Letrozole in the neoadjuvant setting: the P024 trial

Neoadjuvant chemotherapy trials have consistently reported lower response rates in hormone receptor-positive (HR+) breast cancer when compared with HR− cases. Preoperative endocrine therapy has therefore become a logical alternative and has gained considerable momentum from the finding that aromatase inhibitors (AIs) are more effective than tamoxifen for HR+ breast cancer in both the neoadjuvant and adjuvant settings. The most convincing neoadjuvant trial to demonstrate the superiority of an AI versus tamoxifen was the P024 study, a large multinational double-blind trial in postmenopausal women with HR+ breast cancer ineligible for breast-conserving surgery. The overall response rate (ORR) was 55% for letrozole and 36% for tamoxifen (P < 0.001). Significantly more letrozole-treated patients underwent breast-conserving surgery (45 vs. 35%, respectively; P = 0.022). In addition, ORR was significantly higher with letrozole than tamoxifen in the human epidermal growth factor receptor HER1/HER2+ subgroup (P = 0.0004). The clinical efficacy of letrozole in HER2+ breast cancer was confirmed by fluorescent in situ hybridization analysis and was found to be comparable to that of HER2− cases (ORR 71% in both subsets). Biomarker studies confirmed the superiority of letrozole in centrally assessed estrogen receptor-positive (ER+) tumors and found a strong relationship with the degree of ER positivity for both agents. Interestingly, letrozole was effective even in marginally ER+ tumors and, unlike tamoxifen, consistently reduced the expression from estrogen-regulated genes (progesterone receptor and trefoil factor 1). Furthermore, when analyzed by Ki67 immunohistochemistry, letrozole was significantly more effective than tamoxifen in reducing tumor proliferation (P = 0.0009). Thus, neoadjuvant letrozole is safe and superior to tamoxifen in the treatment of postmenopausal women with HR+ locally advanced breast cancer.
Introduction and rationale
Early-stage breast cancer is traditionally treated with an initial surgery such as lumpectomy or mastectomy followed by subsequent adjuvant therapy, including radiotherapy, chemotherapy, and hormone therapy [1, 2]. Numerous studies have investigated the use of preoperative (neoadjuvant) hormonal therapy or chemotherapy prior to surgical intervention, with the goals to improve surgical outcome and obtain long-term disease-free survival (DFS) (see Table 1). The National Surgical Adjuvant Breast and Bowel Project B-18 trial demonstrated that doxorubicin and cyclophosphamide administered as neoadjuvant or adjuvant therapy had equivalent outcomes in terms of both DFS and overall survival (OS) [3]. Adding a taxane in the B-27 trial significantly reduced the local recurrence rate but did not significantly increase DFS or OS [4]. Importantly, neoadjuvant chemotherapy has been shown to increase the rate of breast-conserving surgery (BCS) without adversely affecting DFS or OS [3, 5–7]. Therefore, neoadjuvant chemotherapy has become the standard treatment approach for locally advanced breast cancer and an accepted option for patients with primary operable disease [8].
Table 1Aims of neoadjuvant therapy in different breast cancer populationsPopulationAimsTreatment optionLocally advanced breast cancerPrimary: to improve surgical optionsFit and healthy patients: chemotherapySecondary: to obtain freedom from disease, to gain information on tumor responseUnfit patients with hormone-sensitive disease: endocrine treatmentOperable breast cancer and candidates for adjuvant chemotherapyPrimary: to obtain freedom from diseaseChemotherapy ± OFS and/or AIsSecondary: to improve surgical options, to gain information on tumor responseSequence versus combinationLonger versus shorterOperable breast cancer and candidates for adjuvant endocrine treatment alonePrimary: to improve surgical optionsEndocrine treatment longer versus shorterSecondary: to gain information on tumor responseTamoxifen versus AIsReprinted from [8] with permission from the American Society of Clinical OncologyAIs aromatase inhibitors, NST neoadjuvant systemic therapy, OFS ovarian function suppression
Significant tumor reduction from neoadjuvant chemotherapy occurs in only subsets of breast cancer, however. For example, patients with estrogen receptor-negative (ER−), high-grade, and highly proliferative tumors appear to benefit the most from neoadjuvant chemotherapy [9–11]. In contrast, significantly lower response rates have been reported in patients with ER+ tumors in neoadjuvant chemotherapy trials [12–15]. The German Breast Group demonstrated that the pathologic complete response (pCR) rates were 6.2 versus 22.8% for ER+ and ER− tumors, respectively (odds ratio 3.23, 95% confidence intervals [CIs] 1.91, 5.46; P = 0.0001) [12]. Recent studies indicate that the sensitivity to chemotherapy is likely determined by the underlying gene expression pattern and the molecular subtype of the tumor [16, 17]. The luminal subtype categorized by gene expression profiling, which encompasses most of the ER+ tumors, is less responsive to preoperative chemotherapy than the basal-like and human epidermal growth factor receptor 2-positive (HER2+) cancer. In addition, neoadjuvant chemotherapy is associated with substantial toxicity that may limit its clinical benefits and acceptability, especially in the elderly patient population [13, 18–20]. Thus, there is great need for effective alternatives to cytotoxic chemotherapy in hormone-responsive locally advanced breast cancer.
Endocrine treatment is an attractive alternative to chemotherapy as neoadjuvant or primary systemic therapy for women with hormone-responsive primary breast tumors [21–23]. A study comparing neoadjuvant endocrine therapy with exemestane versus chemotherapy in 152 patients with ER+/progesterone receptor-positive (PgR+) breast cancer found that while efficacy outcomes were comparable in the two groups, chemotherapy treatment was significantly more toxic, confirming that endocrine therapy could be used as an alternative to chemotherapy in older women [24]. Early studies of primary tamoxifen as an alternative to surgery were conducted in older women with hormone-responsive breast cancer who were unfit for chemotherapy [23, 25–27]. While these studies demonstrated a reduction in tumor size with primary tamoxifen, long-term local disease control was found to be poor. The omission of primary surgery resulted in an increased rate of progression, therapeutic intervention, and mortality [28]. Thus, primary tamoxifen treatment is indicated for only the most frail, medically ill, or noncompliant patients [29]. Although primary therapy with tamoxifen was found to be well-tolerated, adverse effects were reported including hot flushes, skin rash, vaginal discharge, breast pain, sleepiness, headache, vertigo, itching, hair loss, cystitis, acute thrombophlebitis, nausea, and indigestion [29]. Furthermore, more serious adverse effects, such as an increased risk of endometrial cancer and thromboembolic events, have been reported in large trials of adjuvant tamoxifen [30].
In view of the limitations of tamoxifen as a single modality treatment, attention has switched to the neoadjuvant use of endocrine therapy to increase the rate of breast conservation [21]. Data from preclinical models [31] and clinical studies in advanced breast cancer [32] predict that aromatase inhibitors (AIs) may be more effective than tamoxifen in the neoadjuvant setting. Furthermore, preclinical and clinical evidence suggests that letrozole may be the most effective AI in this setting [33–35]. In postmenopausal women with endocrine-responsive locally advanced or metastatic breast cancer, first-line treatment with letrozole was shown to be significantly more effective than tamoxifen in terms of response rate (overall response rate [ORR], 30 vs. 20%, P = 0.0006) and time to disease progression (41 vs. 26 weeks) [32]. A phase 1–2 pilot study showed a clinical response rate of 88% (21/24 patients) in postmenopausal women with ER+ locally advanced breast cancer treated with letrozole for 3 months prior to surgery [36]. All patients in the study were eligible for breast conserving surgery following neoadjuvant letrozole.
A large multinational trial, P024, was designed to assess the efficacy of neoadjuvant letrozole compared with tamoxifen in postmenopausal women with hormone receptor-positive (HR+) breast cancer who were ineligible for breast-conserving surgery [21]. The clinical objectives of the trial were to compare response rates and surgical outcomes between the two treatment arms. In addition, the trial provided the opportunity to conduct prospective biomarker studies to explore the biologic basis for response to neoadjuvant endocrine therapy [37]. This review describes the results of the P024 study and the follow-up biomarker studies, focusing on the treatment implications in the neoadjuvant setting.
Trial design and patients
PO24 was a multinational, randomized, double-blind controlled trial comparing letrozole and tamoxifen in postmenopausal women with hormone-responsive primary invasive breast cancer who were not eligible for breast-conserving surgery [21]. The trial was conducted in 55 centers in 16 countries between March 1998 and August 1999. Local ethics review boards approved the protocol and all patients gave written informed consent before study enrollment.
Randomized clinical trial design
Patients were randomly assigned to receive letrozole 2.5 mg or tamoxifen 20 mg administered orally once daily for 4 months prior to scheduled surgery [21]. Patients were considered to have completed the study when they had received 4 months of treatment and had been assessed for surgery. Following surgery, patients were treated at the investigator’s discretion and were followed for 5 years for local recurrence, distant metastasis, and survival.
Patient population
A total of 337 postmenopausal women were enrolled into the trial, and 324 were included in the intent-to-treat population (see Fig. 1). Eligible patients were postmenopausal women with untreated, primary HR+ (≥10% nuclear staining for ER or PgR) invasive breast tumors (stages T2–4a–c, N0–2, M0). The minimum tumor size was ≥3 cm, and all patients were considered inoperable or ineligible for breast-conserving surgery, mostly because of a non-favorable ratio tumor size/breast size. Exclusion criteria included previous exposure to AIs, uncontrolled endocrine or cardiac disease, bilateral or inflammatory breast cancer, distant metastasis, and other malignant disease. In addition, administration of other cancer treatment or hormone replacement therapy was not allowed during study participation.
Fig. 1Patient disposition [21]
Trial end points
The primary end point was ORR, defined as the percentage of patients in each treatment arm with a complete response (CR) or a partial response (PR) as determined by breast palpation [21]. Response categories were CR, PR, no change, progressive disease, or not assessable/not evaluable. The secondary end points were the percentage of patients who underwent breast-conserving surgery and the response rate (CR + PR) determined at 4 months by mammography and by ultrasound [21]. Safety was assessed and adverse events graded according to the National Cancer Institute Common Toxicity Criteria.
Correlative science studies
In parallel, studies were conducted in which tumor biopsies and blood samples were taken from all patients prior to the initiation of therapy and at the end of treatment to generate a database from which changes in molecular markers could be assessed [37–41]. These studies were prospectively designed to assess the biological basis for letrozole efficacy. The specific objectives of the biomarker sub-studies were as follows:To confirm ER and PgR status in a central laboratory. ER and PgR were determined using immunohistochemistry (IHC) performed on pretreatment biopsies; the criterion for minimal ER positivity was 10% positive cells [37]. The Allred histopathological score was applied to further assess the level of ER and PgR expression in both the baseline and surgical specimens [42].To explore relationships between ER and PgR expression levels and response to treatment [37].To examine the relationship between the expression of HER1 and HER2 and the likelihood of primary response [37]. Previous studies have shown that HER2 overexpression may be a predictor of tamoxifen resistance [43, 44], and HER1 has also been linked with endocrine therapy resistance [45]. HER2 IHC was initially scored as 0/+ (negative) or ++/+++ (positive or overexpressed) [46]. Since fluorescent in situ hybridization (FISH) testing has replaced IHC as the gold standard for HER2 assessment, the P024 tumor bank was reassessed using HER1 and HER2 FISH probes, and these data on HER2 gene amplification status were supplemented with 106 tumor samples [40]. Pre- and post-treatment tumor biopsy samples were also analyzed for ER and several indices of ER function, including PgR and trefoil factor 1 (TFF1), HER1 and HER2, and the proliferation marker Ki67 [38].To assess gene expression profiling as a means to further investigate the transcriptional programs that underlie resistance and sensitivity to estrogen deprivation [39]. Gene expression (mRNA) profiles were collected from the tumor biopsies collected prior to surgery, at 1 month of treatment, and at surgery. The analysis was done using an Affymetrix U 133 subA Gene Chip.To measure aromatase expression in tumors before and after 4 months of letrozole or tamoxifen treatment. Aromatase is the key enzyme responsible for estrogen biosynthesis and is present in about 70% of tumors. IHC was performed with a monoclonal aromatase antibody (677) on trial samples prior to and following letrozole or tamoxifen treatment (n = 185); scoring was measured as a proportion of immuno-positive cells and their intensity of reactivity in malignant epithelial, stromal, adipose, and normal compartments [41].
Efficacy
In the intent-to-treat population, 154 patients received letrozole and 170 received tamoxifen. Similar proportions of patients in the letrozole and tamoxifen groups had inoperable tumors (13 and 14%, respectively). Other baseline characteristics were also well-balanced for age, race, HR status, and tumor/nodal stage of disease. More patients in the tamoxifen group (n = 41) discontinued treatment than in the letrozole arm (n = 23). The main reason for premature discontinuation was disease progression.
Clinical response and breast-conserving surgery
Letrozole was consistently superior to tamoxifen for primary and secondary efficacy end points [21]. The ORR was 55% for letrozole versus 36% for tamoxifen (P < 0.001). Median time to response was 66 days in the letrozole group and 70 days in the tamoxifen group. The odds ratio for achieving CR + PR was more than doubled with letrozole (2.23, 95% CI 1.43, 3.50; P = 0.0005). In terms of clinical progression, 12% of patients on letrozole and 17% on tamoxifen progressed, while 24% of letrozole- and 35% of tamoxifen-treated patients had stable disease. Letrozole was also shown to be significantly more effective than tamoxifen when response rates were assessed by mammography (34 vs. 16%, respectively; P < 0.001) and ultrasound (35 vs. 25%, respectively; P = 0.042).
The proportion of patients able to undergo breast-conserving surgery was significantly higher in the letrozole group than in the tamoxifen group (45 vs. 35%, respectively; P = 0.022). Of note, the odds ratio for breast-conserving surgery was 4.56 (P = 0.0001) for patients presenting with T2 tumors compared with all other T stages. The only other factor that increased the odds of undergoing breast-conserving surgery was treatment with letrozole (odds ratio 1.71, P = 0.03).
ER and PgR
A supportive analysis of clinical efficacy data conducted in patients with centrally confirmed ER+ or PgR+ tumors also showed that outcomes were significantly improved with letrozole (n = 124) compared with tamoxifen (n = 126) [37]. In this subgroup, the ORR was 60% for letrozole versus 41% for tamoxifen (P = 0.004), and breast-conserving surgery was performed in 48 versus 36%, respectively (P = 0.036).
There was a linear relationship between ER Allred expression scores and response rates to both letrozole and tamoxifen (see Fig. 2). Tumors with low ER expression were still responsive to letrozole but not to tamoxifen [37]. Of note, letrozole response rates were numerically superior to tamoxifen response rates in every ER Allred category from three to eight, indicating that letrozole is more effective than tamoxifen regardless of the level of ER expression [37]. This finding is important, because of all the AIs, only letrozole has demonstrated clear ORR superiority over tamoxifen in ER-poor tumors. When the effects of letrozole and anastrozole on tumors with low ER values (Allred scores 2–5) were compared in the neoadjuvant setting, only letrozole achieved a significant reduction in cell proliferation in ER-poor tumors [35].
Fig. 2Clinical response rate versus estrogen receptor (ER) Allred score for letrozole and tamoxifen. The P value for a linear logistic model was 0.0013 for letrozole and 0.0061 for tamoxifen according the Wald test. In this analysis, ER−, PgR+ cases (determined by conventional cut points) were excluded. Reprinted from [37] with permission from the American Society of Clinical Oncology
Letrozole, but not tamoxifen, significantly reduced the expression of estrogen-regulated proteins PgR and TFF1 [38]. Average tumor PgR expression decreased dramatically on letrozole treatment (P = 0.0001), and only 4.4% of surgical specimens exhibited an Allred score of between six and eight for PgR expression. The significant decrease in PgR expression with letrozole remained significant in the ER+, HER2+ subpopulation. In contrast, changes in PgR expression with tamoxifen therapy were not consistent, with both increases and decreases in expression frequently observed in the overall and ER+, HER2+ subpopulations. Analysis of changes in TFF1 produced similar conclusions, with letrozole markedly reducing expression (P = 0.0001) and tamoxifen producing no overall trend in either direction.
HER1/HER2 and response
Approximately 15% of tumors were ER+ and overexpressed both HER1 and HER2. The response rate in this patient subgroup was significantly higher with letrozole than with tamoxifen (88 vs. 21%) [37]. The odds ratio for response to letrozole versus tamoxifen was 28 (95% CI 4.5, 177; P = 0.0004). Letrozole was equally effective for HER1/HER2+ and HER1/HER2− tumors, whereas tamoxifen was significantly less effective in HER2+ compared with HER2− tumors (P = 0.045). These data suggest that although HER1 and HER2 status might not be the only explanation for the superiority of letrozole over tamoxifen, overcoming resistance pathways associated with HER1 and HER2 expression is a significant component of the improvement in outcomes associated with letrozole treatment observed in this clinical trial. FISH analysis of tumor samples confirmed the clinical efficacy of letrozole in breast cancers with or without HER2 amplification (ORR 71% in both subsets; P = 0.98). In contrast, tamoxifen-treated tumors with HER2 gene amplification had lower clinical response rates than tamoxifen-treated HER2− tumors (33 vs. 49%, P = 0.49) (see Table 2) [40].
Table 2Analysis of clinical, ultrasound, and mammogram response data according to HER2 FISH status in letrozole-treated patients and tamoxifen-treated patientsResponse CategoryNo. responsesTotal No. (%)No. responsesTotal No. (%)HER2 FISH-positive Tamoxifen-treated patientsHER2 FISH-negative Tamoxifen-treated patientsP-valueaClinical39 (33)4490 (49)0.49Ultrasound 39 (33)2674 (35)0.99Mammography 19 (11)2290 (24)0.68HER2 FISH-positive Letrozole-treated patientsHER2 FISH-negative Letrozole-treated patientsP-valuebClinical1217 (71)131185 (71)0.98Ultrasound817 (47)91170 (54)0.61Mammography716 (44)84178 (47)0.79Reprinted from [40] with permission from the American Society of Clinical OncologyHER2 human epidermal growth factor receptor 2, FISH fluorescence in situ hybridizationaFisher test P-valuebMantel–Haenzel P-value
Biomarkers of tumor proliferation
Letrozole inhibited tumor proliferation, measured by the biomarker Ki67, to a greater extent than tamoxifen (reduction in geometric mean Ki67 level 87 vs. 75%, respectively; P = 0.0009). The differences in Ki67 reduction were also observed in ER+, HER1 and/or HER2 overexpressing tumors (88% for letrozole vs. 45% for tamoxifen, respectively; P = 0.0018) [38]. Changes in the percentage of Ki67-positive cells in HER1/2+ tumors treated with letrozole or tamoxifen are shown in Fig. 3.
Fig. 3A box plot of before and after treatment Ki67 values in the estrogen-receptor-positive, human epidermal growth factor receptor (HER) 1/2+ subset. With letrozole (n = 15), 11 showed a decrease, one exhibited no change, and three showed an increase, of which only one was >2-fold (0.1–0.3%). With tamoxifen (n = 17), ten showed a decrease and seven an increase, of which three were relatively dramatic (9.5–22.7, 20.9–40.7, and 0.1–17.3%). Reprinted from [38] with permission from the American Association for Cancer Research
More recently, it was found that HER2 FISH-positive tumors showed higher histologic grade (P = 0.009), higher pretreatment Ki67 (P = 0.005), and less Ki67 suppression after letrozole when compared with HER2 FISH-negative tumors (P = 0.0001) [40]. Letrozole significantly decreased the geometric mean Ki67 level in HER2 FISH-negative tumors (from 6.25 [95% CI 5.16, 7.58%] to 0.68% [95% CI 0.53, 0.87%]; P = 0.0001), but the decrease in HER2 FISH-positive tumors was blunted (from 14.73 [95% CI 9.67, 22.44%] to 8.1% [95% CI 4.16, 15.75%]; not significant). A similar observation for Ki67 was made in a smaller cohort of tamoxifen-treated tumors [40].
The relationship between cell cycle CR, defined as ≤1% of post-treatment Ki67 staining in the infiltrating component of the tumor, and HER2 status of tumors treated with letrozole was also analyzed [40]. Significantly more HER2 FISH-negative than FISH-positive tumors met the definition of a cell cycle CR at the time of surgery (60 vs. 12%; P = 0.0001). There was a high level of correlation between lack of cell cycle CR and the presence of a positive HER2 FISH test (P = 0.0001) for letrozole- and tamoxifen-treated tumors, which is consistent with the conclusion that HER2 gene amplification generates resistance at the level of cell cycle progression regardless of which endocrine therapy is used [40].
These biomarker findings are discordant with clinical observations that tumor regression is unaffected by HER2 amplification status in patients treated with neoadjuvant letrozole and may imply therapeutic resistance that could manifest later in the clinical course of the disease. Consistent with this finding, Miller and colleagues also reported that neoadjuvant letrozole produces rapid and profound decreases in expression of Ki67 and PgR that do not always correlate with clinical and pathological responses [47].
Gene expression profiling
Preliminary gene expression profiling analysis of biopsies taken pretreatment and 1 month post letrozole treatment showed down-regulation of genes involved in DNA replication and synthesis, cell cycle progression, apoptosis suppression, and tissue invasion [39]. These results illustrate the molecular basis for estrogen-deprivation letrozole therapy which may be useful in the development of predictive models of ER+ breast cancer.
Aromatase
Sufficient pre- and post-treatment tumor material was available from 171 cases (81 on letrozole and 90 on tamoxifen) from the P024 trial for immunohistochemistry analysis of aromatase protein expression [41]. Aromatase was detected in all tumor compartments, with the strongest staining observed in malignant epithelial cells. Median aromatase values did not change significantly with letrozole or tamoxifen treatment; however, changes in score did occur in individual cases, with more noticeable effects observed in letrozole-treated patients [41]. A positive correlation existed between baseline ER and aromatase staining in cancer cells, while a negative correlation was observed between baseline Ki67 and aromatase expression in cancer plus stroma. Baseline aromatase expression did not predict response to letrozole or tamoxifen, or changes in Ki67 induced by treatment. However, negative staining in both stroma and cancer after treatment was strongly associated with fewer cell cycle CR and smaller Ki67 declines with letrozole (but not tamoxifen) treatment [48].
Safety and duration of therapy
P024 demonstrated that letrozole is well-tolerated in the neoadjuvant setting [21]. There were no major tolerability differences between letrozole and tamoxifen, and adverse effects of a similar nature were seen in 57% of patients in each arm [21]. The most common treatment-related adverse event was hot flushes, occurring in 20% of patients in the letrozole group and 24% of patients in the tamoxifen group.
The excellent tolerability, predictable pharmacokinetics, and minimal drug–drug interactions [49, 50] make letrozole a particularly suitable option for older women unable to tolerate or unwilling to accept neoadjuvant chemotherapy and in whom the presence of comorbidities and use of concomitant therapies complicate treatment selection [20]. The median age of patients in P024 treated with letrozole was 68 years, and 46% of patients were at least 70 years old. The feasibility and safety of letrozole was also reported recently from another trial using letrozole as primary systemic therapy in elderly patients (median age 79 years) with breast cancer [51]. In addition, letrozole has been successfully administered to elderly patients (median age of the elderly subgroup 75 years; range 70–96 years) in the advanced breast cancer setting [52].
The favorable safety profile of letrozole also allows for the extension of the neoadjuvant treatment beyond the 4 months used in the P024 trial. In a recent study, 33 postmenopausal women with HR+ breast cancer ineligible for breast-conserving surgery were treated with letrozole for 4 months. Continued administration of letrozole for a further 4 months in responders and patients with stable disease resulted in a statistically significant improvement in tumor size reduction (P = 0.039); ORR was 90% in patients receiving preoperative treatment for longer than 4 months compared with 57% in patients receiving treatment up to 4 months [53].
In another study, 42 patients who were unsuitable for breast-conserving surgery or had refused surgery after responding to initial neoadjuvant therapy with letrozole for 3 months benefited from continuing tumor volume reduction during further letrozole treatment administered for up to 12 months [54]. The median reductions in tumor volume were 52% (95% CI 37, 62) from 0 to 3 months, 57% (95% CI 26, 100) from 3 to 6 months, and 66% (95% CI 22, 100) from 6 to 12 months. Extending the duration of letrozole also improved the CR rate, which increased from 4/42 patients (9.5%) at 3 months to 12/42 (29%) by 6 months and 8/22 (36%) by 12 months.
Conclusions
Neoadjuvant therapy with AIs is a safe and effective treatment option for postmenopausal women who are unwilling or unable to undergo surgery or preoperative chemotherapy [21, 22, 55–57]. Letrozole is significantly more effective than tamoxifen in the neoadjuvant setting in terms of ORR and increased rate of breast-conserving surgery [21, 55].
One of the advantages of using a neoadjuvant strategy is the opportunity to gain information on tumor response early in the course of treatment. Detailed studies correlating clinical response to neoadjuvant therapy with changes in tumor biomarkers and gene expression may ultimately prove useful to tailor therapy for individual patients and to gain a better understanding of the biology of HR+ breast cancer. Correlative studies conducted using tumor samples from P024 have revealed important information about how breast tumors respond to letrozole [37–40]. Letrozole was shown to be significantly more effective than tamoxifen in the inhibition of ER+ tumor proliferation, since letrozole produced a greater reduction in levels of the proliferation biomarker Ki67 [38]. It has been suggested that a greater suppression in proliferation could lead to greater long-term survival in the adjuvant setting. Preliminary data from the Immediate Preoperative Anastrozole Tamoxifen or Combined with Tamoxifen trial have indicated that short-term changes in Ki67 levels, after 2 and 12 weeks, may be a useful predictive marker for relapse-free survival in patients treated with neoadjuvant AI therapy [58]. It has also been suggested that changes in proliferation and concurrent changes in apoptosis may be expected to be more predictive of adjuvant benefit from endocrine therapy than clinical response [59].
Correlative studies have also highlighted the complexity of breast cancer biology and revealed discordance between clinical and biomarker responses [40]. Amplification of HER2 was shown to be associated with a more aggressive breast cancer phenotype and greater resistance to tamoxifen [37, 40]. Clinical response data from the P024 trial have shown that letrozole is equally effective in HER2+ and HER2− tumors, whereas tamoxifen is less effective in HER2+ tumors [37]. These data suggest that letrozole could be a superior option to tamoxifen for postmenopausal women with HER2+, HR+ tumors [37]. However, analysis of proliferation markers has provided evidence of estrogen-independent proliferation of ER+, HER2+ breast cancer despite neoadjuvant letrozole [40]. It appears that cell-cycle regulation is partially or completely estrogen-independent in the majority of primary tumors showing HER2 gene amplification, and patients with such tumors may eventually develop resistance to adjuvant AI therapy. Novel strategies to delay or overcome hormone resistance are described elsewhere in this supplement, in the article, “Femara and the future.”
Gene expression profiling has demonstrated that letrozole targets genes responsible for DNA replication and synthesis, cell cycle progression, apoptosis, and tissue invasion [39]. Research into genetic profiling is continuing, with the aim of developing clinically relevant predictive models that can accurately classify ER+ disease according to likely response to specific neoadjuvant therapies. Predictive models will improve treatment individualization and help to avoid unnecessary treatment-related toxicity in patients unlikely to benefit from systemic therapies [17, 60].
The P024 trial has clearly demonstrated the therapeutic superiority of letrozole over tamoxifen for the neoadjuvant management of primary breast cancer. The trial has also provided the oncology community with a validated research setting within which to gain valuable insights into the molecular features of ER+ breast cancer and its treatment that will help shape new therapies in the years to come.

letrozole
breast cancer
endocrine therapy
aromatase inhibitor
tamoxifen
postmenopausal
neoadjuvant therapy


Why are mineralocorticoid receptor antagonists cardioprotective?

Two clinical trials, the Randomized ALdosterone Evaluation Study (RALES) and the EPlerenone HEart failure and SUrvival Study (EPHESUS), have recently shown that mineralocorticoid receptor (MR) antagonists reduce mortality in patients with heart failure on top of ACE inhibition. This effect could not be attributed solely to blockade of the renal MR-mediated effects on blood pressure, and it has therefore been proposed that aldosterone, the endogenous MR agonist, also acts extrarenally, in particular in the heart. Indeed, MR are present in cardiac tissue, and possibly aldosterone synthesis occurs in the heart. This review critically addresses the following questions: (1) is aldosterone synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR normally, and (3) what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure? Conclusions are that most, if not all, of cardiac aldosterone originates in the circulation (i.e., is of adrenal origin), and that glucocorticoids, in addition to aldosterone, may serve as the endogenous agonist of cardiac MR. MR-mediated effects in the heart include effects on endothelial function, cardiac fibrosis and hypertrophy, oxidative stress, cardiac inotropy, coronary flow, and arrhythmias. Some of these effects occur via or in synergy with angiotensin II, and involve a non-MR-mediated mechanism. This raises the possibility that aldosterone synthase inhibitors might exert beneficial effects on top of MR blockade.
Introduction
The renin-angiotensin-aldosterone system (RAAS) has been viewed conventionally as a circulating system, involved in the regulation of salt, fluid homeostasis and blood pressure. Kidney-derived renin cleaves liver-derived angiotensinogen to form angiotensin (Ang) I in circulating blood (Fig. 1). Angiotensin-converting enzyme (ACE), located at the luminal side of the endothelium, subsequently converts Ang I to Ang II. Ang II exerts its effects via stimulation of Ang II type 1 (AT1) and type 2 (AT2) receptors. Besides acting as a vasoconstrictor via AT1 receptors, Ang II also stimulates the formation of the sodium-retaining hormone aldosterone. Aldosterone mediates its cellular effects by binding to the mineralocorticoid receptor (MR), a member of the steroid/thyroid/retinoid/orphan receptor family of transcription factors.
Fig. 1The renin-angiotensin-aldosterone system. Angiotensin II activates angiotensin II type 1 and type 2 (AT1 and AT2) receptors. AT1 receptor activation in the adrenal results in the synthesis and release of aldosterone, which subsequently exerts ‘genomic’ (after hours, involving protein synthesis) or ‘non-genomic’ (within minutes, not involving protein synthesis) effects through either mineralocorticoid receptors (MR) or other, as yet unidentified, receptors. MR may occur both intracellular and on the cell membrane. Aldosterone upregulates both AT1 and AT2 receptors, and AT1 receptor activation also results in MR stimulation in an aldosterone-independent manner
This classic concept has been updated in the past two decades. It is now believed that some or all of the components of the RAAS are synthesized locally in tissues such as the heart and vessel wall (Danser 2003; Tom et al. 2003). For instance, in the heart, Ang II is synthesized locally by cardiac ACE following uptake of renin and angiotensinogen from the circulation (Danser et al. 1994; van Kats et al. 1998). This Ang II subsequently stimulates cardiac AT1 and AT2 receptors (Batenburg et al. 2004; van Kats et al. 2000). Local synthesis of Ang II at cardiac tissue sites is in agreement with the observation that the beneficial effects of ACE inhibitors in heart failure are, at least in part, independent of their effect on blood pressure (Yusuf et al. 2000).
Traditionally, treatment of heart failure and hypertension has been performed mainly on the basis of the renin-angiotensin system (RAS) rather than the RAAS, with the assumption that aldosterone will be suppressed once Ang II formation is blocked. However, aldosterone formation does not stay suppressed during prolonged RAS blocking therapy. After 3 months of therapy, aldosterone levels start to rise again and they continue to do so due to “Ang II reactivation” or “aldosterone escape” (Farquharson and Struthers 2002; Jorde et al. 2002).
Two clinical trials, the Randomized ALdosterone Evaluation Study (RALES) (Pitt et al. 1999) and the EPlerenone HEart failure and SUrvival Study (EPHESUS) (Pitt et al. 2003), have convincingly indicated that MR antagonists reduce mortality in patients with heart failure and systolic left ventricular dysfunction post-myocardial infarction on top of ACE inhibition. In particular, a reduction in the rate of sudden death was observed. These results draw attention to the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. The benefit of MR antagonists during RAS blockade is not yet fully understood. Their effects cannot be attributed solely to blockade of the renal MR-mediated effects on blood pressure (Pitt et al. 1999, 2003), and it is now generally assumed that aldosterone also acts extrarenally, in agreement with the concept of local RAAS.
Indeed, MR have been demonstrated in the heart, both at the mRNA and protein level (Lombès et al. 1995). Importantly, the enzymes required for the synthesis of aldosterone appear to be expressed in the human heart as well (Young et al. 2001). Together with the fact that Ang II is capable of increasing the aldosterone levels in isolated rat hearts and blood vessels (Silvestre et al. 1998; Takeda et al. 1997), these data suggest that aldosterone, like Ang II, may be formed and act locally in the heart. This review addresses the following questions: (1) is aldosterone really synthesized at cardiac tissue sites, (2) what agonist stimulates cardiac MR, and (3) what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure?
Aldosterone synthesis at cardiac tissue sites?
Aldosterone, a steroid that was originally discovered in 1953, is secreted by the zona glomerulosa cells of the adrenal cortex. The kidney is the major target for adrenal aldosterone to increase sodium (and consequently water) reabsorption and potassium excretion. The production of aldosterone is regulated at two critical enzyme steps: (1) the formation of pregnenolone from cholesterol by the mitochondrial enzyme P450scc (side-chain cleavage), and (2) the conversion of corticosterone to aldosterone by cytochrome P450 11β-hydroxylase 2 (CYP11B2, ‘aldosterone synthase’). Aldosterone synthesis in the adrenal cortex is regulated by Ang II, potassium and, more weakly, sodium and adrenocorticotrophic hormone.
Extra-adrenal aldosterone synthesis has been proposed in heart (Silvestre et al. 1998), brain (Gomez-Sanchez et al. 1997) and vessel wall (Takeda et al. 1995, 1997). The rat heart expresses the steroidogenic acute regulatory (StAR) protein and aldosterone synthase, although at 100-fold lower levels than the adrenal (Casal et al. 2003; Silvestre et al. 1998). The StAR protein facilitates intramitochondrial cholesterol transfer, the rate-limiting step of steroidogenesis. Aldosterone synthase expression has also been proposed in the human heart (Tsybouleva et al. 2004; Young et al. 2001). In support of the functional importance of such expression, net release of aldosterone was observed across the human coronary vascular bed (Nakamura et al. 2004). However, other studies demonstrated the opposite (i.e., cardiac aldosterone extraction) (Hayashi et al. 2003), whereas it was also noted that, in humans, cardiac aldosterone correlated closely with the cardiac levels of renin. Since the latter is exclusively of renal origin (i.e., blood-derived) (Danser et al. 1994, 1997; Saris et al. 2001), its correlation with aldosterone argues against independent aldosterone synthesis at cardiac tissue sites. Furthermore, recent careful studies in rats, paying great attention to the measurement of aldosterone in cardiac tissue, did not confirm the idea of local synthesis of aldosterone in the rat heart (Fiebeler et al. 2005; Gomez-Sanchez et al. 2004). In these studies, the cardiac aldosterone levels were much lower than previously reported by others (Silvestre et al. 1998). Furthermore, the cardiac levels correlated closely with the plasma levels of aldosterone, and they decreased to levels at or below the detection limit after adrenalectomy.
Subsequent perfusion studies with aldosterone in the isolated Langendorff heart (Chai et al. 2006) showed that the steroid rapidly accumulated in cardiac tissue, not only in extracellular (interstitial) fluid but also in a second, as yet unidentified, compartment. At steady state, the aldosterone tissue levels (expressed per g wet weight) were higher than its levels in coronary effluent (expressed per ml). Washout from the second compartment occurred relatively rapid (half life <10 min), suggesting that it represented cell surface-bound rather than internalized aldosterone (Fig. 2). This pattern resembles that of cardiac renin, which also accumulates in extracellular fluid and binds to membrane receptors (Danser et al. 1994; de Lannoy et al. 1997; Saris et al. 2001). After prolonged washout, cardiac aldosterone became undetectable. Thus, on the one hand, the heart displays a large capacity to accumulate aldosterone. This explains why the levels of cardiac aldosterone in rats can be up to 10-fold higher than in serum (Fiebeler et al. 2005). On the other hand, cardiac aldosterone disappears rapidly during perfusion with aldosterone-free buffer. This provides an explanation for the ‘release’ of aldosterone across the coronary vascular bed in humans and rats (Takeda et al. 2000). The majority of cardiac aldosterone, if not all, is however derived from the circulation, i.e., is not synthesized locally, both under normal and pathological conditions (Chai et al. 2006; Fiebeler et al. 2005; Gomez-Sanchez et al. 2004).
Fig. 2Washout of aldosterone from the isolated perfused rat Langendorff heart after its exposure to 10 nmol/l aldosterone for 30 min. Data are modified from Chai et al. (2006)
Activation of cardiac mineralocorticoid receptors by aldosterone?
MRs occur both in Na+-transporting epithelia (e.g., kidney, colon) and non-epithelial tissues such as brain (de Kloet et al. 2000), heart (cardiomyocytes) (Lombès et al. 1995), and blood vessels (endothelial and smooth muscle cells) (Lombès et al. 1992; Oberleithner 2005; Oberleithner et al. 2004). The presence of MR in the cardiovascular system has been confirmed both at the mRNA and protein level in animals as well as in humans (Lombès et al. 1992).
MRs bind mineralocorticoids and glucocorticoids with equal affinity (Kd ≈ 0.5–2 nM) (Arriza et al. 1987; Lombès et al. 1994). Yet, the circulating concentrations of glucocorticoids are several orders of magnitude higher than those of aldosterone (Fig. 3). Selectivity of aldosterone binding to MR is achieved by co-expression of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) (Alzamora et al. 2000). This enzyme converts cortisol (the endogenous glucocorticoid in humans) and corticosterone (the endogenous glucocorticoid in rats) to their non-MR-binding metabolites cortisone and 11-dehydrocorticosterone (Fig. 3). In addition, the off-rate of aldosterone from the MR is five times lower than that of glucocorticoids (Lombès et al. 1994), and thus it is possible that MR also discriminate aldosterone from glucocorticoids, at least in part, independently of 11βHSD2.
Fig. 3Diagram illustrating activation of glucocorticoid and mineralocorticoid receptors (GR, MR) by cortisol (top) or aldosterone (bottom). Selectivity of aldosterone binding to MR is achieved by co-expression of 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2). 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), which acts as a reductase in vivo, reactivates cortisone to cortisol. The affinity constants refer to aldosterone-MR and cortisol-GR binding (Arriza et al. 1987; Hagendorf et al. 2005; Lombès et al. 1994). Note that, in the absence of 11βHSD2, MR bind cortisol with an affinity equal to that for aldosterone
In the kidney, 11βHSD2 expression is high enough to allow selective MR stimulation by aldosterone. In contrast, in the heart, the 11βHSD2 levels are almost negligible (Nagata et al. 2006), and it has therefore been proposed that cardiac MR are occupied by cortisol/corticosterone rather than aldosterone (Funder 2005b; Gomez-Sanchez et al. 2004; Nagata et al. 2006). In fact, the endogenous glucocorticoid levels are high enough to keep all cardiac MR permanently occupied (Funder 2005b). Such permanent occupation does not apply to glucocorticoid receptors, since they have a ≈30-fold lower affinity for cortisol/corticosterone than MR (Fig. 3). Glucocorticoids are assumed to act as antagonists of MR (i.e., they exert no effect following binding to MR) in kidney (Good et al. 2002) and heart (Qin et al. 2003), whereas in the vessel wall, during 11βHSD2 blockade with carbenoxolone, they act as agonists (Alzamora et al. 2000). The latter could relate to the observation that glucocorticoid-MR complexes become activated as a result of the generation of reactive oxygen species (ROS). Under such circumstances, MR antagonists may exert effects by blocking the consequences of glucocorticoid-MR complex activation rather than aldosterone-MR activation (Funder 2005b; Nagata et al. 2006). The increased expression in the failing rat heart of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), which reactivates 11-dehydrocorticosterone to corticosterone, further supports this concept (Nagata et al. 2006).
Genomic versus non-genomic effects
The classical MR-mediated effects of aldosterone are referred to as ‘genomic’ effects. These effects involve binding of aldosterone to intracellular MRs, and the translocation of the steroid-MR complex to the nucleus, where it acts as a transcriptional regulator, inducing protein synthesis (in particular synthesis of the epithelial Na+ channel) after several hours. Genomic effects can be inhibited by agents that block either transcription (e.g., actinomycin D) or translation (e.g., cycloheximide).
In addition to its genomic effects, which occur after hours, aldosterone also exerts rapid effects (within minutes) in various tissues, e.g., heart and vasculature. These effects are usually described as ‘non-genomic’, since they do not involve DNA-directed, RNA-mediated protein synthesis. For instance, aldosterone rapidly affects cardiac inotropy and facilitates both vasodilation and vasoconstriction (Barbato et al. 2002; Liu et al. 2003; Mazak et al. 2004; Michea et al. 2005; Mihailidou et al. 2004; Schmidt et al. 2003). In many (but not all) cases, these effects could not be blocked by the MR antagonist spironolactone, and therefore the existence of a novel (membrane-associated?) aldosterone receptor has been proposed (Funder 2005a; Wehling et al. 1995). However, despite numerous efforts in the past decade, no convincing data toward the characterization of a membrane receptor for aldosterone have been put forward (Funder 2005a; Wehling 2005). Consequently, it is not unlikely that these effects are after all also being mediated via the classical (intracellular) MR (Funder 2005a). If so, an explanation must be provided for the lack of effect of spironolactone toward the rapid aldosterone-induced actions in vitro. Possibly, via modification or dimerization, classical MR can attain an atypical pharmacology, thereby no longer allowing the binding of MR antagonists such as spironolactone. In addition, spironolactone exerts MR-independent effects of its own, i.e., it blocks human Ether-a-Go-Go-Related gene K+ channels (Caballero et al. 2003) and inhibits calcium entry (Cargnelli et al. 2001). Thus, to solve this issue, future studies should make use of alternative MR antagonists such as eplerenone. Eplerenone displays increased selectivity for the MR over other steroid receptors, although its affinity for the MR in vitro is 10- to 20-fold lower than that of spironolactone.
The rapid, non-genomic actions involve activation of the phospholipase C-protein kinase C (PKC)-inositol 1,4,5-trisphosphate (IP3)-1,2 diacylglycerol (DAG) pathway, which leads to an increase in intracellular Ca2+ and stimulation of the Na+/H+ exchanger (Barbato et al. 2004b; Funder 2005a; Liu et al. 2003; Lösel et al. 2002; Mihailidou et al. 2004; Sato et al. 1997). The latter causes a rise in intracellular Na+, which subsequently activates Na+/K+-ATPase. Interestingly, however, when the increase in intracellular Na+ is prevented, aldosterone decreases Na+/K+-ATPase activity in a PKC-dependent manner (Mihailidou et al. 2004). Thus, aldosterone may exert both positive and negative inotropic effects. Other second messenger pathways that have been linked to the rapid effects of aldosterone include mitogen-activated protein (MAP) kinases, ROS and the epidermal growth factor receptor (Jaffe and Mendelsohn 2005; Mazak et al. 2004).
Effects of aldosterone in the cardiovascular system
Substantial evidence has emerged showing that aldosterone induces adverse effects in the cardiovascular system. The co-expression of 11βHSD2 and MR in human heart and blood vessels (Glorioso et al. 2005; Jaffe and Mendelsohn 2005; Lombès et al. 1995), albeit at low levels, supports the concept that these organs possess the cellular machinery required for direct aldosterone action, irrespective of the source of aldosterone.
Endothelial dysfunction
Aldosterone increases the volume and stiffness of endothelial cells and induces gap formation, allowing irregular diffusion pathways for large particles (Oberleithner 2005; Oberleithner et al. 2004). This mechanism could contribute to endothelial dysfunction observed in hyperaldosteronism. The normalization of endothelial function by spironolactone in patients with heart failure supports this concept (Abiose et al. 2004; Macdonald et al. 2004).
Oxidative stress, inflammation, fibrosis and atherosclerosis
A growing number of studies support a specific role of the MR as a mediator of oxidative stress and subsequent inflammation, fibrosis and atherosclerosis. Elevations in circulating aldosterone are accompanied by a pro-inflammatory/fibrogenic vascular phenotype (Ahokas et al. 2005; Blasi et al. 2003; Sun et al. 2002), and since this phenomenon can be blocked by both spironolactone and anti-oxidants (Sun et al. 2002), it appears that aldosterone, via MR, induces oxidative stress. Indeed, aldosterone upregulates various subunits of NADPH oxidase and induces ROS generation in mononuclear and vascular smooth muscle cells (Ahokas et al. 2005; Calo et al. 2004; Mazak et al. 2004; Sun et al. 2002). In addition, aldosterone stimulates collagen synthesis in cardiac fibroblasts (Brilla et al. 1994). Consequently, aldosterone-induced cardiac fibrosis may be due to both direct effects in the heart (mediated via fibroblasts) and indirect peripheral effects (mediated via oxidative stress-activated mononuclear cells) (Ahokas et al. 2005). The MR antagonist eplerenone inhibited atherosclerosis both in monkeys (Takai et al. 2005) and apolipoprotein-E deficient mice (Suzuki et al. 2006) fed a high-cholesterol diet, most likely by attenuating oxidative stress and inflammation.
Cardiac inotropy and coronary flow
In the isolated perfused rat Langendorff heart, aldosterone, like Ang II, rapidly increased left ventricular pressure and decreased coronary flow (Chai et al. 2005a; Moreau et al. 1996). Barbato et al. (2002) observed an increase in cardiac contractility in combination with an increase in coronary flow, but this may relate to the fact that the much larger positive inotropic effects in their study had favoured coronary vasodilation. Spironolactone and eplerenone did not block the inotropic and vasoconstrictor/dilator effects of aldosterone in the rat heart (Barbato et al. 2002; Chai et al. 2005a). In fact, spironolactone exerted similar inotropic effects on top of aldosterone (Barbato et al. 2002; Chai et al. 2005a), and thus it appears that the effects of aldosterone on inotropy and flow occur in a non-MR-mediated manner.
In human myocardial trabeculae, aldosterone induced a negative inotropic response (Fig. 4), in contrast to the well-known positive inotropic effects of Ang II in this preparation (Chai et al. 2005b). The PKC inhibitor chelerythrine chloride, but not spironolactone or eplerenone, blocked this negative inotropic effect, suggesting that it is mediated via a non-MR in a PKC-dependent manner. The aldosterone concentrations required to induce this effect were in the high nanomolar range, i.e. a range that occurred in failing hearts only (Chai et al. 2005b). Hydrocortisone, but not 17β-estradiol, mimicked the effects of aldosterone, although at lower potency (Fig. 4). The contrast between the effects of aldosterone in the rat heart (positive inotropy) and the human heart (negative inotropy) may relate to species differences. However, there are alternative explanations. First, inotropic effects in isolated trabeculae do not necessarily parallel inotropic effects in intact hearts, since the latter also reflect responses on coronary flow (Barbato et al. 2002). Second, similar diametrically differing effects of aldosterone have been observed on flow, either because such effects involve different cells, or because different second messengers are activated depending on the experimental circumstances (Barbato et al. 2004b; Chai et al. 2005a; Mihailidou et al. 2004; Schmidt et al. 2003). Finally, the consequences of PKC-induced regulation of Na+-K+ pump activity are tissue-specific, and range from stimulation to inhibition, as described above (Mihailidou et al. 2004; Therien and Blostein 2000).
Fig. 4Inotropic effects of aldosterone, hydrocortisone and 17β-estradiol in human atrial trabeculae. Left panel original tracing from an experiment with aldosterone (numbers represent -log[aldosterone] in mol/L). Right panel % change from baseline contractile force. Data have been obtained from Chai et al. (2005b)
In human coronary arteries, aldosterone exerted no constrictor or dilator effect by itself. However, prior exposure to 1 μmol/L aldosterone greatly enhanced the constrictor response to Ang II (Chai et al. 2005b). At the second messenger level, this was reflected by an increase in the level of phosphorylated p42/p44 MAP kinase. Hydrocortisone and 17β-estradiol induced similar potentiating effects, but only in the case of aldosterone did these effects occur at the subnanomolar level, i.e., in a physiological range. Future investigations should now address to what degree this potentiation concerns aldosterone-induced endothelial dysfunction (Oberleithner 2005; Oberleithner et al. 2004), and/or an interaction with Ang II at the level of smooth muscle cells, involving some or all of the mediators that have recently been coupled to aldosterone, e.g., the PKC-IP3-DAG pathway, Na+/H+ exchange, Na+/K+-ATPase, p38 MAP kinase, ROS and/or the epidermal growth factor receptor (Jaffe and Mendelsohn 2005; Liu et al. 2003; Mazak et al. 2004). Finally, the possibility of aldosterone-induced, endothelium-dependent, NO-mediated vasodilation, as proposed by several investigators (Liu et al. 2003; Schmidt et al. 2003), needs to be addressed.
Arrhythmias
MR blockade, in addition to standard therapy, reduced sudden death in RALES and EPHESUS (Pitt et al. 1999, 2003). The mechanism responsible for this favorable effect may rely on both renal changes in electrolyte excretion and myocardial fibrosis inhibition. In addition, conditional MR overexpression in the mouse heart, in the absence of aldosteronemia, has been reported to result in severe ventricular arrhythmias (Ouvrard-Pascaud et al. 2005). Apparently, cardiac MR trigger arrhythmias directly, thus providing an additional mechanism through which MR antagonists reduce sudden death in patients. In support of this possibility, spironolactone improved electrophysiological parameters such as QT interval dispersion (Yee et al. 2001), and, in combination with the ACE inhibitor fosinopril, reduced the arrhythmic score post-myocardial infarction (Beck et al. 2001).
Furthermore, both spironolactone and eplerenone improved the condition of the isolated perfused rat Langendorff heart following ischemia and reperfusion, as evidenced by a decrease in infarct size, a decrease in arrhythmia incidence, and an increase in left ventricular pressure recovery (Chai et al. 2005a, 2006) (Fig. 5). Given the virtual lack of aldosterone in the isolated perfused rat heart, it is unlikely that these effects are due to blockade of endogenous aldosterone. In fact, concomitant exposure to 100 nmol/l aldosterone did not further deteriorate the condition of the heart during ischaemia and reperfusion (Chai et al. 2006). A more likely explanation of these findings is therefore that spironolactone and eplerenone had blocked MR activation by endogenous glucocorticoids. Given the 1,000-fold higher levels of corticosterone in the rat heart (Gomez-Sanchez et al. 2004), and assuming that the washout of glucocorticoids resembles that of aldosterone, it can be calculated that, at the time of ischaemia, sufficient glucocorticoid levels are indeed present to allow cardiac MR activation. Such activation might occur particularly under conditions which facilitate ROS generation, such as ischaemia and reperfusion (Funder 2005b; Nagata et al. 2006). Interestingly in this regard, epidemiological observations have recently shown that high-dose corticosteroids increase the risk of developing atrial fibrillation (van der Hooft et al. 2006). The cardioprotective effect of MR antagonism in the Langendorff heart during ischemia and reperfusion cannot be explained on the basis of the vasoconstrictor effect of aldosterone, as proposed by Fujita et al. (2005), since neither spironolactone nor eplerenone are capable of blocking aldosterone-induced vasoconstriction in vitro (Chai et al. 2005a, 2006).
Fig. 5Infarct size (left panel), recovery of left ventricular pressure (LVP) (middle panel), and incidence of arrhythmias (right panel) in rat hearts that were subjected to 45 min left anterior descending coronary artery occlusion, followed by 3 h of reperfusion, after either no pretreatment (C, control) or a 15-min exposure to 100 nmol/l aldosterone (A), 1 μmol/l spironolactone (S), 1 μmol/l eplerenone (E) or 100 nmol/l aldosterone + 1 μmol/L eplerenone (A + E). Data are from Chai et al. (2005a, 2006). *P < 0.05 versus control
Cardiac hypertrophy
Serum aldosterone levels associate with the variability of left ventricular mass (LVM) in both healthy controls and subjects with hypertension (Schunkert et al. 1997). The CYP11B2 C-344T polymorphism associates with circulating aldosterone levels, subjects with the T allele having higher aldosterone levels than those with the CC genotype (Barbato et al. 2004a; Brand et al. 1998; Hautanena et al. 1998; Schunkert et al. 1999; Stella et al. 2004). Given the association between circulating aldosterone and LVM, it is not surprising that the T allele also associates with LVM, both in subjects with hypertension (Stella et al. 2004) and in patients with hypertrophic cardiomyopathy (Chai et al. 2005c). Interestingly, urinary 11β-HSD2 activity correlated directly with LVM in essential hypertension (Glorioso et al. 2005). This suggests that glucocorticoids also take part in the regulation of LVM. Furthermore, two independent investigations have shown that the CYP11B2 C-344T polymorphism is in strong linkage disequilibrium with polymorphisms of the nearby CYP11B1 gene (Ganapathipillai et al. 2005; Keavney et al. 2005). Since CYP11B1 (11β-hydroxylase) is the enzyme catalyzing the final step in the biosynthesis of cortisol, the association with the C-344T polymorphism might not only relate to increased aldosterone levels, but also to reduced 11β-hydroxylase activity (Hilgers and Schmidt 2005). Future studies, involving aldosterone, 11-deoxycortisol and cortisol measurements in serum and/or urine (White and Rainey 2005), should address this possibility.
Interaction with angiotensin II
Ang II stimulates the synthesis and release of aldosterone in the adrenal, and thus MR blockade and/or aldosterone synthase inhibition will exert beneficial effects in Ang II-dependent models (Fiebeler et al. 2005; Virdis et al. 2002). Remarkably, however, aldosterone also appears to exert its effects, at least in part, via Ang II (or its receptors), and both agonists, when applied together, act synergistically (Gonzalez et al. 2005; Mazak et al. 2004; Xiao et al. 2004) (Fig. 1). For instance, in cultured rat aortic smooth muscle cells, nanomolar concentrations of aldosterone enhanced the effect of Ang II on DNA synthesis (Chai et al. 2005a). Unexpectedly, higher (micromolar) aldosterone concentrations reduced DNA synthesis, both in smooth muscle cells and in cardiac myocytes. Such high aldosterone concentrations also reduced collagen synthesis in cardiac fibroblasts. These latter inhibitory effects most likely reflect glucocorticoid receptor activation by aldosterone (Arriza et al. 1987; Sato and Funder 1996). Furthermore, in human coronary artery smooth muscle cells, Ang II stimulated MR-mediated gene expression in an aldosterone-independent manner, suggesting direct MR activation by post-translational modifications such as phosphorylation (Jaffe and Mendelsohn 2005).
Clinical perspective: why are MR antagonists cardioprotective?
The beneficial effects of MR antagonists in heart failure cannot be explained on the basis of their renal and/or blood pressure-lowering effects. Most likely they relate, at least in part, to blockade of a wide range of MR-mediated effects in the heart, including endothelial dysfunction, a decrease in cardiac inotropy and coronary flow, and the induction of fibrosis, hypertrophy, oxidative stress and arrhythmias. Some of these effects occur via or in synergy with Ang II. Given the virtual absence of aldosterone production in the heart, and in view of the low cardiac levels of the cortisol-inactivating enzyme 11β-HSD2, it is unlikely that, under all conditions, aldosterone is the endogenous agonist of cardiac MR. Thus, some of the MR-mediated effects in the heart may be due to MR activation by glucocorticoids, particularly when ROS levels are high. The aldosterone levels in the failing human heart, but not those in the healthy heart, are high enough to be of functional importance. Whether some of the above (acute, ‘non-genomic’) in vitro effects of aldosterone are mediated via a non-MR-mediated mechanism remains to be proven, in particular because no (membrane receptor) candidate has been identified so far that induces these effects. If true, however, aldosterone synthase inhibitors might be expected to exert beneficial effects on top of MR blockade.

mineralocorticoid receptor
aldosterone
inotropy
arrhythmia
non-genomic
cortisol