Antioxidant butylated hydroxyanisole inhibits estrogen‐induced breast carcinogenesis in female ACI rats

Exposure to estrogens is suggested to be a risk factor in human breast cancer development. The mechanisms underlying estrogen‐induced cancer have not been fully elucidated. Both estrogen receptor (ER)‐mediated proliferative processes and ER‐independent generation of oxidative stress are suggested to play important roles in estrogen‐induced breast carcinogenesis. In the current study, we investigated the role of oxidative stress in breast carcinogenesis using the ACI rat model of mammary tumorigenesis. Female ACI rats were treated with 17β‐estradiol (E₂), butylated hydroxyanisole (BHA), or a combination of E₂ + BHA for up to 240 days. Cotreatment of rats with E₂ + BHA reduced estrogen‐induced breast tumor development with tumor incidence of 24%, a significant decrease relative to E₂ where tumor incidence was 82%. Proliferative changes in the breast tissue of E₂ + BHA‐treated animals were similar to those observed in E₂‐treated animals. Tissue levels of 8‐isoprostane, a marker of oxidant stress, as well as the activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase were quantified in the breast tissues of rats treated with E₂ + BHA and compared to activity levels found in E₂‐treated animals and respective age‐matched controls. Cotreatment with BHA inhibited E₂‐mediated increases in 8‐isoprostane levels as well as activities of antioxidant enzymes. In summary, these data suggest that estrogen‐mediated oxidant stress plays a critical role in the development of estrogen‐dependent breast cancers and BHA inhibits E₂‐dependent breast carcinogenesis by decreasing oxidant stress. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:202–211, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20281     

Selective estrogen-induced apoptosis in breast cancer

Antihormone therapy remains the gold standard of care in the treatment of estrogen receptor (ER) positive breast cancer. However, development of acquired long term antihormone resistance exposes a vulnerability to estrogen that induces apoptosis. Laboratory and clinical studies indicate that successful therapy with estrogens is dependent on the duration of estrogen withdrawal and menopausal status of a woman. Interrogation of estradiol (E₂) induced apoptosis using molecular studies indicate treatment of long term estrogen deprived MCF-7 breast cancer cells with estrogen causes an endoplasmic reticulum stress response that induces an unfolded protein response signal to inhibit protein translation. E₂ binds to the ER and mediates apoptosis through the classical genomic pathway. Furthermore, the induction of apoptosis by estrogens is dependent on the conformation of the estrogen–ER complex. In this review, we explore the mechanism and the processes involved in the paradox of estrogen induced apoptosis and the new selectivity of estrogen action on different cell populations that is correctly been deciphered for clinical practice.     

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka’s nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E₂/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E₂/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E₂ signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E₂. Because E₂ stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E₂ to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.     

ERα-negative and triple negative breast cancer: Molecular features and potential therapeutic approaches

Triple negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER-2). TNBC patients account for approximately 15% of total breast cancer patients and are more prevalent among young African, African-American and Latino women patients. The currently available ER-targeted and Her-2-based therapies are not effective for treating TNBC. Recent studies have revealed a number of novel features of TNBC. In the present work, we comprehensively addressed these features and discussed potential therapeutic approaches based on these features for TNBC, with particular focus on: 1) the pathological features of TNBC/basal-like breast cancer; 2) E₂/ERβ-mediated signaling pathways; 3) G-protein coupling receptor-30/epithelial growth factor receptor (GPCR-30/EGFR) signaling pathway; 4) interactions of ERβ with breast cancer 1/2 (BRCA1/2); 5) chemokine CXCL8 and related chemokines; 6) altered microRNA signatures and suppression of ERα expression/ERα-signaling by micro-RNAs; 7) altered expression of several pro-oncongenic and tumor suppressor proteins; and 8) genotoxic effects caused by oxidative estrogen metabolites. Gaining better insights into these molecular pathways in TNBC may lead to identification of novel biomarkers and targets for development of diagnostic and therapeutic approaches for prevention and treatment of TNBC.     

Molecular mechanisms underlying the memory-enhancing effects of estradiol

This article is part of a Special Issue “Estradiol and cognition”.Since the publication of the 1998 special issue of Hormones and Behavior on estrogens and cognition, substantial progress has been made towards understanding the molecular mechanisms through which 17β-estradiol (E₂) regulates hippocampal plasticity and memory. Recent research has demonstrated that rapid effects of E₂ on hippocampal cell signaling, epigenetic processes, and local protein synthesis are necessary for E₂ to facilitate the consolidation of object recognition and spatial memories in ovariectomized female rodents. These effects appear to be mediated by non-classical actions of the intracellular estrogen receptors ERα and ERβ, and possibly by membrane-bound ERs such as the G-protein-coupled estrogen receptor (GPER). New findings also suggest a key role of hippocampally-synthesized E₂ in regulating hippocampal memory formation. The present review discusses these findings in detail and suggests avenues for future study.     

Psoralidin,a coumestan analogue,as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERβ agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERβ. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10 μM was able to induce the maximum reporter gene expression corresponding to that of E₂-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin–ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E₂, and thus, it could act as an ER agonist in the cellular environment.     

Environmental contaminants in pathogenesis of breast cancer

This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer.     

Synthesis,antiproliferative and pro-apoptotic activity of 2-phenylindoles

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC₅₀ values of 2.71 μM and 1.86 μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86.     

Breast cancer aromatase expression evaluated by the novel antibody 677: Correlations to intra-tumor estrogen levels and hormone receptor status

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E₂; estrone, E₁; estrone sulfate, E₁S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E₂ levels comparing 677+ versus 677? tumors, although a non-significant trend towards higher tumor E₁ and E₁S levels was observed in 677+ breast cancers. In contrast, tumor levels of E₂ were significantly higher in ER+ tumors compared to ER? tumors (P < 0.001) and to benign breast tissue from the same breast (P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E₂ (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E₁ (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E₁S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677? tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E₂ were also found to be significantly higher among 677+ compared to 677? tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) (P = 0.015).In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.     

Efficacy of Vitamin D compounds to modulate estrogen receptor negative breast cancer growth and invasion

In estrogen receptor (ER) positive breast cancer cells such as MCF-7 cells, the anti-tumor effects of 1,25(OH)(2)D(3) (1,25D(3)) may be secondary to disruption of estrogen mediated survival signals. If so, then sensitivity to 1,25D(3) mediated growth arrest could be reduced in estrogen independent breast cancer cells. The aim of these studies was to determine the effects of 1,25D(3) and EB1089 on the ER negative, invasive human breast cancer cell line SUM-159PT. 1,25D(3) and EB1089 reduced SUM-159PT cell growth subsequent to elevation of p27 and p21 levels. 1,25D(3) mediated apoptosis of SUM-159PT cells was associated with an enrichment of membrane bound bax, a redistribution of cytochome c from the mitochondria to the cytosol and PARP cleavage. 1,25D(3) and EB1089 also inhibited SUM-159PT cell invasion through an 8 microM Matrigel membrane. In pre-clinical studies, EB1089 dramatically reduced the growth of SUM-159PT xenografts in nude mice. The decreased size of tumors from EB1089 treated mice was associated with decreased proliferation and increased DNA fragmentation. Our data support the concept that Vitamin D(3) compounds trigger apoptosis by mechanisms independent of estrogen signaling. These studies indicate that Vitamin D(3) based therapeutics may be beneficial, alone or in conjunction with other agents, for the treatment of estrogen independent breast cancer.     

Importance of PI3-kinase pathway in response/resistance to aromatase inhibitors

Endocrine therapy has been the most effective treatment modality for hormone receptor positive breast cancer. However, its efficacy has been limited by either de novo or acquired resistance. Recent data indicates that activation of the phosphatidylinositol 3-kinase (PI3K) signaling is associated with the poor outcome luminal B subtype of breast cancer and accompanied by the development of endocrine therapy resistance. Importantly, inhibition of PI3K pathway signaling in endocrine resistant breast cancer cell lines reduces cell survival and improves treatment response to endocrine agents. Interestingly, mutations in PIK3CA, the alpha catalytic subunit of the class IA PI3K, which renders cells dependent on PI3K pathway signaling, is the most common genetic abnormality identified in hormone receptor positive breast cancer. The synthetic lethality observed between estrogen deprivation and PI3K pathway inhibition in estrogen receptor positive (ER+) breast cancer cell lines provides further scientific rational to target both estrogen receptor and the PI3K pathway in order to improve the outcome of ER+ breast cancer.     

Estrogen receptor variants and mutations

There is a large and increasing body of experimental and clinical data supporting the existence of variant estrogen receptor (ER) proteins in both normal and neoplastic estrogen target tissues, including human breast. Therefore, future examination of ER signal transduction and/or measurement of ER protein must take into account variant ER expression. The functions of variant ER proteins, either physiological or pathological, remain unclear, although a role(s) for some ER variants in breast tumorigenesis and breast cancer progression would be consistent with the accumulated data. Possible tissue specific expression leads to the speculation that ER variants may have a role in tissue specific estrogen action. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of estrogen receptor variants and mutations that have been identified to occur naturally in tissues and cell lines.     

Apoptotic action of estrogen

Estrogen as a mitogen stimulates cell proliferation and prevents cell death in many cell types. In patients, estrogen is known to stimulate breast and uterus cancer development. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The comprehensive mechanism by which estrogen induces tumor recession in breast cancer is still unknown, but activation of the Fas/FasL pathways plays a key role in this process. Laboratory studies show that the apoptotic action of estrogen is the major factor leading to cell number decreases in several cell types. The effects of estrogen are estrogen-receptor dependent. In this mini review, we will focus on the latest findings regarding estrogen apoptotic effects in several cell models, including breast cancer cells, and summarize the possible mechanisms involved in these estrogen mediated processes. New potential implications for the pharmacological control of breast cancer with estrogen in post-menopausal women are also discussed.