ER Activity Research Articles

Anatomically‐Specific Actions of Oestrogen Receptor in the Developing Female Rat Brain: Effects of Oestradiol and Selective Oestrogen Receptor Modulators on Progestin Receptor Expression

Steroid hormones largely exert their actions by activating nuclear receptors, which, as transcription factors, powerfully influence fundamental processes of neural development. Often, steroid receptor action demonstrates remarkable specificity under different developmental, anatomical or hormonal conditions. Yet, the mechanisms underlying such specificity are poorly understood. The present study examined the anatomically-specific regulation of progestin receptor (PR) expression by oestrogen receptor (ER) activation in the ventromedial nucleus (VMN) of the hypothalamus and the medial preoptic nucleus (MPN) of the neonatal female rat brain, using the selective oestrogen receptor modulators (SERMs), tamoxifen and ICI 182780 (ICI), in the presence or absence of oestradiol benzoate (EB) treatment. The results demonstrate that PR immunoreactivity (PR-ir) in the neonatal female MPN was significantly increased by EB and this increase was abolished by either tamoxifen or ICI treatment. In contrast, within the VMN of the same animals, EB had no effect on PR-ir and the SERMs only modestly decreased PR-ir. Interestingly, ICI acted as a true antagonist regardless of EB treatment, whereas tamoxifen acted as an ER agonist in the absence of EB in the MPN, but not the VMN, representing one of the first in vivo demonstrations of tissue-specific and oestradiol-independent effects of tamoxifen on ER activation. The present results indicate that PR expression is highly dependent on oestradiol and its receptor in the MPN, although it is independent of both oestradiol and ER activation within the neonatal VMN. These findings demonstrate the anatomically-specific actions of oestradiol and its receptor to induce PR in two brain regions controlling different aspects of female reproductive behaviours in adulthood.

PPARδ Activation Rescues Pancreatic β-Cell Line INS-1E from Palmitate-Induced Endoplasmic Reticulum Stress through Enhanced Fatty Acid Oxidation.

One of the key factors responsible for the development of type 2 diabetes is the loss of functional pancreatic β cells. This occurs due to a chronic exposure to a high fatty acid environment. ER stress is caused by an accumulation of irreversible misfold or unfold protein: these trigger the death of functional pancreatic β cells. PPARδ is an orphan nuclear receptor. It plays a pivotal role in regulating the metabolism of dietary lipids and fats. However, the correlation between PPARδ of fatty acids and ER stress of pancreatic β cells is not quite clear till date. Here, we show that PPARδ attenuates palmitate-induced ER stress of pancreatic β cells. On the other hand, PPARδ agonist inhibits both abnormal changes in ER structure and activation of signaling cascade, which is downstream ER stress. Further, we illustrate that PPARδ attenuates palmitate-induced ER stress by promoting fatty acid oxidation through treatment with etomoxir, an inhibitor of fatty acid oxidation. It dramatically abolishes PPARδ-mediated inhibition of ER stress. Finally, we show that PPARδ could protect pancreatic β cells from palmitate-induced cell death and dysfunction of insulin secretion. Our work elucidates the protective effect of PPARδ on the fatty-acid-induced toxicity of pancreatic β cells.

P1-17-06: A Phase II Trial of the CDK 4/6 Inhibitor PD0332991 in Women with Advanced Breast Cancer.

Abstract Background: Dysregulation of the G1/S checkpoint of the cell cycle is a feature of many breast cancers. PD0332991, a potent oral inhibitor of cyclin-dependent kinases (CDKs) 4 and 6 is well-tolerated and has demonstrated activity in a phase I trial in a variety of solid tumors at a phase II dose of 125 mg daily on a 3 week on/1 week off schedule. Preclinical data suggest that this agent is most active in ER+ (luminal) breast cancers. We are performing a phase II study of PD0332991 in women with advanced breast cancer, one of several parallel disease cohorts under study. Methods: Patients with histologically-confirmed stage IV breast cancer were eligible if they had primary or metastatic tumor which stained positive for retinoblastoma (Rb) protein by immunohistochemistry, disease measureable by RECIST criteria and adequate organ function/performance status. Treatment was initiated with PD0332991 at 125 mg orally, days 1 - 21 of a 28-day treatment cycle. Tumor assessments occurred after every 2 cycles (8-week intervals). The primary objective was to determine the safety and response rates in cohorts of 15 patients per tumor type; 15 patients per arm provided 80% power to detect a 15% (1/15) response rate per disease that would lead to further cohort expansion. Secondary objectives include PK, PD and predictive biomarker assessment. Results: 36 patients were screened, 32 (89%) stained positive for Rb, and 14 have enrolled on study. The only reported toxicites are neutropenia (7 patients, 4 grade 3/4), thrombocytopenia (1 patient, grade 1) and fatigue (1 patient, grade 2). 3 patients (23%) have had dose interruptions and 5 (38%) have had dose-reduction for neutropenia, though no episodes of febrile neutropenia have occurred. Among 11 patients assessable for response to date, there is 1(7%) partial response (PR), 6 (43%) with stable disease (SD) and 4 (29%) with progressive disease (PD). 3 of 6 patients with stable disease have received greater than 6 months of therapy, and these sustained responses have occurred with dosing as low as 50 mg/day. All PR/SD have occurred in patients with ER+ tumors; all PD have been in patients with triple negative (ER-/PR-/Her2-) disease. The cyclin D1 status of all patients are being assessed. Of the 10 ER+ patients, 3 are cyclin D1 amplified, 5 are non-amplified and 2 are pending assessment. 2 in 4 cyclin D1 non-amplified patients had SD, while 2 of 2 evaluable patients with amplification had SD. PK and PD analyses are in progress. Conclusions: PD 0332991 is an extremely well-tolerated, oral CDK 4/6 inhibitor that demonstrates prolonged single-agent activity in ER+ breast cancer patients who have progressed on hormonal therapy. These data have prompted expansion of this breast cancer cohort to further delineate activity and translational studies examining predictors of response are underway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-17-06.

Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E(2)), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E(2), 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E(2) increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects.

Hereditary Thrombocytosis: The Thrombopoietin Receptor c-MPL Can Activate Its Downstream Pathway without Full Glycosylation and Membrane Expression

Abstract 2374Thrombopoiesis is tightly regulated by the interaction between thrombopoietin (TPO) and its receptor c-Mpl. Receptor binding also leads to the clearance of TPO from the plasma thus establishing a negative feedback loop. However, it is still an open question how the receptor activates its downstream pathway. Alternative models posit that ligand binding either results in receptor dimerization in the plasma membrane or leads to conformational change of preformed receptor dimers.Several mutations in the TPO and the c-Mpl receptor genes have been linked to either hereditary thrombocytopenia or thrombocytosis. We focused on mutations in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur. Mutated homozygous c-Mpl R102P and compound heterozygous R102P/F104S receptors cause severe hereditary thrombocytopenia. In contrast, the homozygous c-Mpl P106L mutation was found in patients with hereditary thrombocytosis. We now addressed the question of how the disparate phenotype of mutations in the same domain of the c-Mpl receptor can be explained. We first functionally analyzed and compared normal with mutated R102P, F104S and P106L c-Mpl receptors in transfected HeLa and BA/F3 cells and found that the normal and the F104S c-Mpl receptors are glycosylated normally by the Golgi apparatus and reach the plasma membrane. In contrast, the R102P and P106L mutated receptors are not fully glycosylated, do not reach the plasma membrane and are atypically distributed in the ER.Functional analysis of the TPO/c-Mpl signaling pathway in BA/F3 cells showed decreased phosphorylation of Stat3, Stat5 and Erk1/2 with the R102P and F104S mutants when compared to normal. By contrast, TPO/c-Mpl signaling was up-regulated in cells transfected with the P106L-mutated receptor. Moreover, the P106L mutant, but not the other mutant receptors, enhanced ligand-independent growth of transfected BA/F3 cells.Despite of their opposite function, the TPO plasma levels of patients carrying both, homozygous R102P and P106L mutations were elevated 10 to 20-fold compared to normal and heterozygous individuals. This finding, together with their impaired glycosylation and inability to reach the plasma membrane, suggests that these mutants do not bind and internalize their ligand. TPO binding and degradation thus requires the receptor to be expressed at the plasma membrane, whereas, surprisingly, c-Mpl P106L activated its signaling pathway in a ligand independent fashion. Correct receptor processing and function can thus be separated. This indicates that TPO binding is required for regulation but that the constitutive activation of c-Mpl P106L is a likely direct consequence of premature receptor dimerization in the ER, auto-phosphorylation and subsequent activation of downstream targets. Disclosures:No relevant conflicts of interest to declare.

Preparation, characterization and performance of a novel visible light responsive spherical activated carbon-supported and Er 3+:YFeO 3-doped TiO 2 photocatalyst

A novel spherical activated carbon (SAC) supported and Er 3+:YFeO 3-doped TiO 2 visible-light responsive photocatalyst (Er 3+:YFeO 3/TiO 2-SAC) was synthesized by a modified sol–gel method with ultrasonic dispersion. It was characterized by scanning electron microscope (SEM), energy dispersive X-ray spectroscope (EDS), powder X-ray diffractometer (XRD) and UV–vis diffuse reflectance spectrophotometer (DRS). The photocatalytic activity of Er 3+:YFeO 3/TiO 2-SAC was evaluated for degradation of methyl orange (MO) under visible light irradiation. The effects of calcination temperature and irradiation time on its photocatalytic activity were examined. The experimental results indicated that Er 3+:YFeO 3 could function as an upconversion luminescence agent, enabling photocatalytic degradation of MO by TiO 2 under visible light. The Er 3+:YFeO 3/TiO 2 calcinated at 700 °C showed the highest photocatalytic capability compared to those calcinated at other temperatures. The photocatalytic degradation of MO followed the Langmuir–Hinshelwood kinetic model. Although the photocatalyst showed a good physical stability and could tolerate a shear force up to 25 × 10 −3 N/g, its photocatalytic activity decreased over a four-cycle of reuse in concentrated MO solution, indicating that the decreased activity was ascribed to the fouling of catalyst surface by MO during the degradation process. However, the fouled Er 3+:YFeO 3/TiO 2-SAC could be regenerated through water rinsing-calcination or acid rinsing-calcination treatment.

The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin

Tamoxifen resistance has been largely attributed to genetic alterations in the epithelial tumor cells themselves, such as overexpression of HER-2/Neu. However, in the clinic, only about 15-20% of cases of HER-2/Neu amplification has actually been correlated to the acquisition of endocrine resistance, suggesting that other mechanisms must be involved as well. Using the epithelial LM05-E and the fibroblastic LM05-F cell lines, derived from the estrogen dependent spontaneous M05 mouse mammary tumor, as well as MCF-7 cells, we analyzed whether soluble stromal factors or extracellular matrix components protected against tamoxifen induced cell death. Involvement of signaling pathways was determined by using specific inhibitors and western blot, and phosphorylation of the estrogen receptor alpha by western blot and immunofluorescence. Soluble factors produced by the fibroblastic cells protect the epithelial tumor cells from tamoxifen-induced cell death through a mechanism that involves EGFR and matrix metalloproteinases upstream of PI3K/AKT. Exogenous fibronectin by itself confers endocrine resistance through interaction with β1 integrin and activation of PI3K/AKT and MAPK/ERK 1/2 pathways. The conferred resistance is reversed by blocking β1 integrin. We show also that treatment with both conditioned medium and fibronectin leads to the phosphorylation of the estrogen receptor at serine-118, suggesting stromal factors as modulators of ER activity. Our results show that the tumor microenvironment can modulate tamoxifen resistance, providing an alternative explanation for why patients become refractory to hormone-therapy.

Estrogen receptor-α directly regulates sensitivity to paclitaxel in neoadjuvant chemotherapy for breast cancer

Neoadjuvant chemotherapy (NAC) has become the standard treatment for advanced breast cancer. Several prognostic markers, including estrogen receptor-α (ERα), are used to predict the response to NAC. However, the molecular significance of ERα expression in the efficacy of chemotherapy is not yet fully understood. To examine this issue, we first evaluated ERα transcriptional activity in breast cancer cells derived from pre-NAC specimens using estrogen response element-green fluorescent protein (ERE-GFP) as a reporter gene, and found that, in the cases for which ERα activities determined by GFP expression were not detected or low, pCR (pathological complete response) could be achieved even though ERα protein was expressed. Next, we examined the effects of alterations in ERα expression levels on sensitivity to paclitaxel, a key drug in NAC, by stable expression of ERα in ER-negative SKBR3 cells and by siRNA-mediated down-regulation of ERα in ER-positive MCF-7 cells, and showed that ERα expression and sensitivity to paclitaxel showed an inverse correlation. We also established paclitaxel-resistant MCF-7 cell clones and found that they have higher estrogen-induced ER activity than parent cells. Paclitaxel is a microtubule-stabilizing agent, while HDAC6 (histone deacetylase 6), which we previously identified as an estrogen-regulated gene, enhances cell motility by destabilizing microtubules via deacetylation of α-tubulin. Finally, we demonstrate herein that ERα knockdown in MCF-7 cells prevents deacetylation of α-tubulin, thereby increasing sensitivity to paclitaxel. Taken together, these results suggest that ERα expression directly regulates sensitivity to paclitaxel in NAC for breast cancer via the effect on microtubule stability.

ER and PI3K Independently Modulate Endocrine Resistance in ER-Positive Breast Cancer

Endocrine therapy-resistant estrogen receptor-positive (ER(+)) breast cancer is the most common cause of breast cancer death. Miller and colleagues demonstrate that ligand-independent ER activity promotes the growth of breast cancer cells through CDK4/E2F. As an independent event, the phosphatidylinositol 3-kinase (PI3K) pathway is also upregulated in endocrine therapy-resistant cells. Promising preclinical evidence by several groups for the combination of an inhibitor of ligand-independent ER, fulvestrant, with PI3K inhibition, has led to the activation of trials evaluating this concept.

Repeated and long-term treatment with physiological concentrations of resveratrol promotes NO production in vascular endothelial cells

In the present study, we examined the effect of repeated and long-term treatment with resveratrol on NO production in endothelial cells as a model of routine wine consumption. Repeated treatment with resveratrol for 5 d resulted in an increase in endothelial NO synthase (eNOS) protein content and NO production in human umbilical vein endothelial cell (HUVEC) in a concentration-dependent manner. A significant increase in functional eNOS protein content was observed with resveratrol, even at 50 nm. In contrast, eNOS phosphorylation was not stimulated and inducible NO synthase (iNOS) was not detected after resveratrol treatment. Both eNOS protein and mRNA expression were promoted by 50 nm-resveratrol in a time-dependent manner. Increased eNOS mRNA expression in response to resveratrol was not decreased by an oestrogen receptor (ER) antagonist ICI182780, a PPARα inhibitor MK886 or a sirtuin inhibitor Salermide. However, a combination of ICI182780 and MK886 significantly inhibited resveratrol-induced eNOS mRNA expression. Salermide had no effect even in the presence of ICI182780 or MK886. These results demonstrate that resveratrol within the physiological range increases eNOS mRNA and protein expression through ER and PPARα activation, thereby promoting NO production in endothelial cells. eNOS induction might result from the accumulative effect of nanomolar concentrations of resveratrol. The present study results can account in part for the observation that cardiovascular benefits of red wine are experienced with routine consumption, but not with acute consumption.

Enhancement of sonocatalytic performance of TiO 2 by coating Er 3+:YAlO 3 in azo dye degradation

In this work, the Er 3+:YAlO 3 as an up-conversion luminescence agent was prepared by sol–gel and auto-combustion method. And then the Er 3+:YAlO 3/TiO 2 composite, a novel sonocatalyst, was successfully synthesized by sol–gel method. The key influences such as amount of coating Er 3+:YAlO 3, heat treatment temperature and heat treatment time on the sonocatalytic activity of Er 3+:YAlO 3/TiO 2 composites, as well as the amount of Er 3+:YAlO 3/TiO 2 on the sonocatalytic degradation of organic dye was studied. The results indicated that the sonocatalytic activity of TiO 2 could be enhanced obviously by coating Er 3+:YAlO 3. When 1.0 g/L Er 3+:YAlO 3/TiO 2 composite with 50 wt.% amount of coated Er 3+:YAlO 3 and mixed crystal of TiO 2 film was adopted, the highest degradation efficiency was obtained. In addition, the effect of 1-butanol as OH scavenger on the degradation was studied. It showed that the degradation of Acid red B was mainly caused by hydroxyl radical oxidation. Finally, the sonocatalytic degradation of some other organic dyes revealed that the Er 3+:YAlO 3/TiO 2 composite was a widely useable sonocatalyst in treating various dye wastewaters.

Estrogen receptor activation reduces lipid synthesis in pancreatic islets and prevents β cell failure in rodent models of type 2 diabetes

The failure of pancreatic β cells to adapt to an increasing demand for insulin is the major mechanism by which patients progress from insulin resistance to type 2 diabetes (T2D) and is thought to be related to dysfunctional lipid homeostasis within those cells. In multiple animal models of diabetes, females demonstrate relative protection from β cell failure. We previously found that the hormone 17β-estradiol (E2) in part mediates this benefit. Here, we show that treating male Zucker diabetic fatty (ZDF) rats with E2 suppressed synthesis and accumulation of fatty acids and glycerolipids in islets and protected against β cell failure. The antilipogenic actions of E2 were recapitulated by pharmacological activation of estrogen receptor α (ERα) or ERβ in a rat β cell line and in cultured ZDF rat, mouse, and human islets. Pancreas-specific null deletion of ERα in mice (PERα-/-) prevented reduction of lipid synthesis by E2 via a direct action in islets, and PERα-/- mice were predisposed to islet lipid accumulation and β cell dysfunction in response to feeding with a high-fat diet. ER activation inhibited β cell lipid synthesis by suppressing the expression (and activity) of fatty acid synthase via a nonclassical pathway dependent on activated Stat3. Accordingly, pancreas-specific deletion of Stat3 in mice curtailed ER-mediated suppression of lipid synthesis. These data suggest that extranuclear ERs may be promising therapeutic targets to prevent β cell failure in T2D.

Phase I study of abiraterone acetate (AA) in patients (pts) with estrogen receptor– (ER) or androgen receptor (AR) –positive advanced breast carcinoma resistant to standard endocrine therapies.

2525 Background: Many advanced ER+ breast cancer pts show intrinsic resistance to endocrine treatment with the remainder acquiring resistance. Androgenic steroids upstream of aromatase can drive steroid receptor signaling critical to tumour growth. Evidence also exists for an ERα-/AR+ subset of breast cancers transcriptionally similar to ERα+ disease. We hypothesized that AA, a cytochrome (CYP) 17 inhibitor that irreversibly inhibits androgen and estrogen synthesis, would have anti-tumour activity in ER+ or ER-/AR+ pts. Methods: Post-menopausal women with ER+ or ER-/AR+ advanced breast cancer resistant to >2 lines of hormone therapies were treated in 6-pt cohorts with AA at doses between 250 and 2000mg daily. Results: A total of 25 pts were treated in the phase I trial with 2 pts ongoing on study. Median time on treatment was 1.8 mths (range 0.7 – 11.6 mths). There were no dose limiting toxicities. The majority of adverse events (AEs) were Common Toxicity Criteria grade 1 or 2: fatigue, nausea, anorexia, dyspnoea, palpitations, dizziness and flushes. Hypokalemia was frequent, due to secondary mineralocorticoid syndrome: grade 3/4 hypokalemia occurred in 4 pts. This was effectively managed with potassium supplementation, hydrocortisone (20 mg mane, 10 mg nocte) and eplerenone (50-200 mg) with no clinical consequences. Other grade 3 AEs were neutropenia and reduced left ventricular ejection fraction (1 pt) and exercise-induced hypotension with dizziness (1 pt). At the 2000mg dose 5/5 subjects had suppression of estradiol, testosterone, DHEA and DHEAS to below the lower limit of detection of the assay. Two pts (both ER+/AR+) continued on study beyond 11 mths, one of whom achieved a radiological partial response and 80% reduction in serum CA15.3 from baseline. Pharmacokinetic data will be presented. Conclusions: AA is well tolerated in advanced breast cancer pts with preliminary evidence of antitumour activity. The predominant AEs are mechanism-based (hypokalemia) and can be managed expectantly, although careful monitoring of potassium levels is recommended.

Insulin-like growth factor-1 and 17β-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WT1 regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17β-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 µM ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 µM LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 µM SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Cellular and molecular actions displayed by estrone on vascular endothelium

In this work we provide evidence that estrone “per se” modulates cellular endothelial growth and survival, events that play key roles in the development of vascular disease. Moreover, under oxidative stress conditions the hormone prevented apoptosis triggered by hydrogen peroxide. Although estrone did not affect E-selectin and VCAM-1 mRNAs synthesis, the hormone prevented the expression of these adhesion molecules induced by the proinflammatory agent LPS. The steroid partially attenuated leukocyte adhesion not only under basal conditions but also in the presence of LPS. Using ICI182780 compound as estrogen receptor antagonist, and PD98059 as MAPK inhibitor we obtained evidence that the mitogenic action of estrone involved the participation of ER and MAPK transduction pathway activation. The presence of estradiol impaired the effect of estrone on cell proliferation and vasoactive production. These results suggest that estrone exhibits a remarkable biological action on endothelial cells, modulating vasoactive production, proliferation, apoptosis, and cell adhesion events.

Abstract 2290: Crosstalk between ER and PI3K signaling pathways in human luminal type breast cancer and its therapeutic implications

Abstract Background: We have recently shown that the luminal B subtype breast cancer (BC) is associated with PI3K molecular signature. Breast tumors of the luminal B subtype generally have lower ER expression levels and/or activity and are more aggressive and less sensitive to endocrine therapy when compared to the luminal A tumors. To further investigate the role of PI3K signaling in BC luminal types and endocrine resistance, we have characterized an array of luminal A and B cell lines, as well as their endocrine resistant derivatives, to examine how modulation of the PI3K pathway can influence ER activity and endocrine sensitivity. Materials and Methods: We focused on endocrine resistant derivatives of two luminal A (ZR75-1 and BT483) and two luminal B (ZR75-B and CAMA-1) cell lines. Resistant clones were developed by long-term (>6 months) endocrine treatment [estrogen deprivation (ED) or ED plus Tamoxifen (Tam)]. IC50 values of the PI3K inhibitor BEZ-235 were determined in the parental and the endocrine resistant lines, and qRT-PCR was performed to measure the effect of the inhibitor on mRNA levels of ER and its regulated genes (e.g. PR, CAV1, IGF1R and IRS1). Cells were also treated (4 days) with Tam, BEZ-235, or the combination and growth inhibition assays were conducted to assess the drugs efficacy. Inducible PTEN knockdown clones were established in MCF7L and BT483 cell lines. Endocrine sensitivity of these clones was studied in vitro and in vivo, using xenograft mouse models. Results: Ten days of treatment showed that all parental lines were sensitive to endocrine therapy. In contrast, stabilized endocrine resistant cell lines showed similar cell growth rate under Tam or ED treatment compared to control E2 treatment (p>0.05). ZR75-1 endocrine resistant derivatives acquired higher sensitivity to BEZ-235 treatment compared to the parental (p<0.05); a finding that is currently being confirmed in other cell line models. BEZ-235 increased expression of ER and its targeted genes in all 4 parental cell lines (p<0.05). BEZ-235 and Tam administered together additively suppressed cell growth in all 4 parental cell lines (p<1E-5). PTEN knockdown significantly increased the levels of pAkt and decreased the expression of ER and its regulated genes (PR and IGF1R). In vivo xenograft mouse models showed that PTEN knockdown renders tumors continued growth under ED, in contrast to the regression of parental/control tumors under this endocrine regimen (p<0.05). Discussion: The inverse association between PI3K and ER pathways in luminal BC cell lines validates the relevance of these models for further studies. Endocrine resistant phenotype may be partially caused by activated PI3K signaling. Our data suggest that inhibition of PI3K signaling can restore ER levels and/or activity, thereby reversing the aggressive and hormonal-refractory phenotype of the tumors, and achieving improved endocrine therapy response. 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 2290. doi:10.1158/1538-7445.AM2011-2290

Abstract 1200: BP1 regulates ERα activity in MCF-7 cells

Abstract Introduction. BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with estrogen receptor negativity – 100% of ERα negative (hereafter called ER-) tumors expressed BP1, while 73% of ER positive (ER+) tumors expressed BP1; this difference was statistically significant (p=0.03). Initially most tumors are hormone dependent and are sensitive to antiestrogens. Over time, many tumors progress to hormone-independence. One possible mechanism by which this might occur is reduction of ER protein. Here we present data indicating that high BP1 expression decreases ER protein levels in ER+ cells grown in the absence of serum. Since it is not currently understood what factors may cause ER+ cells to become more ER negative, it is important to determine the role of BP1 in reducing ER protein levels. Results. Our data suggest that MCF-7 ER+ cells engineered to overexpress BP1, when grown in the absence of serum, exhibit decreased ER protein compared with control cells containing an empty vector and grown under the same conditions. This is not a transcriptional effect since there was no change in ER mRNA levels in the presence of high levels of BP1. There was also a decrease in ER immunostaining in cells overexpressing BP1 grown without serum. Decreased ER protein levels correlated with decreased expression of pS2, a known downstream target of ER, demonstrating that there is less functional ER protein in cells overexpressing BP1 when grown in the absence of serum. We speculate that regulation of ER levels by BP1 is post-translational. Several pathways that control ER protein stability are being evaluated in cells grown with or without serum. Phosphorylation of Akt on Ser473, the site that is known to phosphorylate and stabilize ER protein, is being investigated to determine whether BP1 regulates phospho Akt levels. MAPK signaling can result in activation of ER in the absence of estrogen, but we did not see any evidence that BP1 is involved in the MAPK pathway. p300 is known to acetylate and stabilize ER protein; experiments are underway to determine whether BP1 directly regulates p300 expression since there are two potential BP1 binding sites in the p300 gene. Conclusions. We hypothesize that BP1 overexpression leads to reduced ER protein in ER positive cells. This could result in decreased sensitivity to antiestrogens in those cells. 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 1200. doi:10.1158/1538-7445.AM2011-1200

Abstract 545: Laminin induces tamoxifen resistance through alfa 6 integrin in breast cancer cells

Abstract Even though it is now well established that the tumor microenvironment plays a central role in modulating tumor progression and response to therapy, its function as a regulator of endocrine resistance in breast cancer has not yet been thoroughly explored. We previously showed that fibronectin through beta 1 integrin induces tamoxifen resistance in breast cancer cells. The aim of this study was to determine whether other extracellular matrix components induce tamoxifen resistance and what mechanisms are involved. We found that laminin, but not collagen IV, induced tamoxifen resistance in both estrogen receptor positive (ER+) human MCF-7 and mouse LM05-E breast cancer cells (P<0.001). To understand what signaling pathways were involved, cells were treated for 48 hours with estradiol (10−8M) and 4-OH-tamoxifen (10−6M) with the addition of either the MAPK/ERK1/2 inhibitor PD98059 (10µM) or the PI3K/AKT inhibitor LY294002 (10µM). Only LY294002 reversed the protective effect of laminin (P<0.001). Next to determine what integrin was mediating endocrine resistance, cells were pre-incubated with either an alfa 6 blocking antibody (GoH3, 0.15µM) or a beta 1 integrin blocking antibody (AIIB2, 0.15µM). The alfa 6 blocking antibody interfered with tamoxifen resistance induced by laminin (P<0.01), but not the beta 1 integrin blocking antibody. To establish whether laminin or fibronectin regulated the transcriptional activity of ER we carried out luciferase reporter assays by transiently transfecting the cells with 0.2µg of a pSV40-ERE-Luc plasmid together with 0.04µg pRL-TK-Renilla as a control. As expected, estradiol 10−8M induced a significant stimulation of ER activity (P<0.001) which was counteracted by the addition of 10−6M 4-OH-tamoxifen (P<0.001). Cells were then additionally treated with either laminin or fibronectin (30µg/ml) to test whether on their own, or in the presence of estradiol and/or tamoxifen they could regulate ER activity. No regulation of the transcriptional activity of ER was found, even though we have previously shown that fibronectin stimulates the phosphorylation of ER-α in serine-118. Our results show that laminin, like fibronectin, induces tamoxifen resistance in breast cancer cells through the specific binding to alfa 6 integrin, and that the PI3K/AKT pathway mediates the protective effect downstream. However, the mechanism does not seem to involve regulation of the transcriptional activity of ER. These results suggest that integrin-matrix interactions could be a valuable molecular target for breast cancer patients that develop tamoxifen resistance. This work is supported by grants from the Susan G. Komen Foundation for the Cure (BCTR0600341), and FONCYT (PICT 2008-0325) to M.S. 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 545. doi:10.1158/1538-7445.AM2011-545

Abstract 5253: A model of tumor dormancy in Luminal A breast cancer

Abstract Introduction: The clinical course and biologic features of metastasis vary extensively depending on the type of cancer. Latency is one of the most important variables in metastasis. Late-onset recurrences are frequently found in Luminal A (ER+/PR+/Her2-) breast cancer patients. In both ER+ tumors and ER+ breast cancer cell lines, tumor cells preferentially disseminate to the bone marrow where they are capable of remaining dormant. The identification of mechanisms that support the survival of disseminated cancer cells (DTCs) has clear implications for understanding the biology of latent metastatic disease. Method: ER+ breast cancer cell lines expressing a GFP luciferase vector (for in vivo bioluminescent monitoring of tumor growth and dissemination) were orthotopically injected in the mammary fat pad (MFP) of mice. DTCs were isolated and characterized from the bone marrow and lungs after several months. Results: The isolated DTCs from the bone and lung do not proliferate, are resistant to both chemo and hormonal therapy (HT) and are endowed with a highly invasive/disseminating capacity (loss of E-Cadherin and gain of EMT features). Moreover, these DTCs exhibit decreased expression of ER compared to the cancer cells within the primary tumor. Since ER activity is required for ER+ breast cancer cell growth in vitro and in vivo, our data suggest that loss of ER activity can be a mechanism of tumor dormancy in Luminal A breast cancer. Conclusion: An understanding of the consequences of ER activity in terms of tumor dormancy and dissemination potential of ER+ breast cancer cells has implications for improving the treatment of latent metastatic disease. 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 5253. doi:10.1158/1538-7445.AM2011-5253

Abstract 4882: Determining endocrine receptor activity using quantitative automated image analysis and Redistribution® technology

Abstract Endocrine hormone function has been shown to regulate numerous oncogenic processes from tumor progression, promotion, and dependence to prevention, diagnosis, and treatment. Notably, estrogen and androgen activity in breast and prostate cancers have been used to diagnose steroid hormone dependence and determine therapy options. Endocrine disruptors are compounds that mimic estrogen or androgen steroid activity and affect the endocrine system by altering hormone function, providing possible therapies for endocrine-dependent cancers. Screening complex chemical libraries for endocrine disruptors and investigating the molecular and cellular effects of existing treatment strategies is accelerated by using high throughput procedures to assess endocrine activity and potential cancer therapeutic efficacy. Redistribution® technology can be used to monitor the localization of GFP-tagged proteins in response to extra-cellular stimuli such as treatment with steroid compounds or activation of signaling cascades. Redistribution® GFP-tagged steroid receptors such as estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and androgen receptor (AR) form nuclear foci in response to hormone stimulation that can be easily monitored and quantitated by automated image analysis, thereby establishing a high-throughput screen for both agonists and antagonists of ER and AR activation. Additionally, multiplexed cell and nuclear phenotypes such as receptor kinetics, cellular health, and cell cycle effects can be monitored simultaneously following direct or indirect receptor stimulation. Here we utilize Redistribution® cell lines and known endocrine agonist and antagonists to establish a screen for ER and AR disruptors. For ER screens, cells were treated with agonists including 17β-estradiol and bisphenol A with and without antagonists such as fulvestrant (ICI 182,780) and tamoxifen. AR cells were treated with dihydrotesterone (5α-androstan-17β-ol-3-one) or progesterone agonists in the presence or absence of the AR antagonist mifepristone (RU 486). Following fixation and Hoechst staining, plates were analyzed using a quantitative automated imaging platform to evaluate features such as nuclear translocation (for AR only), nuclear GFP foci count, area, and intensity, and overall nuclear morphology on a cell-by-cell basis. EC50 values were determined from dose-dependent response curves of the treatments to establish hierarchical receptor activation comparisons for both ER and AR. Z-factors for these features indicate that using this technology gives a robust assay with high reproducibility. This data suggests that ER and AR Redistribution® cell lines can be effectively utilized with high throughput automated image analysis to screen for potential steroid hormone agonists and antagonists that would increase cancer therapeutic efficacy and specificity. 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 4882. doi:10.1158/1538-7445.AM2011-4882