ERα-positive Breast Cancer Cell Lines Research Articles

MiR-491-5p functions as a tumor suppressor by targeting JMJD2B in ERα-positive breast cancer

The involvement of miR-491-5p in breast cancer development is unclear. This study showed that miR-491-5p is significantly downregulated in ERα-positive breast cancer tissues and cell lines and is generally hypermethylated in ERα-positive breast cancer. MiR-491-5p overexpression significantly suppressed estrogen signaling and estrogen-stimulated proliferation of breast cancer cells. Furthermore, the histone demethylase JMJD2B was identified as a direct target of miR-491-5p. The ectopic expression of JMJD2B abrogated the phenotypic changes induced by miR-491-5p in breast cancer cells. Collectively, our data indicate that miR-491-5p plays a tumor suppressor role in the development and progression of breast caner and may be a novel therapeutic target against ERα-positive breast cancer.

A kinetic model identifies phosphorylated estrogen receptor‐α (ERα) as a critical regulator of ERα dynamics in breast cancer

Receptor levels are a key mechanism by which cells regulate their response to stimuli. The levels of estrogen receptor-α (ERα) impact breast cancer cell proliferation and are used to predict prognosis and sensitivity to endocrine therapy. Despite the clinical application of this information, it remains unclear how different cellular processes interact as a system to control ERα levels. To address this question, experimental results from the ERα-positive human breast cancer cell line (MCF-7) treated with 17-β-estradiol or vehicle control were used to develop a mass-action kinetic model of ERα regulation. Model analysis determined that RNA dynamics could be captured through phosphorylated ERα (pERα)-dependent feedback on transcription. Experimental analysis confirmed that pERα-S118 binds to the estrogen receptor-1 (ESR1) promoter, suggesting that pERα can feedback on ESR1 transcription. Protein dynamics required a separate mechanism in which the degradation rate for pERα was 8.3-fold higher than nonphosphorylated ERα. Using a model with both mechanisms, the root mean square error was 0.078. Sensitivity analysis of this combined model determined that while multiple mechanisms regulate ERα levels, pERα-dependent feedback elicited the strongest effect. Combined, our computational and experimental results identify phosphorylation of ERα as a critical decision point that coordinates the cellular circuitry to regulate ERα levels.

Abstract 3341: Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer

Abstract The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is a critical survival cascade in tumor progression. Our previous study demonstrated that combinational treatment of a potent PI3K inhibitor, LY294002 (LY), and DNA methyltransferase inhibitor, 5’-Aza-deoxycytidine (DAC), compounds effectively reduce tumor formation in the immunodeficiency mouse model, suggesting that PI3K/AKT signaling suppresses tumorigenesis through the epigenetic modulation. While tumor size of the LY-treated xenografts is dramatically decreased compared to the control group, the tumor growth cannot be completely attenuated by either LY or LY/DAC treatment, indicating that there is an alternative route for tumor survival. Here, we performed a microarray analysis to identify LY-mediated expression profiling on an ERα-positive breast cancer cell line, T-47D, the cell line applied in the previous xenograft experiments. A subset of 24 LY-activated genes associated with better clinical outcome are found to be not only direct targets of PI3K/AKT but involved in estrogen (E2)/ERα signaling by ingenuity pathway analysis, suggesting the involvement of ERα signaling in PI3K/AKT-mediated tumor growth. To clarify their relationship, pharmacological experiments using an ERα antagonist, ICI 182, 780 (ICI) and LY coupled with E2 was conducted. We observed that E2 stimulation induces both AKT and ERα activation in a time-dependent manner. Either ICI or LY treatment cannot repress the activation levels of AKT or ERα, respectively. Though, either single or combined inhibitor treatment can effectively restore E2-suppressed gene expression. These findings indicate that cross-talk between PI3K/AKT and ERα signaling cascades may cooperatively regulate cell growth and associated gene expression. In addition, transcription factor motif analysis showed that signal transducer and activator of transcription 3 and 6 (STAT3/6) binding motifs are enriched with highest prediction score on the promoter regions of the examined AKT-directed targets. Inhibitors prohibit E2-mediated assembly of STAT3/6 with ERα and STAT3/6 recruitment onto the promoter regions of target loci using co-immunoprecipitation (co-IP) and chromatin IP-qPCR. Moreover, repressive histone marks, H3K27me3 and H3K9me3, are found to occupy on the same regions as STAT3/6 upon E2 treatment. DAC treatment can effectively restore E2-induced suppression of AKT-directed target genes. Further association studies are ongoing to interrogate whether STAT3/6 is part of repressive transcription complex with H3K27me3 and H3K9me3. In summary, we propose that cross-talk of ERα with PI3K/AKT signaling represents the potential function switch of ERα from genomic binding to no-genomic binding. Combined treatment of PI3K/AKT inhibitors with hormone therapy may shed a light of therapeutic strategy to patients with aggressive or metastatic breast tumors. Citation Format: Hang-Kai Hsu, Pei-Yin Hsu, Yao Wang, Zelton D. Sharp, Tim H.-M. Huang, Victor X. Jin. Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2014-3341

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

IL-13Rα2 mediates PNR-induced migration and metastasis in ERα-negative breast cancer.

Emerging evidence has linked photoreceptor cell-specific nuclearreceptor (PNR/NR2E3), an orphan nuclear hormone receptor, to human breast cancer. PNR was shown to be a transcriptional activator of estrogen receptor-α (ERα) in ERα-positive breast cancer cell linesand high-level expression of PNR correlates with favorable response of ERα-positive breast cancer patients to tamoxifen. Interestingly, gene expression microarray study shows that PNR regulates distinct genes from those regulated by ERα, suggesting that PNR could have ERα-independent functions. Herein, we investigated the function of PNR in ERα-negative breast cancer cells. Our results showed that PNR-induced cell migration and metastasis of ERα-negative breast cancer cells both in vitro and in vivo, and the effect was attributed to the upregulation of interleukin (IL)-13Rα2, a high-affinity receptor for IL-13 that regulates tumor growth, invasion and metastasis of various human cancers. Mechanistically, PNR activated transcription of IL-13Rα2 through direct recruitment to IL-13Rα2 promoter. Upon stimulation with IL-13, IL-13Rα2 increased the extracellular signal-regulated kinases 1 and 2phosphorylation, which led to breast cancer migration and metastasis. The IL-13 triggered signal cascade was specific to IL-13Rα2, as the closely related IL-13Rα1 was not regulated by PNR. IL-13Rα2 is a novel tumor antigen that is overexpressed in a variety of solid tumor types. This study presents the first evidence that PNR could promote ERα-negative breast cancer metastasis through activation of IL-13Rα2-mediated signaling pathway.

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

MicroRNA-34a Suppresses Cell Proliferation by Targeting LMTK3 in Human Breast Cancer MCF-7 Cell Line

Breast cancer remains the leading cause of cancer mortality in females, and about 70% of the primary breast cancer patients are diagnosed ERα-positive, which is the most common type of breast cancer. MicroRNA-34a (miR-34a) has been shown to be a master regulator of tumor suppression in many types of cancers including breast cancer. However, the role of miR-34a in ERα-positive breast cancer has not been elucidated. Here, we find that in MCF-7, which is an ERα-positive breast cancer cell line, miR-34a is remarkably downregulated after E2 treatment. Overexpression of miR-34a by lentivirus suppresses cell proliferation, S phase ratio, and tumor formation in an E2-dependent manner in vitro. According to the mRNA sequence, lemur tyrosine kinase 3 (LMTK3), which is an important regulator of estrogen receptor alpha (ERα), is a predicted target of miR-34a. This is confirmed by dual luciferase reporter assay and the decrease of LMTK3 mRNA and protein levels after overexpression of miR-34a. Moreover, miR-34a overexpression decreases AKT signaling pathway and increases ERα phosphorylation status. Taken together, these results suggest that miR-34a inhibits breast cancer proliferation by targeting LMTK3 and might be used as an anti-ERα agent in breast cancer therapy.

RNF115/BCA2 E3 Ubiquitin Ligase Promotes Breast Cancer Cell Proliferation through Targeting p21Waf1/Cip1 for Ubiquitin-Mediated Degradation

The E3 ubiquitin ligase RING finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), has previously been reported to be overexpressed in estrogen receptor α (ERα)-positive breast tumors and to promote breast cell proliferation; however, its mechanism is unknown. In this study, we demonstrated that silencing of BCA2 by small interfering RNAs (siRNAs) in two ERα-positive breast cancer cell lines, MCF-7 and T47D, decreases cell proliferation and increases the protein levels of the cyclin-dependent kinase inhibitor p21Waf/Cip1. The protein stability of p21 was negatively regulated by BCA2. BCA2 directly interacts with p21 and promotes p21 ubiquitination and proteasomal degradation. Knockdown of p21 partially rescues the cell growth arrest induced by the BCA2 siRNA. These results suggest that BCA2 promotes ERα-positive breast cancer cell proliferation at least partially through downregulating the expression of p21.

Transcription factor Ets1 cooperates with estrogen receptor α to stimulate estradiol-dependent growth in breast cancer cells and tumors.

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm3/day) compared to control MCF7 tumors (2.52±1.70 mm3/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.

Abstract 509: RNF115/BCA2 E3 ubiquitin ligase promotes breast cancer cell proliferation through targeting p21Waf1/Cip1 for ubiquitin-mediated degradation .

Abstract The E3 ubiquitin ligase Ring Finger Protein 115 (RNF115), also known as Breast Cancer Associated gene 2 (BCA2), has previously been reported to be overexpressed in estrogen receptor (ERα) positive breast tumors and to promote breast cell proliferation; however, the mechanism is unknown. In this study, we demonstrated that silencing of BCA2 by siRNA in two ERα-positive breast cancer cell lines MCF7 and T47D decreases cell proliferation and increases the protein levels of the cyclin-dependent kinases inhibitor p21Waf/Cip1. The protein stability of p21 was negatively regulated by BCA2. BCA2 directly interacts with p21 and promotes p21 ubiquitination and proteasomal degradation. Knockdown of p21 partially rescues the cell growth arrest induced by the BCA2 siRNA. These results suggest that BCA2 promotes ERα-positive breast cancer cell proliferation at least partially through down-regulating the expression of p21. This study was supported by National Nature Science Foundation of China (81072162, 81120108019, and U1132605) . Citation Format: Zehua Wang, Arun Seth, Ceshi Chen. RNF115/BCA2 E3 ubiquitin ligase promotes breast cancer cell proliferation through targeting p21Waf1/Cip1 for ubiquitin-mediated degradation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 509. doi:10.1158/1538-7445.AM2013-509

Abstract 1316: Estrogen receptor-α dictates the subcellular localization of ERK5 to control differential proliferation and invasion programs of breast cancer cells.

Abstract Mitogen activated protein kinases (MAPKs) are key players in relaying extracellular signals to the cell nucleus and are often overexpressed in aggressive and endocrine-resistant breast cancers. Our previous studies (Madak-Erdogan et al. Mol Cell Biol 31:226,2011) revealed that one such kinase, ERK2, was recruited to distal enhancers of estradiol(E2)-regulated genes together with estrogen receptor alpha (ERα) and collaborated with ERα to regulate gene expression and cell proliferation programs in response to hormone. In the current study, we identified ERK5, a close relative of ERK2, as a factor that is recruited to the transcription start site (TSS) of E2-stimulated genes. Use of dominant negative and constitutively active forms, as well as small molecule inhibitors of ERK5 and MEK5, revealed that activation of ERK5 by upstream kinase MEK5 and activity of ERK5 itself was required for recruitment of the kinase to the chromatin. Immunofluorescence studies demonstrated that ERK5 localized to transcription factories in the nucleus upon cell treatment with E2. Inhibitors of ERK5 completely abrogated E2-dependent cell proliferation and colony formation for all of the ERα-positive breast cancer cell lines studied. In contrast to what we observed in ERα-positive cells, in ERα-negative breast cancer cell lines we did not detect any nuclear ERK5; instead, we found ERK5 localized to the actin cytoskeleton, especially to sites where substantial remodeling takes place. Upon expression of ERα in these ERα-negative cells, we observed re-localization of ERK5 to the nucleus and a decrease in actin reorganization associated with reduced cell motility and invasion. Abrogation of ERα expression using siRNA in ERα-positive cell lines caused loss of nuclear ERK5 and relocalized this kinase to the actin cytoskeleton. Our studies with tamoxifen resistant cell lines revealed that ERK5 localization to the nucleus was reduced and localization to the actin cytoskeleton increased as resistance progressed, suggesting a correlation between ERα activity, ERK5 localization and motility of breast cancer cells. Our data reveal that ERα elicits nuclear localization of ERK5, diminishing ERK5 localization to regions of high actin remodeling, thereby possibly accounting for the generally lower metastatic potential that is characteristic of many ERα-positive breast cancer cells. (Supported by grants T32 ES007326 and P50 AT006268 from NIH, and a grant from The Breast Cancer Research Foundation.) Citation Format: Zeynep Madak Erdogan, Anna Bergamaschi, Rosa Ventrella, Hailing Lu, Benita S. Katzenellenbogen. Estrogen receptor-α dictates the subcellular localization of ERK5 to control differential proliferation and invasion programs of breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1316. doi:10.1158/1538-7445.AM2013-1316

HDAC3 regulates stability of estrogen receptor α mRNA

Estrogen receptor alpha (ERα) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ERα expression in ERα-positive breast cancer cell lines, but the molecular mechanisms are poorly understood.Here, we showed that HDAC inhibitors decrease the stability of ERα mRNA, and that knockdown of HDAC3 decreases the stability of ERα mRNA and suppresses estrogen-dependent proliferation of ERα-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ERα-positive tumors are higher than those in ERα-negative tumors, thus suggesting that HDAC3 is necessary for ERα mRNA stability, and is involved in the estrogen-dependent proliferation of ERα-positive tumors.

A transformation in the mechanism by which the urokinase receptor signals provides a selection advantage for estrogen receptor-expressing breast cancer cells in the absence of estrogen

Binding of urokinase-type plasminogen activator (uPA) to its receptor, uPAR, in estrogen receptor-α (ERα) expressing breast cancer cells, transiently activates ERK downstream of FAK, Src family kinases, and H-Ras. Herein, we show that when uPAR is over-expressed, in two separate ERα-positive breast cancer cell lines, ERK activation occurs autonomously of uPA and is sustained. Autonomous ERK activation by uPAR requires H-Ras and Rac1. A mutated form of uPAR, which does not bind vitronectin (uPAR-W32A), failed to induce autonomous ERK activation. Expression of human uPAR or mouse uPAR but not uPAR-W32A in MCF-7 cells provided a selection advantage when these cells were deprived of estrogen in cell culture for two weeks. Similarly, MCF-7 cells that express mouse uPAR formed xenografts in SCID mice that survived and increased in volume in the absence of estrogen supplementation, probably reflecting the pro-survival activity of phospho-ERK. Autonomous uPAR signaling to ERK was sensitive to the EGFR tyrosine kinase inhibitors, Erlotinib and Gefitinib. The transition in uPAR signaling from uPA-dependent and transient to autonomous and sustained is reminiscent of the transformation in ErbB2/HER2 signaling observed when this gene is amplified in breast cancer. uPAR over-expression may provide a pathway for escape of breast cancer cells from ERα-targeting therapeutics.

Tartrazine and sunset yellow are xenoestrogens in a new screening assay to identify modulators of human oestrogen receptor transcriptional activity

Primary biliary cirrhosis (PBC) is a cholestatic liver disease of unknown cause that occurs most frequently in post-menopausal women. Since the female sex hormone oestrogen can be cholestatic, we hypothesised that PBC may be triggered in part by chronic exposure to xenoestrogens (which may be more active on a background of low endogenous oestrogen levels seen in post-menopausal women). A reporter gene construct employing a synthetic oestrogen response element predicted to specifically interact with oestrogen receptors (ER) was constructed. Co-transfection of this reporter into an ER null cell line with a variety of nuclear receptor expression constructs indicated that the reporter gene was trans-activated by ERα and ERβ, but not by the androgen, thyroid, progesterone, glucocorticoid or vitamin D receptors. Chemicals linked to PBC were then screened for xenoestrogen activity in the human ERα-positive MCF-7 breast cancer cell line. Using this assay, the coal-derived food and cosmetic colourings – sunset yellow and tartrazine – were identified as novel human ERα activators, activating the human ER with an EC50% concentration of 220 and 160nM, respectively.

The Estrogen-Regulated Transcription Factor PITX1 Coordinates Gene-Specific Regulation by Estrogen Receptor-Alpha in Breast Cancer Cells

The estrogen receptor α (ERα) is a master regulator of gene expression and works along with cooperating transcription factors in mediating the actions of the hormone estradiol (E2) in ER-positive tissues and breast tumors. Here, we report that expression of paired-like homeodomain transcription factor (PITX1), a tumor suppressor and member of the homeobox family of transcription factors, is robustly up-regulated by E2 in several ERα-positive breast cancer cell lines via ERα-dependent interaction between the proximal promoter and an enhancer region 5' upstream of the PITX1 gene. Overexpression of PITX1 selectively inhibited the transcriptional activity of ERα and ERβ, while enhancing the activities of the glucocorticoid receptor and progesterone receptor. Reduction of PITX1 by small interfering RNA enhanced ERα-dependent transcriptional regulation of a subset of ERα target genes. The consensus PITX1 binding motif was found to be present in 28% of genome-wide ERα binding sites and was in close proximity to estrogen response elements in a subset of ERα binding sites, and E2 treatment enhanced PITX1 as well as ERα recruitment to these binding sites. These studies identify PITX1 as a new ERα transcriptional target that acts as a repressor to coordinate and fine tune target-specific, ERα-mediated transcriptional activity in human breast cancer cells.

Abstract 1426: Cyclooxygenase-2 (COX-2) utilizes Jun N-terminal kinases to induce invasion but not tamoxifen resistance in MCF-7 breast cancer cells

Abstract Elevated Cyclooxygenase-2 (COX-2) expression in breast tumors had been associated with a lower survival rate in patients with Estrogen Receptor α (ERα)-positive tumors. We propose that COX-2 reduces survival rate in breast cancer patients with ERα-positive tumors because COX-2 decreases tamoxifen sensitivity and increases invasive activity of breast cancer cells. We demonstrated that transfection of the COX-2 gene in the tamoxifen-sensitive, ERα-positive MCF-7 breast cancer cell line decreased the cells’ sensitivity to tamoxifen by about 5-fold, and increased their invasive activity by about 3-fold. Furthermore, we demonstrated that COX-2 utilizes PGE2 to activate Protein Kinase C (PKC) to induce these tumorigeneic effects. However, which downstream factors are recruited by the COX-2/PGE2/PKC pathway to induce tamoxifen resistance and breast cancer cell invasion are not known. Here we report that COX-2 increases the phosphorylation of Jun N-terminal kinase (JNK), but not that of extracellular signal regulated kinases (ERK1,2), p38 mitogen activated protein kinase, or Akt. Inhibition of JNK by a chemical inhibitor or siRNA led to decreased COX-2-mediated invasion. Yet, JNK inhibition did not affect COX-2-mediated tamoxifen resistance. We propose that JNK is a promising target to block invasion in patients whose tumors are ERα-positive and present high levels of COX-2. 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 1426. doi:10.1158/1538-7445.AM2011-1426

Response of Estrogen Receptor-Positive Breast Cancer Tumorspheres to Antiestrogen Treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.

Abstract PD05-05: Hormone-Independent Breast Cancer Cells Maintain an ERα — Dependent, E2F-Directed Transcriptional Program

Abstract In order to discover mechanisms of escape from hormone dependence, we established four ERα-positive human breast cancer cell lines after long-term estrogen-deprivation (LTED). Growth of MCF-7/LTED and HCC-1428/LTED cells was inhibited by the ER downregulator fulvestrant and by ER siRNA, suggesting continued dependence upon estrogen-independent ER signaling. Fulvestrant also prevented the emergence of hormone-independent MCF-7, HCC-1428, and BT-474 cells, but not ZR75- 1, MDA-361, and T47D cells. Gene expression analysis revealed that ER modulates a transcriptional program directed by the E2F proteins, a family of transcription factors derepressed by CDK4/6-mediated phosphorylation of Rb. A set of 1,003 genes with expression altered ≥1.5-fold (P<0.05) by fulvestrant in MCF-7/LTED and HCC-1428/LTED cells was used to query the TRANSFAC database. Five of the top ten sets of genes which share a transcription factor binding motif ± 2 kb of transcription start sites contained E2F motifs (all p<10-12). From these, we derived an expression signature of 24 genes with no cell cycle association. This signature was predictive of response of 48 ER+ primary tumors to 2 weeks of neoadjuvant letrozole as assessed by Ki67 IHC. In a separate set of experiments, we screened a siRNA library targeting 779 kinases and found that CDK4 is required for MCF-7/LTED cell growth. Inhibition of CDK4/6 using the small molecule PD-0332991 or CDK4 siRNA phenocopied the inhibitory effect of fulvestrant on MCF-7/LTED and HCC-1428/LTED cells, implying that the primary growth-promoting effect of ligand-independent ER is activation of the Rb/E2F pathway. CDK4 inhibition also suppressed the hormone-independent growth of 4 other ER+ breast cancer lines. We previously demonstrated that phosphatidylinositol-3 kinase (PI3K) signaling is required for the escape of ER+ breast cancer cells from hormone dependence. Thus, we postulated that treatment with the PI3K inhibitor BKM120 (Novartis) would synergize with fulvestrant to suppress hormone-independent cell growth. Indeed, these drugs exerted a synergistic inhibitory effect in 6/8 ER+ breast cancer cell lines. Finally, we injected MCF-7 cells and 14-day-release 17P-estradiol pellets into female athymic mice. After 19-29 days, mice bearing tumors ≥100 mm3 were randomized to treatment with vehicle, fulvestrant, BKM120, or the combination. While BKM120 and fulvestrant individually slowed tumor growth, the combination induced near-complete tumor regression. Fulvestrant downregulated ER and PR levels, while BKM120 decreased P-AKT-S473, P-AKT-T308, P-S6-S240/244 (by immunoblotting), and [18F]fluorodeoxyglucose (FDG) tumor uptake as measured by PET, indicative of suppressed ER and PI3K signaling, respectively. These findings collectively suggest that 1) a fraction of ER+ breast cancer cells engage ERα in a ligand-independent manner to drive cell growth, 2) pharmacological downregulation of ER may be effective in a fraction of ER+ tumor cells that escape estrogen deprivation, 3) CDK4/6 inhibition suppresses the hormone-independent growth of fulvestrant-sensitive and-insensitive ER+ breast cancer cells, and 4) resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both the ER and PI3K pathways. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-05.

Abstract 4603: Role of endoxifen as an antiestrogen: Effects on oncogene expression and polyamine pathway

Abstract The nonsteroid antiestrogen tamoxifen [trans-1-(4-b-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene] is the most commonly used endocrine treatment for estrogen receptor α (ERα)-positive breast cancer in pre- and post-menopausal women, and it has helped to reduce breast cancer death rate by one third. Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. It is metabolized to 4-hydroxytamoxifen and N-desmethyltamoxifen by the action of CYP2D6 and CYP3A4/5 enzymes, respectively. N-desmethyltamoxifen and 4-hydroxy-tamoxifen are further converted to endoxifen by the action of these enzymes. Steady state levels of serum tamoxifen/metabolite are achieved within 4 weeks of continuous administration of the drug. The therapeutic efficacy of tamoxifen is determined by the distribution of the drug into tissues and the availability of the parent drug and its active metabolites in target tissues. Recent reports indicate that endoxifen might act as an antiestrogen. In order to examine the mechanism of action of endoxifen on an ERα-positive breast cancer cell line, MCF-7, we determined the effects of 100, 250, 500 and 1000 nM concentrations of this agent on cell growth, c-myc oncogene expression and activity of the polyamine biosynthetic enzymes. MCF-7 breast cancer cells were treated for 1, 3 or 5 days with endoxifen. Endoxifen had a growth inhibitory effect on MCF-7 cells by day 3 and 5 of treatment, as measured by cell number. Estradiol increased cell number by >2-fold compared to untreated control by day 5 of treatment, whereas endoxifen inhibited the estrogenic effect. c-myc gene expression was increased by a significant 2- to 3-fold by 4 nM estradiol in 2 and 4 hours of treatment, while 250 and 500 nM endoxifen suppressed the increase in c-myc gene expression, as measured by real-time qPCR experiment. In the absence of estradiol, endoxifen slightly (1.5 fold) increased c-myc gene expression. Endoxifen decreased ornithine decarboxylase (ODC) and S-adenosine methyl decarboxylase (AdoMetDC) activities in a concentration- and time-dependent manner; however, it had little effect on spermidine/spermine N1-acetyl transferase (SSAT) activity. The change in biosynthetic enzyme activities were reflected in polyamine pools, as decreased putrescine and spermidine levels. There were no changes in spermine level. As expected, treatment of the cells with 4 nM estradiol induced ODC and AdoMetDC activities, and slightly decreased SSAT activity. Addition of endoxifen in combination with 4 nM estradiol led to even more dramatic decrease in ODC and AdoMetDC activities and reduction of putrescine and spermidine levels. These results support our hypothesis that endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol in ERα-positive breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4603.