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 resistance mechanisms in model systems in vivo

Aromatase inhibitors (AIs) have now been shown to be more effective than the anti-estrogen (AE) tamoxifen and have few side effects in ER+ breast cancer patients. However, some patients may not respond and resistance to treatment may develop in others. To investigate the mechanisms involved in the loss of sensitivity of the tumors to AIs, we have studied athymic mice with tumors grown from human estrogen receptor (ER) positive breast cancer cells (MCF-7) stably transfected with aromatase (MCF-7Ca). Treatment with letrozole upregulated Her-2 after four weeks despite continued responsiveness of tumor growth to letrozole. Furthermore, the level of Her-2 protein in letrozole refractory tumors was found to be six fold higher than the control tumors. Cells isolated from these tumors also had increased levels of Her-2 along with lower expression of ERα and aromatase and apparent estradiol independent growth. When Her-2 was inhibited by trastuzumab (antibody against Her-2) ERα levels in the cells were restored indicating that Her-2 is a negative regulator of ERα. This interaction between Her-2 and ER suggests that inhibition of both the Her-2 and estrogen signaling pathways is required to prolong the responsiveness of the tumors to endocrine therapies. Thus, when treatment with trastuzumab and letrozole was combined, ER was restored and tumor growth markedly inhibited compared to treatment with either drug alone. These findings demonstrate that tumor cells under the stress of treatment can adapt and utilize alternate pathways. Thus, when letrozole treatment was stopped, tumor Her-2 levels declined and ER levels were restored to those of hormone sensitive tumors. A second course of letrozole treatment inhibited tumors growth to the same extent and for as long as the initial treatment. These and other strategies to restore aromatase and ERα resulting in sensitivity to hormone therapy could be of substantial benefit to patients who have acquired resistance to AIs.     

Aromatase inhibitors and xenograft studies

Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p < 0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p < 0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer.     

The development, application and limitations of breast cancer cell lines to study tamoxifen and aromatase inhibitor resistance

Estrogen plays important roles in hormone receptor-positive breast cancer. Endocrine therapies, such as the antiestrogen tamoxifen, antagonize the binding of estrogen to estrogen receptor (ER), whereas aromatase inhibitors (AIs) directly inhibit the production of estrogen. Understanding the mechanisms of endocrine resistance and the ways in which we may better treat these types of resistance has been aided by the development of cellular models for resistant breast cancers. In this review, we will discuss what is known thus far regarding both de novo and acquired resistance to tamoxifen or AIs. Our laboratory has generated a collection of AI- and tamoxifen-resistant cell lines in order to comprehensively study the individual types of resistance mechanisms. Through the use of microarray analysis, we have determined that our cell lines resistant to a particular AI (anastrozole, letrozole, or exemestane) or tamoxifen are distinct from each other, indicating that these mechanisms can be quite complex. Furthermore, we will describe two novel de novo AI-resistant cell lines that were generated from our laboratory. Initial characterization of these cells reveals that they are distinct from our acquired AI-resistant cell models. In addition, we will review potential therapies which may be useful for overcoming resistant breast cancers through studies using endocrine resistant cell lines. Finally, we will discuss the benefits and shortcomings of cell models. Together, the information presented in this review will provide us a better understanding of acquired and de novo resistance to tamoxifen and AI therapies, the use of appropriate cell models to better study these types of breast cancer, which are valuable for identifying novel treatments and strategies for overcoming both tamoxifen and AI-resistant breast cancers.     

Heat shock 70 kDa protein 5/glucose-regulated protein 78 “AMP”ing up autophagy

Breast cancer is one of the most prevalent cancers in women, with more than 240,000 new cases reported in the United States in 2011. Classification of breast cancer based upon hormone and growth factor receptor profiling shows that approximately 70% of all breast cancers express estrogen receptor-α. Thus, drugs that either block estrogen biosynthesis (aromatase inhibitors like Letrozole), or compete with estrogen for estrogen receptor (ER) binding (selective ER modulators including tamoxifen; TAM) and/or cause ER degradation (selective estrogen receptor downregulators such as fulvestrant), are among the most prescribed targeted therapeutics for breast cancer. However, overall clinical benefit from the use of these drugs is often limited by resistance; ER⁺ breast cancers either fail to respond to endocrine therapies initially (de novo resistance), or they respond and then lose sensitivity over time (acquired resistance). While several preclinical studies postulate how antiestrogen resistance occurs, for the most part, the molecular mechanism(s) of resistance is unknown.     

Summary of aromatase inhibitor trials: the past and future

Antagonizing estrogen by inhibition of aromatase has become a mainstay of adjuvant endocrine therapy in women with hormone receptor positive (ER+) breast cancer. Recent trials have shown an incremental gain for the AIs over tamoxifen when given as an up-front alternative to tamoxifen, but additionally added benefit is achieved by giving them in sequence with tamoxifen after either an early switch (2–3 years) or as a late switch (5 years). The true clinical implications of accelerated bone resorption from AIs is becoming better understood and its management defined. AI minimally effect quality of life. The chronic relapsing nature of ER+ breast cancer implies long term therapy will be of benefit in selected patients. Outstanding issues under investigation include optimal duration of endocrine therapy, optimal sequence, optimal agents and whether combining anti-estrogens will yield advantage. The role of AIs is also under investigation in premenopausal women in combination with ovarian function suppression. Identifying prognostic and predictive factors of endocrine therapy is important as is the identification and overcoming of resistance mechanisms. Both tumor and host signatures are being pursued to this end. Optimizing, expanding and extending endocrine therapy is likely to add further to patient outcome.     

What do we know about the mechanisms of aromatase inhibitor resistance?

Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. Yet, as with all prolonged drug therapy, resistance to aromatase inhibitors does develop. To date, the precise mechanism responsible for resistance to aromatase inhibitors is not completely understood. In this paper, several mechanisms of de novo/intrinsic resistance and acquired resistance to AIs are discussed. These mechanisms are hypothesized based on important findings from a number of laboratories.

To better understand this question, our lab has generated, in vitro, breast cancer cell lines that are resistant to aromatase inhibitors. Resistant cell lines were generated over a prolonged period of time using the MCF-7aro (aromatase overexpressed) breast cancer line. These cell lines are resistant to the aromatase inhibitors letrozole, anastrozole and exemestane and the anti-estrogen tamoxifen, for comparison. Two types of resistant cell lines have been generated, those that grow in the presence of testosterone (T) which is needed for cell growth, and resistant lines that are cultured in the presence of inhibitor only (no T). In addition to functional characterization of aromatase and ER in these resistant cell lines, microarray analysis has been employed in order to determine differential gene expression within the aromatase inhibitor resistant cell lines versus tamoxifen, in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. We anticipate that our studies will generate important information on the mechanisms of AI resistance. Such information can be valuable for the development of treatment strategies against AI-resistant breast cancers.     

Estradiol receptors in breast cancer cells: Associated co-factors as targets for new therapeutic approaches

Estrogen receptors α (ERα) and β (ERβ) are nuclear receptors which transduce estradiol (E2) response in many tissues including the mammary gland and breast cancers (BC). They activate or inhibit specific genes involved in cell cycle progression and cell survival through multiple enzyme activities leading to malignant transformation. Hormone therapy (antiestrogens (AEs) and aromatase inhibitors (AIs) have been widely used to block the mitogenic action of E2 in patients with ER-positive BC. ERs act in concert with numerous other proteins outside and inside the nucleus where co-activators such as histone modifying enzymes help reaching optimum gene activation. Moreover, E2-mediated gene regulation can occur through ERs located at the plasma membrane or G protein-coupled estrogen receptor (GPER), triggering protein kinase signaling cascades. Classical AEs as well as AIs are inefficient to block the cascades of events emanating from the membrane and from E2 binding to GPER, leading patients to escape anti-hormone treatments and hormone therapy resistance. Many pathways are involved in resistance, mostly resulting from over-expression of growth factor membrane receptors, in particular the HER2/ErbB2 which can be inhibited by specific antibodies or tyrosine kinases inhibitors. Together with the Hsp90 molecular chaperone machinery, a complex interplay between ERs, co-activators, co-repressors and growth factor-activated membrane pathways represents potent targets which warrant to be manipulated alone and in combination to designing novel therapies. The discovery of new potential targets arising from micro array studies gives the opportunity to activate or inhibit different new ER-modulating effectors for innovative therapeutic interventions.     

Steroidal aromatase inhibitors inhibit growth of hormone-dependent breast cancer cells by inducing cell cycle arrest and apoptosis

Different hormonal therapies are used for estrogen receptor positive (ER⁺) breast cancers, being the third-generation of aromatase inhibitors (AIs), an effective alternative to the classical tamoxifen. AIs inhibit the enzyme aromatase, which is responsible for catalyzing the conversion of androgens to estrogens. In this study, it was evaluated the effects of several steroidal AIs, namely 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), on cell proliferation, cell cycle progression and cell death in an ER⁺ aromatase-overexpressing human breast cancer cell line (MCF-7aro). All AIs induced a decrease in cell proliferation and these anti-proliferative effects were due to a disruption in cell cycle progression and cell death, by apoptosis. AIs 1 and 16 caused cell cycle arrest in G₀/G₁, while AIs 12 and 13a induced an arrest in G₂/M. Moreover, it was observed that these AIs induced apoptosis by different pathways, since AIs 1, 12 and 13a activated the apoptotic mitochondrial pathway, while AI 16 induced apoptosis through activation of caspase-8. These results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer cells and will also highlight the importance of AIs as inducers of apoptosis in hormone-dependent breast cancers.     

Potentiation of paclitaxel effect by resveratrol in human breast cancer cells by counteracting the 17β-estradiol/estrogen receptor α/neuroglobin pathway

Neuroglobin (NGB), an antiapoptotic protein upregulated by 17β-estradiol (E2), is part of E2/estrogen receptor α (ERα) pathway pointed to preserve cancer cell survival in presence of microenvironmental stressors including chemotherapeutic drugs. Here, the possibility that resveratrol (Res), an anticancer plant polyphenol, could increase the susceptibility of breast cancer cells to paclitaxel (Pacl) by affecting E2/ERα/NGB pathway has been evaluated. In MCF-7 and T47D (ERα-positive), but not in MDA-MB 231 (ERα-negative) nor in SK-N-BE (ERα and ERβ positive), Res decreases NGB levels interfering with E2/ERα-induced NGB upregulation and with E2-induced ERα and protein kinase B phosphorylation. Although Res treatment does not reduce cell viability by itself, this compound potentiates Pacl proapoptotic effects. Notably, the increase of NGB levels by NGB expression vector transfection prevents Pacl or Res/Pacl effects. Taken together, these findings indicate a new Res-based mechanism that acts on tumor cells impairing the E2/ERα/NGB signaling pathways and increasing cancer cell susceptibility to chemotherapeutic agent.     

The effects of all-trans retinoic acid on estrogen receptor signaling in the estrogen-sensitive MCF/BUS subline

Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17β-estradiol (E₂) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E₂-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E₂-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E₂, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.     

Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer

This article describes the origins and evolution of "antiestrogenic" medicines for the treatment and prevention of breast cancer. Developing drugs that target the estrogen receptor (ER) either directly (tamoxifen) or indirectly (aromatase inhibitors) has improved the prognosis of breast cancer and significantly advanced healthcare. The development of the principles for treatment and the success of the concept, in practice, has become a model for molecular medicine and presaged the current testing of numerous targeted therapies for all forms of cancer. The translational research with tamoxifen to target the ER with the appropriate duration (5 years) of adjuvant therapy has contributed to the falling national death rates from breast cancer. Additionally, exploration of the endocrine pharmacology of tamoxifen and related nonsteroidal antiestrogen (e.g. keoxifene now known as raloxifene) resulted in the laboratory recognition of selective ER modulation and the translation of the concept to use raloxifene for the prevention of osteoporosis and breast cancer. However, the extensive evaluation of tamoxifen treatment revealed small but significant side effects such as endometrial cancer, blood clots and the development of acquired resistance. The solution was to develop drugs that targeted the aromatase enzyme specifically to prevent the conversion of androstenedione to estrone and subsequently estradiol. The successful translational research with the suicide inhibitor 4-hydroxyandrostenedione (known as formestane) pioneered the development of a range of oral aromatase inhibitors that are either suicide inhibitors (exemestane) or competitive inhibitors (letrozole and anastrozole) of the aromatase enzyme. Treatment with aromatase inhibitors is proving effective and is associated with reduction in the incidence of endometrial cancer and blood clots when compared with tamoxifen and there is also limited cross resistance so treatment can be sequential. Current clinical trials are addressing the value of aromatase inhibitors as chemopreventive agents for postmenopausal women.     

FOXA1: a transcription factor with parallel functions in development and cancer

When aberrant, factors critical for organ morphogenesis are also commonly involved in disease progression. FOXA1 (forkhead box A1), also known as HNF3α (hepatocyte nuclear factor 3α), is required for postnatal survival due to its essential role in controlling pancreatic and renal function. In addition to regulating a variety of tissues during embryogenesis and early life, rescue experiments have revealed a specific role for FOXA1 in the postnatal development of the mammary gland and prostate. Activity of the nuclear hormone receptors ERα (oestrogen receptor α) and AR (androgen receptor) is also required for proper development of the mammary gland and prostate respectively. FOXA1 modulates ER and AR function in breast and prostate cancer cells, supporting the postulate that FOXA1 is involved in ER and AR signalling under normal conditions, and that some carcinogenic processes in these tissues stem from hormonally regulated developmental pathways gone awry. In addition to broadly reviewing the function of FOXA1 in various aspects of development and cancer, this review focuses on the interplay of FOXA1/ER and FOXA1/AR, in normal and cancerous mammary and prostate epithelial cells. Given the hormone dependency of both breast and prostate cancer, a thorough understanding of FOXA1's role in both cancer types is critical for battling hormone receptor-positive disease and acquired anti-hormone resistance.