ERα Ser118 Research Articles

Abstract P3-10-09: Gene deletion of IRS1 inhibited IGF-I, insulin and estradiol stimulated MCF-7L breast cancer cell growth

Abstract Targeting of the type I insulin-like growth factor receptor (IGF-IR) has not been successful in breast cancer. Data suggest the highly homologous insulin receptor (IR) may be an alternate growth stimulatory pathway used by cancer cells. Since both receptors phosphorylate the insulin receptor substrate-1 (IRS-1) protein as an immediate consequence of ligand binding, disruption of both receptors could be accomplished by suppression of IRS-1. To test this hypothesis, we created a doxycycline-inducible IRS-1 shRNA lentiviral construct to infect hormone dependent MCF-7 breast cancer cells. IRS-1 shRNA downregulation resulted in decreased responses to IGF-I, insulin, and estradiol in monolayer and anchorage independent growth assays. Decreased IRS-1 levels also suppressed estradiol stimulated gene expression and estrogen receptor binding to DNA. Xenograft growth was also inhibited by induction of IRS-1 shRNA. These data show IRS-1 is a critical regulator of endocrine responsive breast cancer. To further study the critical effects of IRS1 in ER positive breast cancer biology, CRISPR technology was used to delete IRS1 from MCF-7L cells. We analyzed 5 CRISPR IRS1 gene edited clones. Two different commercial available IRS1 antibodies were used to examine IRS1 protein expression. Clones #16, #20 did not show any IRS1 expression, while clones #22, #33 and #38 showed truncated IRS1 expression. IGFIRβ and Insulin R levels were comparable to the parent MCF-7L cells in all the clones, while IRS2 and ERα levels were significantly lower in clone #16. IRS phosphorylation, detected by PY20 antibody, was significantly lower in clone #16. The downstream signaling measured by pAKT and pErk1,2 did not change in all clones compared to those in MCF-7L. In all the clones, ERα ser118 phosphorylation by estradiol stimulation (10 nM) and ERα downregulation by fulvestrant treatment (100 nM) were not affected by IRS1 knockout. IGF-I (5 nM), insulin (10 nM) and estradiol (10 nM) stimulated monolayer cell growth in clone #16 was completely inhibited, while other clones were partially inhibited. In contrast, growth in clone #22 was not inhibited due to extremely high IRS2 expression and compensation. In conclusion, when the IRS1 gene was deleted, multiple growth stimulatory ligands (IGF-I, insulin and estradiol) were inhibited in breast cancer cells. However, IRS-2 compensated for IRS-1 gene deletion in one of the clones we studied. Thus, targeting the IRS proteins may inhibit multiple ligands that cause growth stimulation in ER positive breast cancer therapy. Citation Format: Xihong Zhang, Douglas Yee. Gene deletion of IRS1 inhibited IGF-I, insulin and estradiol stimulated MCF-7L breast cancer cell growth [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 P3-10-09.

PIK3CA mutation, reduced AKT serine 473 phosphorylation, and increased ERα serine 167 phosphorylation are positive prognostic indicators in postmenopausal estrogen receptor-positive early breast cancer.

Although endocrine therapy is the most important treatment option in estrogen receptor (ER)-positive breast cancer, new strategies, such as molecular targeted agents together with endocrine therapy are required to improve survival. PIK3CA is the most frequent mutated gene in ER-positive early breast cancers, and PIK3CA mutation status is reported to affect activation of AKT and ERα. Moreover, recent studies demonstrate that patients had a better prognosis when tumors expressed ER, androgen receptor (AR), and vitamin D receptor (VDR). In this study, we examined expression of AR and VDR, phosphorylation of AKT serine (Ser) 473 (AKT phospho-Ser473) and ERα Ser167 (ERα phospho-Ser167) by immunohistochemistry in ER-positive, HER2-negative early breast cancer. PIK3CA gene mutations were also detected in genomic DNA extracted from tumor blocks. Correlations between these biological markers, clinicopathological factors and prognosis were analyzed. Levels of AKT phospho-Ser473 were significantly higher in premenopausal women than in postmenopausal women. In contrast, AR expression was significantly higher in postmenopausal women than in premenopausal women. PIK3CA mutations were detected in 47% in premenopausal women and 47% in postmenopausal women. Postmenopausal women with PIK3CA wild-type tumors had significantly worse disease-free survival than patients with PIK3CA mutant tumors. Low levels of AKT phospho-Ser473 and high levels of ERα phospho-Ser167 were strongly associated with increased disease-free survival in postmenopausal women. Evaluation of ERα activation, in addition to PIK3CA mutation status, might be helpful in identifying patients who are likely to benefit from endocrine therapy alone versus those who are not in postmenopausal ER-positive early breast cancer.

Abstract P4-08-07: PIK3CA mutation, reduced AKT serine 473 phosphorylation, and increased ERα serine 167 phosphorylation are positive prognostic indicators in postmenopausal estrogen receptor-positive, HER2-negative early breast cancer

Abstract Background: Endocrine therapy is the most important treatment option for women with estrogen receptor (ER)-positive breast cancer. We recently reported that approximately two-thirds of patients who relapsed within 5 years had received anthracyclins and/or taxanes as adjuvant or neoadjuvant chemotherapy in addition to adjuvant endocrine therapy. New strategies, such as signal transduction inhibitors together with endocrine therapy are required to improve survival. PIK3CA mutations are detected in almost 40% of early ER-positive breast cancers, and are therefore the most frequent genetic alterations in this subtype. PIK3CA mutation status is reported to affect activation of AKT and ERα. Moreover, recent studies demonstrate that patients had a better prognosis when tumors expressed ER, androgen receptor (AR), and vitamin D receptor (VDR). Methods: Expression of AR and VDR, phosphorylation of AKT serine (Ser) 473 (AKT phospho-Ser473) and ERα Ser167 (ERα phospho-Ser167) were examined by immunohistochemistry in ER-positive, HER2-negative early breast cancer tissues. Seventeen mutations in exons 1, 4, 7, 9, and 20 of the PIK3CA gene were detected in genomic DNA extracted from formalin-fixed paraffin-embedded tumor blocks. Correlations between these biological markers and clinicopathological factors and prognosis were analyzed separately in pre- and postmenopausal women. Results: Levels of AKT phospho-Ser473 were significantly higher in premenopausal women (n = 62) than in postmenopausal women (n = 152) (P < 0.0001 and P = 0.014, respectively). In contrast, expression levels of AR were significantly higher in postmenopausal women than in premenopausal women (P < 0.0001). In premenopausal women, 26 tumors (43%) had a single mutation of PIK3CA gene, and 3 tumors (5%) had mutations at two sites. In postmenopausal women, 64 tumors (44%) had a single PIK3CA mutation, 6 tumors (4%) had mutations at two sites, and one tumor (1%) had mutations at three sites. In premenopausal women, wild type PIK3CA was associated with smaller tumor size, higher ER expression levels, and lower AR expression levels when compared with women in the same cohort with PIK3CA mutant tumors. In postmenopausal women, patients with PIK3CA wild-type tumors had higher Ki67 labeling index, higher AKT phospho-Ser473, and lower ERα phospho-Ser167 when compared to patients with PIK3CA mutant tumors. Postmenopausal women with PIK3CA wild-type tumors had significantly worse disease-free survival than patients with PIK3CA mutant tumors (P = 0.007). In contrast, PIK3CA mutation status was not correlated with survival in premenopausal women. Low levels of AKT phospho-Ser473 and high levels of ERα phospho-Ser167 were strongly associated with increased disease-free survival in postmenopausal women (P = 0.016 and P = 0.0016, respectively). Conclusion: ERα activation, in addition to PIK3CA mutation, may be biomarkers for highly endocrine-responsive tumors. This would facilitate the selection of postmenopausal ER-positive breast cancer patients who are likely to benefit from endocrine therapy alone from those who are not. Citation Format: Ishida N, Hatanaka Y, Baba M, Hagio K, Okada H, Hatanaka KC, Matsuno Y, Yamashita H. PIK3CA mutation, reduced AKT serine 473 phosphorylation, and increased ERα serine 167 phosphorylation are positive prognostic indicators in postmenopausal estrogen receptor-positive, HER2-negative early breast cancer [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 P4-08-07.

Estradiol suppresses phosphorylation of ERα serine 167 through upregulation of PP2A in breast cancer cells.

Aromatase inhibitors (AIs) are effective endocrine therapeutics for postmenopausal women with estrogen receptor (ER)α-positive breast cancer. However, the efficacy of the treatment is often limited by the onset of AI resistance, owing to the phosphorylation of ERα serine 167 (Ser167). Previous studies have indicated that hyperactivation of the phosphoinositide-3 kinase/RAC serine/threonine-protein kinase signaling pathway occurs in AI-resistant breast cancer models, which coincides with elevated levels of ERα phosphorylation at Ser167. The tumor suppressor serine/threonine-protein phosphatase 2A (PP2A) regulates the phosphatidylinositol 3-kinase/RAC serine/threonine-protein kinase signaling pathway. A previous study indicated that PP2A inhibition decreased ERα Ser167 phosphorylation and estradiol (E2)-independent cell growth. The present study investigated the potential relevance of PP2A in E2 deprivation-resistant MCF-7 cells. E2 depletion reduced the susceptibility of MCF-7 cells to inhibitors of mechanistic target of rapamycin (mTOR) and significantly increased ERα Ser167 phosphorylation and decreased expression of PP2A. Conversely, long-term E2-deprived (LTED) MCF-7 cells, a model of AI-resistant breast cancer, exhibited decreased ERα Ser167 phosphorylation and further upregulation of PP2A in E2-containing medium. The PP2A activator forskolin (FSK) significantly inhibited LTED cell proliferation by increasing the effect of everolimus (Eve), an mTOR inhibitor. In summary, the present study provides further evidence that PP2A represents a therapeutic target for AI-resistant breast cancer.

Abstract PD2-05: Inhibition of mutant HER2 results in synthetic lethality when combined with ER antagonists in ER+/HER2 mutant human breast cancer cells

Abstract Background: Human epidermal growth factor receptor 2 (ERBB2 or commonly known as HER2) missense mutations have been reported in 2-4% of breast cancers and occur primarily in the absence of HER2 gene amplification. Based on TCGA, approximately 60% of these tumors are hormone-dependent and express estrogen receptor (ER) a. Among ER+ breast cancers with HER2 missense mutations, more than 80% are in the HER2 kinase domain. We examined herein whether ER+/HER2 mutant breast cancer cells are resistant to anti-estrogen therapies and, thus, whether they should be treated with combined ER and HER2 inhibitors. Methods: Three common HER2 activating mutations (G309A, L755S, V777L) and wild type (WT) HER2 were incorporated into ER+ MCF7 cells using AAV-mediated homologous recombination. The isogenic incorporation of a heterozygous mutation more accurately represents primary human tumors as compared to transfection and overexpression of exogenous vectors. We examined cell viability and ER transcriptional activity, using an ERE-luciferase reporter, in response to estrogen deprivation and treatment with fulvestrant (a selective ER downregulator) and neratinib (an irreversible, pan-HER tyrosine kinase inhibitor), either alone or in combination. Signaling downstream mutant HER2 was examined by immunoblot analysis. In vivo anti-tumor efficacy of fulvestrant ± neratinib is currently being assessed in ovariectomized athymic mice bearing MCF7/HER2V777L xenografts. Results: MCF7 cells containing HER2 kinase missense mutations (L755S and V777L), but not cells with HER2WT or an extracellular domain mutation (G309A), were able to proliferate exponentially in estrogen-free medium. MCF7/HER2L755S and MCF7/HER2V777L were also resistant to 1 mM fulvestrant, despite fulvestrant's ability to downregulate ER in these cells. Additionally, MCF7/HER2L755S and MCF7/HER2V777L showed increased levels of pERK and p70S6K. Treatment with 200 nM neratinib potently inhibited growth of MCF7/HER2L755S and MCF7/HER2V777L in estrogen-free conditions and resensitized them to fulvestrant while partially downregulating HER2 levels. Addition of 1 nM estradiol markedly rescued all three HER2 mutant cells from neratinib-induced cell death suggesting that the inhibition of both ER and mutant HER2 is required for tumor cell apoptosis. Using ERE-luc reporter assays, neratinib did not inhibit basal or estrogen-induced ER transcriptional activity or ERα Ser118 phosphorylation, thus not supporting HER2 mutation-to-ER crosstalk in these genetically engineered cells. This result also suggests that the ER and HER2 mutant pathways can operate independently and it is the dual pathway inhibition that results in synthetic lethality. Conclusions: These data suggest that, in ER+ breast cancers and similar to HER2 gene amplification, HER2 kinase domain mutations induce resistance to antiestrogen therapies. Therefore, we propose simultaneous therapeutic targeting of both ER and HER2 signaling pathways is required for maximal inhibition of ER+ breast cancers also harboring HER2 activating mutations, as is currently being investigated in the phase II SUMMIT trial (NCT01953926). Citation Format: Croessmann S, Zabransky DJ, Cutler, Jr. RE, Lalani AS, Park BH, Arteaga CL. Inhibition of mutant HER2 results in synthetic lethality when combined with ER antagonists in ER+/HER2 mutant human breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-05.

Hyaluronate synthase-2 overexpression alters estrogen dependence and induces histone deacetylase inhibitor-like effects on ER-driven genes in MCF7 breast tumor cells

In breast carcinoma cells, high levels of hyaluronan (HA) and its CD44 receptor are frequently associated with alteration in estrogen signaling. We demonstrate that stable hyaluronate synthase 2 (HAS2) overexpression in estrogen receptor α (ERα) -positive MCF7 cells oppositely altered estrogen dependence of cell growth and its sensitivity towards antiestrogens. Albeit without effect on ERα expression and estradiol binding properties, HAS2 overexpression increased ERα Ser118 phosphorylation as well as transcriptional activity of estrogen in an ERE-luciferase reporter gene assay. However, HAS2 overexpression induced partial silencing of E2 driven-genes without affecting the magnitude of regulation by estradiol. This effect was associated with half-reduction in the activity of nuclear histone deacetylases (HDACs) through a post-translational mechanism likely consecutive to the enhanced expression of the histone acetyl-transferase EP300. In conclusion, increase in HA/CD44 interactions may contribute, through an HDAC inhibitor-like and ER-independent mechanism, to the silencing of estrogen–driven genes in breast carcinoma.

Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes

Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM–10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated).

Non-tumorigenic epithelial cells secrete MCP-1 and other cytokines that promote cell division in breast cancer cells by activating ERα via PI3K/Akt/mTOR signaling

Efforts in understanding the role of the microenvironment in the development of breast cancer have focused on tumor-stroma cross-talk, but the possibility that normal epithelial cells might also play a role in tumor progression has received little attention. Here, we show that non-tumorigenic human mammary epithelial cells (MCF10A and HMEC) secrete factors able to enhance the proliferation of estrogen receptor α (ERα) positive breast cancer cells (MCF7 and T47D) and suppress their ability to undergo apoptosis. Conditioned medium (CM) derived from MCF10A and HMEC cells was capable of activating ERα in a hormone-independent way, by phosphorylating ERα on Ser167. Co-exposure with PI3K and mTORC1 inhibitors significantly reduced the ERα Ser167 phosphorylation and suppressed the proliferation-enhancing effects of both 10A-CM and HMEC-CM on MCF7 cells. We show that MCF10A and HMEC secrete numerous cytokines, among them MCP-1, which was one of the most prevalent. MCP-1 was shown to have a role in the effects elicited by the 10A-CM. It activated the ERα by phosphorylating Ser167 via the PI3K/Akt/mTORC1 signaling pathway, an effect which was further confirmed by silencing the MCP-1 receptors, CCR2 and CCR4. To our knowledge, this is the first time MCP-1 has been shown to contribute to ERα signaling activation.These data suggest that normal mammary cells could have the capability of supporting the proliferation of breast cancer cells via paracrine interactions. A better understanding of the role of these cells may be useful for designing strategies for the prevention of tumor progression at early stages.

Xenoestrogens Alter Estrogen Receptor (ER) α Intracellular Levels

17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

LXRβ/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity

Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRβ has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRβ to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRβ and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRβ- and ERα-dependent processes. These studies demonstrate that LXRβ has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.

Phosphorylation of Estrogen Receptor α at Serine 118 Directs Recruitment of Promoter Complexes and Gene-Specific Transcription

Phosphorylation of estrogen receptor α (ERα) is important for receptor function, although the role of specific ERα phosphorylation sites in ERα-mediated transcription remains to be fully evaluated. Transcriptional activation by ERα involves dynamic, coordinate interactions with coregulators at promoter enhancer elements to effect gene expression. To determine whether ERα phosphorylation affects recruitment of unique protein complexes at gene-specific promoters, changes in ERα Ser118 phosphorylation were assessed for effects on receptor and coregulator recruitment and transcription of ERα-regulated genes. Chromatin immunoprecipitation assays to measure promoter association found a 17β-estradiol (E2)-dependent recruitment of ERα at 150 min to ERα-regulated promoters, whereas ERα phosphorylated at Ser118 was dissociated from promoters after E2 treatment. Mutation of Ser118 to alanine (S118A) altered unliganded and ligand-induced association of ERα and p160 coregulators with ERα target promoters when compared with wild-type (WT)-ERα transfection. S118A and WT-ERα exhibited a similar level of recruitment to the estrogen response element-driven pS2 promoter and induced pS2 mRNA after E2 treatment. Although WT-ERα was recruited to c-myc and cyclin D1 promoters after E2 treatment and induced mRNA expression, S118A exhibited reduced interaction with c-myc and cyclin D1 promoters, and E2 did not induce c-myc and cyclin D1 mRNA. In addition, S118A resulted in increased recruitment of steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and activated in breast cancer-1 to pS2, c-myc, and cyclin D1 irrespective of the presence of E2. Together, these data indicate that site specific phosphorylation of ERα directs gene-specific recruitment of ERα and transcriptional coregulators to ERα target gene promoters.

Abstract 5180: The neuroreticular axis modulates cell signaling kinases in bone metastasis

Abstract Bone metastasis is a main cause of cancer associated pain and mortality. The activation of osteoblasts and osteoclasts by cancer cells generates a vicious cycle of bone destruction and increased tumor growth, causing pain, fractures and spinal cord compression. In this study the microenvironment of human bone metastasis is being examined from a new point of view: the neuroreticular axis. The sympathetic nervous system regulates the homing and survival of bone marrow immune stem cells within a structural bone network called the neuroreticular complex. The neuroendocrine axis permits stem cells to survive in the bone microenvironment, regulates B and T cells and influences mesenchymal stem cells (MSC). Estrogen/androgen ratio is a fundamental regulator of bone development and turnover, inducing osteoblast or osteoclast differentiation. Platelet-derived serotonin (5-HT) is involved in bone metabolism via release from small intestine enterochromaffin cells to induce bone lysis or neuronal release, supporting bone consolidation. The aims are a) identify activated kinase pathway interconnections in each bone metastasis from different primary tumors to identify predictive/prognostic biomarkers, b) identify targets for molecular targeted therapies. 142 bone metastases frozen samples from different types of primary carcinomas (13 head and neck, 45 urogenital, 16 gastro enteric, 14 breast, 25 lung, 7 melanoma and 22 unknown carcinomas) and 24 from primary sarcomas were lysed using Covaris adaptive focused acoustic technology. Levels of 100 post-translationally modified cell signaling kinases were quantified by Reverse Phase Protein Microarray (RPMA). RPMA provides a quantitative “circuit map” of pathways that are activated/deactivated in a sample of tumor cells obtained from a biopsy. The metastasis originating from different primary tumors showed similar levels of cell signaling across tissue types for the majority of proliferation, invasion and adhesion pathway proteins analyzed. Sex hormone receptor expression was found to be similar among the metastasis samples from different carcinoma types, while bone metastasis from sarcomas showed a statistically significant higher expression compared to those originating from carcinomas (ERα Ser118 p<0.001, PR Ser190 p<0.005). No differences were found in serotonin levels among the bone metastasis derived from different tumor types (carcinomas and sarcomas). This data suggests that the bone microenvironment influences the signaling profiles of the metastatic tumor (seed vs. soil hypothesis). Therapies targeting bone metastasis are actually under evaluation but with poor success on prolonging patient survival. The results of this study may provide the opportunity to develop therapies targeting the bone microenvironment via an unexplored pathway, the neuroendocrine axis. 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 5180. doi:10.1158/1538-7445.AM2011-5180

Expression of Estrogen Receptor Beta and Phosphorylation of Estrogen Receptor Alpha Serine 167 Correlate with Progression-Free Survival in Patients with Metastatic Breast Cancer Treated with Aromatase Inhibitors

Aromatase inhibitor (AI) is widely used as an endocrine treatment in postmenopausal patients with hormone receptor-positive breast cancer. To identify useful prognostic factors for patients with metastatic breast cancer treated with AI therapy, we investigated the association between several hormone receptor-related factors and prognosis. The expressions of estrogen receptor-α (ERα), ERβ, progesterone receptor, the phosphorylation of ERα serine 118 (Ser118) and ERα Ser167 were examined using immunohistochemical techniques for the primary tumors of 41 patients with metastatic breast cancer who received first-line AI therapy after relapse. To assess the associations of protein expression and phosphorylation levels with progression-free survival (PFS), the levels of each factor were categorized into low and high values at optimal cutoff points. In univariate analysis, high ERα expression and high ERα Ser167 phosphorylation correlated with longer PFS (p = 0.016 and 0.013, respectively). In multivariate analysis, low ERβ expression and high ERα Ser167 phosphorylation correlated with longer PFS (p = 0.031 and 0.004, respectively). Patients with both low ERβ expression and high ERα Ser167 phosphorylation had longer PFS than the others (p = 0.0107). These data suggest that the expression of ERβ and phosphorylation of ERα Ser167 may be useful prognostic factors in patients with metastatic breast cancer who received first-line AI therapy.

Predictors of Response to Exemestane as Primary Endocrine Therapy in Estrogen Receptor-Positive Breast Cancer.

Abstract Endocrine therapy is the most important treatment of choice for estrogen receptor-positive breast cancer. Potential mechanisms for resistance to endocrine therapy involve estrogen receptor (ER)-coregulatory proteins and cross-talk between ER and other growth factor-signaling networks. However, the factors and pathways responsible for endocrine therapy resistance, particularly resistance to aromatase inhibitors, have not been clearly established. Sixteen postmenopausal patients with ERΑ-positive primary breast cancer were treated daily with 25 mg of exemestane (an aromatase inhibitor) for 6 months. Expressions of ERΑ, ERΒ, progesterone receptor (PgR), androgen receptor (AR), AIB1, aromatase, EGFR, HER2, Ki67, cyclin D1, p53, Bcl2, Stat5 and IGFBP5, and phosphorylations of ERΑ serine (Ser) 118, ERΑ Ser167, Akt Ser473 and p44/42 MAPK threonine (Thr) 202/ tyrosine (Tyr) 204 were examined by immunohistochemistry on pretreatment tumor biopsies and posttreatment surgical specimens. Analyses were made to test for correlations with response to exemestane. Of the 16 patients, 7 responded and 9 retained stable disease. High-level expression of AIB1 and phosphorylation of Akt Ser473 were significantly associated with a better response to exemestane, suggesting that these factors could be considered as predictors of exemestane response. Expressions of ERΑ, ERΒ, PgR, aromatase, Ki67, cyclin D1 and p53, and phosphorylations of ERΑ Ser118, ERΑ Ser167 and p44/42 MAPK Thr202/Tyr204 were decreased, whereas expressions of Stat5 and IGFBP5 were increased in posttreatment specimens compared to the values in pretreatment biopsies. Thus, the analysis of factors involved in the estrogen-dependent growth-signaling pathways may be useful in identifying patients responsive to exemestane. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2041.

Abstract A31: mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Abstract Purpose of the Study: Phosphorylation of estrogen receptor α (ERα) Ser167 is associated with development of endocrine resistance, and is a prognostic marker for the disease. Hereby we dissect the signaling pathways responsible for Ser167 phosphorylation. Experimental Procedures: MCF7 cells were grown in standard media. For experiments, cells were deprived of serum for 24h, and were pre-treated with 20ng/mL rapamycin for 30 min and/or 10 µM U0126 for 2h, and then stimulated with 100nM insulin or 100ng/mL PMA. Cell extracts were prepared, and proteins were resolved on SDS-PAGE, followed by immunoblots. For the siRNA studies, double-stranded RNAs for RSK1 and RSK2 were transfected using Lipofectamine2000 (Invitrogen). At 72h post-transfection, cells were lysed using 1X Passive Lysis Buffer (Promega). For luciferase reporter assays, cells were transfected with plasmids encoding for firefly luciferase under control of three ERE, and control renilla luciferase. At 48h post-transfection, cells were harvested and relative luciferase activity was measured using the Dual Luciferase Reporter Assay System and Glomax 20/20 luminometer (Promega). Data: mTOR/S6K1 and MAPK/RSK contribute non-overlapping inputs into ERα activation via Ser167 phosphorylation. The degree of input of each pathway depends on the nature of the activating signal. In unsynchronized cells that are growing in the presence of serum or in cells acutely stimulated with insulin, which preferentially activated the mTOR/S6K1 pathway, we observed that the MAPK/RSK pathway has a minor contribution to Ser167 phosphorylation. However, the MAPK/RSK contribution is more evident following acute stimulation with PMA, which activates both the mTOR/S6K1 and MAPK/RSK pathways. In response to PMA we observed significant rapamycin-insensitive Ser167 phosphorylation, which was U0126-sensitive. Using RNAi against RSK1/2 isoforms we demonstrated that this rapamycin-insensitive phosphorylation is mediated by RSK. In addition to being sensitive to different stimuli, the mTOR and MAPK pathways are also activated with different kinetics. S6K1 and RSK perform a non-redundant role with regard to ERα Ser167 phosphorylation, and are responsible for a biphasic phosphorylation pattern. The early-activated RSK acts in a priming role, while the late-responding S6K1 is responsible for the maintenance of high-level phosphorylation. Estrogen receptor is an important clinical target in treatment of breast cancer. However, resistance to anti-estrogen therapy often develops. One of the mechanisms of resistance is through mitogenic estrogen-independent activation of ERα. Thus, a question arises whether dual inhibition of the mTOR and MAPK pathways may result in a better clinical response. We investigated the effect of a combination of rapamycin and U0126 on transcriptional activity mediated by ERα. We demonstrated that the combination produces a greater inhibition of ERα activity than each agent alone. This strategy may be effective for combating resistance to anti-estrogen or endocrine therapy. Conclusions: The data presented here reveal that the effect of rapamycin therapy in breast cancer could be augmented by combination with MEK inhibitors, which would target two major pathways leading to ERα activation and stimulation of breast cancer cell proliferation. Citation Information: Cancer Res 2009;69(23 Suppl):A31.

Endocrine therapy resistance can be associated with high estrogen receptor α (ERα) expression and reduced ERα phosphorylation in breast cancer models

Hormone-dependent estrogen receptor (ER)-positive breast cancer cells may adapt to low estrogen environments such as produced by aromatase inhibitors. In many instances, cells become insensitive to the effects of estrogen but may still retain dependence on ER. We have investigated the expression, function, and activation of ERalpha in two endocrine-resistant MCF-7 models to identify mechanisms that could contribute to resistance. While MCF-7/LCC1 cells are partially estrogen dependent, MCF-7/LCC9 cells are fully estrogen insensitive and fulvestrant and tamoxifen resistant. In both MCF-7/LCC1 and MCF-7/LCC9 cell lines, high expression of ERalpha was associated with enhanced binding to the trefoil factor 1 (TFF1) promoter in the absence of estrogen and increased transcription of TFF1 and progesterone receptor. In contrast to the observations derived from hypersensitive and supersensitive models, these cells were truly estrogen independent; nevertheless, removal of ERalpha by siRNA, or fulvestrant, a specific ER downregulator, inhibited growth indicating dependence on ERalpha. In the absence of estrogen, neither ERalpha Ser118 nor Ser167 were phosphorylated as frequently found in other ligand-independent cell line models. Addition of estrogen activated ERalpha Ser118 in MCF-7 and LCC1 cells but not in LCC9 cells. We suggest that the estrogen-independent growth within these cell lines is accounted for by high levels of ERalpha expression driving transcription and full estrogen independence explained by lack of ERalpha activation through Ser118.

An Inherent Role of Integrin-Linked Kinase-Estrogen Receptor α Interaction in Cell Migration

Integrin-linked kinase (ILK) and estrogen receptor (ER)-alpha modulate cell migration. However, the crosstalk between ERalpha and ILK and the role of ILK in ERalpha-mediated cell migration remain unexplored. Here, we report that ILK participates in ERalpha signaling in breast cancer cells. We found that ILK binds ERalpha in vitro and in vivo through a LXXLL motif in ILK. Estrogen prevented ERalpha-ILK binding, resulting in phosphatidylinositol 3-kinase (PI3K)-dependent increase in ILK kinase activity. Furthermore, the regulation of ERalpha-ILK interaction was dependent on the PI3K pathway. Unexpectedly, transient knockdown or inhibition of ILK caused hyperphosphorylation of ERalpha Ser(118) in an extracellular signal-regulated kinase/mitogen-activated protein kinase pathway-dependent manner and an enhanced ERalpha recruitment to the target chromatin and gene expression, a process reversed by overexpression of ILK. Compatible with these interactions, estrogen regulated cell migration via the PI3K/ILK/AKT pathway with stable ILK overexpression hyperactivating cell migration. Thus, status of ILK signaling may be an important modifier of ER signaling in breast cancer cells and this pathway could be exploited for therapeutic intervention in breast cancer cells.

Phosphorylation of estrogen receptor α serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer

IntroductionEndocrine therapy is the most important treatment option for women with hormone-receptor-positive breast cancer. The potential mechanisms for endocrine resistance involve estrogen receptor (ER)-coregulatory proteins and crosstalk between ER and other growth factor signaling networks. However, the factors and pathways responsible for endocrine resistance are still poorly identified.MethodsUsing immunohistochemical techniques, we focused on the expression and phosphorylation of hormone receptors themselves and examined the phosphorylation of ER-α Ser118 and ER-α Ser167 and the expression of ER-α, ER-β1, ER-βcx/β2, progesterone receptor (PR), PRA, and PRB in the primary breast carcinomas of 75 patients with metastatic breast cancer who received first-line treatment with endocrine therapy after relapse.ResultsPhosphorylation of ER-α Ser118, but not Ser167, was positively associated with overexpression of HER2, and HER2-positive tumors showed resistance to endocrine therapy. The present study has shown for the first time that phosphorylation of ER-α Ser167, but not Ser118, and expression of PRA and PRB, as well as ER-α and PR in primary breast tumors are predictive of response to endocrine therapy, whereas expression of ER-β1 and ER-βcx/β2 did not affect response to the therapy. In addition, patients with either high phosphorylation of ER-α Ser167, or high expression of ER-α, PR, PRA, or PRB had a significantly longer survival after relapse.ConclusionThese data suggest that phosphorylation of ER-α Ser167 is helpful in selecting patients who may benefit from endocrine therapy and is a prognostic marker in metastatic breast cancer.

Elevated ERK1/ERK2/estrogen receptor cross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation

The knowledge that steroids play a pivotal role in the development of breast cancer has been exploited clinically by the development of endocrine treatments. These have sought to perturb the steroid hormone environment of the tumour cells, predominately by withdrawal or antagonism of oestrogen. Unfortunately, the beneficial actions of existing endocrine treatments are attenuated by the ability of tumours to circumvent the need for steroid hormones, whilst in most cases, retaining the nuclear steroid receptors. The mechanisms involved in resistance to estrogen deprivation are of major clinical relevance for optimal treatment of breast cancer patients and the development of new therapeutic regimes. We have shown that long-term culture of MCF7 cells in medium depleted of oestrogen (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of both ERalpha phosphorylated on Ser(118) and ERK1/ERK2. Our data suggest elevated ERK1/ERK2 activity results wholly or in part from enhanced ERBB2 expression in the LTED cells. These cells showed greater sensitivity to the tyrosine kinase inhibitor ZD1839 in both ERalpha-mediated transcription and growth assays compared with the wt-MCF7. Similarly the MEK inhibitor U0126 decreased basal ERalpha-mediated transcription and proliferation in the LTED cells by 50% and reduced their sensitivity to the proliferative effects of E2 10-fold, whilst having no effect on the wild type (wt). However, complete suppression of ERK1/ERK2 activity in the LTED cells did not inhibit ERalpha Ser(118) phosphorylation suggesting that the cells remained ligand-dependent. This was further confirmed by the increased sensitivity of the LTED cells to the growth suppressive effects of ICI 182,780 and suggested that the LTED cells remained wholly or partially dependent on oestrogen receptor (ER)/oestrogen responsive elements directed growth. These findings suggest that treatments targeted at growth factor signalling pathways may be useful in patients acquiring resistance to oestrogen deprivation with aromatase inhibitors and that the pure anti-oestrogen ICI 182,780 may also be effective by blocking or destabilizing ER and hence disrupting cross-talk.

The interaction of the ER with ERBB2 and PI3K results in elevated levels of AKT and p90RSK in tamoxifen-resistant MCF-7 cells

Despite advances in endocrine therapies, the majority of patients receiving tamoxifen will eventually relapse while retaining functional estrogen receptors. We have previously shown, using an MCF-7 cell line (TamR) resistant to the anti-proliferative effects of tamoxifen, that elevated levels of phosphorylated AKT and p90RSK lead to an apparent ligand-independent phosphorylation of ERα ser167. Analysis of the growth factor receptors in these cells indicated elevated levels of both phosphorylated ERBB2 and total ERBB3, which we postulated formed heterodimers and activated the PI3 kinase pathway leading to elevated AKT. However, our recent data suggest that elevated AKT results from an interaction between ERα and ERBB2. This association is knocked out by treatment with the pure anti-estrogen ICI 182,780 and is absent in the WT parental cell line. Similarly, we have demonstrated an association between the p85 subunit of PI3K and ERα in TamR but not in WT cells. Treatment of the cell lines with the specific AKT inhibitor SH6 and the MEK1/2 inhibitor U0126 caused greater decrease in cell proliferation and concomitant ERα-directed transactivation in the TamR cells versus the WT, confirming that these pathways are integral to the TamR phenotype. To establish whether p90RSK or AKT was responsible for the phosphorylation of the estrogen receptor at ser167, TamR and WT cells were treated with SH6, U0126 or a combination of the two. Blocking either pathway individually had little effect on ERα ser167phosphorylation. However, a combination of the two inhibitors resulted in almost complete loss of phosphorylation. These data were confirmed using siRNA technology to suppress MAPK and AKT expression. Taken together these data suggest that, in this setting, the ER functions via a non-genomic mechanism, associating with ERBB2 and PI3K at the cell membrane leading to activation of both p90RSK and AKT. This in turn leads to phosphorylation of ERα ser167, ultimately regulating cell growth via genomic mechanisms. Although several of these complexes have previously been postulated, to our knowledge this is the first demonstration of this phenomenon in a tamoxifen-resistant cell line.