MCF-7aro Cells Research Articles

Cannabidiol as a Promising Adjuvant Therapy for Estrogen Receptor-Positive Breast Tumors: Unveiling Its Benefits with Aromatase Inhibitors.

Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype, with aromatase inhibitors (AIs) being one of the therapeutic drug types used in the clinic. However, endocrine resistance may develop after prolonged treatment, and different approaches, such as combining endocrine and targeted therapies, have been applied. Recently, we demonstrated that cannabidiol (CBD) induces anti-tumor actions in ER+ breast cancer cells by targeting aromatase and ERs. Considering this, we studied, in vitro, whether CBD when combined with AIs could improve their effectiveness. MCF-7aro cells were used and the effects on cell viability and on the modulation of specific targets were investigated. CBD when combined with anastrozole (Ana) and letrozole (Let) caused no beneficial effect in comparison to the isolated AIs. In contrast, when combined with the AI exemestane (Exe), CBD potentiated its pro-cell death effects, abolished its estrogen-like effect, impaired ERα activation, and prevented its oncogenic role on the androgen receptor (AR). Moreover, this combination inhibited ERK1/2 activation, promoting apoptosis. The study of the hormonal microenvironment suggests that this combination should not be applied in early stages of ER+ breast tumors. Contrary to Ana and Let, this study highlights the potential benefits of combining CBD with Exe to improve breast cancer treatment and opens up the possibility of new therapeutic approaches comprising the use of cannabinoids.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERβ and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Mitochondrial stress adaptation promotes resistance to aromatase inhibitor in human breast cancer cells via ROS/calcium up-regulated amphiregulin-estrogen receptor loop signaling.

Many breast cancer patients harbor high estrogen receptor (ER) expression in tumors that can be treated with endocrine therapy, which includes aromatase inhibitors (AI); unfortunately, resistance often occurs. Mitochondrial dysfunction has been thought to contribute to progression and to be related to hormone receptor expression in breast tumors. Mitochondrial alterations in AI-resistant breast cancer have not yet been defined. In this study, we characterized mitochondrial alterations and their roles in AI resistance. MCF-7aro AI-resistant breast cancer cells were shown to have significant changes in mitochondria. Low expressions of mitochondrial genes and proteins could be poor prognostic factors for breast cancer patients. Long-term mitochondrial inhibitor treatments-mediated mitochondrial stress adaptation could induce letrozole resistance. ERα-amphiregulin (AREG) loop signaling was activated and contributed to mitochondrial stress adaptation-mediated letrozole resistance. The up-regulation of AREG-epidermal growth factor receptor (EGFR) crosstalk activated the PI3K/Akt/mTOR and ERK pathways and was responsible for ERα activation. Moreover, mitochondrial stress adaptation-increased intracellular levels of reactive oxygen species (ROS) and calcium were shown to induce AREG expression and secretion. In conclusion, our results support the claim that mitochondrial stress adaptation contributes to AI resistance via ROS/calcium-mediated AREG-ERα loop signaling and provide possible treatment targets for overcoming AI resistance.

The anti-cancer potential of crotoxin in estrogen receptor-positive breast cancer: Its effects and mechanism of action

Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype of breast cancer. Currently, aromatase inhibitors (AIs) are used as first-line treatment option in this type of tumors, however they cause several side effects, which is why new therapeutic approaches are demanding. The South American rattlesnake Crotalus durissus terrificus produces a venom enriched in several bioactive substances, like phospholipases A2 (PLA2). One of those is crotoxin, a β-neurotoxin, that has already been reported for its anti-cancer properties in different cancers. Recently, its clinical interest has emerged and, in fact, a clinical trial in patients with advanced cancer is underway. Considering this, in this work, we studied the biological mechanisms behind the anti-cancer effects of crotoxin B (CTX) in an ER+ aromatase-overexpressing breast cancer cell line (MCF-7aro cells). Results revealed that CTX impairs MCF-7aro cells growth, through a cell cycle arrest at G2/M phase, inhibition of ERK1/2 pathway and by apoptosis through activation of caspase-8. In addition, it can be considered a safe natural compound as did not affect non-cancerous cells and only showed anti-growth effects in breast cancer cells. Therefore, this study represents an important landmark to better understand the effects and mechanisms of action of crotoxin in ER+ breast cancer.

Unveiling the mechanism of action behind the anti-cancer properties of cannabinoids in ER+ breast cancer cells: Impact on aromatase and steroid receptors

Breast cancer is the leading cause of cancer-related death in women worldwide. In the last years, cannabinoids have gained attention in the clinical setting and clinical trials with cannabinoid-based preparations are underway. However, contradictory anti-tumour properties have also been reported. Thus, the elucidation of the molecular mechanisms behind their anti-tumour efficacy is crucial to better understand its therapeutic potential. Considering this, our work aims to clarify the molecular mechanisms underlying the anti-cancer properties of the endocannabinoid anandamide (AEA) and of the phytocannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), in estrogen receptor-positive (ER+) breast cancer cells that overexpress aromatase (MCF-7aro). Their in vitro effects on cell proliferation, cell death and activity/expression of aromatase, ERα, ERβ and AR were investigated. Our results demonstrated that cannabinoids disrupted MCF-7aro cell cycle progression. Unlike AEA and THC that induced apoptosis, CBD triggered autophagy to promote apoptotic cell death. Interestingly, all cannabinoids reduced aromatase and ERα expression levels in cells. On the other hand, AEA and CBD not only exhibited high anti-aromatase activity but also induced up-regulation of ERβ. Therefore, all cannabinoids, albeit by different actions, target aromatase and ERs, impairing, in that way, the growth of ER+ breast cancer cells, which is dependent on estrogen signalling. As aromatase and ERs are key targets for ER+ breast cancer treatment, cannabinoids can be considered as potential and attractive therapeutic compounds for this type of cancer, being CBD the most promising one. Thus, from an in vitro perspective, this work may contribute to the growing mass of evidence of cannabinoids and cannabinoids-based medicines as potential anti-cancer drugs.

Discovery of a multi-target compound for estrogen receptor-positive (ER+) breast cancer: Involvement of aromatase and ERs

Despite intense research, breast cancer remains the leading cause of cancer-related death in women worldwide, being estrogen receptor-positive (ER+) the most common subtype. Nowadays, aromatase inhibitors (AIs), the selective estrogen receptor modulator (SERM) tamoxifen and the selective estrogen receptor down-regulator (SERD) fulvestrant are used as therapeutic options for ER+ breast cancer, since they interfere directly with the production of estrogens and with the activation of estrogen-dependent signaling pathways. Despite the success of these treatments, the occurrence of resistance limits their clinical efficacy, demanding the development of novel therapies. Recently, multi-target compounds emerged as promising therapeutic strategies for ER+ breast cancer, as they can potentially modulate several important targets simultaneously. In line with this, in this work, the anti-cancer properties and multi-target action of 1,1-Bis(4-hydroxyphenyl)-2-phenylbut-1-ene, tamoxifen bisphenol (1,1-BHPE), were evaluated in an ER+ breast cancer cell model (MCF-7aro cells). Molecular docking analysis predicted that 1,1-BHPE was able to bind to aromatase, ERα and ERβ. In vitro studies showed that, although it did not present anti-aromatase activity, 1,1-BHPE reduced aromatase protein levels and interfered with ERα and ERβ signaling pathways, acting as an ERα antagonist and inducing ERβ up-regulation. Through these mechanisms, 1,1-BHPE was able to impair breast cancer growth and induce apoptosis. This represents an important therapeutic advantage because the main players responsible for estrogen production and signaling are modulated by a single compound. To the best of our knowledge, this is the first study describing the anti-cancer properties of 1,1-BHPE as a multi-target compound specific for ER+ breast cancer.

Abstract P6-04-19: Estrogen receptor β agonist S-Equol promotes letrozole sensitivity of endocrine therapy resistant breast cancer cells by upregulating FOXO3 expression

Abstract Background: Breast cancer (BC) is the most frequently diagnosed cancer and the primary leading cause of cancer deaths in women worldwide. Although the clinical outcome of BC patients has been considerably improved, resistance to endocrine and chemotherapy treatments contributes to BC relapse and mortality. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Recent studies suggested that estrogen receptor beta (ERβ) and its ligand S-Equol promote suppression of BC progression. However, the mechanisms by which S-equol inhibits growth of BC cells and enhance therapy response remains unknown. Here, we examined the mechanisms by which S-equol contribute to BC growth inhibition in hormone therapy sensitive and resistant BC cells. Methods: We evaluated the therapeutic efficacy of S-equol in therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells. Expression levels of target genes were evaluated in both cell lines by global RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses, qRT-PCR and Western blot. ERβ knock down cell lines were generated by CRISPR-Cas9 technology. Xenograft and patient derived BC explants (PDEX) were used for preclinical evaluation of the antiproliferative activity of S-equol. Results: S-equol reduced the growth of endocrine sensitive and resistant BC models in vitro and in vivo. Treatment of ER-positive PDEX with S-equol decreased the proliferation compared to untreated tumors. RNA-seq analysis of S-equol treated BC cells showed modulation of ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis showed that S-equol treatment upregulated 438 proteins and downregulated 429 proteins in MCF7-Aro cells compared to controls. Whereas, DIA analysis in Letrozole-resistant cells revealed that S-equol upregulated 572 proteins and downregulated 481 proteins compared to controls. Reactome pathway analysis revealed that S-equol treatment modulated the proteins involved in DNA replication, cell cycle, proteins regulating p53 activity and VEGF-VEGFR2 pathway. Our mechanistic studies confirmed that treatment with S-equol upregulated FOXO3 and downregulated FOXM1. Using CRISPR-Cas9 mediated ERβ knockout cells, we confirmed that S-equol could not modulate forkhead box transcription factors in BC cells lacking ERβ compared to parental cells. Conclusions: Collectively, our results suggest that S-equol exhibits its antitumor activity by targeting forkhead box transcription factors and that S-equol might be an effective treatment strategy in treating / preventing breast cancer recurrence. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294). Citation Format: Kumaraguruparan Ramasamy, Shaorong Chen, Suryavathi Viswanadhapalli, Rong Li, Ratna K Vadlamudi, Rajeshwar R Tekmal. Estrogen receptor β agonist S-Equol promotes letrozole sensitivity of endocrine therapy resistant breast cancer cells by upregulating FOXO3 expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-19.

Effects of new C6-substituted steroidal aromatase inhibitors in hormone-sensitive breast cancer cells: Cell death mechanisms and modulation of estrogen and androgen receptors

Estrogen receptor-positive (ER+) breast cancers require estrogens for their growth. Aromatase inhibitors (AIs) are considered the first-line therapy for this type of tumours. Despite the well-established clinical benefit of this therapy, the search for novel potent AIs that present higher efficacy and fewer side effects is still demanded. Thus, taking into account the known interactions of the natural substrate, androstenedione, within the aromatase active-site, a range of new steroidal compounds have been designed, synthesized and studied by our group. In this work, it was evaluated in MCF-7aro, an ER+ breast cancer cell line that overexpress aromatase, the anti-aromatase efficacy and the biological effects of eight new AIs: 6α-methyl-5α-androst-3-en-17-one (1a), 6α-methyl-3α,4α-epoxy-5α-androstan-17-one (3a), 6α-methylandrost-4-ene-3,17-dione (9), 6α-allylandrosta-1,4-diene-3,17-dione (13), 6α-allylandrost-4-ene-3,17-dione (15), 6α-allylandrost-4-en-17-one (17), 6β-hydroxyandrost-4-ene-3,17-dione (19) and 6α-hydroxyandrost-4-ene-3,17-dione (20). Their anti-cancer properties were elucidated, as well as, the dependence of their mechanism of action on aromatase inhibition and/or on steroid receptors modulation, such as estrogen and androgen receptors, which are key targets for this type of cancer. Results demonstrate that the studied AIs present high anti-aromatase activity, disrupt MCF-7aro cell cycle progression and induce apoptosis, through the mitochondrial pathway. Compounds 1a, 3a, 9, 13, 15 and 17 exhibited an aromatase-dependent effect on cells and, interestingly, steroids 9 and 13 displayed the ability to decrease aromatase protein levels without affecting CYP19A1 mRNA levels. Furthermore, the effects of compounds 1a, 3a and 15 were dependent on ER and on AR modulation, whereas compounds 9 and 19 were only dependent on AR modulation. From a clinical point of view, these actions can be considered as a therapeutic advantage for this type of tumours. Thus, new promising AIs that impair ER+ breast cancer cell growth, by acting on aromatase, and even, on ER and AR were discovered. Furthermore, new insights on the most favourable structural modifications in the steroidal core structure were provided, helping to a more rational drug design of new and potent AIs.

Abstract 1008: S-Equol inhibits breast cancer growth by regulating phosphorylation status of estrogen receptor β

Abstract Background: Breast cancer is the most common malignancy in females and second most common cause of cancer related mortality in women. Since 70% of all breast cancers are estrogen receptor-positive (ER+ve), endocrine therapy such as anti-estrogens or aromatase inhibitors, targeting the estrogen receptor (ER) pathway is the most common treatment used for ER+ve breast cancers. However, patients will develop de novo or acquired resistance to therapy, leads to tumor progression, and metastasis. It is well documented that ERβ functions as tumor suppressor in different cancers including breast cancer. We recently showed that phosphorylation status of ERβ is important for its antitumor activity. However, little is known about the role of ERβ phosphorylation status in hormone therapy and resistance; therefore, we investigated whether phosphorylation status of ERβ has role in overcoming hormone therapy resistance in ER+ve breast cancers. Experimental design: To elucidate the importance of phosphorylation status of ERβ, we used CRISPR-Cas9 system to knockout ERβ in MCF7-Aro (therapy-sensitive) and Letrozole resistant (MCF7aro-LTLT) cells. Several mutant clones were identified for both MCF7-Aro cells and LTLT cells and the depletion of ERβ protein in both cell clones was confirmed by immunoblotting. The parental and knockout cells with or without treatment of S-equol were analyzed for cell proliferation, protein (Western) and RNA (RT-qPCR) analysis. Results: First we analyzed the cell proliferation in parental (MCF7 Aro and LTLT) and ERβ knock out cells. The proliferation rate is increased in the ERβ knockout cells compared to the parental cells. Treatment with ERβ agonist S-Equol to the parental cells inhibited the cell proliferation whereas in the knock out cells, the effect of S-equol is compromised. RNA-seq analysis of S-equol treated parental cells showed the downregulation of ERβ target genes involved in tumor progression and resistance to hormone therapies. In contrast, compared to parental cells, ERβ knock out cells showed diverse effects to S-equol treatment. RT-qPCR analysis revealed that S-Equol could not modulate the ERβ-target genes in ERβ knock out cells compared to parental cells. Conclusions: Our findings provide evidence that phosphorylation status of ERβ is important for elucidating its antitumor activity in therapy-resistant cells. The differential effects of S-equol on parental and ERβ knockout cells suggest that the antiproliferative action of S-equol is partly mediated by ERβ. We believe that our ongoing studies may further validate the role of phosphorylation status of ERβ by using both ERβ agonists and phosphorylation-regulating compounds in both therapy sensitive and resistant cells. Citation Format: Kumaraguruparan Ramasamy, Cathy Samayoa, Shaorong Chen, Rong Li, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. S-Equol inhibits breast cancer growth by regulating phosphorylation status of estrogen receptor β [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1008.

Abstract P5-04-16: Total estrogenic activity during neoadjuvant therapy with letrozole and exemestane – An intra-patient cross-over comparison using the AroER tri-screen

Abstract Background. Aromatase inhibitors (AIs), letrozole (Femarâ / Femaraâ) and exemestane (Aromasinâ), are widely used anti-hormonal drugs for breast cancer. Both compounds strongly reduce circulating estradiol levels in postmenopausal women. In the setting of metastatic breast cancer, these drugs may be used after another, causing new responses in selected patients by switching to the alternative drug after progressing on the first choice. This well-known ”lack of cross resistance” has been recognized for some time and is documented by several clinical trials. However, the precise explanation for this clinical observation is still unknown. The availability of mechanistic information may lead to an improved strategy against hormone-sensitive breast cancer. Patients and methods. NEO-LET-EXE was a neoadjuvant, randomized, open-label, intra-patient cross-over trial. Postmenopausal patients with estrogen receptor (ER) positive (>50%), HER-2 negative, locally advanced breast cancer were enrolled. Sequential blood samples (obtained at baseline, after 2 months and 4 months of treatment) were available from 29 patients. All patients were randomized to sequential treatment starting with either letrozole (2.5 mg o.d.) or exemestane (25 mg o.d) for 2 months followed by another 2 months on the alternative compound. The total estrogenic activities in the collected blood samples were determined using AroER tri-screen assay developed by the Chen laboratory. The assay utilizes MCF-7aro ERE cells which contain both aromatase and ER. The samples were assayed in the presence as well as the absence of letrozole, to estimate relative contributions of estrogen and estrogen+androgen to the activities, respectively. Results. Using the highly sensitive AroER tri-screen assay, estrogenic activity were detected at three time points in all blood samples. Importantly, a significantly higher total estrogenic activity was found during therapy with exemestane compared to letrozole in 23 out of 26 patients. Only in three patients, the activity was higher during therapy with letrozole compared to exemestane. When letrozole was included in the assay, the estrogenic activities in most samples collected during exemestane treatment were further reduced, suggesting that low levels of androgen were present in samples from exemestane treatment. Four samples collected after exemestane treatment and three after letrozole treatment had higher activities than baseline samples when assay was carried out with letrozole. Discussion. Our results suggest the AroER tri-screen to be a very sensitive method to estimate the overall estrogen-mediated activity in human samples. Significant higher levels of estrogenic activity in human serum were observed during exemestane than those during letrozole treatment. Our observations, that additional letrozole could reduce further the estrogen activity in the exemestane-treated samples, demonstrate probably residual aromatase activity during therapy with exemestane alone.In addition to distinguish the effects of exemestane and letrozole, our results also demonstrate that the assay can also potentially detect the effects of estrogenic mimics. Citation Format: Bahrami N, Chang G, Kanaya N, Sauer T, Gravdehaug B, Chen S, Geisler J. Total estrogenic activity during neoadjuvant therapy with letrozole and exemestane – An intra-patient cross-over comparison using the AroER tri-screen [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-16.

Abstract P1-09-09: Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance

Abstract Estrogen plays an important role in the initiation and progression of breast cancer (BCa). Approximately, 70% of breast tumors are estrogen receptor (ER) positive at the time of presentation. Endocrine therapy using aromatase inhibitors (AI), or anti-estrogen (AE) molecules are widely used for treating ER+ve BCa. However, their efficacy is limited by intrinsic and acquired therapy resistance and most patients develop resistance to these drugs. The transcriptional effects of estrogen are mediated by two ERs (ERα and ERβ) and both are expressed in normal breast tissue. Unlike ERα, ERβ functions as tumor suppressor. However, role of ERβ specific agonists in the prevention of BCa progression remains elusive. In this study, we investigated the effectiveness of two ERβ agonists (S-Equol and LY500307) in the prevention of BCa progression using endocrine therapy sensitive (MCF7-aro) and letrozole resistant (MCF7-aro-LTLT) cells. Our results demonstrated that treatment with ERβ agonists inhibit short-and long-term growth of both endocrine therapy sensitive and resistant BCa cells. In addition, ERβ agonists treatment inhibited invasion and migration of both MCF7-aro and MCF7-aro-LTLT cells. Importantly, cell cycle analysis revealed that ERβ agonists induced cell cycle arrest. Our gene microarray analysis demonstrated that both ERβ agonists significantly modulated genes involved in the cell cycle progression, DNA replication and cell death pathways. Further, gene enrichment analysis of differentially expressed genes revealed that genes involved in the cell cycle checkpoints emerged as significant pathway modulated by ERβ agonists treatment in MCF7-aro cells. Interestingly, in letrozole-resistant MCF7-aro cells, DNA replication was significantly affected by ERβ agonists treatment. Pathway analysis also identified enrichment for chemokine signaling pathways. We confirmed pathway analysis by qRT-PCR and western blot analysis. Accordingly, treatment of in vivo syngeneic xenografts with ERβ agonists significantly inhibited BCa progression. Collectively, these results from this study suggest that ERβ agonists have potential to prevent the progression of BCa progression. Citation Format: Ramasamy K, Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-09.

Abstract 3457: Evaluating estrogen receptor β agonists for the treatment of breast cancer

Abstract Breast cancer is the main cause of cancer-associated mortality in women worldwide. The estrogen receptors play a critical role in normal mammary gland development as well as breast cancer. Over two thirds of all breast cancers express high levels of Estrogen Receptor α (ERα) and are depend on estrogen for growth. Current endocrine therapies aim to decrease estrogen levels with the use aromatase inhibitors or to inhibit ERα signaling with the use of anti-estrogens. Although these treatment options are initially effective, a significant number of patients will present with recurrent disease. Therefore, there is a critical need to identify targeted therapies which will inhibit tumor progression and circumvent therapy resistance. In addition to focusing on ERα, emerging evidence has shed light on the importance of Estrogen Receptor β in breast cancer. Unlike ERα, ERβ has tumor suppressing activity, therefore targeting ERβ with selective agonists may provide therapeutic benefit in the treatment of breast cancer. The objective of this study was to investigate the effects of selective ERβ agonists in distinct breast cancer models. Liquiritigenin and S-Equol are selective plant-derived ERβ agonists currently in clinical trials for the relief of symptoms associated with menopause, and for the treatment of benign prostate hyperplasia. Additionally, LY500307, a synthetic ERβ agonist is currently in clinical trials and has been shown to be well tolerated. To test the effects of ERβ agonists on growth, we carried out cell growth assays using various cell lines. MCF7aro and ZR-75aro cells were used as models of post-menopausal breast cancer given their ability to synthesize their own estrogens. A letrozole resistant cell line was used to determine if ERβ agonists could inhibit the growth of therapy resistant cells. Additionally, we used a syngeneic tumor mouse model to investigate if ERβ agonist could inhibit tumor growth in a model with an intact immune system. Our results demonstrate that both, plant-derived and synthetic, ERβ agonists can inhibit the growth of breast cancer cells, including those that over express aromatase, and are resistant to letrozole. The synthetic ERβ agonist was able to inhibit growth at much lower doses, overcoming a critical barrier presented by the plant derived agonists. Our mechanistic studies show that ERβ agonists can alter ERα and ERβ levels, which shift the α/β ratio to a more tumor suppressive state. We also found that these compounds affect cell cycle through cyclin D1 and p27. Additionally, we also saw induction of apoptosis and increased p53 levels. Our studies evaluated the effects of ERβ agonists in various breast cancer cell lines, and our results suggest that targeting ERβ may be a valuable therapeutic option for the treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen Krishnegowda, Ratna Vadlamudi, Rajeshwar Tekmal. Evaluating estrogen receptor β agonists for the treatment of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2015-3457

MicroRNA-125b upregulation confers aromatase inhibitor resistance and is a novel marker of poor prognosis in breast cancer.

IntroductionIncreasing evidence indicates that microRNAs (miRNAs) are important players in oncogenesis. Considering the widespread use of aromatase inhibitors (AIs) in endocrine therapy as a first-line treatment for postmenopausal estrogen receptor α–positive breast cancer patients, identifying deregulated expression levels of miRNAs in association with AI resistance is of utmost importance.MethodsTo gain further insight into the molecular mechanisms underlying the AI resistance, we performed miRNA microarray experiments using a new model of acquired resistance to letrozole (Res-Let cells), obtained by long-term exposure of aromatase-overexpressing MCF-7 cells (MCF-7aro cells) to letrozole, and a model of acquired anastrozole resistance (Res-Ana cells). Three miRNAs (miR-125b, miR-205 and miR-424) similarly deregulated in both AI-resistant cell lines were then investigated in terms of their functional role in AI resistance development and breast cancer cell aggressiveness and their clinical relevance using a cohort of 65 primary breast tumor samples.ResultsWe identified the deregulated expression of 33 miRNAs in Res-Let cells and of 18 miRNAs in Res-Ana cells compared with the sensitive MCF-7aro cell line. The top-ranked Kyoto Encyclopedia of Genes and Genomes pathways delineated by both miRNA signatures converged on the AKT/mTOR pathway, which was found to be constitutively activated in both AI-resistant cell lines. We report for the first time, to our knowledge, that ectopic overexpression of either miR-125b or miR-205, or the silencing of miR-424 expression, in the sensitive MCF-7aro cell line was sufficient to confer resistance to letrozole and anastrozole, to target and activate the AKT/mTOR pathway and to increase the formation capacity of stem-like and tumor-initiating cells possessing self-renewing properties. Increasing miR-125b expression levels was also sufficient to confer estrogen-independent growth properties to the sensitive MCF-7aro cell line. We also found that elevated miR-125b expression levels were a novel marker for poor prognosis in breast cancer and that targeting miR-125b in Res-Let cells overcame letrozole resistance.ConclusionThis study highlights that acquisition of specific deregulated miRNAs is a newly discovered alternative mechanism developed by AI-resistant breast cancer cells to achieve constitutive activation of the AKT/mTOR pathway and to develop AI resistance. It also highlights that miR-125b is a new biomarker of poor prognosis and a candidate therapeutic target in AI-resistant breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0515-1) contains supplementary material, which is available to authorized users.

Exemestane metabolites: Synthesis, stereochemical elucidation, biochemical activity and anti-proliferative effects in a hormone-dependent breast cancer cell line

Exemestane is a third-generation steroidal aromatase inhibitor that has been used in clinic for hormone-dependent breast cancer treatment in post-menopausal women. It is known that exemestane undergoes a complex metabolization, giving rise to some already identified metabolites, the 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-βHE) and the 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione (6-HME). In this study, four metabolites of exemestane have been analyzed, three of them were synthesized (6β-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-βHE (4)) while one was acquired, the 6-HME (6). The stereochemistry of the epoxide group of 2 and 3 has been unequivocally elucidated for the first time on the basis of NOESY experiments. New structure–activity relationships (SAR) have been established through the observation that the substitution of the double bonds by epoxide groups led to less potent derivatives in microsomes. However, the reduction of the C-17 carbonyl group to a hydroxyl group originating 17-βHE (4) resulted in a significant increase in activity in MCF-7aro cells when compared to exemestane (IC50 0.25 μM vs 0.90 μM, respectively). All the studied metabolites reduced MCF-7aro cells viability in a dose and time-dependent manner, and metabolite 3 was the most potent one. Altogether our results showed that not only exemestane but also its main metabolites are potent aromatase inhibitors and reduce breast cancer cells viability. This suggests that exemestane efficacy may also be due to the active metabolites that result from its metabolic transformation. Our results emphasize the importance of performing further studies to expand our understanding of exemestane actions in breast cancer cells.

Abstract 1602: Effects of Letrozole and radiotherapy on cell cycle and death in a breast cancer cell line.

Abstract Preclinical and clinical studies have shown that hormone receptor-positive tumors have molecular, biological and clinical differences. Tamoxifen is the standard endocrine therapy in premenopausal women, whereas in ER (+), postmenopausal patients the optimal adjuvant hormonal therapy is aromatase inhibitors either as initial therapy or after tamoxifen treatment. Studies have shown that concurrent use of tamoxifen sensitizes cells to radiotherapy (RT) and increases RT induced pulmonary fibrosis. We aimed to investigate biological effects of the concurrent use of letrozole with radiotherapy on the breast cancer cells; using cell lines MCF7, which does not express the aromatase enzyme; and MCF7aro which is a aromatase expressing subclone. Cells were exposed to letrozole at varying doses of 100, 500, 1000 nM; for 24, 72 and 144 hours. Irradiations were performed using a cobalt-60 source with doses 2 and 4 Gy. Cell death was evaluated by measuring caspase 3 activation and by cell surface annexin V / PI staining. Beclin expression levels were determined by western blot. Cell cycle analyses were performed by DNA content staining/flow cytometry. All experiments were done in triplicates. Caspase-3 and annexin-V / PI results showed no significant difference for early apoptosis, late apoptosis and necrosis between the letrozole treated and untreated aromatase non-expressing MCF7 cells. In the aromatase expressing MCF7aro cells, we observed that there was a general reduction in cell death in cells treated with letrozole; with a trend towards apoptosis as a cell death modality rather than necrosis. We also observed increased beclin protein levels -which may imply an increase in autophagy- in MCF7 cells which have received 4 Gy irradiation, independent of the presence of letrozole indicating an acute stress response when expose to high doses of radiation. On the other hand beclin levels were increased in all experimental groups of MCF-7aro cells independent of the presence letrozole and/or radiation dose which supports the notion of an autophagic response under stress. The presence of the aromatase gene itself may be a positive factor for cell survival under stress conditions. Cell cycle analyses revealed 5% more cells accumulated at G1 in letrozole treated MCF7aro cells. Our preliminary findings suggest a reduction of cell death in hormone-sensitive, receptor-positive and aromatase enzyme expressing cells, after concurrent use of letrozole and RT; with apoptosis being the primary cell death modality. This observation correlates with our findings that autophagy may have also increased (based on beclin protein levels) in these cells. It is still not clear whether letrozole treated aromatase expressing cells have a survival advantage when treated concurrently with letrozole and RT. More extensive studies are needed to be able to evaluate the effects of the concurrent use of letrozole and radiotherapy on tumor cell death. Citation Format: Burcu Durmak Isman, Dilek Gokturk, Zeynep Sercan, Halil Ates, Zafer Karaguler, Ilknur Bilkay Gorken. Effects of Letrozole and radiotherapy on cell cycle and death in a breast cancer cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2013-1602

Abstract 546: GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis.

Abstract Endocrine therapies such as letrozole or fulvestrant are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but patients ultimately relapse. Phosphatidylinositol 3-kinases (PI3K) regulate breast tumor cell growth, migration and survival. The alpha isoform of PI3K is frequently mutated and activated in HR+ breast cancer and has been implicated in resistance to endocrine therapies. PI3K inhibitors are therefore attractive for combination with endocrine therapies. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that GDC-0032 has increased activity against PI3K alpha isoform (PIK3CA) mutant and HER2-amplified cancer cell lines. Single agent and combination studies were carried out to determine if GDC-0032 enhances the anti-tumor activity of endocrine therapies in human breast cancer models. MCF7 cells (Estrogen Receptor positive (ER+), PI3K alpha E545K mutant) were transfected with the aromatase gene and stable clones (MCF7-ARO) were selected that are capable of converting androstenedione to estrogen in culture. When grown in the presence of androstenedione, MCF7-ARO cells were more reliant on estrogen for growth. Under these conditions the cells were treated with GDC-0032 in combination with endocrine therapies and assayed for cellular viability, modulation of PI3K pathway and ER pathway markers and apoptosis induction. The combination of GDC-0032 and endocrine therapies decreased the cellular viability of MCF7-ARO cells and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggests a mechanism of action for the combination. The data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for HR+ breast cancer treatment in the clinic. Citation Format: Jeffrey J. Wallin, Jane Guan, Rebecca Hong, Carol O'Brien, Timothy Wilson, Michelle Nannini, Deepak Sampath, Marcia Belvin, Lori S. Friedman. GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 546. doi:10.1158/1538-7445.AM2013-546

Abstract 1312: The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways.

Abstract Estrogen and estrogen receptors (ERα and ERβ) play an important role in breast cancer. Currently, antiestrogen (AE) targeted to ERα and aromatase inhibitors (AI), which block local and systemic estrogen production, are used in the treatment of breast cancer. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, in women with advanced breast cancer, essentially all breast cancers develop resistance to these two classes of agents. Moreover, the side effects associated with currently used AI limit the long-term utility of them as chemopreventative agents. Therefore there is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (LIQ), from the plant Glycyrrhizae uralensis and an active component of MF101, and S-equol, from the soy isoflavone daidzein, are currently being tested to treat vasomotor hot flashes associated with menopause. In addition, S-equol is also being tested as a treatment for BPH in men. However, the exact mechanism of action of these compounds is not clear. In this study we sought to determine the mechanism by which ERβ acts as a tumor suppressor, and whether these ERβ agonists could resensitize Letrozole-resistant breast cancer cells. MCF7aro and LTLT-ca cells were used as models of hormone-responsive and hormone-resistant breast cancer respectively. MTT assay was used to determine the half maximal inhibitory concentrations of the ERβ agonists. Upon treatment, our results show changes in the levels of p53, NFkB, cyclin D1, KLF5 and others using qRT-PCR and western blot analysis. The effects of ERβ agonists on the activation of these genes were confirmed using specific gene promoter-luciferase reporter gene constructs. Specificity of ERβ-mediated actions was confirmed using a siRNA approach in addition to other methods and knockdown efficiency was confirmed through RT-PCR. Interestingly, our results show that the plant-derived ERβ-agonists, LIQ and S-equol, not only affect the growth of breast cancer cells and tumors but treatment increases the expression of p53 and others, and upon ERβ knockdown this effect is ablated. This study suggests that ERβ acts as a tumor suppressor by regulating the expression of p53 and others involved in cell proliferation. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers, as well as provides rationale for new preventive approaches to decrease the incidence of breast cancer. Citation Format: Cathy Samayoa, Anil Kotha, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar Rao Tekmal. The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2013-1312

Effects of steroidal aromatase inhibitors on sensitive and resistant breast cancer cells: Aromatase inhibition and autophagy

Several therapeutic approaches are used in estrogen receptor positive (ER+) breast cancers, being one of them the use of aromatase inhibitors (AIs). Although AIs demonstrate higher efficacy than tamoxifen, they can also exhibit de novo or acquired resistance after prolonged treatment. Recently, we have described the synthesis and biochemical evaluation of four steroidal AIs, 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), obtained from modifications in the A-ring of the aromatase substrate, androstenedione. In this study, it was investigated the biological effects of these AIs in different breast cancer cell lines, an ER+ aromatase-overexpressing human breast cancer cell line (MCF-7aro cells), an estrogen-receptor negative (ER−) human breast cancer cell line (SK-BR-3 cells), and a late stage of acquired resistance cell line (LTEDaro cells). The effects of an autophagic inhibitor (3-methyladenine) plus AIs 1, 12, 13a or exemestane in LTEDaro cells were also studied to understand the involvement of autophagy in AI acquired resistance. Our results showed that these steroids inhibit aromatase of MCF-7aro cells and decrease cell viability in a dose- and time-dependent manner. The new AI 1 is the most potent inhibitor, although the AI 12 demonstrates to be the most effective in decreasing cell viability. Besides, and in advantage over exemestane, AIs 12 and 13a also reduced LTEDaro cells viability. The use of the autophagic inhibitor allowed AIs to diminish viability of LTEDaro cells, presenting a similar behavior to the sensitive cells. Thus, inhibition of autophagy may sensitize hormone-resistant cancer cells to anti-estrogen therapies.

The aromatase inhibitor letrozole enhances the effect of doxorubicin and docetaxel in an MCF7 cell line model

<p>Introduction: Post-menopausal women with estrogen receptor (ER) positive breast cancer receive adjuvant chemotherapy and endocrine therapy sequentially since tamoxifen may antagonise the cytotoxicity of chemotherapy drugs. With increased use of aromatase inhibitors (AIs) in place of tamoxifen, the potential use of concomitant chemo-endocrine treatments with the AI letrozole, before clinical trials are undertaken, requires evaluation. </p> <p>Methods: MCF7-aro cells expressing the aromatase gene were treated with letrozole, doxorubicin and docetaxel. The effects of different drug concentrations, drug combinations and scheduling on cytotoxicity and aromatase activity were investigated. Key receptor, cell cycle regulation and apoptosis proteins were examined by immunoblotting.</p> <p>Results: Administration of letrozole with either doxorubicin or docetaxel resulted in increased levels of cytotoxicity under all treatment schedules (add in, sequential or simultaneous drug administration) with the greatest anti-proliferative effect observed using concomitant treatment (letrozole first with chemotherapy added in). The inhibitory effect of letrozole on aromatase activity was unchanged by the addition of doxorubicin or docetaxel. Letrozole treatment resulted in decreased HER2 expression and addition of doxorubicin and docetaxel to letrozole led to elevated ER-ß levels.</p> <p>Conclusions: <em>In vitro</em>, letrozole, unlike tamoxifen, enhances the cytotoxicity of both doxorubicin and docetaxel. This supports the prospect of trials using letrozole with chemotherapy in postmenopausal women with ER positive breast cancer. </p>

Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G0/G1 or in G2/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.