Aromatase inhibitors for therapy of advanced breast cancer

In postmenopausal women with advanced breast cancer, numerous phase III trials have been performed comparing the third-generation non-steroidal aromatase inhibitors (NS-AIs) anastrozole and letrozole and the steroidal AI (S-AI) exemestane in the “first-line” setting against tamoxifen and in the “second-line” setting against megestrol acetate. In both settings, the AIs were at least as efficacious or superior in some endpoints with a preferable toxicity profile including a lower incidence of thrombotic events. Relatively small differences in potency between the three AIs have been identified and it has not been demonstrated that these differences have clinical implications. The recent establishment of the value of AIs in the adjuvant setting for postmenopausal women will impact on their utilization in advanced disease. In premenopausal women the third-generation AIs have not been studied as monotherapy and there is a paucity of data in combination with ovarian function suppression in the advanced disease setting. The main area of future investigations for the AIs in premenopausal women will be in the adjuvant therapy setting in combination with suppression of ovarian function.     

Aromatase inhibitors: assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression

The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors.     

Aromatase inhibitors versus tamoxifen for management of postmenopausal breast cancer in the advanced disease and neoadjuvant settings

The third-generation aromatase inhibitors anastrozole, exemestane and letrozole have become firmly established as the agents of choice in patients with tamoxifen-resistant tumors. Large, well-conducted, double-blind clinical trials directly comparing the non-steroidal aromatase inhibitors anastrozole and letrozole with tamoxifen in the advanced disease setting have matured. Based on these trials, there is sufficient evidence to choose one of these agents over tamoxifen because of a superior time to disease progression and acceptable toxicity which includes a lower incidence of thromboembolic complications. Information for the steroidal aromatase inhibitor exemestane will be forthcoming from a phase III trial which has completed accrual. Consistent with the findings in the advanced disease setting, a double-blind trial comparing letrozole with tamoxifen in the neoadjuvant setting revealed superiority for letrozole in terms of clinical response rate. This provides a strong impetus for further study of the aromatase inhibitors in the preoperative setting.     

Mechanisms of resistance to aromatase inhibitors

Aromatase inhibitors are rapidly becoming the first choice for hormonal treatment of steroid receptor positive breast cancer in postmenopausal women. An understanding of the resistance mechanisms to these agents is, therefore, important for the appropriate delivery of treatment to responsive patients and the rational development of new agents targeted at the resistance pathways. De novo resistance appears to be a quantitative rather than qualitative phenomenon with virtually all oestrogen receptor positive tumours showing an anti-proliferative response to the aromatase inhibitor anastrozole. While the expression of type 1 growth factor receptors reduces response to tamoxifen this appears to have little detrimental effect on response to aromatase inhibitors. Studies of acquired resistance in vitro have indicated that acquisition of hypersensitivity to oestrogenic stimulation is a key mechanism that is dependent on enhanced cross-talk of growth factor and oestrogen signaling pathways. Collection of resistant biopsy tissues from patients is important to determine if this mechanism is clinically relevant.     

Role of aromatase in endometrial disease

Aromatase is the key enzyme for estrogen biosynthesis. It is normally expressed in the human ovary, skin, adipose tissue and brain. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE₂. This results in local production of estrogen, which induces PGE₂ formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17β-hyroxysteroid dehydrogenase (17β-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE₂ in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of post-menopausal endometriosis using an aromatase inhibitor.     

Aromatase in human lung carcinoma

Lung cancer is the leading cause of cancer mortality in both women and men worldwide but gender differences exist in their clinical and biological manifestations. In particular, among life time non-smoker, female are far more likely to develop lung carcinoma than male. Recent studies demonstrated that estrogens are synthesized in situ in both male and female lung cancers through aromatase, suggesting that sex steroid may contribute to the pathogenesis and development of lung carcinoma. In addition, human lung carcinomas have been recently demonstrated to be frequently associated with expression of estrogen receptors in both male and female patients and a lower expression of aromatase was reported to be associated with better prognosis. Preclinical studies further demonstrated that aromatase inhibitor (AI) suppressed the lung tumor growth both in vitro and in vivo. These findings all suggest a potential role of intratumoral aromatase in biological behavior of non-small cell lung cancer (NSCLC), the most common form of human lung malignancy. Therefore, AIs may become viable therapeutic options for disease management in NSCLC patients but further studies are definitely required to obtain a better understanding of the potential roles of intratumoral aromatase expression as a predictive biomarker for clinical outcome in these NSCLC patients.     

Endocrine effects of aromatase inhibitors and inactivators in vivo: review of data and method limitations

The so-called “third-generation” aromatase inhibitors/inactivators have become standard first-line endocrine therapy for postmenopausal women in the metastatic setting. In addition, these compounds, administered as monotherapy or in sequence with tamoxifen, are likely to become standard adjuvant therapy in most countries in the near future. In contrast to the SERMs, aromatase inhibitors may be assessed for their biochemical efficacy in vivo either by measuring their ability to suppress plasma and tissue estrogen levels or, alternatively, by measuring their ability to inhibit the conversion of tracer-labelled androstenedione into estrone. While contemporary methods for estrogen measurement (with the exception of estrone sulphate) lack the sensitivity to measure plasma estrogen levels during treatment with the most potent compounds, in vivo aromatase inhibition can be determined with a much better sensitivity. Thus, in a joint program conducted by the Royal Marsden Hospital, London and our team in Bergen, we were able to reveal profound differences between first- and second-generation aromatase inhibitors, causing 50–90% aromatase inhibition, and the three third-generation compounds, causing >98% inhibition of total body aromatization.     

Overview of adjuvant trials of aromatase inhibitors in early breast cancer

The third-generation aromatase inhibitors are an important class of drugs for use in adjuvant therapy for postmenopausal women with resected estrogen receptor positive breast cancer. Multiple large prospective randomized trials have established their value in this setting and provided guidance for their use in clinical management. This review will outline the trials that have provided evidence on the value of the aromatase inhibitors in the adjuvant setting as well as the ongoing trials that will expand our knowledge of how to use them most effectively.     

Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators

Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E₁) and estradiol (E₂) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E₁, E₂ and estrone sulfate (E₁S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E₁S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized.     

Estrogens and cell death in murine uterine luminal epithelium

Summary The luminal epithelium of adult ovariectomized mice responds to estradiol-17 with a synchronised wave of DNA synthesis and mitosis. Estriol, however, although producing a similar DNA-synthetic and mitotic response fails to cause an increase in cell number owing to a wave of cell death occurring at mitosis. In the present study it was shown that cells died by two different routes. The majority died by apoptosis but, unusually, a minority also died by necrosis. In the apoptotic cells the cytoplasm became dense, the endoplasmic reticulum and nuclear cisternae dilated; chromatin became marginated the nucleus shrank and became deeply infolded and contorted. Apoptosis, however, was uncharacteristic in that the nucleus failed to fragment, form caps or show disruption before the cells died by membrane rupture. Furthermore, the cells were frequently lost in sheets from the epithelium into the lumen. Part of the biochemical explanation for this onset of cell death comes from the accelerated loss from the tissue of estriol when compared to estradiol-17. This resulted in a decline in protein and rRNA biosynthesis and a failure to complete ribosomal maturation. Evidence in favour of this explanation came from experiments that showed a return to the estradiol-17 level of response and an inhibition of cell death when the occupancy of the estriol receptor was maintained.     

The characterization of human uterine smooth muscle cells in culture

Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro. Among the common features present were: (a) abundant bundles of 60--70 A myofilaments; (b) branched mitochondria; (c) stacks of cisternae of rough endoplasmic reticulum; (d) caveolae intracellulares; (e) nexuses. Other features included ovoid nuclei, a well developed Golgi apparatus and abundant free ribosomes. The subcultured cells exhibited features of dedifferentiation in the log phase of growth and at post-confluency. However, the post-confluent cells showed characteristics indicating redifferentiation back towards their in vivo morphology. Smooth muscle cells isolated from endometrial curettings may provide a useful model for biochemical and pharmacological studies of a cell type derived from a hormonal target tissue as the cells "age" in culture.     

Icon immunoconjugate treatment results in regression of red lesions in a non-human primate (Papio anubis) model of endometriosis

Endometriosis is a common condition in reproductive-aged women characterized by ectopic endometrial lesions of varied appearance, including red, white, blue, black or powder burn coloration, which contribute to chronic pain and infertility. The immunoconjugate molecule (Icon) targets Tissue Factor, a transmembrane receptor for Factor VII/VIIa that is aberrantly expressed in the endothelium supporting ectopic endometrial tissue. Icon has been shown to cause regression of endometriosis in a murine model of disease but prior to this study had not been tested in non-human primates. This study evaluated Icon as a novel treatment for endometriosis in non-human primates (Papio anubis) using an adenoviral vector (AdIcon) delivery system. Female baboons (n?=?15) underwent surgical induction of endometriosis. After laparoscopic confirmation of endometriosis lesions 6-weeks post-surgery, the treatment group (n?=?7) received weekly intraperitoneal injections of viral particles carrying the sequence for Icon, resulting in expression of the protein, while the control group (n?=?8) received no treatment. Icon preferentially reduced the number and volume of red vascularized lesions. Icon may present a novel treatment for endometriosis by degrading red vascularized lesions, likely by targeting tissue factor aberrantly expressed in the lesion vasculature.     

Neurotransmitter-Mediated Regulation of Brain Aromatase: Protein Kinase C- and G-Dependent Induction

Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α₁-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α₁-agonist phenylephrine than in control neurons, whereas prazosin, an α₁-antagonist, suppressed this increase, and ligands for α₂- or β-adrenergic receptors did not exert any influence. The profile of α₁-adrenergic receptor subtypes during actual development in vivo suggested that the α_1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via α₁ (possibly α_1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.     

Aromatase and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

Effects of newly synthetized isoquinoline derivatives on rat uterine contractility and ROCK II activity

Protein kinases have an important role in signal transduction in the cellular system via protein phosphorylation. RhoA activated Rho-kinases have a pivotal role in the regulation of smooth muscle contraction. ROCK I and ROCK II phosphorylate myosin-phosphatase and myosin-kinase, which induces contraction in the myometrium. Several studies have investigated the affinity of isoquinoline alkaloids (HA-1077, H1152P) to Rho-kinases, and these compounds notably inhibited the Ca²⁺-independent process.We measured the efficiency of 25 original, newly synthesized isoquinoline derivatives for the Rho-kinase activity using Rho-associated kinase activity assay and determined their effects on the non-pregnant, 20-day pregnant and parturient rat myometrial contraction in vitro.The IC₅₀ values of 11 from among the 25 derivatives were significantly lower on the oxytocin-induced non-pregnant rat uterine contraction compared with Y-27632 and fasudil, although their maximal inhibitory effects were weaker than those of Y-27632 and fasudil. We measured the effects of 11 isoquinoline molecules with significant IC₅₀ values on ROCK II activity. We found two isoquinolines out of 11 compounds (218 and 852) which decreased the active ROCK II level similarly as Y-27632. Then we found that 218 and 852 relaxed the 20th-day pregnant and parturient rat uterus with greater potency as compared with fasudil.The majority of the synthesized isoquinoline derivatives have uterus relaxant effects and two of them significantly suppress the Rho-kinase mediated myosin light chain phosphorylation. Our results may suggest that the isoquinoline structure has a promising prospect for the development of new and effective inhibitors of uterine contractions in preterm birth.     

Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase

Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp³⁰⁹ in substrate binding and catalysis. These studies predicted an elevated pK_a for Asp³⁰⁹ and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp³⁰⁹ with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp³⁰⁹ based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pK_a of 8.2 observed for androstenedione binding to the side chain of Asp³⁰⁹. To our knowledge, this work represents the first experimental assignment of a pK_a value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.     

Comparison of Aromatase Activity in Human Prostatic,Testicular and Placental Tissues

Abstract

The aromatase enzyme was quantified by the release of tritiated water from [1β-³H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0μM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC₅₀ values of 6.4, 0.17 and 0.0017 μM. respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075μM, respectively).