Estrogen Receptor In Breast Cancer Cells Research Articles

Molecular docking and ADMET studies to predict the anti-breast cancer effect of aloin by targeting estrogen and progesterone receptors

The existing anti-cancer drugs are known for their adverse effects. Hence, it calls for an urgent need to explore the alternative therapies that are safer and efficient with no or minimal side effects. This approach points towards the utilization of traditional medicinal herbs to formulate naturally-derived drugs. So, aloin is a bioactive compound of Aloe Vera and known to exhibit an anticancerous effect on different cancer types. However, limited studies are indicating its possible interaction with estrogen receptors (ER) and progesterone receptors (PGR), that are the most targeted receptors in breast cancer therapeutics. Our study aims to predict the possible interactions of ER and PGR with aloin. We also analysed the drug likeliness properties of aloin for its anti-breast cancer activity. Hence, we performed molecular docking study of aloin against both these receptors using AutodockVina. Drug-like properties and bioactivity score for aloin was also predicted, using Molinspiration tool. The results suggest that aloin exhibit better interaction with ER than PGR with a binding affinity of −8.0 Kcal/mol. Molinspiration assay further suggested the aloin to carry drug-like property and behave as a good ligand for nuclear receptors. Our study indicates that aloin may behave as a potential anti-breast cancer agent by targeting the ER in breast cancer cells.

Abstract A39: Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A

Abstract Breast cancers are divided into different subtypes based on receptor expression status: estrogen receptor (ER), progesterone receptor (PR) and Her2. The luminal A subtype (ER+PR+Her2-) is most often associated with a good prognosis while the triple-negative (TN) cancers (ER-PR-Her2-) have a poor prognosis. ER+ cancer cells depend on estrogen for their growth and can be treated with drugs to block its effects. While most human breast cancers express ER alpha, some tumors are hormone independent despite the presence of ER and others become ER negative over time. Myeloid cells are known to affect tumor development, and infiltration of tumor-associated macrophages is associated with a worse prognosis. Theoretically, however, macrophages can be both beneficial and bad for the patient. Proinflammatory M1 macrophages (CD68+ CD163-) can eliminate cancer cells and promote antitumor immune responses while anti-inflammatory M2 macrophages (CD163+CD68+) or anti-inflammatory myeloid cells (CD68-CD163+) can promote tumor progression by inhibiting proinflammatory immune responses and induce wound-healing processes. This includes angiogenesis and matrix degradation, both of which promote metastatic spread and possibly even EMT. Macrophages have also been proposed to downregulate ER in breast cancer cells through an unknown mechanism. Due to the recent reports suggesting that macrophages can induce EMT and downregulate ER, we evaluated the effect of human primary macrophages on ER expression in breast cancer cells. Immunohistochemistry analyses of breast cancer tumor xenografts, established with co-transplantation of primary human myeloid cells together with ER+ breast cancer cells in NSG mice, showed association of monocytes with downregulation of ER-receptor expression. Using primary human monocyte-derived macrophage cultures In vitro, we show that the different subtypes of macrophages have unique cytokine secretion profiles with M1 macrophages secreting significantly higher levels of TNF alpha compared to M2 macrophages. ER alpha positive MCF-7 and ER alpha negative MDA-MB-231 breast cancer cells were cultured in macrophage condition media, and both Western blot and qPCR analysis showed downregulation of ER was caused by M1 macrophage-derived TNF alpha that inhibits FoxO3A transcription of ER alpha. We propose that proinflammatory macrophages (M1), despite being tumoricidal, may have direct effects on endocrine resistance in breast cancer patients. Our findings suggest that different macrophage subtypes have various and unique impacts on breast cancer progression, and that proinflammatory macrophages, despite being tumoricidal, may have unwanted and direct effects on endocrine resistance mechanisms in breast cancer patients. Citation Format: Frida Björk Gunnarsdottir, Catharina Hagerling, Caroline Bergenfelz, Camilla M. Rydberg, Meliha Mehmeti, Roni Allaoui, Sofie Mohlin, Sven Påhlman, Daniel Bexell, Karin Leandersson. Inflammatory macrophage derived TNF alpha induces downregulation of estrogen receptor alpha in breast cancer cells by inactivation of Foxo3A [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A39.

SUN-016 27-Hydroxycholesterol a Selective Estrogen Receptor Modulator: Profiling 27-Hydroxycholesterol Effects on Estrogen Receptor Activity in Mice

27-Hydroxycholesterol (27-HC) is the most prevalent metabolite of cholesterol in circulation, and also the first identified endogenous selective estrogen receptor (ER) modulator (SERM) (1). Previously, we found that 27-HC acts as an agonist of ER in breast cancer cells, while it acts as an antagonist of ER in endothelial cells in the cardiovasculature. Yet, potential agonistic or antagonistic effects of 27-HC on ER activity in other tissues and cell types remain largely unknown. Here, we examined the effects of 27-HC on ER activity using an ER responsive element (ERE)-luciferase transgenic mice, which have the expression plasmid that contain ERE, linked to a luciferase gene. This mouse model expresses luciferase proteins upon activation of ER, which could be further quantified and visualized by luciferase assay, and imaging respectively. To remove any artifacts from estrogens in the diet and endogenous production at the ovary, the mice were fed an estrogen depleted diet, and female mice were ovariectomized at 8 weeks of age. Both male and female mice were injected subcutaneously with either physiological dose of 27-HC (20 mg/kg body weight), 5ug/kg of 17b-estradiol (E2), E2 plus 27-HC, or vehicle control, at 9am of the day of sacrifice, imaged by in vivo imaging system after treating with luciferin (150mg/kg body weight) intraperitoneally at 3pm, and sacrificed at the same day, at age of 10 weeks. Tissues were collected, homogenized, and measured for luciferase activity through luciferase assay (Promega). The tissue extraction method was also determined to make sure that the luciferase activities are comparable among tissues. The level of ER activity in the presence of E2 was used as a positive control, and upon E2 treatment, ER activity was significantly increased in all analyzed tissues compared to vehicle treatment, with highest levels of ER activity in liver, testis, ovaries, and brain. Interestingly, the 27-HC treatment decreased the ER activity significantly in many tissues both in male and females, suggesting the existence of basal ER activity and an important antagonistic role of 27-HC. Yet, 27-HC showed agonistic effect and the levels of ER activity was even higher than E2 treatments in some tissues, showing a potential important role of 27-HC in these tissues. This study shed light upon potential effects of 27-HC on ER activity in various tissues, that can open the doors for further studies on 27-HC SERM characteristics. Reference:(1) Umetani, Michihisa, et al. Nature medicine 13.10 (2007): 1185.

Abstract P4-04-16: Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production

Abstract Introduction: Obesity is associated with a worse breast cancer prognosis, particularly in estrogen receptor alpha (ERα) positive, postmenopausal patients. Resistance to aromatase inhibitor treatment may be one mechanism mediating this effect, as obese patients have a lower response rate to this class of drugs and higher breast tissue aromatase expression. We hypothesized that obesity-associated circulating factors promote breast cancer cell cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production, resulting in an elevation in pre-adipocyte aromatase expression that enhances breast cancer cell estrogen receptor activity and proliferation. Methods: We utilized an in vitro model of the obese patient’s tumor microenvironment in which cultured MCF-7 breast cancer cells and pre-adipocytes were exposed to pooled serum from obese (OB; BMI≥30.0 kg/m2) or normal weight (N; BMI 18.5-24.9 kg/m2) postmenopausal women. Results: Exposure to OB versus N patient sera significantly increased MCF-7 cell COX-2 expression and PGE2 production. This was coupled with 89% greater pre-adipocyte aromatase expression following culture in conditioned media (CM) from MCF-7 cells exposed to OB versus N patient sera, a difference nullified by treatment of the MCF-7 cells with the COX-2 inhibitor celecoxib during CM generation. Previous analysis of the sera revealed significantly higher interleukin 6 (IL-6) concentrations in the OB versus N patient samples. Depletion of IL-6 from the sera resulted in neutralization of the difference between OB and N CM-stimulated aromatase expression. N CM generated with the addition of IL-6 treatment at the time of N patient sera exposure also induced pre-adipocyte aromatase expression that was statistically equivalent to the OB CM condition. Finally, CM from pre-adipocyte/MCF-7 cell co-cultures exposed to OB patient sera stimulated greater MCF-7 and T47D breast cancer cell ERα activity and proliferation in comparison to N. Conclusions: This study indicates that obesity-associated systemic IL-6 indirectly enhances pre-adipocyte aromatase expression via increased breast cancer cell PGE2 production, thereby promoting greater cancer cell ERα activity and proliferation. Investigation regarding the efficacy of a COX-2 inhibitor/aromatase inhibitor combination therapy in the obese postmenopausal patient population is warranted. Citation Format: Laura W Bowers, Andrew J Brenner, Stephen D Hursting, Rajeshwar R Tekmal, Linda A deGraffenried. Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-16.

Adipocyte hypoxia promotes epithelial-mesenchymal transition-related gene expression and estrogen receptor-negative phenotype in breast cancer cells.

The development of breast cancer is linked to the loss of estrogen receptor (ER) during the course of tumor progression, resulting in loss of responsiveness to hormonal treatment. The mechanisms underlying dynamic ERα gene expression change in breast cancer remain unclear. A range of physiological and biological changes, including increased adipose tissue hypoxia, accompanies obesity. Hypoxia in adipocytes can establish a pro-malignancy environment in breast tissues. Epidemiological studies have linked obesity with basal-like breast cancer risk and poor disease outcome, suggesting that obesity may affect the tumor phenotype by skewing the microenvironment toward support of more aggressive tumor phenotypes. In the present study, human SGBS adipocytes were co-cultured with ER-positive MCF7 cells for 24 h. After co-culture, HIF1α, TGF-β, and lectin-type oxidized LDL receptor 1 (LOX1) mRNA levels in the SGBS cells were increased. Expression levels of the epithelial-mesenchymal transition (EMT)-inducing transcription factors FOXC2 and TWIST1 were increased in the co-cultured MCF7 cells. In addition, the E-cadherin mRNA level was decreased, while the N-cadherin mRNA level was increased in the co-cultured MCF7 cells. ERα mRNA levels were significantly repressed in the co-cultured MCF7 cells. ERα gene expression in the MCF7 cells was decreased due to increased HIF1α in the SGBS cells. These results suggest that adipocytes can modify breast cancer cell ER gene expression through hypoxia and also can promote EMT processes in breast cancer cells, supporting an important role of obesity in aggressive breast cancer development.

Abstract P1-06-01: NSAID use attenuates breast cancer recurrence in obese women: Role of prostaglandin-aromatase interactions

Abstract Introduction: Obesity is associated with a worse breast cancer prognosis, with the most prominent effects seen in hormone responsive postmenopausal patients. It has also been linked to elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the pro-inflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. Consequently, we hypothesized that non-steroidal anti-inflammatory drug (NSAID) use decreases estrogen receptor (ER) positive breast cancer recurrence in the obese population via inhibition of PGE2-mediated local aromatase expression. Methods: Four-hundred and forty women treated for invasive, ER positive breast cancer at San Antonio area clinics were retrospectively classified according to NSAID use, body mass index category (Normal Weight (NW): 18.5-24.9 kg/m2; Overweight (OW): 25.0-29.9 kg/m2; Obese (OB): ≥30.0 kg/m2), and disease recurrence. To examine the role of obesity-induced local aromatase expression in the link between NSAID use and disease recurrence, we utilized an in vitro model of obesity in which we exposed macrophages to pooled sera samples from NW or OB postmenopausal breast cancer patients. Adipose stromal cells’ (ASC) aromatase expression was measured following exposure to conditioned media (CM) collected from these sera-exposed macrophages. ER activity and in vitro measures of cancer aggression were assessed in MCF-7 and T47D breast cancer cells cultured in CM from sera-exposed macrophage/ASC co-cultures. Results: Within our patient population, which had an average BMI in the obese range, NSAID users had significantly lower recurrence rates (p = 0.05) and their time to disease progression was delayed by almost 28 months in comparison to nonusers. Our in vitro studies demonstrated that growth in OB macrophage CM significantly enhances ASC aromatase expression in comparison to NW (p<0.05), while macrophage treatment with celecoxib during the generation of CM neutralizes the difference between OB and NW. This was correlated with significantly greater macrophage PGE2 production following OB versus NW sera exposure (p<0.05). In addition, CM from macrophage/ASC co-cultures exposed to OB patient sera stimulates more breast cancer cell ER activity, proliferation, and S phase activity, and these differences are eliminated by the addition of an aromatase inhibitor during the generation of CM. We also plan to examine how the co-culture CM impacts breast cancer cell expression of a panel of genes related to cancer aggression. Conclusions: Our results indicate that NSAID use can improve the recurrence rate for hormone-responsive breast cancer patients, particularly those with an elevated BMI. The in vitro model suggests that obesity-related enhancement of PGE2-induced local aromatase expression and estrogen production may be a key mechanism mediating this effect. Further studies designed to examine the clinical benefit of NSAID use in the obese breast cancer patient population are warranted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-01.

Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.

A co-ordinated interaction between CTCF and ER in breast cancer cells

BackgroundCCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor that is involved in both intra- and interchromasomal looping. Recent research has shown a role for CTCF in estrogen receptor (ER) biology, at some individual loci, but a multi-context global analysis of CTCF binding and transcription activity is lacking.ResultsWe now map CTCF binding genome wide in breast cancer cells and find that CTCF binding is unchanged in response to estrogen or tamoxifen treatment. We find a small but reproducible set of CTCF binding events that overlap with both the nuclear receptor, estrogen receptor, and the forkhead protein FOXA1. These overlapping binding events are likely functional as they are biased towards estrogen-regulated genes, compared to regions lacking either CTCF or ER binding. In addition we identify cell-line specific CTCF binding events. These binding events are more likely to be associated with cell-line specific ER binding events and are also more likely to be adjacent to genes that are expressed in that particular cell line.ConclusionThe evolving role for CTCF in ER biology is complex, but is likely to be multifunctional and possibly influenced by the specific genomic locus. Our data suggest a positive, pro-transcriptional role for CTCF in ER-mediated gene expression in breast cancer cells. CTCF not only provides boundaries for accessible and 'protected' transcriptional blocks, but may also influence the actual binding of ER to the chromatin, thereby modulating the estrogen-mediated gene expression changes observed in breast cancer cells.

CtIP, a BRCA1 binding protein, is a transcriptional corepressor of estrogen receptor α.

Abstract Abstract #606 The role of estrogen and estrogen receptor (ER) transcriptional regulation in breast carcinogenesis is indisputable. In the last few years there has been dramatic progress in understanding how receptor transcriptional coactivator and corepressor complexes operate.
 We demonstrated that CtIP (CtBP interacting protein) is downregulated in tamoxifen resistant breast cancer lines. Furthermore, CtIP protein expression correlates with response to neo-adjuvant endocrine therapy and patients with progressive disease express lower CtIP protein in their primary breast carcinomas than those who respond. CtIP expression directly correlates with ER alpha expression, and inversely correlates with disease free survival and breast cancer metastasis. Importantly, silencing endogenous CtIP confers tamoxifen resistance in tamoxifen sensitive breast cancer cells and re-expression restored tamoxifen sensitivity in resistant breast cancer cells (Wu et al. Molec. Cancer Res. 2007; 5: 1285-1295). Our findings indicate that CtIP silencing is a novel mechanism for the development of tamoxifen resistance in breast cancer.
 CtIP interacts with the BRCT domains of BRCA1 where most mutations occur in familial BRCA1 breast cancers. CtIP also interacts with the transcriptional corepressor CtBP. Emerging evidence suggests that CtIP is involved in transcriptional repression and may be a tumor suppressor gene. We hypothesize that CtIP plays a key role in repression of ER transcription and such repressive function is linked to the inhibitory growth effects exerted by tamoxifen. We further speculate that CtIP inhibition of ER transcription is mediated by its ability to bridge BRCA1 with CtBP to form a transcriptional corepressor complex able to modulate ER activity via the interaction between BRCA1 and ER and that CtIP is in fact required for BRCA1 to exert its transcriptional repression activity on ER. We will present our work showing that CtIP, BRCA1 and CtBP form a multiprotein complex, that requires CtIP, and associates with ER in breast cancer cells. More importantly, we will show that BRCA1, CtIP and CtBP associate with ER on the ER-dependent promoter of the pS2 gene in the absence of ligand. Stimulation by ligand leads to dissociation of the complex and ER-dependent gene expression. Furthermore, silencing CtIP expression using RNAi in E2-starved MCF-7 cells led to an increase of basal transcription of the pS2 gene. CtIP was also observed to repress E2 stimulation of ERE-luciferase in transfected MCF-7 cells. We are currently characterizing the role of the CtIP corepressor complex in ER-transcriptional activity. We will present the results of our efforts to identify specific ER-dependent promoters repressed by the CtIP corepressor. In addition, we will present results of studies aimed at characterizing CtIP-specific changes in chromatin remodeling and ER-cofactor recruitment at ER-dependent promoters. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 606.

Peroxisome proliferator-activated receptor-gamma agonist, troglitazone has anticancer effect on breast cancer cells

Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat as they do not respond to hormone therapy. In an attempt to determine if expressing the human estrogen receptor in an ectopic manner could restore the hormone responsiveness of these cells, we have expressed the human ER in ER-negative MDA-MB 231 breast cancer cells using a recombinant adenovirus gene delivery system that allows high level expression of ER in essentially all cells. In these cells, the ER was correctly translated, had a wild type hormone binding affinity (Kd=0.6 nM), bound well to estrogen response element-containing DNA, and showed an activation pattern of estrogen response element-reporter gene activity by estrogen and antiestrogens very similar to that observed in MCF-7 breast cancer cells containing endogenous ER (stimulation by estrogen, no stimulation by the antiestrogens trans-hydroxytamoxifen or ICI 164 384, and blockade of estradiol stimulation by trans-hydroxytamoxifen or ICI 164 384). Intriguingly, estradiol stimulation of these cells was also able to induce expression of pS2, an estrogen regulated gene considered to be a favorable prognostic marker for endocrine therapy in ER-positive breast cancer cells. Expression of the ER had no effect by itself on the proliferation rate of MDA-MB 231 cells. However, treatment of the ER-containing cells with estradiol or with the pure antiestrogen ICI 164 384 suppressed proliferation of the cells while the antiestrogen trans-hydroxytamoxifen had little effect on proliferation; and cotreatment with trans-hydroxytamoxifen reversed the estradiol- or ICI 164 384-evoked suppression of proliferation. To understand the mechanism underlying the inhibition of proliferation by estradiol, we examined the expression of several growth related endogenous genes. c-Myc protooncogene expression was strongly inhibited by treatment with estradiol as was expression of BRCA1 and BRCA2 genes, which is in agreement with their mitogenic-dependent expression, while expression of β-actin, a housekeeping gene, was not affected by hormone treatment. Together, these data suggest that reexpressing the human ER in breast cancer cells that no longer express this protein renders them sensitive to hormone treatment. The ability of the antiestrogen ICI 164 384 to suppress the proliferation of ER-negative breast cancer cells that reexpress ER might be useful ultimately as an endocrine gene therapy approach for controlling the growth of ER-negative breast cancer cells. The application of recombinant adenoviruses expressing the human ER presents interesting features which might be used as a basis for designing more powerful and effective treatments for ER-negative breast cancers.

Signal cross-talk between estrogen receptor alpha and beta and the peroxisome proliferator-activated receptor gamma1 in MDA-MB-231 and MCF-7 breast cancer cells

We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) is expressed and transcriptionally responsive to both synthetic and natural ligands in a variety of human breast cancer cells. We also observed significant differences in basal and ligand-mediated transactivation of PPARγ in cells with variable expression of the estrogen receptor. While previous reports indicate that PPARγ can mediate the expression of estrogen target genes, no data have suggested that estrogen receptor (ER) expression can alter the transcriptional regulation of PPARγ target gene expression. Here we have demonstrated that the expression of either ERα or β, but not the androgen or aryl hydrocarbon receptors, lowers both basal and stimulated PPARγ-mediated reporter activity. Interestingly, the presence of an ER antagonist does not inhibit this response while estradiol treatment further inhibits the ligand-stimulated transactivation of PPARγ in cells expressing ERα but not ERβ. Cells transfected with ERα deletion mutants demonstrate that the DNA binding domain of the ER is required to repress PPAR transactivation in these cells. Finally, using RNase protection assays we show that the inhibition of PPAR function is not due to a decrease in the expression of PPARγ. These data suggest that signal cross talk exists bidirectionally between PPARγ and ER in breast cancer cells.

Expression of human estrogen receptor using an efficient adenoviral gene delivery system is able to restore hormone-dependent features to estrogen receptor-negative breast carcinoma cells

Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat as they do not respond to hormone therapy. In an attempt to determine if expressing the human estrogen receptor in an ectopic manner could restore the hormone responsiveness of these cells, we have expressed the human ER in ER-negative MDA-MB 231 breast cancer cells using a recombinant adenovirus gene delivery system that allows high level expression of ER in essentially all cells. In these cells, the ER was correctly translated, had a wild type hormone binding affinity ( K d=0.6 nM), bound well to estrogen response element-containing DNA, and showed an activation pattern of estrogen response element-reporter gene activity by estrogen and antiestrogens very similar to that observed in MCF-7 breast cancer cells containing endogenous ER (stimulation by estrogen, no stimulation by the antiestrogens trans-hydroxytamoxifen or ICI 164 384, and blockade of estradiol stimulation by trans-hydroxytamoxifen or ICI 164 384). Intriguingly, estradiol stimulation of these cells was also able to induce expression of pS2, an estrogen regulated gene considered to be a favorable prognostic marker for endocrine therapy in ER-positive breast cancer cells. Expression of the ER had no effect by itself on the proliferation rate of MDA-MB 231 cells. However, treatment of the ER-containing cells with estradiol or with the pure antiestrogen ICI 164 384 suppressed proliferation of the cells while the antiestrogen trans-hydroxytamoxifen had little effect on proliferation; and cotreatment with trans-hydroxytamoxifen reversed the estradiol- or ICI 164 384-evoked suppression of proliferation. To understand the mechanism underlying the inhibition of proliferation by estradiol, we examined the expression of several growth related endogenous genes. c- Myc protooncogene expression was strongly inhibited by treatment with estradiol as was expression of BRCA1 and BRCA2 genes, which is in agreement with their mitogenic-dependent expression, while expression of β-actin, a housekeeping gene, was not affected by hormone treatment. Together, these data suggest that reexpressing the human ER in breast cancer cells that no longer express this protein renders them sensitive to hormone treatment. The ability of the antiestrogen ICI 164 384 to suppress the proliferation of ER-negative breast cancer cells that reexpress ER might be useful ultimately as an endocrine gene therapy approach for controlling the growth of ER-negative breast cancer cells. The application of recombinant adenoviruses expressing the human ER presents interesting features which might be used as a basis for designing more powerful and effective treatments for ER-negative breast cancers.