; n e i t h e r d o c o n c e n t r a t i o n s o f i n d i v i d u a l c y t o k i n e s w i t h b r e a s t extracts a n d fluids always c o r r e late weil w i t h q u a n t i t a t i v e effects o n a r o m a t a s e . T h e r e m a y b e c o n f o u n d i n g factors s u c h as the n e e d to m e a s u r e b o t h cytokines a n d t h e i r r e c e p t o r s - - s y n e r gic effects o f I L - 6 a n d its s o l u b l e r e c e p t o r are s p e c t a c u l a r in c o m p a r i s o n w i t h t h e m o d e s t effects o f e i t h e r a g e n t a l o n e ( S i n g h et al. [25] a n d t h e p r e s e n t studies). H o w e v e r , it was n o t a b l e t h a t w h e n I L - 6 was a d d e d to cyst fluids w h i c h a l o n e h a d t h e p o t e n t i a l to i n d u c e a r o m a t a s e , t h e c y t o k i n e failed to p r o d u c e e n h a n c i n g effects. It s e e m s likely t h a t o t h e r c y t o k i n e s m a y p l a y i m p o r t a n t roles a n d their i d e n t i t y m a y b e crucial in u n d e r s t a n d i n g the r e g u l a t i o n o f a r o m a t a s e in t h e b r e a s t - - especially as t h e r e is e v i d e n c e t h a t a r o m a tase t r a n s c r i p t i o n w i t h i n t h e b r e a s t m a y utilize m u l tiple p r o m o t e r s w h i c h involve a l t e m a t i v e splicing m e c h a n i s m s [26]. I n t e r m s o f c o n t r o l s at t h e level o f e n z y m e activity the possibility that endogenous activators/inhibitors m a y exist w i t h i n the b r e a s t h a s a l r e a d y b e e n a l l u d e d to. C o n f i r m a t i o n o f the e x i s t e n c e o f s u c h m o d i f i e r s is w o r t h w h i l e b e c a u s e o f t h e p o t e n t i a l benefits f r o m the b l o c k a d e o f t u m o u r a r o m a t a s e in o e s t r o g e n - s e n s i t i v e cancers. F o r t h e m o m e n t , s y n t h e t i c a r o m a t a s e i n h i b i tors fulfil this n e e d a n d have a n e s t a b l i s h e d role in the treatment of postmenopausal patients with breast cancer [27]. T h e r e is no d o u b t i n g t h a t s o u r c e s o f o e s t r o gen b i o s y n t h e s i s in s u c h w o m e n are p e r i p h e r a l , b u t c o n t r o v e r s y still exists as to w h e t h e r a r o m a t a s e w i t h i n the b r e a s t a n d t u m o u r s has m o r e r e l e v a n c e t h a n m o r e d i s t a n t sites. I r r e s p e c t i v e o f this, b r e a s t tissues f o r m useful t e s t - b e d s for a r o m a t a s e i n h i b i t o r s a n d m a y p r o vide a d d i t i o n a l i n f o r m a t i o n b e y o n d t h a t o b t a i n e d

201

f r o m classical s y s t e m s such as p l a c e n t a l m i c r o s o m e s . F o r e x a m p l e , the b r e a s t m a y reflect d r u g u p t a k e a n d m e t a b o l i c activities w i t h i n p e r i p h e r a l tissues m o r e realistically. E v i d e n c e is also g r a d u a l l y a c c r u i n g t h a t a r o m a t a s e in c e r t a i n t u m o u r s m a y d i s p l a y a different sensitivity to inhibitors. I n t h e p r e s e n t p a p e r , d a t a are p r e s e n t e d r e g a r d i n g the a p p a r e n t resistance to 4h y d r o x y a n d r o s t e n e d i o n e w i t h the r e t e n t i o n o f sensitivity to a m i n o g l u t e t h i m i d e - l i k e inhibitors. S u c h a p h e n o t y p e has also b e e n g e n e r a t e d b y i n t r o d u c i n g p o i n t m u t a t i o n s into t h e c D N A e n c o d i n g for the a r o m a t a s e e n z y m e [28]. H o w e v e r , t h e use o f singles t r a n d c o n f o r m a t i o n p o l y m o r p h i s m analysis ( S S C P ) a n d the d i r e c t s e q u e n c i n g o f P C R p r o d u c t s has n o t s h o w n a m a j o r m u t a t i o n in a n y e x o n o f the C Y P 1 9 gene in t u m o u r s r e s i s t a n t to 4 - h y d r o x y a n d r o s t e n e d i o n e [29]. I n c o n c l u s i o n , as is s h o w n in Fig. 10, d a t a have b e e n p r e s e n t e d to s h o w t h a t a r o m a t a s e activity w i t h i n t h e b r e a s t is p r i m a r i l y a s s o c i a t e d with m a l i g n a n t a n d a d i p o s e tissue. T h e level o f activity is v a r i a b l e a n d is i n f l u e n c e d b y t r a n s c r i p t i o n a l activity w h i c h a p p e a r s to b e r e g u l a t e d , at least in p a r t , b y factors w o r k i n g t h r o u g h the J A K - S T A T p a t h w a y . T h e p r o d u c t i o n o f s u c h factors m a y b e locally h i g h in the vicinity o f m a l i g n a n t t u m o u r s , a n d c o u l d a c c o u n t for t h e h i g h e r levels o f a r o m a t a s e d i s p l a y e d b y p a r t i c u l a t e fractions a n d p r i m a r y c u l t u r e s f r o m a d i p o s e tissue a d j a c e n t to t u m o u r s . A r o m a t a s e activity m a y also b e c o n t r o l l e d b o t h b y e n d o g e n o u s a n d e x o g e n o u s inhibitors. F u r t h e r k n o w l e d g e o n the r e g u l a t i o n o f a r o m a t a s e w i t h i n the b r e a s t has t h e r a p e u t i c p o t e n t i a l for p a t i e n t s with b r e a s t cancer.

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