Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts

Journal of Steroid Biochemistry & Molecular Biology 79 (2001) 165–172

Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts夽 D. Parish a , A. Purohit a , A. Singh a , J. Rosankiewicz a , M.W. Ghilchik b , M.J. Reed a,∗ a

Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary’s Hospital, London W2 1NY, UK b The Breast Clinic, St. Mary’s Hospital, London W2 1NY, UK

Abstract The cytokine interleukin-6 (IL-6) and its soluble receptor (IL-6sR) can act synergistically to stimulate aromatase activity in cultured stromal fibroblasts derived from breast tissues. In this study, a 16 amino acid peptide, AROHIB, has been used in an attempt to block the ability of IL-6 plus IL-6sR to stimulate aromatase activity in stromal fibroblasts. Pre-incubation of cells with AROHIB for a 3-h period before the addition of IL-6 and IL-6sR resulted in a marked (67%) reduction in the ability of these factors to stimulate aromatase activity. AROHIB was found to be rapidly degraded when exposed to MCF-7 breast cancer cells or fibroblasts. Analysis by FAB-MS was used to identify the site of peptide cleavage. Subsequently, a series of 10 amino acid peptides, DP1–DP4, were designed, synthesised and tested for their ability to resist proteolytic degradation and to inhibit IL-6 plus IL-6sR-stimulated aromatase activity. Peptide DP2, a modified version of the active fragment of AROHIB, had N-acetyl and C-amino terminal protection and an internal d-amino acid (instead of l form) at the site of proteolytic cleavage. Using cells cultured in the presence of 2% stripped foetal calf serum, peptide DP2 resulted in a 74% reduction in cytokine-stimulated aromatase activity. Under serum-free conditions, peptides DP1–DP3 showed modest inhibitory properties. Results from this study suggest that it may be possible to develop small peptides to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Interleukin-6 (IL-6); Cytokine; Breast cancer; Aromatase

1. Introduction The conversion of androstenedione to oestrone in peripheral tissues, by the aromatase enzyme complex, is the exclusive source of oestrogen synthesis in post-menopausal women. Major sites for the location of this enzyme include adipose tissue and normal and malignant breast tissues [1,2]. Studies to investigate the origin of oestrogens in normal and malignant breast tissues have revealed that a significant proportion originates via in situ synthesis from androstenedione [3,4]. Regulation of aromatase gene expression is now known to be mediated in a tissue-specific manner and there is evidence that expression is controlled via different promoters in normal and malignant breast tissues [5,6]. In normal breast tissue and adipose tissue expression is regulated mainly by promoter (P) I.4 which is responsive to cytokines [7]. In malignant breast tissue, there is an increase in usage of PII and PI.3 [8]. Regulation of expression by PII and I.3 夽 Proceedings of the Symposium: ‘Aromatase 2000 and the Third Generation’ (Port Douglas, Australia, 3–7 November 2000). ∗ Corresponding author. Tel.: +44-207-886-1738; fax: +44-207-886-1790. E-mail address: [email protected] (M.J. Reed).

is influenced by cAMP and factors such as prostaglandin E2 (PGE2 ) which increase cAMP levels [6]. Recent evidence, however, suggests that fibroblasts from normal or malignant breast tissues show a similar stimulatory response to factors that regulate aromatase expression via PI.4 or PI.3 [9]. Thus, differences in levels of regulatory molecules and not intrinsic differences of fibroblasts is a likely explanation for the variation in promoter usage in different tissues. As oestrogens have a major role in supporting the growth of hormone-dependent breast tumours, a number of potent aromatase inhibitors have been developed and are now in clinical use [10,11]. While the results from clinical trials with the latest generation of aromatase inhibitors are very encouraging [12], it is apparent that such compounds give rise to an almost total block of oestrogen synthesis in all tissues. As oestrogens also have many other important physiological functions in the body, including the regulation of bone formation, it may be difficult to use such inhibitors in a long-term therapeutic or preventative setting. In addition, a number of adverse side-effects are associated with the use of current aromatase inhibitors [13]. Research into the control of aromatase activity in stromal fibroblasts derived from tissue proximal to breast tumours or reduction mammoplasties has identified interleukin-6 (IL-6),

0960-0760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 0 7 6 0 ( 0 1 ) 0 0 1 5 5 - 8

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tumour necrosis factor alpha and PGE2 as important regulatory factors [6,14,15]. Furthermore, the ability of IL-6 to stimulate aromatase activity in these cells is markedly potentiated by the soluble form of its receptor (IL-6sR) [16]. IL-6 is produced in large amounts by breast tissue explants, fibroblasts derived from breast tissues and cells of the immune system [17]. IL-6sR is produced by malignant, but not normal, fibroblasts and also by cells of the immune system [16]. Like other cytokines IL-6 acts by binding to a membrane receptor (IL-6R), but this receptor or its soluble form, has to associate with a signal transducing receptor (gp130) in order to exert its effects [18,19]. By blocking the ability of IL-6 and its receptor to associate with the gp130 signal transducing receptor it should be possible to inhibit IL-6-stimulated aromatase activity. As IL-6 is produced by breast tissues and invading macrophages, such inhibition could give rise to the ability to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner. To explore this possibility we have used a 16 amino acid (aa) peptide, 249 Y16T264 , that was previously reported to block the ability of IL-6 to stimulate the growth of B9 cells [20]. Initial investigations suggested that this peptide underwent rapid proteolytic degradation. A series of modified 10 aa peptides were therefore synthesised and tested in an attempt to achieve a greater degree of inhibition of cytokine-stimulated aromatase activity.

Table 1 Peptide sequences

2. Materials and methods

2.4. Aromatase assay

2.1. Tissue samples

Aromatase activity was measured in intact fibroblast monolayers using [1␤-3 H] androstenedione (15–30 Ci/mmol, NEN, Du Pont, Stevenage, Herts, UK) over a 3–20 h period [14]. The number of cells were measured using a Coulter Counter.

Samples of breast adipose tissue proximal to breast tumours (i.e. ‘normal’ breast tissue) were collected from women undergoing lumpectomy for the removal of breast tumours. The study was approved by the Hospital Ethics Committee and the samples were collected after obtaining women’s consent to the investigation. 2.2. Fibroblast culture Resected tissues were minced with sterile scalpels and incubated in Eagle’s modified minimum essential medium (EMEM) for 18–24 h at 37 ◦ C with collagenase (200 ␮g/ml). The dispersed cells were harvested by centrifugation and washed twice with medium to remove collagenase. Dispersed cells were seeded into 25 cm2 culture flasks and allowed to attach. Cells were grown to confluence in EMEM containing Hepes buffer (20 mmol), 10% foetal calf serum (FCS) and supplements [14]. Cells were routinely passaged two to three times after which replicate 25 cm2 culture flasks were seeded with fibroblasts and grown to confluency. The cells were washed once with phosphate buffered saline and the medium was replaced with serum-free, phenol red-free EMEM for 24 h. Treatments were then added in this medium ± 2% stripped FCS for 48 h and included IL-6 ± its soluble receptor (R&D Systems Ltd., Abingdon,

Peptide

Sequence

AROHIB

NH2 –Tyr–Arg–Leu–Arg–Phe–Glu–Leu–Arg– Tyr–Arg–Ala–Glu–Arg–Ser–Lys–Thr–COOH NH2 –Tyr–Arg–Leu–Arg–Phe–Glu– Leu–Arg–Tyr–Arg–COOH CH3 CONH–Tyr–Arg–Leu–Arg–(D-Phe)–Glu– Leu–Arg–Tyr–Arg–CONH2 CH3 CONH–Tyr–Arg–Leu–Arg–Phe–Glu–Leu–Arg– Tyr–Arg–CONH2 NH2 –Leu–Glu–Tyr–Arg–Leu–Arg–Tyr– Arg–Phe–Arg–COOH

DP1 DP2 DP3 DP4

Oxon, UK). The 16 aa peptide (AROHIB) was synthesised and purified by GENSYS (Cambridge, UK) while other peptides were synthesised by the Advanced Biotechnology Centre, Imperial College School of Medicine. The aa sequences of the peptides used are shown in Table 1. 2.3. MCF-7 breast cancer cell culture MCF-7 breast cancer cells were initially used to investigate the degradation of AROHIB and were cultured as previously described [16].

2.5. Peptide degradation In order to monitor peptide degradation, I125 labelled peptides were added to confluent MCF-7 cells or fibroblasts. Peptides were labelled using I125 (Amersham International, Aylesbury, Bucks, UK) using chloramine T. Cells were exposed to labelled peptides and aliquots of medium were removed after 1, 4 and 24 h. Peptides were extracted from culture medium using Sep Pak reverse phase columns with 80% acetonitrile as eluent and subsequently subjected to HPLC analysis. For this, samples were applied to a C18 PepSep reverse phase HPLC column (Pharmacia) and eluted with a gradient from 0.1% trifluoroacetic acid (TFA) to 65% acetonitrile, 0.1% TFA over a 45-min period. Eluent fractions were collected at 30 s intervals and radioactivity in the fraction was measured using a gamma counter. To identify peptide fragments aliquots of medium were removed 4 h after addition of AROHIB or peptides DP1–DP4 and peptides isolated using a Sep Pak column. The eluent containing peptides was concentrated to 10 ␮l in a 10:1 thioglycerol/TFA matrix and subjected to FAB-MS (VG 40-250, positive ion mode).

D. Parish et al. / Journal of Steroid Biochemistry & Molecular Biology 79 (2001) 165–172

Fig. 1. Inhibition of IL-6 (50 ng/ml) and IL-6 soluble receptor (IL-6sR, 100 ng/ml) stimulated aromatase activity in cultured fibroblasts by peptide AROHIB. Cells were cultured in phenol red-free, serum-free medium in the presence of dexamethasone (100 nM). Cells were pre-incubated with AROHIB (125 ␮M) for 3 h before the addition of IL-6 and IL-6sR (mean ± S.D., n = 3).

2.6. Statistics The significance of differences in aromatase activity in treated and control cells was assessed using Student’s t-test.

3. Results and discussion Evidence from previous investigations indicating that IL-6 plus its soluble receptor could markedly stimulate aromatase

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Fig. 2. Inhibition of IL-6 and IL-6sR-stimulated aromatase activity in cultured fibroblasts by AROHIB (60–150 ␮M). Condition used are as indicated in the legend to Fig. 1 (mean ± S.D., n = 3).

activity in fibroblasts, derived from breast adipose tissue, prompted a search for a method to specifically block stimulation via this route. Breast explants, fibroblasts derived from breast tissues and cells of the immune system are all important sources of IL-6 and IL-6sR [17]. Thus, blocking stimulation of aromatase activity via this route could be used to develop a specific method of inhibiting aromatase stimulation within the breast. In initial experiments, the ability of IL-6 plus its soluble receptor to markedly stimulate aromatase activity in proximal fibroblasts was confirmed (Fig. 1). The 16 aa peptide AROHIB did, on its own, cause a small increase in aromatase activity although the mechanism by which this might occur

Fig. 3. Effect of AROHIB on IL-6 plus IL-6sR-stimulated aromatase activity in cultured fibroblasts. AROHIB (125 ␮M) was added with no pre-incubation period before addition of IL-6 and IL-6sR, after a 3-h pre-incubation period prior to the addition of IL-6 and IL-6sR or with subsequent further additions of AROHIB at 12 and 24 h. Culture conditions were as indicated in the legend to Fig. 1 (mean ± S.D., n = 3).

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is not known. It is possible that peptides from part of the IL-6R are able to interact with the gp130 signal transduction receptor. Pre-incubation of fibroblasts with AROHIB for a 3 h period prior to the addition of IL-6 plus IL-6sR caused a significant (67%) reduction in the ability of these factors

to stimulate aromatase activity (Fig. 1). In a dose response study (60–150 ␮M AROHIB) the degree of inhibition of IL-6 plus IL-6sR-stimulated aromatase activity (40–55%) was similar at the three concentrations tested (Fig. 2). In a further experiment, the necessity for a 3 h pre-incubation

Fig. 4. I125 labelled AROHIB was incubated with MCF-7 breast cancer cells or fibroblasts for 1, 4 or 24 h and the isolated peptides separated on a C18 reverse phase HPLC. The elution position of intact AROHIB is indicated with an arrow.

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period with AROHIB prior to the addition of IL-6 plus IL-6sR was demonstrated. The simultaneous addition of AROHIB with IL-6 plus IL-6sR, without a pre-incubation period, resulted in a modest increase in aromatase activity compared with the addition of IL-6 plus IL-6sR (Fig. 3). Pre-incubation of fibroblasts with AROHIB for 3 h again resulted in a significant reduction in stimulated aromatase activity. However, the subsequent addition of AROHIB to cells at 12 or 24 h did not result in any further reduction in the ability of the IL-6 plus IL-6sR to stimulate aromatase activity. This finding suggests that once the ability of IL-6 receptor complex to interact with the gp130 signal transduction protein is blocked the effect is relatively long lasting. While these initial studies showed that AROHIB could significantly reduce cytokine-stimulated aromatase activity, inhibition was not complete. In the original investigation, in which this peptide was used to block IL-6 induced B9 cell growth, a greater degree of inhibition was observed [20]. This suggested the possibility that in cells derived from breast tissues AROHIB might be subjected to rapid proteolytic degradation. To examine such a possibility, MCF-7 breast cancer cells were initially employed to study the degradation of AROHIB. As shown in Fig. 4, although at 1 h no significant degradation of AROHIB had occurred, by 4 h considerable degradation was evident and this was complete by 24 h. A similar pattern of AROHIB degradation was detected when the peptide was exposed to fibroblasts (Fig. 4).

FAB-MS analysis was subsequently employed to determine the site in AROHIB where proteolytic cleavage occurred. After incubating AROHIB with MCF–7 cells for 4 h, peptide fragments were isolated from the medium using a Sep Pak column and subjected to FAB-MS analysis. Only two molecular ions were detected which corresponded to the molecular weights of the protonated form of AROHIB 1–4 and AROHIB 5–16, suggesting that a single proteolytic cleavage had occurred between Arg4 –Phe5 (Fig. 5). Having identified the point at which AROHIB was being cleaved, a series of peptides were designed, synthesized and tested in an attempt to develop a peptide that was more resistant to proteolytic cleavage than AROHIB (Table 1). DP1 is a 10 aa peptide and was the shortest peptide previously shown to retain the ability to block IL-6 induced growth of B9 cells [20]. DP2 is a similar peptide but with N-acetyl and C-amino terminal protection and an internal d aa (instead of l form) at the site of proteolytic cleavage. DP3 is a peptide with the same aa sequence as DP1 but with N- and C-terminal protection. DP4 is a random peptide incorporating the same amino acids used for the synthesis of DP1–DP3. The ability of peptides DP1–DP4 to inhibit IL-6 plus IL-6sR-stimulated aromatase activity was again examined using fibroblasts derived from tissue proximal to a breast tumour. Peptides were added for a 3 h pre-incubation period before the addition of IL-6 plus IL-6sR. In the presence of 2% SFCS and dexamethasone, DP1, DP3 and DP4 had no

Fig. 6. Inhibition of IL-6 plus IL-6sR-stimulated aromatase activity by peptides DP1–DP4: (a) peptides (125 ␮M) were added 3 h prior to the addition of IL-6 (50 ng/ml) plus IL-6sR (100 ng/ml) to cells cultured in the presence of 2% stripped foetal calf serum (2% SFCS) plus dexamethasone (100 nM); (b) as for the previous experiment with the exception that cells were cultured in phenol red-free, serum-free medium (mean ± S.D., n = 3).

D. Parish et al. / Journal of Steroid Biochemistry & Molecular Biology 79 (2001) 165–172

inhibitory effect on the ability of IL-6 plus IL-6sR to stimulate aromatase activity (Fig. 6). In contrast DP2, the peptide with N- and C-terminal protection and an internal d aa inhibited cytokine-stimulated aromatase activity by 74%. A similar experiment was also carried out under serum-free conditions (Fig. 6). Under these conditions, DP1–DP3 all showed a modest inhibitory effect on the ability of IL-6 plus IL-6sR to stimulate aromatase activity (29–42%), with the DP2 peptide again being the most potent. However, the degree of inhibition exerted by DP2 using serum-free conditions (42%) was somewhat lower than that detected in the presence of 2% SFCS (74%). As found in the presence of 2% SFCS, the peptide DP4 containing a random aa sequence had no significant inhibitory effect on stimulated aromatase activity. The degradation of peptides DP1–DP4 was also examined by exposing I125 labelled peptides to fibroblasts (Fig. 7). The shorter form of AROHIB, DP1 and DP3 were more resistant

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to degradation than AROHIB with significant amounts of the peptides remaining at 24 h. The protection at the N- and C-terminals of the DP3 peptide reduced the rate of degradation compared to that of DP1 which had no terminal protection. The peptide DP4 which has a random aa sequence was almost completely degraded by 1 h. For peptide DP2, the most potent inhibitory peptide in this series, the rate of degradation did not seem to differ greatly from that of peptides DP1 or DP3. However, in contrast to the degradation of peptide DP1 two intermediate peptide fragments were evident at 4 h and were still detectable at 24 h. As only peptide DP2 appeared to inhibit cytokine-stimulated aromatase activity in 2% SFCS it is possible that these intermediary peptide fragments possess inhibitory properties. It is not readily apparent why only the DP2 peptide was active in 2% SFCS whereas peptides DP1–DP3 all showed modest inhibitory activity under serum-free condition. The possibility that serum alone might degrade the peptides was

Fig. 7. I125 labelled peptides, DP1–DP4 were incubated for 24 h with fibroblasts and the resulting peptides were isolated and separated on a c18 reverse phase HPLC. The elution position of the intact peptides is indicated with an arrow.

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examined, but no such degradation was detected (data not shown). However, it is possible that factors in serum might stimulate fibroblasts to secrete proteases which are able to degrade (or activate) the peptides. Under serum-free conditions, peptides DP1–DP3 showed modest activity. This may be accounted for by the fact that in the absence of serum proteases capable of peptide degradation are not produced. In the presence of serum, only peptide DP2 was active. This may be accounted by serum inducing proteases which not only rapidly degrade peptides DP1 and DP3 but activate peptide DP2 to an intermediate form. Further studies are currently in progress to determine the role of proteases in regulating the activity of these peptides. The development of tissue-specific methods of inhibiting aromatase activity remains an important therapeutic goal. Results from the present study have shown that using small 10–16 aa peptides, it is possible to significantly attenuate the ability of IL-6 plus its soluble receptor to stimulate aromatase activity in fibroblasts derived from the breast tissue. While evidence was obtained that the peptides undergo proteolytic degradation, as yet, there is no information as to which proteases may be involved. Identification of the proteases could lead to the use of an appropriate protease inhibitor in combination with a peptide to potentiate the duration of the inhibitory effects. It may also be possible to develop a peptide mimetic, based on peptide DP2, as a means of blocking cytokine-stimulated aromatase activity. As an alternative approach, it may be possible to use IL-6 receptor super antagonists, such as Sant 7 [21], to inhibit cytokine-stimulated aromatase activity. Having identified a small peptide with the ability to block cytokine-stimulated aromatase activity, it should be feasible to identify peptides that are resistant to proteolytic degradation as a novel mean to inhibit aromatase activity in a tissue-specific manner.

[4]

[5]

[6]

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[9]

[10]

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Acknowledgements [17]

This research was supported by Sterix Ltd. [18]

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