The study reported here was designed to determine whether a phytoestrogen-containing soy extract (SSE) could negate/overwhelm the inhibitory effects of ICI 182 780 on the growth of estrogen-sustained human breast cancer xenografts (MCF-7), in ovariectomized athymic mice. As expected, estradiol-supplemented tumors did not grow over the study period in ICI 182 780-treated females; concomitant administration of 50 mg/kg per day SSE slightly potentiated the inhibitory activity of the drug, while at 100 mg/kg per day, SSE partially negated ICI 182 780 activity. In keeping with these in vivo outcomes, we observed that the level of cyclin D1 (and progesterone receptor) in MCF-7 xenografts was considerably reduced by ICI 182 780, an effect enhanced by concomitant treatment with 50 SSE, but reduced by the higher dosage (i.e. 100 mg/kg per day). Thrombospondin-1 (TSP-1) and kallikrein 6 (KLK6) levels were also reduced following ICI 182 780, although to a lesser degree; again, combined anti-estrogen and SSE produced a dose-dependent regulation in TSP-1 and KLK6 tumor level, with a further reduction in the mRNA gene expression at 50 SSE (compared with ICI 182 780) and a partial reversion of the drug-induced down-regulation at 100 mg/kg per day. No modulation was detected in the serum concentration of IGF-1 (a potent mitogen for estrogen receptor-positive breast cancer cell lines) either upon treatment with ICI 182 780 or concomitant administration of the anti-estrogen with SSE. In conclusion, results from this study raise concerns about the consumption of isoflavone supplements in conjunction with ICI 182 780 therapy, in postmenopausal women with estrogen-dependent breast cancer.
Daniela Gallo, Elisabetta Mantuano, Manuela Fabrizi, Cristiano Ferlini, Simona Mozzetti, Ilaria De Stefano, and Giovanni Scambia
Giorgio Secreto, Paola Muti, Milena Sant, Elisabetta Meneghini, and Vittorio Krogh
Five years of adjuvant therapy with anti-estrogens reduce the incidence of disease progression by about 50% in estrogen receptor-positive breast cancer patients, but late relapse can still occur after anti-estrogens have been discontinued. In these patients, excessive androgen production may account for renewed excessive estrogen formation and increased risks of late relapse. In the 50% of patients who do not benefit with anti-estrogens, the effect of therapy is limited by de novo or acquired resistance to treatment. Androgen receptor and epidermal growth factor receptor overexpression are recognized mechanisms of endocrine resistance suggesting the involvement of androgens as activators of the androgen receptor pathway and as stimulators of epidermal growth factor synthesis and function. Data from a series of prospective studies on operable breast cancer patients, showing high serum testosterone levels are associated to increased risk of recurrence, provide further support to a role for androgens in breast cancer progression. According to the above reported evidence, we proposed to counteract excessive androgen production in the adjuvant setting of estrogen receptor-positive patients and suggested selecting postmenopausal patients with elevated levels of serum testosterone, marker of ovarian hyperandrogenemia, for adjuvant treatment with a gonadotropins-releasing hormone analogue (medical oophorectomy) in addition to standard therapy with anti-estrogens. The proposed approach provides an attempt of personalized medicine that needs to be further investigated in clinical trials.
S Chen, D Zhou, T Okubo, Y C Kao, and C Yang
Aromatase has been shown to be expressed at a higher level in human breast cancer tissue than in normal breast tissue, by means of enzyme activity measurement, immunocytochemistry, and RT-PCR analysis. Cell culture including MCF-7 breast cancer cells, animal experiments using aromatase-transfected breast cancer cells, and transgenic mouse studies have demonstrated that estrogen production in situ plays a more important role than circulating estrogens in breast tumor promotion. In addition, tumor aromatase is believed to be able to stimulate breast cancer growth through both autocrine and paracrine pathways, as demonstrated by a three-dimensional cell culture study. RT-PCR and gene transcriptional studies have revealed that the aromatase promoter is switched from a glucocorticoid-stimulated promoter, I.4, in normal tissue to cAMP-stimulated promoters, I.3 and II, in cancerous tissue. Recently, we identified and characterized a cAMP-responsive element (CREaro) upstream from promoter I.3 by DNA deletion and mutational analyses. Our results from promoter functional analysis also demonstrated an interaction between the CREaro and the silencer element (S1) that was identified previously in our laboratory. In the presence of cAMP, the positive regulatory CREaro can overcome the action of the silencer on the function of promoter I.3. On the basis of results generated from our own and other laboratories, we propose that, in normal breast adipose stromal cells and fibroblasts, aromatase expression is driven by promoter I.4 (glucocorticoid dependent), and that the action of promoters I.3 and II is suppressed by the silencer negative regulatory element. However, in cancer cells and surrounding adipose stromal cells, the cAMP level increases, and aromatase promoters are switched to cAMP-dependent promoters - I.3 and II. Furthermore, we applied the yeast one-hybrid screening method to search for proteins interacting with the silencer element, S1. The major protein identified was ERRalpha-1; however, SF-1, which is present in the ovary, is not detected in breast cancer tissue. Using a reporter plasmid with the aromatase genomic fragment containing promoter I.3 and S1, in breast cancer SK-BR-3 cells, ERRalpha-1 was found to have a positive regulatory function. It is believed that the silencer element in the human aromatase gene may function differently in different tissues, as a result of distinct expression patterns of transcription factors.
H Sasano, S Sato, K Ito, A Yajima, J Nakamura, M Yoshihama, K Ariga, T J Anderson, and W R Miller
It is very important to examine the influence of inhibition of in situ estrogen production on the pathobiology of human sex steroid-dependent tumors in order to understand the clinical effects of aromatase inhibitors. We have examined the biological changes before and after aromatase inhibitor treatment in vitro (endometrial and ovarian cancer) and in vivo (breast cancer). First, we analyzed these changes using histoculture of 15 human endometrial cancers and 9 ovarian cancers. Five of the fifteen endometrial cancers and four of the nine ovarian cancers demonstrated decreased [3H]thymidine uptake or Ki67 labeling index after 14alpha-hydroxy-4-androstene-3,6,17-trione (NKS01) treatment. In ovarian cancer cases, the responsive cases tended to be associated with higher aromatase and estrogen receptor alpha (ER) expression compared with the other cases but this was not seen in the endometrial cancer cases. There were no changes in ER and aromatase expression before and after NKS01 treatment in either ovarian or endometrial cancer cases. We then studied the same primary human breast tumors before and after aminoglutethimide (AMG, n=3) and 4-hydroxyandrostenedione (4-OHA, n=3) treatment. Tumor aromatase activity increased in 3 cases and decreased or was unchanged in 3 cases but aromatase immunoreactivity in stroma and adipocytes was unaltered in 5 cases. There were no changes in the ER labeling index before or after treatment. Five of the six cases including the responsive cases tended to be associated with decreased cell proliferation or Ki67 expression and increased apoptosis when examined by the TUNEL method. These results indicate that aromatase inhibitors may exert their effects on human breast and other cancers through decreasing proliferation and increasing apoptosis, possibly without altering ER status.
G Olt and R Mortel
Francesmary Modugno, Robin Laskey, Ashlee L Smith, Courtney L Andersen, Paul Haluska, and Steffi Oesterreich
Ovarian cancer is the sixth most common cancer worldwide among women in developed countries and the most lethal of all gynecologic malignancies. There is a critical need for the introduction of targeted therapies to improve outcome. Epidemiological evidence suggests a critical role for steroid hormones in ovarian tumorigenesis. There is also increasing evidence from in vitro studies that estrogen, progestin, and androgen regulate proliferation and invasion of epithelial ovarian cancer cells. Limited clinical trials have shown modest response rates; however, they have consistently identified a small subset of patients that respond very well to endocrine therapy with few side effects. We propose that it is timely to perform additional well-designed trials that should include biomarkers of response.
R R Tekmal, N Kirma, K Gill, and K Fowler
To test directly the role of breast-tissue estrogen in initiation of breast cancer, we have developed the aromatase-transgenic mouse model and demonstrated for the first time that increased mammary estrogens resulting from the overexpression of aromatase in mammary glands lead to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Continued overexpression of aromatase that leads to increased breast-tissue estrogen contributes to a number of epigenetic changes in mammary tissue such as alteration in the regulation of genes involved in apoptosis, activation of genes involved in cell cycle and cell proliferation, and activation of a number of growth factors. Our current studies show aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Preneoplastic and neoplastic changes induced in mammary glands as a result of aromatase overexpression can be completely abrogated with the administration of the aromatase inhibitor, letrozole. Consistent with complete reduction in hyperplasia, we have also seen downregulation of estrogen receptor and a decrease in cell proliferation markers, suggesting aromatase-induced hyperplasia can be treated with aromatase inhibitors. Our studies demonstrate that aromatase overexpression alone, without circulating estrogen, is responsible for the induction of breast hyperplasia and these changes can be abrogated using aromatase inhibitors.
E Enmark and J Å Gustafsson
The recent discovery of a second estrogen receptor, ER&B;, shows that the mechanisms behind the effects of estrogen are far more complicated than previously assumed, and gives unique opportunities to gain a better understanding of these phenomena. ER&B; is expressed in many important target tissues for estrogen, and a better insight into the respective mechanisms of action of ERalpha and ER&B; might give clues concerning the etiology and pathogenesis of for example prostate or ovarian cancer. Development of ERalpha and ER&B; specific ligands may furthermore open up interesting new possibilities for treatment of e.g. postmenopausal symptoms and breast cancer. In this review, we will try to summarize what is known sofar about estrogen receptor &B;, with some emphasis on the human receptor and its expression. We will furthermore try so summarize what is known about different isoforms of the receptor, in view of what is known about isoforms and variants of other receptors, in particular estrogen receptor alpha (ERalpha) and the progesterone receptor (PR).
This study was supported by a grant from the Swedish Cancer Society.
A Brodie, Q Lu, Y Liu, and B Long
The potential of aromatase (estrogen synthetase) within the breast to provide a significant source of estrogen mediating tumor proliferation is suggested by studies reporting 4- to 6-fold higher estrogen levels in tumors than in plasma of postmenopausal patients with breast cancer. Recent studies in our laboratory have identified aromatase and its mRNA in tumor epithelial cells using immunocytochemistry and in situ hybridization. In addition, significant aromatase activity, which was stimulated 7-fold by dexamethasone, was measured in metastatic cells isolated from a breast cancer patient. Increase in proliferation, as measured by proliferating cell nuclear antigen immunostaining in tumor sections and by thymidine incorporation into DNA in response to testosterone, was observed in histocultures of breast cancer samples. This latter effect could be inhibited by 4-hydroxyandrostenedione. These results imply that intratumoral aromatase has functional significance and may be an important target for successful inhibitor treatment of breast cancer patients. To investigate treatment strategies with aromatase inhibitors and antiestrogens, we developed an intratumoral aromatase model to simulate the hormone responsive postmenopausal breast cancer patient. Tumors of estrogen receptor positive human breast carcinoma cells (MCF-7) transfected with the human aromatase gene are grown in ovariectomized nude mice. These cells synthesize sufficient estrogen to stimulate tumor formation. We have utilized this model to investigate the effects on tumor growth of the antiestrogens, tamoxifen and ICI 182780, and the aromatase inhibitors, letrozole and anastrozole (arimidex), alone and in combination. Both the aromatase inhibitors and the antiestrogens were effective in suppressing tumor growth. However, letrozole was significantly more effective than the antiestrogens. When the aromatase inhibitors were combined with the antiestrogen, tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Furthermore, the results do not suggest any benefit from combining tamoxifen with the pure antiestrogen, ICI 182780. Thus sequential use of these agents is likely to be more advantageous to the patient in terms of longer duration of effective treatment.
S A Khan, D Bhandare, and R T Chatterton Jr
Recent developments in breast epithelial sampling techniques (nipple fluid aspiration, ductal lavage, and random fine needle aspiration) provide new opportunities for the acquisition of hormonal and cellular biomarker data in asymptomatic women, and thereby the possibility of developing a unified vision of how the hormonal environment of the breast may interact with the cellular expression of proteins, and with other evolving candidate markers of breast cancer risk. The purpose of this review is to integrate available information regarding cellular and breast fluid biomarkers of hormone action on the breast, to identify candidate biomarkers for studies of breast cancer risk and prevention. These include the estrogen receptors α andβ, markers of proliferative and apoptotic response, and protein markers of estrogen action in breast cells and nipple fluid. Studies of breast hormone levels in nipple aspiration fluid (NAF) show that estrone sulphate is present in large quantities in the normal breast, while the differences in serum ovarian steroids that are seen in pre- and postmenopausal women are blunted in NAF. The variability of several estradiol precursors in NAF over time is relatively small, a useful attribute of potential biomarkers of breast cancer risk, particularly if they are reversible with intervention in Phase 2 prevention trials. These studies are already providing new insights into the hormonal etiology of breast cancer, and should lead to the identification of robust, reversible biomarkers for use in breast cancer prevention studies.