Aromatase

Aromatase catalyzes the conversion of androgens to estrogens through a series of monooxygenations to achieve the 19-desmolation and aromatization of the neutral steroid ring-A structure. We have separated two forms of aromatase, a major (P2a) and a minor (P3) form, from human term placenta through solubilization and chromatography. Partially purified aromatase in each form was immunoaffinity chromatographed to give a single band (SDS-PAGE) cytochrome P-450 of 55 kDa, utilizing a mouse monoclonal anti-human placental aromatase cytochrome P-450 IgGi (MAb3-2C2) which is capable of suppressing placental aromatase activity. The purified cytochrome P-450 showed specific aromatase activity of 25-30 nmol/min per mg with Km of 20-30 nM for androstenedione on reconstitution with NADPH-cyt P-450 reductase and dilauroyl L-alpha-phosphatidylcholine. This one step represents a higher than 100-fold purification with maintenance of the same Km. The stability analysis showed a half-life of more than 5 yr for solubilized aromatase and 2 months for the aromatase cytochrome P-450 on storage at -90 degrees C. Contrary to the recent claim that estrogen biosynthesis by reconstituted human placental cytochrome P-450 is by trans-diaxial 1 alpha,2 beta-hydrogen elimination, all of our partially purified forms and reconstituted aromatase synthesized estrogens by cis-1 beta, 2 beta-hydrogen elimination. Use of purified aromatase and [19-3H3, 4-14C]androstenedione led us to discover a metabolic switching by aromatase to 2 beta-hydroxylation of androgen. Results of the MAb3-2C2 suppression of aromatase activity in different species and tissues including human, baboons, horses, cows, pigs and rats indicated the presence of various isozymes of aromatase.     

Aromatase, aromatase inhibitors, and breast cancer

Estrogens are known to be important in the growth of breast cancers in both pre and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also in innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs. Article from the special issue on 'Targeted Inhibitors'.     

Aromatase inhibitors--socio-economical issues

Novel, third-generation aromatase inhibitors are currently implemented for treatment of postmenopausal breast cancer in the metastatic and adjuvant setting and, potentially, for breast cancer prevention. Introduction of novel therapeutic strategies to large patient groups may add significant costs to health care budgets, forcing institutions to focus entirely on costs or the cost-utility of implementing such novel strategies. Breast cancer is the most frequent cancer in the female population in western societies, and its incidence is currently increasing in other parts of the world as well. Due to the proven efficacy and limited side effects of endocrine therapy in the adjuvant setting, the indications for use have been successively broadened. Currently, the majority of postmenopausal women treated for an estrogen-receptor positive breast cancer will be offered adjuvant endocrine therapy; thus, a general change of practice may cause significant implications to healthcare costs. This may relate to direct drug costs as well as indirect costs related to prevention of side effects, like additional use of bisphosphonates to prevent enhanced bone loss. The aim of this paper is to overview these considerations and put them into perspective by simple illustrations taken from current cost estimates.     

Aromatase inhibitors--socioeconomical issues

With costs of health care in general and for cancer therapy in particular escalating due to implementation of novel compounds, there is an increasing focus on therapy costs in most countries. A common way of assessing therapeutic utility versus cost is by assessing cost per additional life year gained or cost per additional quality-adjusted life year (QALY) gained with a novel therapy. While endocrine therapy in general is associated with low costs, the fact that aromatase inhibitors are administered over several years to each patient in the adjuvant setting, together with the substantial number of postmenopausal breast cancer patients that are candidates for adjuvant treatment with aromatase inhibitors, advocates critical examination of cost–utilities related to implementation of such therapy in the adjuvant setting. While cost–utility estimates for treatment with aromatase inhibitors in the adjuvant setting look favorable, the estimates are sensitive to variations with respect to long-term benefits but also side effects. For patient groups with a low-risk of relapse but also patients with a limited life expectancy due to high age, cost–utility estimates may exceed the upper limits generally proposed for costs per quality-adjusted life year gained.     

Aromatase inhibitors--gene discovery

Microarray analysis of tumour RNA is an extremely powerful tool which allows global gene expression to be measured. When used in combination with neoadjuvant treatment protocols in which therapy is given with the primary tumour within the breast, sequential biopsies may be analysed and results correlated with clinical and pathological response. In the present study, a neoadjuvant protocol has been used, administering the third generation inhibitor, letrozole, for 3 months and subjecting RNA extracted from biopsies taken before and after 10–14 days of treatment to microarray analysis. The objectives were to discover: (i) genes that change with estrogen deprivation (the only known biological effect of letrozole is to inhibit aromatase activity and reduce endogenous estrogens in postmenopausal women) and (ii) genes whose basal, on treatment or change in expression differ between tumours which are either responsive or resistant to treatment (so that predictive indices of response/resistance may be developed).

Early changes in gene expression were identified by comparing paired tumour core biopsies taken before and after 14 days treatment in 58 patients using three different approaches based on frequency of changes, magnitude of changes and SAM analysis. All three approaches showed a greater number of genes were down-regulated than up-regulated. Merging of the data produced a total of 143 genes which were subject to gene ontology and cluster analysis. The ontology of the 91 down-regulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, up-regulated genes were associated with organ development and extra-cellular matrix turnover and regulation.

Clinical response was assessable in 52 patients; 37 (71%) tumours were classified as clinical responders (>50% reduction in volume at 3 months). Microarray analysis of pre- and 14-day biopsies identified 291 covariates (84 baselines, 72 14-day and 135 changes) highly predictive of response status. A similarity matrix using the covariates showed responding tumours have a similar genetic profile which was dissimilar to non-responding cancers whereas non-responsive cases were distinctive from each other. Changed genes predicting for response showed no concordance with those changed significantly by treatment in the overall group.     

Aromatase inhibition by flavonoids

Several synthetic flavones were found to inhibit the aromatization of androstenedione to estrone catalyzed by human placental microsomes. Twenty-one compounds were tested and the IC50 of the most active were: flavone, 10 microM; 7-hydroxyflavone, 0.5 microM; 7,4'-dihydroxyflavone, 2.0 microM; flavanone, 8.0 microM; and 4'-hydroxyflavanone, 10 microM. Most of the others had IC50 values ranging from 80 to greater than 200 microM. These findings show that 4'-hydroxylation results in either no change or very little change in IC50 for flavanone, isoflavone and isoflavanone as well as other ring A hydroxylated flavones. Derivatives of flavone with a hydroxyl substituent at position 5, 6 and 7 were also screened. 7-Hydroxyflavone (11) was the most effective competitive inhibitor (IC50 = 0.5 microM) with an apparent Ki value of 0.25 microM. Compound 11 also induced a change in the absorption spectrum of the aromatase cytochrome P-450 which is indicative of substrate displacement. The relative binding affinities of the flavonoid analogs were determined and only ring A adn ring B dihydroxylated analogs were found to bind to the estrogen receptor.     

Aromatase and its inhibitors

Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNA_arom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive, human breast carcinoma cells transfected with the human aromatase gene. Thus, the cells synthesized estrogen which stimulated tumor formation. Both aromatase inhibitors and antiestrogens were effective in suppressing tumor growth in this model. However, letrozole was more effective than tamoxifen. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. This suggests that sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.     

Aromatase inhibitors and bone

The decrease in estrogen levels with the use of aromatase inhibitors results in an increase in the rate of bone remodelling. This result in an acceleration of bone loss, and probably to an increase in the risk of fractures. The risk of fracture is particularly high in the older woman and in the woman with a low bone mineral density. We have a number of proven treatments for the treatment of postmenopausal osteoporosis and it is likely that some of these, particularly bisphosphonates, could be effective at preventing bone loss with aromatase inhibitors.     

Subject Index Vol. 1, Issue 3, 2002

Phytochemistry Reviews -     

Aromatase inhibitors and arthralgia

In several large adjuvant clinical trials it has been demonstrated that substitution (eventually addition) of aromatase inhibitors (AIs) provides an improved outcome of endocrine-sensitive breast cancer over tamoxifen alone. Nevertheless, arthralgia induced by the AIs is one of the most frequent side effects in hormonal therapy. It is characterized by tenosynovial changes and is more frequent in patients in clinical practice than previously appreciated in adjuvant clinical trials. AI-related arthralgia may be related to estrogen deprivation, but estrogen replacement is not an option for these women. Therefore standard painkillers, NSAIDs (COX2 inhibitors), week opioids and other interventions (vitamin D, calcium, bisphosphonates, exercise, acupuncture, complementary and alternative approaches, eventually switch to another endocrine drug) are used for managing this treatment-related side effect, and improve adherence and quality of life among breast cancer survivors.