Ligand-independent Activation Of ERα Research Articles

Genomic profiling in low grade serous ovarian cancer: Identification of novel markers for disease diagnosis and therapy

ObjectivesLow grade serous ovarian cancer (LGSOC) differs from high grade serous in terms of pathogenesis, molecular, genetic, and clinical features. Molecular studies have been hampered by small sample sizes, heterogenous histology, and lack of comprehensive testing. We sought to molecularly profile LGSOC in a homogenously tested, histologically confirmed cohort. MethodsUsing hot-spot and whole exome next generation sequencing (NGS), fusion gene analysis interrogating RNA, fragment analysis, in situ hybridization and/or immunohistochemistry, 179 specimens were evaluated by Caris Life Sciences (Phoenix, AZ). A second independent histologic review confirmed histology in 153 specimens. ResultsMost frequently mutated genes (5% or greater) were members of the mitogen-activated protein kinase (MAPK) pathway: KRAS (23.7%, n = 36), NRAS (11.2%, n = 19), NF1 (7.9%, n = 5), and BRAF (6.6%, n = 10). Class III mutations were seen in 3 of 10 BRAF mutations while 7 were Class I V600E. Overall, estrogen and progesterone receptor expression was 80.2% (n = 130) and 27.8% (n = 45), respectively. Of those that were hormone negative, nearly 50% contained KRAS or NF1 mutations. None were NRAS mutated. Markers of response to immunotherapy were low to absent. ConclusionBRAF mutations were seen to be lower than those traditionally reported. With increased MAPK activation resulting in ligand independent activation of ERα, a role of combination therapy with hormonal and targeted therapy should be considered as 49.2% of hormone negative specimens were KRAS or NF1 mutated. Absence of immunotherapy biomarkers suggest limited benefit to immunotherapeutic agents.

Deoxynivalenol enhances estrogen receptor alpha-induced signaling by ligand-independent transactivation

Deoxynivalenol (DON), which is one of the prevalent mycotoxins in food and feeds, exerts adverse effects on animal and human health. These effects are mainly associated with its ribotoxic properties, although few studies suggest the involvement of other mechanisms of action. To assess the ability of DON to disrupt estrogen signaling, we conducted an in vitro study using MCF-7 and MDA-MB-231 cells. After 72h, DON reduced cell viability in both cell lines, thus highlighting its well-known cytotoxic effect. However, after 6h, DON increased the expression of estrogen-responsive genes, hence demonstrating the stimulation of estrogen signaling by this mycotoxin after a short-term exposure. This effect was partially reversed by siRNA-mediated silencing of ERα expression and by 4-hydroxytamoxifen (ERα antagonist), but neither by G36 (GPER antagonist) nor by the siRNA-mediated silencing of PPARγ2 expression. Moreover, DON exposure induced an increase in the level of ERα phosphorylation at serine 167. Furthermore, when combined with zearalenone (a naturally co-occurring mycotoxin recognized as an endocrine disruptor), DON increased the expression of estrogen-responsive genes to a greater extent than each individual compound taken separately. Taken together, our results suggest, for the first time, that DON can disrupt estrogen signaling through the ligand-independent activation of ERα.

Nexus between PI3K/AKT and Estrogen Receptor Signaling in Breast Cancer.

Simple SummaryBreast cancers are broadly classified into two subtypes: estrogen receptor-positive and estrogen receptor-negative. Approximately 70% of breast cancers are estrogen receptor-positive and this type of breast cancer is more common in postmenopausal women. Estrogen receptor-positive breast cancers are treated with a class of drugs called anti-estrogens. While the majority of tumors respond to this class of drugs, disease recurs in approximately 30% of cases, sometimes even 20 years after initial diagnosis. This review highlights efforts to understand why tumors recur despite effective treatments and outcome of these efforts in the development of new combination therapies. At least three new types of combination therapies that delay progression of recurrent tumors are in clinical use.Signaling from estrogen receptor alpha (ERα) and its ligand estradiol (E2) is critical for growth of ≈70% of breast cancers. Therefore, several drugs that inhibit ERα functions have been in clinical use for decades and new classes of anti-estrogens are continuously being developed. Although a significant number of ERα+ breast cancers respond to anti-estrogen therapy, ≈30% of these breast cancers recur, sometimes even after 20 years of initial diagnosis. Mechanism of resistance to anti-estrogens is one of the intensely studied disciplines in breast cancer. Several mechanisms have been proposed including mutations in ESR1, crosstalk between growth factor and ERα signaling, and interplay between cell cycle machinery and ERα signaling. ESR1 mutations as well as crosstalk with other signaling networks lead to ligand independent activation of ERα thus rendering anti-estrogens ineffective, particularly when treatment involved anti-estrogens that do not degrade ERα. As a result of these studies, several therapies that combine anti-estrogens that degrade ERα with PI3K/AKT/mTOR inhibitors targeting growth factor signaling or CDK4/6 inhibitors targeting cell cycle machinery are used clinically to treat recurrent ERα+ breast cancers. In this review, we discuss the nexus between ERα-PI3K/AKT/mTOR pathways and how understanding of this nexus has helped to develop combination therapies.

Abstract 1242: Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer

Abstract Estrogen receptor alpha (ERα) signaling plays a critical role in the development and progression of hormone receptor-positive (HR+) breast cancer. One mechanism of tumor progression of HR+ breast cancer is phosphorylation of ERα by non-receptor protein kinases, leading to ligand-independent activation of ERα. Mitotic kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast cancer and can phosphorylate ERα on serine 305, increasing the ability of ERα to induce transcription of target genes implicated in tumorigenesis. In addition, PAK1 phosphorylates and activates AURKA and its functional partners, suggesting possible synergistic activity in tumors overexpressing both kinases. We tested the hypothesis that combined inhibition of AURKA and PAK1 would synergistically suppress tumor growth of HR+ breast cancer cells in vitro and in vivo. Treatment of T47D HR+ breast cancer cell line with the PAK1 inhibitor FRAX1036, combined with AURKA inhibitor alisertib, improved cell killing in comparison to single agents. FRAX1036 and alisertib were then evaluated in an orthotopic xenograft model using BT474 HR+ breast cancer cell line. Ten million BT474 cells were implanted into the mammary fat pads of 6-week old NSG mice simultaneously with implantation of estrogen pellets to facilitate growth of xenografts. Mice received 21 days of treatment with FRAX1036 20mg/kg daily and alisertib 10 or 15 mg/kg BID, alone or in combination. We observed exceptional inhibition of tumor growth in the combination treatment groups. Combination of FRAX1036 20mg/kg daily with alisertib 15mg/kg BID was the most effective, with partial or near-complete responses that continued through 21 days of treatment. This combination resulted in significantly better inhibition of tumor growth than alisertib 15mg/kg BID alone or FRAX1036 20mg/kg daily alone (p = 0.003 and p = 1.29e-6, respectively). Similarly, combination treatment of FRAX1036 20mg/kg daily with alisertib 10mg/kg BID was significantly better then alisertib alone or FRAX1036 alone in the corresponding doses (p = 0.002 and p = 3.52e-7, respectively). Analysis of H&E stained sections and Ki-67 proliferation index of BT474 xenograft tumors indicated a highly significant decrease in viable tumor cells and replacement by stroma after combination treatment compared to treatment with single agents or with control. Caspase-3 IHC analysis showed significantly greater proportion of apoptotic cells per tumor area in the group of mice treated with FRAX1036 and Alisertib 15mg/kg BID combination comparing to control (p = 0.008). We conclude that combining FRAX1036 and alisertib is a novel and promising approach in HR+ breast cancer. Citation Format: Elena Shagisultanova, Vladislav Korobeynikov, Anna Nikonova, Jonathan Chernoff, Virginia Borges, Erica Golemis. Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1242.

Abstract 4561: Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer

Abstract The cocaine- and amphetamine-regulated transcript (CART) was first discovered as a peptide up-regulated by the administration of cocaine and amphetamine to rats. CART peptides are involved in regulating physiological processes, such as feeding, reward and neuro-protection. However, recent studies have associated high CART expression with worse overall survival in patients with small-bowel carcinoid tumours [1] and estrogen receptor-positive (ER+), lymph node-negative breast cancer [2]. Interestingly, in ER+ breast cancer, CART was also shown to be associated with poor patient response to tamoxifen, suggesting CART may play a role in conferring tamoxifen resistance [2]. The aim of this study was to elucidate the mechanism(s) by which CART signals in ER+ breast cancer. In order to test whether CART could mediate the ligand-independent activation of ER-alpha, MAP-Kinase pathway activation and levels of downstream gene-targets of ER-alpha were assessed post CART stimulation. Additionally, the ability of CART to activate ER-alpha using three LXD motifs (nuclear receptor co-activator recognition motifs) present within the CART sequence was also assessed. This was achieved through selectively mutating these motifs and testing whether CART still possessed the ability to activate ER-alpha using an ERE-Dual luciferase reporter assay. Treatment of cells with CART demonstrated an increase in MAP-Kinase activity through the detection of increasing phosphorylated ERK levels. An increase in the phosphorylation of ER-alpha at serine 118 (a phosphorylation site thought to be involved in tamoxifen resistance) was also evident following CART stimulation. CART stimulation also resulted in an increase in levels of the progesterone receptor, a known ER-alpha gene target. Mutagenesis of each LXD motif within CART resulted in significant decreases in ER-alpha activity, suggesting a potential structure-function relationship between CART and ER-alpha. In conclusion, we suggest that CART can activate ER-alpha in a ligand-independent manner through the MAP-Kinase pathway, and also potentially through specific LXD motifs within its sequence. Further investigation into the relationship between CART and ER-alpha will help us gain a better understanding of, not only the potential structure-function relationship between CART and ER-alpha, but also the role CART plays with regards to tamoxifen resistance in ER+ breast cancer. [1] Landerholm K et al., Expression of cocaine- and amphetamine-regulated transcript is associated with worse survival in small bowel carcinoid tumors. Clin Cancer Res. 2012 Jul 1;18(13):3668-76 [2] DJ Brennan, DP O’Connor et al., The cocaine- and amphetamine-regulated transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer. Oncogene 2011 Dec 5. Doi: 10.1038/onc.2011.519 Citation Format: Brian Mooney, Sudipto Das, Rut Klinger, William Gallagher, Darran O’Connor. Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4561.

Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases.

Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.

What came first—the chicken or the egg? Ligand-independent activation of ERα in KRas mutant endometrial cancer

What came first—the chicken or the egg? Ligand-independent activation of ERα in KRas mutant endometrial cancer

Evidence for Ligand-Independent Activation of Hippocampal Estrogen Receptor-α by IGF-1 in Hippocampus of Ovariectomized Rats.

In the absence of ovarian estrogens, increased levels of estrogen receptor (ER)α in the hippocampus are associated with improvements in cognition. In vitro evidence indicates that under conditions of low estrogen, growth factors, including Insulin-Like Growth Factor 1 (IGF-1), can activate ERα and regulate ERα-mediated transcription through mechanisms that likely involve modification of phosphorylation sites on the receptor. The goal of the current work was to investigate a role for IGF-1 in ligand-independent activation of ERα in the hippocampus of female rats. Ovariectomized rats received a single intracerebroventricular infusion of IGF-1 and hippocampi were collected 1 or 24 hours later. After 1 h, IGF-1 increased hippocampal levels of phosphorylated ERα at serine 118 (S118) as revealed by Western blotting. Coimmunoprecipitation revealed that at 1 hour after infusion, IGF-1 increased association between ERα and steroid receptor coactivator 1, a histone acetyltransferase that increases transcriptional activity of phosphorylated ERα. IGF-1 infusion increased levels of the ERα-regulated proteins ERα, choline acetyltransferase, and brain-derived neurotrophic factor in the hippocampus 24 hours after infusion. Results indicate that IGF-1 activates ERα in ligand-independent manner in the hippocampus via phosphorylation at S118 resulting in increased association of ERα with steroid receptor coactivator 1 and elevation of ER-regulated proteins. To our knowledge, these data are the first in vivo evidence of ligand-independent actions of ERα and provide a mechanism by which ERα can impact memory in the absence of ovarian estrogens.

Synergistic Activation of ERα by Estrogen and Prolactin in Breast Cancer Cells Requires Tyrosyl Phosphorylation of PAK1

Serine/threonine kinase PAK1 is activated by estrogen and plays an important role in breast cancer. However, the integration of PAK1 into the estrogen response is not fully understood. In this study, we investigated the mechanisms underlying the hormone-induced activation of estrogen receptor (ERα, ESR1). We show that estrogen activated PAK1 through both the ERα and GPER1 membrane receptors. Estrogen-dependent activation of PAK1 required the phosphorylation of tyrosine residues by Etk/Bmx and protein kinase A (PKA) within an assembled signaling complex comprising pTyr-PAK1, Etk/Bmx, the heterotrimer G-protein subunits Gβ1, Gγ2, and/or Gγ5, PAK-associated guanine nucleotide exchange factor (βPIX, ARHGEF7), and PKA. Moreover, the PKA RIIβ subunit is a direct target of PAK1, and thus in response to estrogen, the activated pTyr-PAK1 complex reciprocally potentiated PKA activity, suggesting a positive feedback mechanism. We also demonstrate that PKA phosphorylated Ser305-ERα in response to estrogen, but pTyr-PAK1 phosphorylated Ser305-ERα in response to prolactin (PRL), implying that maximal ERα phosphorylation is achieved when cells are exposed to both PRL and estrogen. Furthermore, S305-ERα activation led to enhanced phosphorylation of Ser118-ERα and promoted cell proliferation and tumor growth. Together, these data strongly support a critical interplay between PRL and estrogen via PAK1 and suggest that ligand-independent activation of ERα through PRL/PAK1 may impart resistance to anti-estrogen therapies. Cancer Res; 76(9); 2600-11. ©2016 AACR.

Abstract LB-148: Inflammatory cytokines alter the sensitivity of breast cancer cells to endocrine treatments

Abstract In breast cancer, infiltration of macrophages establishes an inflammatory tumor microenvironment, which can foster an extremely aggressive and therapy resistant tumor. Although inflammatory cytokines are known to modulate the transcriptional potential of estrogens and contribute to an endocrine-resistant state in cell-based model systems, the underlying molecular mechanisms remain poorly defined. We have used the human breast cancer cell-line MCF-7, an estrogen receptor α(ERα) positive and inflammatory cytokine sensitive cell line, to define the global impact inflammatory cytokines on the ERα cistrome and ERα-dependent transcriptional activity using next-generation sequencing methods. IL-1β and TNFα treatments establish an ERα cistrome that substantially overlaps with the E2-dependent ERα cistrome and results in ERα-dependent, ligand-independent activation of gene expression. Cytokine stimulation of MCF-7 cells results in phosphorylation of ERα at S305 which drives the ligand-independent activation of ERα. Pharmacological inhibition of IKKα/β blocks cytokine-dependent S305 phosphorylation, inhibits the cytokine-dependent ERα cistome, and abolishes cytokine-dependent gene activation. Additionally, S305 phosphorylation by cytokine treatments blocks the ability of tamoxifen to inhibit the expression of a subset of E2-dependent target genes and mutation of S305 to alanine restores tamoxifen sensitivity in the presence of IL-1β or TNFα. Collectively, these results provide molecular details dictating how inflammatory cytokines activate the transcriptional potential of ERα and drive an endocrine-resistant state in human breast cancer cells. In addition, they highlight the potential for anti-inflammatory therapies to be utilized in the treatment of patients with tamoxifen-resistant breast cancer. Citation Format: Joshua D. Stender, Irida Kastrati, Maayan Yakir, Jonna Frasor, Christopher K. Glass. Inflammatory cytokines alter the sensitivity of breast cancer cells to endocrine treatments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-148. doi:10.1158/1538-7445.AM2014-LB-148

Flavonoids from Herba epimedii selectively activate estrogen receptor alpha (ERα) and stimulate ER-dependent osteoblastic functions in UMR-106 cells

Total flavonoids in Herba epimedii (HEP) have been demonstrated to protect against bone loss and bone deterioration associated with estrogen deficiency without exerting any uterotrophic effects. However, it is unclear how flavonoids in HEP exert their protective effects on bone and if different flavonoids exert estrogenic actions in bone cells via similar mechanism of actions. The present study aims to investigate the bone anabolic effects of four major flavonoids isolated from HEP, namely icariin, baohuoside-I, epimedin B and sagittatoside A as well as the mechanism involved in mediating their estrogenic actions in rat osteoblastic-like UMR-106 cells. All tested compounds significantly stimulated the cell proliferation rate, alkaline phosphate (ALP) activity and osteoprotegerin (OPG)/receptor activator of nuclear factor κ-B ligand (RANKL) mRNA expression in UMR-106 cells and their effects could be abolished by co-incubation with 10−6M ICI 182,780. None of the flavonoids exhibited binding affinities toward ERα and ERβ. However, sagittatoside A selectively activated estrogen response element (ERE)-luciferase activity via ERα. In addition, icariin and sagittatoside A induced ERα phosphorylation at serine 118 residue. Taken together, our results indicated that all four flavonoids from HEP stimulated ER-dependent osteoblastic functions in UMR-106 cells, but only two of them appeared to exert their actions by ligand-independent activation of ERα. Our study provides evidence to support the hypothesis that the estrogen-like protective effects on bone by flavonoids are mediated via mechanisms that are distinct from the classical actions of estrogen.

P4-01-08: The Role of Ser991 PELP1 Phosphorylation in Therapy Resistance and Metastasis of Breast Cancer.

Abstract BACKGROUND: Growth factor induced activation of MAPK pathway is suggested to play a critical role both in invasive and hormone therapy resistant breast cancer via ligand independent activation of ERα. Proline Glutamic acid Leucine Rich Protein (PELP1) is a well established ER-coregulator that plays a critical role in ER's nuclear and extra-nuclear functions. PELP1 functions as a proto-oncogene and deregulation of PELP1 expression is linked to increased proliferation, metastasis and therapy resistance. The objective of this study is to examine, whether growth factor signaling modulates PELP1 mediated ER coactivation functions via MAPK pathway. METHODS: Bioinformatics and phosphopeptide specific antibodies were employed in this study. In vitro kinase assays and MAPK inhibitors were used to confirm MAPK phosphorylation of PELP1. Site directed mutants and ERE reporter gene assays were used to demonstrate the significance of MAPK site in PELP1. Growth factor and estrogen regulation of PELP1 phosphoryaltion at Ser991 was validated in therapy sensitive (MCF7, ZR75) and resistant (MCF-7 HER2, MCF7-Tam and MCF-7Ca-LTLT cells using PELP1 specific phosphoantibody. Breast cancer model cells stably expressing PELP1 mutant or PELP1 peptides encompassing MAPK phosphorylation was used to test the significance of this MAPK site in PELP1 using proliferation, migration, and invasion assays. IHC analysis using PELP1 phospho antibodies was performed using murine xenograft tumors and human breast tumor arrays (TMA). RESULTS: Growth factors (EGF, Heregulin) induced Ser991 phosphorylation of PELP1 in murine mammary epithelial cells and also in human breast cancer cell lines. Growth factor mediated Ser991PELP1 phosphorylation was abrogated by ERK1/2 MAPK pathway inhibitors including PD98059 and U0126. In ERE-reporter luciferase assays using ZR75 cells, PELP1 (WT) but not the ser991Ala mutant of PELP1 corroborated growth factor-ER crosstalk. Therapy resistant model cells (MCF-7-Tam, MCF-7-LTLT-ca and MCF-7-HER2) exhibited enhanced Ser991PELP1 phosphoryation compared to parental MCF7. TAT-Peptide encompassing Ser991 inhibited growth factor mediated phosphorylation of PELP1 and revealed a potent ability to inhibit the growth of therapy resistant cells. Xenograft tumors of hormone therapy model cell lines showed increased Ser991PELP1 phosphorylation and enhanced nuclear localization compared to hormonal sensitive xenograft tumors. IHC analysis of human breast invasive TMA revealed enhanced PELP1 ser991 phosphorylation in advanced metastatic tumors with prominent nuclear/nucleolar localization compared to normal and benign tumors. CONCLUSIONS: In this study, we identified that growth factor-MAPK pathway modulate ER coregulator PELP1 functions via phosphorylation and demonstrated that Ser991 phosphorylation of PELP1 has the potential to govern the growth Factor-ER cross talk leading to therapy resistance and metastatic phenotype of breast cancer. Peptides encompassing Ser991PELP1 site and Phosphoantibodies targeting Ser991 represent a novel therapeutic target and diagnostic markers in breast cancer. Supported by DOD Fellowship W81XWH-09-1-0010 (BCN) and DOD W81XWH-08-1-0604 (RKV) Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-08.

Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

BackgroundEstrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites.ResultsPreviously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor.ConclusionThese novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.

Molecular characterization of aromatase inhibitor-resistant, tamoxifen-resistant and LTEDaro cell lines

To determine potential genes involved in mediating resistance to aromatase inhibitors (AIs), a microarray study was performed using MCF-7aro (aromatase overexpressing) cells that are resistant to letrozole (T + LET R), anastrozole (T + ANA R) and exemestane (T + EXE R), as well as LTEDaro and tamoxifen-resistant (T + TAM R) lines for comparison. Based on hierarchical clustering, estrogen-responsive genes were found to be differentially expressed in AI-resistant lines versus LTEDaro and T + TAM R. Additional genome-wide analysis showed that gene expression profiles of the non-steroidal AI-resistant lines were most closely correlated and that T + EXE R lines exhibit differing profiles. Also, LTEDaro and T + TAM R lines are inherently different from expression profiles of AI-resistant lines. Further characterization of these resistant lines revealed that T + LET R, T + ANA R and LTEDaro cells contain a constitutively active estrogen receptor α (ERα) that does not require the ligand estrogen for activation. Ligand-independent activation of ERα does not activate identical estrogen-responsive gene profiles in AI-resistant lines as in LTEDaro lines, thereby establishing differing mechanisms of resistance. This ligand-independent activation of ER was not observed in the parental cell lines MCF-7aro, T + EXE R or T + TAM R cells. Based on the steroidal structure of EXE, our laboratory has shown that this AI has weak estrogen-like properties, and that EXE resistance involves an ER-dependent crosstalk with EGFR growth factor signaling. Recent studies in our laboratory pertaining to pre-clinical models of AI treatment revealed that intermittent use of EXE delays the onset of acquired resistance in comparison to continuous treatment. Specific molecular mechanisms involved in intermittent use of EXE are currently being explored, based on microarray gene expression profiling. Lastly, our laboratory has initiated a study of microRNAs and their potential role in regulating target genes involved in AI-resistance. Overall, we propose a model of acquired resistance that progresses from hormone-dependence (T + TAM R and T + EXE R) to hormone-independence (T + LET R and T + ANA R), eventually resulting in hormone-independence that does not rely on conventional ER signaling (LTEDaro).

Estrogen Receptor-α Phosphorylated at Ser118 Is Present at the Promoters of Estrogen-Regulated Genes and Is Not Altered Due to HER-2 Overexpression

Detection of estrogen receptor (ER)-alpha phosphorylated at Ser(118) (P-Ser(118)-ER-alpha) may be an indicator of an intact ligand-dependent ER-alpha in breast tumors in vivo and may predict responsiveness to endocrine therapy. The current study addresses whether P-Ser(118)-ER-alpha is functionally involved in ER target gene transcription and if this is modulated by HER-2 overexpression. Using chromatin immunoprecipitation analysis, P-Ser(118)-ER-alpha was found associated with the promoters of several estrogen-regulated genes in MCF-7 breast cancer cells 30 minutes following estrogen treatment. Coactivators AIB1 and p300 were coimmunoprecipitated with P-Ser(118)-ER-alpha following estrogen treatment. The overexpression of HER-2 protein in MCF-7 cells did not affect estrogen induction of phosphorylation of Ser(118) or its presence at the promoters of several estrogen-regulated genes. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) pathway, had no effect on P-Ser(118)-ER-alpha. The lack of effect of HER-2 overexpression on P-Ser(118)-ER-alpha expression in cell models is supported by similar levels of expression of P-Ser(118)-ER-alpha in ER(+)/HER-2-overexpressing and ER(+)/HER-2(-) breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], and IkappaB kinase-alpha (IKK-alpha; BAY-11-7082), we show that IKK-alpha, but not Cdk7, is at least in part involved in estrogen-mediated phosphorylation at Ser(118) in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser(118)-ER-alpha in estrogen-regulated signaling and do not support the hypothesis that resistance of breast tumors to tamoxifen therapy involves ligand independent activation of ER-alpha due to constitutive phosphorylation of Ser(118) by constitutive activation of MAPK pathway.