Ligand-independent Activation Of Estrogen Receptor Research Articles

Repeated exposure to 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) accelerates ligand-independent activation of estrogen receptors in long-term estradiol-deprived MCF-7 cells

It was previously identified that there may be an active metabolite of bisphenol A (BPA), 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). An in vitro system was developed to detect MBP toxicity to the Michigan Cancer Foundation-7 (MCF-7) cells that had been repeatedly exposed to a low dose of the metabolite. MBP profoundly activated estrogen receptor (ER)-dependent transcription as a ligand, with an EC50 of 2.8 nM. Women are continuously exposed to numerous estrogenic environmental chemicals; but their susceptibility to these chemicals may be significantly altered after menopause. Long-term estrogen-deprived (LTED) cells, which display ligand-independent ER activation, are a postmenopausal breast cancer model derived from MCF-7 cells. In this study, we investigated the estrogenic effects of MBP on LTED cells in a repeated exposure in vitro model. The results suggest that i) nanomolar levels of MBP reciprocally disrupt the balanced expression of ERα and ERβ proteins, leading to the dominant expression of ERβ, ii) MBP stimulates ERs-mediated transcription without acting as an ERβ ligand, and iii) MBP utilizes mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling to evoke its estrogenic action. Moreover, the repeated exposure strategy was effective for detecting low-dose estrogenic-like effects caused by MBP in LTED cells.

Plasma-Based Longitudinal Evaluation of ESR1 Epigenetic Status in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer.

BackgroundEndocrine therapy (ET) is the mainstay of treatment for hormone receptor-positive human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer; however, adaptive mechanisms emerge in about 25–30% of cases through alterations in the estrogen receptor ligand-binding domain, with a consequent ligand-independent estrogen receptor activity. Epigenetic-mediated events are less known and potentially involved in alternative mechanisms of resistance. The aim of this study was to test the feasibility of estrogen receptor 1 (ESR1) epigenetic characterization through liquid biopsy and to show its potential longitudinal application for an early ET sensitivity assessment.MethodsA cohort of 49 women with hormone receptor-positive HER2-negative MBC was prospectively enrolled and characterized through circulating tumor DNA using methylation-specific droplet digital PCR (MS-ddPCR) before treatment start (BL) and after 3 months concomitantly with computed tomography (CT) scan restaging (EV1). ESR1 epigenetic status was defined by assessing the methylation of its main promoters (promA and promB). The most established cell-free tumor DNA (ctDNA) factors associated with ET resistance [ESR1 and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations] were assessed through next-generation sequencing. Associations were tested through Mann–Whitney U test, matched pairs variations through Wilcoxon signed rank test, and survival was analyzed by log-rank test.ResultsThe ET backbone was mainly based on aromatase inhibitors (AIs) (70.83%) in association with CDK4/6 inhibitors (93.75%). Significantly lower promA levels at baseline were observed in patients with liver metastases (P = 0.0212) and in patients with ESR1 mutations (P = 0.0091). No significant impact on PFS was observed for promA (P = 0.3777) and promB (P = 0.7455) dichotomized at the median while a ≥2-fold increase in promB or in either promA or promB at EV1 resulted in a significantly worse prognosis (respectively P = 0.0189, P = 0.0294). A significant increase at EV1 was observed for promB among patients with PIK3CA mutation (P = 0.0173). A trend was observed for promB in ESR1 wild-type patients and for promA in the ESR1 mutant subgroup.ConclusionThe study proofed the concept of an epigenetic characterization strategy based on ctDNA and is capable of being integrated in the current clinical workflow to give useful insights on treatment sensitivity.

Abstract PD7-11: Therapeutic strategy for ESR1 mutation driven-endocrine resistance in ER-positive breast cancers

Abstract Background: Endocrine therapy is used in estrogen receptor (ER)-positive breast cancers, however, 25% of these patients are at risk of distant relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been validated to be acquired during the development of endocrine resistance. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity and emerges subclonally during aromatase inhibitor treatment. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to a novel checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in steroid deprived conditions. Cell cycle analysis and apoptosis were examined by flow cytometry annnexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar and MTT assays. Replication stress was identified by RPA32 and gamma-H2AX foci formation assay. For in vivo studies, MCF-7 ESR1 Y537S mutant cells were injected into female athymic nude mice with 17β-estradiol (E2) supplemented water. When tumors reached 350 mm3, tamoxifen (20 mg/kg; s.c.; three times a week), fulvestrant (200 mg/kg; s.c; once a week) and/or PF477736 Chk1 inhibitor (7.5 mg/kg; i.p.; twice a day and twice a week) was treated without E2. Results: ESR1 Y537S mutant cells accumulated approximately 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen-deprived (ED) conditions. BrdU incorporation also increased about 2.5-fold, however, apoptosis was decreased about 60 % compared with wild-type ER parental cells. ESR1 Y537S mutant cells induced significant replication stress, showing increased RPA32 foci together with increased gamma H2AX foci, a marker of DNA double-stranded breaks. ChIP-seq analysis revealed binding sites on ATR and CHEK1 genomic locations. ATR/Chk1-mediated checkpoint signaling was activated in ESR1 Y537S mutant cells, and was repressed with fulvestrant, tamoxifen, or ESR1 siRNA treatment. The Chk1 inhibitor, PF477736, sensitized MCF-7 expressing the ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen, and the ER degrader AZD9496 in cell proliferation assays. In MCF-7 ESR1 Y537S mutant xenograft and patient derived mouse models, tamoxifen treatment combined with the Chk1 inhibitor PF477736 repressed primary xenograft tumor doubling times (P=0.038, Wilcoxon test). Treatment of mutant tumors with PF477736 together with fulvestrant significantly inhibited the frequency of distant lung metastases by 80% (P=0.0031, t-test), suggesting that these combinations may be useful in second line treatment of metastatic breast cancer patients resistant to endocrine therapies. Conclusion: These preclinical results suggest that ESR1 mutant tumors have a therapeutic vulnerability to combination endocrine therapy with cell cycle checkpoint kinase inhibitors. These data demonstrate that this new therapeutic approach may be useful to restore endocrine sensitivity in metastatic breast cancer patients with ESR1 mutation driven-endocrine resistance. Citation Format: Kim J-A, Dustin D, Gu G, Corona-Rodriguez A, Edwards D, Coarfa C, Keyomarsi K, Fuqua SA. Therapeutic strategy for ESR1 mutation driven-endocrine resistance in ER-positive breast cancers [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 PD7-11.

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

Ginsenoside Rg1 activates ligand-independent estrogenic effects via rapid estrogen receptor signaling pathway

BackgroundGinsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor (ER) via mitogen-activated protein kinase–mediated pathway. Our study aimed to delineate the mechanisms by which Rg1 activates the rapid ER signaling pathways. MethodsER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidney HEK293 cells were treated with Rg1 (10−12M, 10−8M), 17ß-estradiol (10−8M), or vehicle. Immunoprecipitation was conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or their inhibitors were applied. ResultsRg1 rapidly induced ERα translocation to plasma membrane via caveolin-1 and the formation of signaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator of nongenomic activity of ER (MNAR), ERα, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase (MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interfering RNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in these cells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor (EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). The increase in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could be abolished by G15. ConclusionGinsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containing signalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independently and exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogen receptor.

Abstract P4-04-08: Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers

Abstract Background: Although estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy, 25% of these patients are at risk of relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been identified which induce resistance to endocrine therapy. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity. It is known that ER regulates the cell cycle in a ligand-dependent manner. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in phenol red minus medium containing 5% charcoal-dextran treated serum for 5 days to remove exogenous hormones. Cell cycle and apoptosis were examined by Flow cytometry and Annexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell senescence was determined using beta-galactosidase assays. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar or MTT assays. Results: Levels of p53 and apoptosis pathway proteins were significantly elevated in Y537S ESR1 mutant cells using RNA expression and reverse-phase protein microarrays. The ATM/ATR and Chk1/Chk2 mediated checkpoint signaling, the upstream pathway of p53, was activated in ESR1 Y537S mutation, which was repressed with fulvestrant treatment. ESR1 Y537 mutant cells accumulated about 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen deprived (ED) condition. BrdU incorporation was also increased about 2.5 fold compared with parental cells in estrogen-free medium. In addition, ESR1 Y537 mutant cells expressed a DNA double-strand break marker, gamma-H2AX protein in ED condition. Apoptosis and senescence were observed in ESR1 Y537S mutant cells in regular medium, however, apoptosis was not shown in ED medium. Chk1 inhibitor, PF477736 sensitized MCF-7 expressing ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen and AZD9496. Conclusion: Combination therapy with cell cycle checkpoint kinase inhibitor may lead better prognosis in ER mutation driven-endocrine resistance in postmenopausal breast cancers. Citation Format: Kim J-A, Fuqua SAW. Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers [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 P4-04-08.

ISY15-4 - Mechanisms of resistance to hormonal therapy in breast cancer

Luminal-type breast cancers are preferentially treated with hormonal therapy, while not a few patients relapse. The mechanisms of the hormone refractoriness have been investigated by numerous studies so far. However, it has not been fully clarified yet. We have been investigating the mechanisms using cancer specimens and cell lines by monitoring transcription activity of estrogen receptor (ER). We so far established six different types of cell lines which mimic aromatase inhibitor (AI) resistance from ERE-GFP introduced ER-positive breast cancer cell lines. They revealed that multiple and alternative ER activating pathways were involved in the resistance. The mechanisms are currently proposed to be three categories, such as (1) an aromatase-independent estrogen-producing pathway, (2) estrogen-independent ER function, and (3) ER-independent growth signaling. The established cell lines of category (1) showed increased expression of steroid metabolizing enzymes to produce agonist for ER. The category (2) was ligand-independent activation of ER, mainly via intracellular phosphorylation signaling pathway, which has been well investigated so far in many laboratory. The category (3) showed decreased ER expression or loss of ER function. Some of them were depended no androgen signaling pathway. As a study for therapeutic approach for AI-resistance, the effect of SERD, mTOR inhibitor, pan- or specific-PI3K inhibitors, and CDK4/6 inhibitor on these resistant cell lines were explored. Furthermore, fulvestrant-resistant cell lines, evelorimus-resistant cell lines, PI3K inhibitor-resistant cell lines, and CDK4/6 inhibitor-resistant cell lines were established and examined the efficacy of the other drugs and their cross-resistances. The results of these investigation will provide useful information for overcoming the hormonal therapy resistance.

The changing role of ER in endocrine resistance

Estrogen receptor (ER) is expressed in approximately 70% of newly diagnosed breast tumors. Although endocrine therapy targeting ER is highly effective, intrinsic or acquired resistance is common, significantly jeopardizing treatment outcomes and minimizing overall survival. Even in the presence of endocrine resistance, a continued role of ER signaling is suggested by several lines of clinical and preclinical evidence. Indeed, inhibition or down-regulation of ER reduces tumor growth in preclinical models of acquired endocrine resistance, and many patients with recurrent ER+ breast tumors progressing on one type of ER-targeted treatment still benefit from sequential endocrine treatments that target ER by a different mechanism. New insights into the nature and biology of ER have revealed several mechanisms sustaining altered ER signaling in endocrine-resistant tumors, including deregulated growth factor receptor signaling that results in ligand-independent ER activation, unbalanced ER co-regulator activity, and genomic alterations involving the ER gene ESR1. Therefore, biopsies of recurrent lesions are needed to assess the changes in epi/genomics and signaling landscape of ER and associated pathways in order to tailor therapies to effectively overcome endocrine resistance. In addition, more completely abolishing the levels and activity of ER and its co-activators, in combination with selected signal transduction inhibitors or agents blocking the upstream or downstream targets of the ER pathway, may provide a better therapeutic strategy in combating endocrine resistance.

Cross‐talk between the Growth Factor and Estrogen Signaling Pathways in Control of Breast Cancer

The specific hypothesis underlying our proposed research is that positive crosstalk between the growth factor and estrogen signaling pathways is the basis of the growth advantage of Estrogen Receptor (ER)‐positive breast cancer cells, and is the driving mechanism for ligand‐independent ER activation and endocrine therapy escape. First, we have observed that in ER‐positive breast cancer cells the expression of the gene RPS6KB1, encoding S6K1, is regulated by estrogen/ER. While many studies have addressed the regulation of S6K1 kinase activity, very little is known regarding the transcriptional regulation of S6K1 expression. We determined that ER cooperates with other transcription factors, including GATA‐3 and ERRα in regulating expression of the S6K1 gene. Second, it has been shown that ER phosphorylated as a consequence of growth factor signaling is a strong marker of endocrine treatment response. Phosphorylation is important for ER transactivational activity, and ligand‐independent phosphorylation of this site may be involved in development of endocrine resistance in vivo. While several kinases have been implicated in phosphorylation of ER, the exact mechanism of these events is unclear. We characterize the contribution of mTORC1 to phosphorylation of ER, its effect on transcriptional activity of ER, and the role in endocrine resistance.

New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

Relation between outcomes and expression of estrogen receptor-α phosphorylated at Ser(167) in endometrioid endometrial cancer.

Both ligand-dependent and ligand-independent activation of estrogen receptor (ER)α is modulated by receptor phosphorylation and results in activation of the ERα-dependent pathways that are involved in endometrioid endometrial cancer (EEC) pathogenesis. It is also known that the mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) and MAPK/p90 ribosomal S6 kinase (RSK) signaling pathways coordinately regulate phosphorylated-ERα at Ser167 (p-Ser167-ERα). However, the expression of p-Ser167-ERα in EEC and its prognostic role in ECC is largely unexplored. The purpose of the present study was to investigate the expression of p-Ser167-ERα in ECC and its relationship with prognosis. Immunohistochemical staining of primary EEC surgical specimens (n = 103) was carried out using antibodies specific for p-Ser167-ERα and for p-mTOR/p-S6K1 and p-MAPK/p-RSK. The correlation of p-Ser167-ERα expression with clinicopathological features and survival of ECC was studied. Patients that were positive for nuclear p-Ser167-ERα had significantly shorter relapse-free survival, and although the result was not significant, levels of nuclear p-Ser167-ERα tended to be higher in advanced-stage ECC patients. Nuclear p-Ser167-ERα was significantly positively correlated with p-MAPK and p-S6K1, and with significantly shorter relapse-free survival in EEC.

Differential ERα-mediated rapid estrogenic actions of ginsenoside Rg1 and estren in human breast cancer MCF-7 cells

Recent studies indicated that both estren and Rg1 appear to be able to activate mitogen-activated protein kinase (MAPK) pathway in estrogen responsive cells. Rg1 could lead to MAPK activation through ligand-independent activation of estrogen receptor (ER), while estren could activate the Src-MAPK pathway in an ERE-independent manner. Thus, it is important to understand the mechanistic insights on the difference in transcriptional activation between estren and Rg1. The present study also addressed the differential abilities of Rg1 and estren in terms of the ability to activate ER and the ability to induce ER translocation in MCF-7 cells. Our data indicated that Rg1 could increase pS2 gene expression, and could recruit the co-activator steroid receptor co-activator-1 (SRC-1) to the pS2 promoter. Rg1 could also induce ERα nuclear translocation as well as ERα phosphorylation at Ser118 principally in the cytoplasm in MCF-7 cells. We deduced that estren induced ERE-dependent transcriptional activity and activated ERα at Ser118 occurred in the nucleus of MCF-7 cells. However, it was found to decrease pS2 gene expression and failed to induce the recruitment of SRC-1 to the pS2 promoter in MCF-7 cells. Our results suggest that the abilities of Rg1 and estren to regulate pS2 gene expression, to recruit co-activators as well as to induce sub-cellular distribution of ERα are dramatically different.

Abstract A068: Targeting Akt reactivation following mTOR inhibition in breast cancer treatment

Abstract Breast cancer is the second leading cause of cancer related deaths in women and up to 60% of diagnosed cases are estrogen receptor (ER)-positive. ER-positive status is associated with a poor prognosis. While ER-positive cancers could be targeted with endocrine therapy, such as aromatase inhibitors, resistance often develops. Therefore, there is a need to better understand molecular contributions of other signaling pathways with the goal of treating patients with personalized combination therapies. The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell cycle progression and proliferation and is a target of a naturally occurring inhibitor rapamycin. The 40S ribosomal S6 Kinase 1 (S6K1) is one of the best characterized downstream targets of mTORC1. The mTORC1/S6K1 pathway is often hyperactivated in breast cancer, promoting cell growth and proliferation. S6K1 is encoded by the RPS6KB1 gene, located on the 17q23 chromosomal region. While this region is amplified in several types of cancer, multi-copy amplicons are found specifically in breast cancer, underscoring the importance of S6K1 in this disease. Importantly, there is a direct link between the mTORC1/S6K1 and ER signaling pathways. S6K1 directly phosphorylates and activates ER, promoting ligand-independent ER activation and endocrine resistance. Based on this data, current FDA approved treatment of advaced ER-positive breast cancer combines mTORC1 inhibitors with endocrine therapy. Some of the concerns that arise with the use of mTORC1 inhibitors (rapamycin analogs or rapalogs) is that they promote induction of autophagy and relieve the negative feedback signaling to Akt effected by S6K1, which promotes oncogenic Akt signaling, survival, and further stimulates mTORC1/S6K1 signaling, which may contribute to acquired drug resistance and patient relapse. Our research focuses on identifying agents that could be combined with mTORC1 inhibitors to block S6K1 while maintaining negative feedback signaling to Akt. Our results show that such combination treatment does in fact prevent rapamycin-induced upregulation of Akt while maintaining inhibition of the S6K1 signaling pathway, as assayed by analysis of S6K1 targets PDCD4, eIF4B and S6. Moreover, the combination treatment inhibits autophagy and promotes apoptosis. Thus, the combined inhibition of S6K1 and Akt may prevent or reverse drug resistance in breast cancer. Citation Format: Anya Alayev, Rose B. Snyder, Marina K. Holz. Targeting Akt reactivation following mTOR inhibition in breast cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A068.

Estrogen receptor α phosphorylated at Ser167: Distribution and prognostic implications in endometrial cancer.

e15539 Background: Both ligand-dependent and ligand-independent activation of estrogen receptor (ER)α is modulated by receptor phosphorylation, which is enhanced by ligand binding. Similar to Akt, ERα is activated through phosphorylation at Ser167, resulting in activation of the ERα-dependent pathways that are involved in endometrioid endometrial cancer (EEC) pathogenesis. It is known that the mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) and mitogen-activated protein kinase (MAPK)/p90 ribosomal S6 kinase (RSK) signaling pathways coordinately regulate phosphorylated- (p-) ERα. The purpose of this study was to determine the importance of Ser167 phosphorylation in EEC progression. Methods: Immunohistochemical staining of primary EEC surgical specimens (n=103) was carried out using antibodies specific for p-ERα and for p-Akt, p-mTOR/S6K1 and p-MAPK/RSK. and for p-Akt, p-mTOR/S6K1 and p-MAPK/RSK. Results: Patients positive for nuclear Ser167 phosphorylation had significantly shorter relapse free survival. Althoughthe result was not significant, levels of nuclear p- tended to be higher in advanced-stage ECC patients. Nuclear p-ERα was significantly positively correlated with p-MAPK and p-S6K1. Conclusions: The amount of nuclear ERa Ser167 phosphorylation is associated with p-MAPK and p-S6K1, and may be important in EEC progression and outcome.

Abstract 1738: Histone deacetylase inhibitor mediated reversal of acquired endocrine therapy resistance in breast cancer cell lines

Abstract Although nearly 70% of breast cancers are estrogen receptor (ER) positive, endocrine therapy using the selective ER modulator tamoxifen or the selective ER down regulator fulvestrant have proved to be beneficial in less than 50% of these patients. Moreover, most ER positive patients that initially respond to tamoxifen eventually acquire resistance. Numerous studies have focused on determining the mechanism of anti-estrogen resistance which involve cellular changes such as variation in uptake/metabolism of tamoxifen, expression of altered ER proteins, ligand-independent ER activation, loss of ER expression and/or variation in recruitment of cofactors and altered cross-talk with other growth factor signaling pathways. Recently, an alternative strategy to modulate ER signaling has been the use of histone deacetylase (HDAC) inhibitors. Preclinical studies have shown that with the addition of HDAC inhibitors, the anti-tumor activity of tamoxifen is potentiated. In these cells, apoptotic cell death is induced, which cannot be rescued by estrogen, indicating the potential to reverse hormone therapy resistance, a strategy currently being evaluated in several clinical trials. Our laboratory is interested in exploring the potential of HDAC inhibition for the reversal of anti-estrogen resistance in ER-positive breast cancer. To this end, we have generated several MCF-7 cell lines resistant to various levels of tamoxifen; up to 10 micromolar. Our preliminary data demonstrate the addition of an HDAC inhibitor to tamoxifen reverses resistance. Significant effects on proliferation and viability are observed in these cells with clinically relevant concentrations of pan-HDAC inhibitors. Furthermore, with the addition of an HDAC inhibitor to tamoxifen, colony formation is attenuated. Initial characterization of these resistant cells indicated a several fold higher growth rate than the parental cells but a similar growth rate in presence and absence of the ligand estrogen, suggesting the possibility of loss of estrogen-dependence. Both the parental and resistant cells, in presence of estrogen, show a stabilization of the estrogen receptor protein by tamoxifen, which is not altered by the HDACi, suggesting an effect that goes beyond modulation of ER protein level. Tamoxifen or the combination in the resistant cells do not decrease Cyclin D1 protein level as is seen in parental cells, suggesting the involvement of pathways not involving ER. Our previous data using siRNA against HDAC2 indicate sensitization of ER positive breast cancer cells to tamoxifen-mediated apoptosis suggesting the importance of HDAC2. We plan to study the importance of ER, in resistant cells with varied ER status, in mediating the hormone therapy response and to identify the potential involvement of other signaling pathways, using both HDAC inhibitors and selectively depleting HDAC2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2011-1738

Absence of ligand-independent transcriptional activation of the estrogen receptor via the estrogen response element in pituitary lactotrophs in primary culture

The estrogen receptor (ER) is a ligand-activated transcription factor that enhances gene expression by binding to specific regulatory DNA sequences called estrogen response elements (EREs). In some cell lines, the ER is also activated in a ligand-independent manner by multiple signaling pathways. In this study, we developed a novel adenovirus-mediated assay for promoter activation, termed LASETA, which we then used to examine whether ligand-independent activation of the ER occurred in normal pituitary lactotrophs in primary culture. In the LASETA adenovirus vector, the loxP-flanked stop sequence was deleted by prolactin (PRL) promoter-regulated expression of Cre recombinase. This led to lactotroph-specific expression of a reporter gene driven by an ERE-containing promoter. Estrogen-induced expression of the reporter protein luciferase in LASETA was specific for lactotrophs and was ER-dependent. LASETA was shown to be reliable even with varying Cre recombinase expression levels, which were caused by changes in PRL promoter activity. Using LASETA, we observed no change in ERE-mediated ER activity in the absence of estrogen after treatment of normal lactotrophs with agents such as insulin-like growth factor-1, epidermal growth factor, the adenylate cyclase activator forskolin, the extracellular signal-regulated kinase kinase inhibitor U0126, and the protein kinase A inhibitor H89. The ERE-mediated ligand-independent ER activity was induced by the growth factors and forskolin in the somatolactotroph tumor cell line GH4C1 cells. These results suggest that ERE-mediated ligand-independent activation of ER does not occur in normal lactotrophs in primary culture, and is a phenomenon likely restricted to transformed cells.

Genome-Wide Analysis of Aromatase Inhibitor-Resistant, Tamoxifen-Resistant, and Long-Term Estrogen-Deprived Cells Reveals a Role for Estrogen Receptor

Acquired resistance to either tamoxifen or aromatase inhibitors (AI) develops after prolonged treatment in a majority of hormone-responsive breast cancers. In an attempt to further elucidate mechanisms of acquired resistance to AIs, MCF-7aro cells resistant to letrozole (T+LET R), anastrozole (T+ANA R), and exemestane (T+EXE R), as well as long-term estrogen deprived (LTEDaro) and tamoxifen-resistant (T+TAM R) lines were generated. This is the first complete panel of endocrine therapy-resistant cell lines, which were generated as multiple independent biological replicates for unbiased genome-wide analysis using affymetrix microarrays. Although similarities are apparent, microarray results clearly show gene signatures unique to AI-resistance were inherently different from LTEDaro and T+TAM R gene expression profiles. Based on hierarchical clustering, unique estrogen-responsive gene signatures vary depending on cell line, with some genes up-regulated in all lines versus other genes up-regulated only in the AI-resistant lines. Characterization of these resistant lines showed that LTEDaro, T+LET R, and T+ANA R cells contained a constitutively active estrogen receptor (ER)alpha that does not require estrogen for activation. This ligand-independent activation of ER was not observed in the parental cells, as well as T+EXE R and T+TAM R cells. Further characterization of these resistant lines was performed using cell cycle analysis, immunofluorescence experiments to visualize ER subcellular localization, as well as cross-resistance studies to determine second-line inhibitor response. Using this well-defined model system, our studies provide important information regarding differences in resistance mechanisms to AIs, TAM, and LTEDaro, which are critical in overcoming resistance when treating hormone-responsive breast cancers.

Mechanism of estrogen-induced apoptosis in breast cancer cells: Role of the NF-κB signaling pathway

The ability of sex steroid hormones to up-regulate the apoptotic signaling proteins is well documented; however, the apoptotic potential of sex hormones is not remarkable and fully compensated by their growth stimulatory action to target cells. In the present study using the long-term cultivation of estrogen-dependent MCF-7 breast cancer cells in steroid-free medium, we have established a cell subline, designed as MCF-7/LS, which was characterized by the resistance to growth stimulatory estradiol action and hypersensitivity to estrogen-induced apoptosis. We have demonstrated that estrogen treatment of the cells does not influence on the level of TNF-R1 or Fas, but dramatically decreases the transcriptional activity of NF-kappaB. Importantly, the MCF-7/LS cells, which are insensitive to growth stimulatory estrogen action, retain the ability to decrease in the NF-kappaB activity in response to estrogen stimulus. Furthermore, the significant increase in the basal (in the absence of ligand) estrogen receptor (ER)-dependent transcriptional activity in the MCF-7/LS cells was revealed and reciprocal transcriptional antagonism between ER and NF-kappaB was demonstrated. Finally, we proved the possible involvement of phosphatidylinositol-3 kinase (PI3K) in the ligand-independent ER activation. In general, the results presented suggest that long-term growth of MCF-7 breast cancer cells in steroid-free medium is accompanied with the increase in the basal ER-dependent transcriptional activity as well as the maintenance of the negative regulatory loop ER-NF-kappaB. The latter may be considered as one of the factors resulting in a disbalance between pro- and anti-apoptotic pathways and enhancement in estrogen apoptotic action in the cells.

Estrogenic activity of procymidone in rainbow trout ( Oncorhynchus mykiss) hepatocytes: a possible mechanism of action

It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 μM (with 17β-estradiol [E2] 20 μM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 μM or ICI 182,780 1 μM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (α-tocopherol 30 μM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by α-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with α-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with α-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK.

Ligand-independent activation of estrogen receptor alpha by XBP-1.

The estrogen receptor (ER) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that XBP-1 mRNA expression is associated with ERalpha status in breast tumors. However, the role of XBP-1 in ERalpha signaling remains to be elucidated. More recently, two forms of XBP-1 were identified due to its unconventional splicing. We refer to the spliced and unspliced forms of XBP-1 as XBP-1S and XBP-1U, respectively. Here, we report that XBP-1S and XBP-1U enhanced ERalpha-dependent transcriptional activity in a ligand-independent manner. XBP-1S had stronger activity than XBP-1U. The maximal effects of XBP-1S and XBP-1U on ERalpha transactivation were observed when they were co-expressed with full-length ERalpha. SRC-1, the p160 steroid receptor coactivator family member, synergized with XBP-1S or XBP-1U to potentiate ERalpha activity. XBP-1S and XBP-1U bound to the ERalpha both in vitro and in vivo in a ligand-independent fashion. XBP-1S and XBP-1U interacted with the ERalpha region containing the DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, including the N-terminal basic region leucine zipper domain (bZIP) and the C-terminal activation domain. The bZIP-deleted mutants of XBP-1S and XBP-1U completely abolished ERalpha transactivation by XBP-1S and XBP-1U. These findings suggest that XBP-1S and XBP-1U may directly modulate ERalpha signaling in both the absence and presence of estrogen and, therefore, may play important roles in the proliferation of normal and malignant estrogen-regulated tissues.