ER-positive Human Breast Cancer Cell Research Articles

Beauvericin, produced by Fusarium oxysporum inhibits bisphenol A-induced proliferation of human breast cancer cell line by regulating ERα/p38 pathway

Beauvericin (BEA) is a cyclic depsipeptide secondary metabolite of Fusarium species. It causes chemical hazards in food products and exists in an environment containing soil and various food types. On the other hand, the purified BEA has various biological activities and is regarded as a potential candidate for pharmaceutical research. This study was performed to assess the anti-proliferation activity of BEA against human breast cancer cells by regulating the estrogen receptor-alpha (ERα)/p38 pathway. TA and BA assays verified that BEA is a completed ER antagonist. Additionally, BEA suppressed cell proliferation in the anti-proliferation assay involving ER-positive human breast cancer cells co-treated with BPA and BEA. In respect to an anti-proliferation activity, the BPA-induced phosphorylation of p38 protein was inhibited in the presence of BEA. These results suggested that BEA exerts inhibitory potentials on endocrine disrupting effect and possibly acts as a natural therapeutic material for human estrogen hormonal health.

Abstract P4-09-11: Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors

Abstract Background CDK4/6 inhibitors are the latest and highly specific CDK inhibitors approved for metastatic and early estrogen receptor (ER)-positive breast cancer patients, improving the survival outcomes. Prior preclinical research has indicated various aberrations of cell cycle regulation and activated oncogenic signaling pathway as the mechanisms of resistance to CDK4/6 inhibitors. However, there is currently no available biomarker except ER and human epidermal growth factor receptor 2 (HER2) in clinical practice. The aim of our study is to elucidate diverse mechanisms of acquired resistance of ER-positive breast cancers to CDK4/6 inhibitors and develop personalized medicine in ER-positive breast cancer patients. Materials and methods To generate resistant lines, the ER-positive human breast cancer cell lines MCF7 and T47D were treated individually with increasing concentrations of the CDK4/6 inhibitors palbociclib and ribociclib until a target concentration of 3μM for six months. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin at 37°C in a 5% CO2 incubator. Western blot analysis was performed for whole cell extract proteins from resistant cells to CDK4/6 inhibitors and parental cells. Colony formation assay was done to observe cell viability. Results Western blot analysis showed elevated FGFR2, CDK6, pS6RP, and cyclin E1 in resistant cells compared to parental cells. Specifically, cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, while FGFR2 and CDK6 in T47D palbociclib- and ribociclib-resistant cells. Notably, CDK6 was upregulated in MCF7 ribociclib-resistant cells, whereas increased cyclin E1 was detected in T47D palbociclib-resistant cells, respectively. Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in generated resistant cell lines. Conclusions The tumors harboring resistance to CDK4/6 inhibitors have heterogenous mechanisms of therapeutic resistance to these agents. The novel combination treatment of targeted molecular therapy and CDK4/6 inhibition will lead development of personalized medicine for ER-positive breast cancer based on elucidation of diverse resistant mechanisms to CDK4/6 inhibition. Western blot analysis of lysates from MCF-7 cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) Cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, whereas CDK6 was upregulated especially in MCF7 ribociclib-resistant cells. Western blot analysis of lysates from T47D cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) FGFR2 and CDK6 were increased in T47D palbociclib- and ribociclib-resistant cells, whereas increased cyclin E1 was detected especially in T47D palbociclib-resistant cells. Viability in MCF7 ribociclib-resistant and parental cells by colony formation assay Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in MCF7 resistant-cells. Citation Format: Yoshie Kobayashi, Yusuke Motoi, Mutsumi Fujimoto, Hideo Shigematsu. Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-11.

Inhibitors of Keap1-Nrf2 protein-protein interaction reduce estrogen responsive gene expression and oxidative stress in estrogen receptor-positive breast cancer

Estrogen contributes to the development of breast cancer through estrogen receptor (ER) signaling and by generating genotoxic metabolites that cause oxidative DNA damage. To protect against oxidative stress, cells activate nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream cytoprotective genes that initiate antioxidant responses and detoxify xenobiotics. Nrf2 activation occurs by inhibiting the protein-protein interaction (PPI) between Nrf2 and its inhibitor Keap1, which otherwise targets Nrf2 for ubiquitination and destruction. In this study, we examined a series of novel direct inhibitors of Keap1-Nrf2 PPI in their role in promoting the availability of Nrf2 for antioxidant activity and attenuating estrogen-mediated responses in breast cancer. ER-positive human breast cancer cells MCF-7 were treated with 17β-estradiol (E2) in the presence or absence of selected Keap1-Nrf2 PPI inhibitors. Keap1-Nrf2 PPI inhibitors suppressed the mRNA and protein levels of estrogen responsive genes induced by E2 exposure, such as PGR. Keap1-Nrf2 PPI inhibitors caused significant activation of Nrf2 target genes. E2 decreased the mRNA and protein level of the Nrf2 target gene NQO1, and the Keap1-Nrf2 PPI inhibitors reversed this effect. The reversal of E2 action by these compounds was not due to binding to ER as ER antagonists. Further, a selected compound attenuated oxidative stress induced by E2, determined by the level of a biomarker 8-oxo-deoxyguanosine. These findings suggest that the Keap1-Nrf2 PPI inhibitors have potent antioxidant activity by activating Nrf2 pathways and inhibit E2-induced gene and protein expression. These compounds may serve as potential chemopreventive agents in estrogen-stimulated breast cancer.

BPTF promotes the progression of distinct subtypes of breast cancer and is a therapeutic target.

To assess the biomarker and functional role of the chromatin remodeling factor, bromodomain PHD finger transcription factor (BPTF), in breast cancer progression. BPTF copy number was assessed using fluorescence in situ hybridization. BPTF expression was regulated in breast cancer cells by shRNA/siRNA-mediated gene silencing and BPTF cDNA overexpression. The effects of regulating BPTF expression were examined on key oncogenic signaling pathways and on breast cancer cell proliferation, apoptosis, and cell cycle progression, as well as in xenograft models. The consequences of pharmacological bromodomain inhibition, alone or in combination with other targeted agents, on breast cancer progression were assessed in culture and in xenograft models. BPTF copy number was gained in 34.1% and separately amplified in 8.2% of a breast cancer tissue cohort. Elevated BPTF copy number was significantly associated with increasing patient age and tumor grade and observed in both ER-positive and triple-negative breast cancer (TNBC) subtypes. BPTF copy number gain and amplification were also observed in The Cancer Genome Atlas (TCGA) breast cancer cohort. Stable shRNA-mediated silencing of BPTF significantly inhibited cell proliferation and induced apoptosis in TNBC and ER-positive human breast cancer cell lines. BPTF knockdown suppressed signaling through the phosphoinositide 3 kinase (PI3K) pathway, including reduced expression of phosphorylated AKT (Ser473), phosphorylated GSK-β (Ser9), and CCND1. These findings were confirmed following transient BPTF knockdown by a distinct siRNA in TNBC and ER-positive breast cancer cells. Stable suppression of BPTF expression significantly inhibited the in vivo growth of TNBC cells. Conversely, BPTF cDNA overexpression in TNBC and ER-positive breast cancer cells enhanced breast cancer cell proliferation and reduced apoptosis. BPTF targeting with the bromodomain inhibitor bromosporine, alone or in combination with the PI3K pathway inhibitor gedatolisib, produced significant anti-tumor effects against TNBC cells in vitro and in vivo. These studies demonstrate BPTF activation in distinct breast cancer subtypes, identify pathways by which BPTF promotes breast cancer progression, and suggest BPTF as a rational target for breast cancer therapy.

Carbidopa suppresses estrogen receptor-positive breast cancer via AhR-mediated proteasomal degradation of ERα.

Estrogen receptor-α (ERα) promotes breast cancer, and ER-positive cancer accounts for ~ 80% of breast cancers. This subtype responds positively to hormone/endocrine therapies involving either inhibition of estrogen synthesis or blockade of estrogen action. Carbidopa, a drug used to potentiate the therapeutic efficacy of L-DOPA in Parkinson's disease, is an agonist for aryl hydrocarbon receptor (AhR). Pharmacotherapy in Parkinson's disease decreases the risk for cancers, including breast cancer. The effects of carbidopa on ER-positive breast cancer were evaluated in cell culture and in mouse xenografts. The assays included cell proliferation, apoptosis, cell migration/invasion, subcellular localization of AhR, proteasomal degradation, and tumor growth in xenografts. Carbidopa decreased proliferation and migration of ER-positive human breast cancer cells in vitro with no significant effect on ER-negative breast cancer cells. Treatment of ER-positive cells with carbidopa promoted nuclear localization of AhR and expression of AhR target genes; it also decreased cellular levels of ERα via proteasomal degradation in an AhR-dependent manner. In vivo, carbidopa suppressed the growth of ER-positive breast cancer cells in mouse xenografts; this was associated with increased apoptosis and decreased cell proliferation. Carbidopa has therapeutic potential for ER-positive breast cancer either as a single agent or in combination with other standard chemotherapies.

Towards replacement of animal tests with in vitro assays: a gene expression biomarker predicts in vitro and in vivo estrogen receptor activity

High-throughput transcriptomics (HTTr) has the potential to support efforts to reduce or replace some animal tests. In past studies, we described a computational approach utilizing a gene expression biomarker consisting of 46 genes to predict estrogen receptor (ER) activity after chemical exposure in ER-positive human breast cancer cells including the MCF-7 cell line. We hypothesized that the biomarker model could identify ER activities of chemicals examined by Endocrine Disruptor Screening Program (EDSP) Tier 1 screening assays in which transcript profiles of the same chemicals were examined in MCF-7 cells. For the 62 chemicals examined including 5 chemicals examined in this study using RNA-Seq, the ER biomarker model accuracy was 1) 97% for in vitro reference chemicals, 2) 76–85% for guideline uterotrophic assays, and 3) 87–88% for guideline and nonguideline uterotrophic assays. For the same chemicals, these accuracies were similar or slightly better than those of the ToxCast ER model based on 18 in vitro assays. The performance of the ER biomarker model indicates that HTTr interpreted using the ER biomarker correctly identifies active and inactive ER reference chemicals. As part of the HTTr screening program the approach could rapidly identify chemicals with potential ER bioactivities for additional screening and testing.

Synergy of Electrical Pulses and Black Pepper (Piper nigrum) Extracts for Effective Breast Cancer Treatment: An In Vitro Model Study

In this research, the effectiveness of electrical pulse-mediated Piper nigrum (Black pepper) extracts against MCF-7, ER-positive human breast cancer cells were investigated. Initially, P. nigrum extracts were extracted by using the conventional (Soxhlet) and electrical pulse-assisted method. We assessed the phytochemicals present in both extracts by phytochemical screening tests and compared their total phenol contents and antioxidant activity with the conventional extract. FTIR and GCMS for untreated and treated extracts were also measured and compared. Electrically enhanced extracts (EE) resulted in an improvement of ∼41% in its antioxidant activity and ∼24% increase in their total phenol contents than untreated extracts. The cytotoxic activity of both extracts was determined by MTT proliferation assay in a dose-dependent manner. The results show that electrical pulse-assisted extracts have enhanced the bioactive compounds, with respect to untreated extracts, significantly promoted total phenols and antioxidant activity, which is correlated with increased MCF7 cytotoxicity. Furthermore, to enhance the uptake of extracts against plasma membranes, electrical pulses of different parameters (EP1 and EP2) were used. The results indicate cell viabilities of 27% with raw extract only, 12% with EE only, 7% with (EP1 + EE) and 6% (EP2 + EE) after 72h, indicating the potential of the synergetic effect of electrical pulses (EP) and enhanced extract (EE). On comparison, the enhanced extract showed viability of two times less than raw extracts, whereas when it is mediated with electrical pulses, there is a marked reduction in cell viability. This clearly indicates that the application of electrical pulses aids in achieving antioxidant and phenolic-rich extracts. Furthermore, this treatment is more appropriate to achieve maximum cell death, with less concentration of the extract to treat breast cancer.

Flavonoid calycopterin triggers apoptosis in triple-negative and ER-positive human breast cancer cells through activating different patterns of gene expression.

Breast cancer is the most common cause of death related to cancer in women, and several studies proved that flavonoids could induce apoptosis in this cancer through different pathways. Calycopterin is a flavonoid which was shown to induce preferential antiproliferative effects on some cancers; however, no information is available on its effect on breast cancer. Therefore, in this paper, the apoptotic effect of calycopterin and its underlying mechanism in two different breast cancer cells, MDA-MB-231 and MCF7 cell lines were investigated. MTT assay showed that calycopterin reduced the proliferation of both cancer lines with no adverse effect on normal cells. The clonogenic assay showed that calycopterin treatments decreased the colony numbers and sizes, and wound healing assay demonstrated the inhibition of migration in both cancer cells. Cell cycle and annexin/PI analyses indicated that calycopterin augmented sub-G1 population and annexin/PI-positive cells. Gene expression revealed that Bax/Bcl2 increased in the MDA-MB-231 cell line, while no change was observed in that of the MCF7 line. Expression of gene caspase-8 was augmented in both lines, although increased expression of caspase-3 was found just in MDA-MB-231 cells. Our results validated the apoptotic effect of calycopterin on both breast cancer lines with more potency on triple-negative ones.

Arctigenin inhibits the activation of the mTOR pathway, resulting in autophagic cell death and decreased ER expression in ER-positive human breast cancer cells.

Arctigenin, a member of the Asteraceae family, is a biologically active lignan that is consumed worldwide due to its several health benefits. However, its use may pose a problem for patients with estrogen receptor (ER)α-positive breast cancer, since studies have shown that arctigenin is a phytoestrogen that exerts a proliferative effect by binding to the ER. Thus, in this study, we examined the effect of arctigenin on ERα-positive MCF-7 human breast cancer cells to determine whether the consumption of arctigenin is safe for patients with breast cancer. First, we found that arctigenin inhibited the viability of the MCF-7 cells, and colony formation assay confirmed that this effect was cytotoxic rather than cytostatic. The cytotoxic effects were not mediated by cell cycle arrest, apoptosis, or necroptosis, despite DNA damage, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage and phosphorylated H2A.X. An increase in lipidated LC3, a marker of autophagosome formation, was observed, indicating that autophagy was induced by arctigenin, which was found to be triggered by the inhibition of the mechanistic target of rapamycin (mTOR) pathway. We then examined the effects of arctigenin on ERα expression and determined whether it affects the sensitivity of the cells to tamoxifen, as tamoxifen is commonly used against hormone-responsive cancers and is known to act via the ERα. We found that treatment with arctigenin effectively downregulated ERα expression, which was found to be a consequence of the inhibition of the mTOR pathway. However, treatment with arctigenin in combination with tamoxifen did not affect the sensitivity of the cells to tamoxifen, but instead, exerted a synergistic effect. On the whole, our data indicate that the phytoestrogen, arctigenin, mainly targeted the mTOR pathway in ERα-positive MCF-7 human breast cancer cells, leading to autophagy-induced cell death and the downregulation of ERα expression. Furthermore, the synergistic effects between arctigenin and tamoxifen suggest that the consumption of arctigenin is not only safe for patients with hormone-sensitive cancers, but may also be an effective co-treatment.

One-Step 18F-Labeling of Estradiol Derivative for PET Imaging of Breast Cancer.

Positron emission tomography (PET) imaging is a useful method to evaluate in situ estrogen receptor (ER) status for the early diagnosis of breast cancer and optimization of the appropriate treatment strategy. The 18F-labeled estradiol derivative has been successfully used to clinically assess the ER level of breast cancer. In order to simplify the radiosynthesis process, one-step 18F-19F isotope exchange reaction was employed for the 18F-fluorination of the tracer of [18F]AmBF3-TEG-ES. The radiotracer was obtained with the radiochemical yield (RCY) of ~61% and the radiochemical purity (RCP) of >98% within 40 min. Cell uptake and blocking assays indicated that the tracer could selectively accumulate in the ER-positive human breast cancer cell lines MCF-7 and T47D. In vivo PET imaging on the MCF-7 tumor-bearing mice showed relatively high tumor uptake (1.4~2.3 %D/g) and tumor/muscle uptake ratio (4~6). These results indicated that the tracer is a promising PET imaging agent for ER-positive breast cancers.

Estrogenic activity of zearalenone, α-zearalenol and β-zearalenol assessed using the E-screen assay in MCF-7 cells

Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and β-zearalenol, β-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and β-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and β-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.

Biological impact of advanced glycation endproducts on estrogen receptor-positive MCF-7 breast cancer cells

Diabetes mellitus potentiates the risk of breast cancer. We have previously described the pro-tumorigenic effects of advanced glycation endproducts (AGEs) on estrogen receptor (ER)-negative MDA-MB-231 breast cancer cell line mediated through the receptor for AGEs (RAGE). However, a predominant association between women with ER-positive breast cancer and type 2 diabetes mellitus has been reported. Therefore, we have investigated the biological impact of AGEs on ER-positive human breast cancer cell line MCF-7 using in vitro cell-based assays including cell count, migration, and invasion assays. Western blot, FACS analyses and quantitative real time-PCR were also performed. We found that AGEs at 50–100μg/mL increased MCF-7 cell proliferation and cell migration associated with an enhancement of pro-matrix metalloproteinase (MMP)-9 activity, without affecting their poor invasiveness. However, 200μg/mL AGEs inhibited MCF-7 cell proliferation through induction of apoptosis indicated by caspase-3 cleavage detected using Western blotting. A phospho-protein array analysis revealed that AGEs mainly induce the phosphorylation of extracellular-signal regulated kinase (ERK)1/2 and cAMP response element binding protein-1 (CREB1), both signaling molecules considered as key regulators of AGEs pro-tumorigenic effects. We also showed that AGEs up-regulate RAGE and ER expression at the protein and transcript levels in MCF-7 cells, in a RAGE-dependent manner after blockade of AGEs/RAGE interaction using neutralizing anti-RAGE antibody. Throughout the study, BSA had no effect on cellular processes. These findings pave the way for future studies investigating whether the exposure of AGEs-treated ER-positive breast cancer cells to estrogen could lead to a potentiation of breast cancer development and progression.

BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human breast cancer in vitro and in vivo

BackgroundTamoxifen (TAM) has been widely used for the treatment of estrogen receptor (ER)-positive breast cancer and its combination with other therapies is being actively investigated as a way to increase efficacy and decrease side effects. Here, we evaluate the therapeutic potential of co-treatment with TAM and BreastDefend (BD), a dietary supplement formula, in ER-positive human breast cancer.MethodsCell proliferation and apoptosis were determined in ER-positive human breast cancer cells MCF-7 by MTT assay, quantitation of cytoplasmic histone-associated DNA fragments and expression of cleaved PARP, respectively. The molecular mechanism was identified using RNA microarray analysis and western blotting. Tumor tissues from xenograft mouse model were analyzed by immunohistochemistry.ResultsOur data clearly demonstrate that a combination of 4-hydroxytamoxifen (4-OHT) with BD lead to profound inhibition of cell proliferation and induction of apoptosis in MCF-7 cells. This effect is consistent with the regulation of apoptotic and TAM resistant genes at the transcription and translation levels. Importantly, TAM and BD co-treatment significantly enhanced apoptosis, suppressed tumor growth and reduced tumor weight in a xenograft model of human ER-positive breast cancer.ConclusionBD sensitized ER-positive human breast cancer cells to 4-OHT/TAM treatment in vitro and in vivo. BreastDefend can be used in an adjuvant therapy to increase the therapeutic effect of tamoxifen in patients with ER-positive breast cancer.

Abstract 4555: Inhibition of CDK1/2 but not CDK4/6 cooperates with tamoxifen to induce apoptosis

Abstract As essential drivers of cell cycle progression and transcription, cyclin-dependent kinases (CDKs) often become dysregulated during tumorigenesis, thus motivating extensive efforts towards development of CDK inhibitors as a new class of promising anticancer agents with clear CDK specificity including those targeting mainly CDK4 and CDK6 (e.g. PD0332991, LY2835219) vs. those primarily targeting CDK1 and CDK2 (e.g. BMS265246, Dinaciclib). Given there different cyclin dependencies and mitotic phase mechanisms, including the relative cell cycle independence of some CDKs, specific CDK inhibitors are expected to exhibit different anticancer activities, side effects, and interactions with other anticancer agents, and require different clinical biomarkers. With regard to breast cancers in which at least 75% overexpress estrogen receptor (ER, alpha isoform), it is particularly important to establish the type of CDK inhibitor most effective in combination with anti-estrogenic agents like tamoxifen or aromatase inhibitors. To preclinically determine the potential added efficacy of specific CDK inhibition in combination with anti-estrogen therapy, the ER-positive human breast cancer cell line MCF7 was used to compare the relative efficacy of CDK1/2 inhibition vs. CDK4/6 inhibition in combination with 4-hydroxytamoxifen (Tam) in promoting breast cancer cell death (apoptosis). These studies were also designed based on our recent evidence that CDK1/2 inhibition, but not CDK4/6 inhibition, suppressed transcriptional upregulation of ER inducible genes by impairing ER hinge domain phosphorylation at Ser294, suggesting that CDK1/2 inhibition would preferentially cooperate with Tam to impair ER-positive breast cancer viability by their convergent suppression of ligand-dependent ER transcription. We confirmed this hypothesis by demonstrating that CDK1/2 inhibitors, but not CDK4/6 inhibitors, suppressed ligand induced ER phosphorylation at Ser294, without altering Ser118 phosphorylation which remained constant despite either CDK1/2 or CDK4/6 inhibition. Consistent with its ability to suppress ER phosphorylation at Ser294, CDK1/2 inhibition, in combination with Tam, enhanced MCF7 apoptosis, while CDK4/6 inhibition had no such cooperative effect. These ER-positive breast cancer cell line and molecular studies should guide further preclinical and clinical efforts in evaluating the optimal choice of CDK inhibitor for use in combination with endocrine agents. These findings also suggest that clinical emphasis should be placed on the evaluation of CDK1/2 inhibitors in combination with anti-estrogens like tamoxifen, where loss of ER phosphorylation at Ser294 can also be used as an early biomarker of response to monitor this therapeutic combination. Citation Format: Gary K. Scott, Daniel Rothschild, Ravneet Kaur, Christopher Benz. Inhibition of CDK1/2 but not CDK4/6 cooperates with tamoxifen to induce apoptosis. [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 4555. doi:10.1158/1538-7445.AM2014-4555

CAPER, a novel regulator of human breast cancer progression

CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.

Starfish polysaccharides downregulate metastatic activity through the MAPK signaling pathway in MCF-7 human breast cancer cells

We investigated the effects of starfish (Asterina pectinifera) polysaccharides on metastatic activity in MCF-7 estrogen receptor (ER)-positive human breast cancer cells. In wound healing assay, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration was dose-dependently decreased by the starfish polysaccharides (PS). Transcription of aromatase, which catalyzes estrogen synthesis from androgen, was reduced by PS. Also, transcription of TPA-induced cyclooxygenase-2 (COX-2), which enhances breast cancer progression and metastasis via the increase of prostaglandin E2 biosynthesis, was downregulated by the PS in a dose-dependent manner. PS decreased the expression and activity of matrix metalloproteinase (MMP)-9, an important factor in the degradation of basement membrane and extracellular matrix in the metastasis process. In contrast, mRNA expression of tissue inhibitor of matrix metalloproteinase (TIMP)-1, a MMP inhibitor, was increased by 10-120 μg/ml of PS but not that of TIMP-2. We also found that PS reversed the phosphorylations of p38, ERK and JNK but not IκBα and NF-κB. These results demonstrate that PS successfully inhibits PKC-mediated cell migration and metastatic activities in MCF-7 ER-positive human breast cancer cells via downregulation of MMP-9 activity mediated by TIMP-1 upregulation and inhibition of aromatase and COX-2 expression. Also, COX-2 and MMP-9 expressions are attenuated through the inhibition of AP-1 transcription activity via the downregulation of c-Jun expression regulated by p38, ERK and JNK signaling. In conclusion, the present investigation shows that PS may prevent COX-2- and MMP-9-mediated metastatic activities in MCF-7 ER-positive breast cancer cells through the downregulation of MAPK signaling pathways.

Abstract 4409: The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro.

Abstract Tamoxifen is the most widely used agent in the world for antihormonal therapy of Estrogen Receptor (ER)-positive breast cancer for both pre- and postmenopausal patients. Tamoxifen is a pro-drug and needs to be metabolically activated to a more potent antiestrogen with a higher affinity for the ER. Initially 4-hydroxytamoxifen (4OHT) was the active metabolite of interest, but more recently 4-hydroxy-N-desmethyltamoxifen (endoxifen) is also of significant interest. The role of endoxifen in therapy remains controversial, however both 4OHT and endoxifen exist in two isomeric forms- cis and trans. Previously, it was shown that cis-isomers of tamoxifen metabolites have weak anti-estrogenic properties using in vitro models available (Jordan VC et al, Endocrinology, 1987, 122(4); 1449-54). However, studies show that an increase of cis-isomers of 4OHT in animals and patients may contribute to tamoxifen resistance (Osborne CK et al, JNCI, 1991, 83; 1477-82; Osborne CK et al, J Clin Oncol, 1992, 10; 304-10). Thus, it is important to know the estrogenic properties of clinically relevant tamoxifen metabolites. To document the anti-estrogenic profiles of both geometric isomers of two major hydroxylated metabolites of tamoxifen, isomerically stable fixed-ring derivatives of 4OHT and endoxifen were synthesized. We have assessed the anti-estrogenic properties of these compounds using cell proliferation assays in selectively cloned hypersensitive ER-positive human breast cancer cell lines MCF-7:WS8 and T47D:A18. Also to assess to estrogenic/anti-estrogenic properties of synthesized compounds on transcriptional level, we have used real-time PCR of rat Prolactin (Prl) mRNA isolated from GH3 rat pituitary cell line. Our results indicate that the fixed-ring trans-4OHT and trans-Endoxifen are potent antiestrogens and equivalent to each other, and to their non-fixed-ring counterparts in both cell proliferation assays and Prl gene activity, as they do not possess any agonistic activities by themselves and are able to inhibit the estrogenic actions of 0,1nM 17β-estradiol (E2) completely (IC50 1nM). However, cis-isomers of the fixed-ring compounds are very weakly estrogenic by themselves but are very weak anti-estrogens in combination with 0,1nM E2 in all assays. Conclusions: the weak estrogen-like activity and low potency of the cis-isomers is unlikely to influence the growth of breast cancer during long-term therapy. The high potency of trans-endoxifen and 4OHT will dominate the control of the tumor growth. Support: This study (PYM) was supported by Susan G. Komen for the Cure International Postdoctoral Fellowship under award number SAC100009 (VCJ). Citation Format: Philipp Y. Maximov, Cynthia Myers, Daphne J. Fernandes, V. Craig Jordan. The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro. [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 4409. doi:10.1158/1538-7445.AM2013-4409

Inferring coregulation of transcription factors and microRNAs in breast cancer

Both transcription factors (TFs) and microRNAs (miRNAs) regulate gene expression. TFs activate or suppress the initiation of the transcription process and miRNAs regulate mRNAs post-transcriptionally, thus forming a temporally ordered regulatory event. Ectopic expression of key transcriptional regulators and/or miRNAs has been shown to be involved in various tumors. Therefore, uncovering the coregulation of TFs and miRNAs in human cancers may lead to the discovery of novel therapeutics.We introduced a two-stage learning fuzzy method to model TF–miRNA coregulation using both genomic data and verified regulatory relationships. In Stage 1, a learning (adaptive) fuzzy inference system (ANFIS) combines two sequence alignment features of TF and target by learning from verified TF–target pairs into a sequence matching score. Next, a non-learning FIS incorporates a sequence alignment score and a correlation score from paired TF–target gene expression to output a Stage 1 fuzzy score to infer whether a TF–target regulation exists. For significant TF–target pairs, in Stage 2, similar to Stage 1, we first infer whether a miRNA regulates each common target by an ANFIS, which incorporates their sequences and known miRNA–target relationships to output a sequence score. Next, an FIS incorporates the Stage 1 fuzzy score, Stage 2 sequence score and gene expression correlation score of a miRNA–target pair to determine whether TF–miRNA coregulation exists. We collected 54 (8) TF–miRNA–target triples validated in ER-positive (ER-negative) breast cancer cell lines in the same article, and they were used as positives. Negative examples were constructed for Stage 1 (Stage 2) by pairing TFs (miRNAs) with human housekeeping genes found in the literature; both positives and negatives were used to train ANFISs in the training step.This two-stage fizzy algorithm was applied to predict 54 (8) TF–miRNA coregulation triples in ER-positive (ER-negative) human breast cancer cell lines, and resulted in true-positive rates of 0.55 (0.74) and 0.57 (0.75) using 3-fold and n-fold cross validations (CVs), respectively. False-positive rate bound was 0.07 (0.13) for ER-positive (ER-negative) breast cancer using both 3-fold and n-fold CVs. Interestingly, among the 62 coregulatroy triples from ER-positive/negative breast cancer cells, about 72% have TF- and coregulatory miRNA expression simultaneously greater or less than the corresponding medians, while in the remaining 28% of TFs and their coregulatory miRNAs are conversely expressed. The proposed fuzzy algorithm performed well in identification of TF–miRNA coregulation triples in human breast cancer. After being trained by the corresponding verified coregulatory triples and genomic data, this algorithm can be applied to uncover novel coregulation in other cancers in the future.

Abstract 2054: The BH3-only protein, Noxa, is upregulated by estrogen and promotes cell cycle progression in estrogen receptor-positive breast cancer cells

Abstract According to the American Cancer Society, breast cancer is the second leading cause of cancer-related deaths in American women, with more than 200,000 new cases diagnosed annually. In addition, the vast majority of breast cancers are initially hormone-dependent and 70% are estrogen receptor (ER)-positive (Colditz, J. Natl. Cancer Inst., 1998; Hankinson et al., Breast Cancer Res., 2004; and Harvey et al., J. Clin. Oncol., 1999), highlighting the importance of estrogen signaling pathways in breast oncogenesis. It has been well-established that estrogen/17β-estradiol/E2 stimulates cell growth and inhibits apoptosis in ER-positive breast cancer cells; however, recent reports have demonstrated that E2 paradoxically induces the expression of Noxa/PMAIP1, a BH3-only pro-apoptotic member of the Bcl-2 family. In the present study, we report on the mechanisms by which E2 upregulates Noxa expression and the associated cellular effects on apoptosis and cell cycle progression in ER-positive MCF7 human breast cancer cells. We demonstrate that c-Myc, ERα, and E2F1 are involved in the transcriptional upregulation of Noxa following E2 treatment. Specific knockdown of c-Myc and ERα protein expression using siRNA technology inhibited E2-mediated induction of Noxa expression. Furthermore, E2 also promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, suggesting that these two proteins are indeed mediators of Noxa upregulation following E2 treatment. Although p53 is an important regulator of Noxa transcription under conditions of cellular stress, our data demonstrate that E2-mediated upregulation of Noxa is a p53-independent process, as knocking down p53 with specific siRNA did not block E2-mediated induction of Noxa. Consistent with this result, E2 treatment failed to induce the recruitment of p53 to the NOXA promoter in ChIP assays. Interestingly, E2-mediated upregulation of Noxa was associated with cell cycle progression but not with apoptosis under normal, unstressed cellular conditions. Cell cycle analysis by flow cytometry showed that siRNA-mediated knockdown of Noxa caused cell cycle arrest in G0/G1-phase and significantly delayed the G1-to-S-phase transition following E2 treatment. Collectively, these data indicate that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells, suggesting a novel role for the BH3-only protein Noxa, outside of its traditional role as an apoptosis sensitizer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2054. doi:1538-7445.AM2012-2054

Role of ERRF, a Novel ER-Related Nuclear Factor, in the Growth Control of ER-Positive Human Breast Cancer Cells

Whereas estrogen-estrogen receptor α (ER) signaling plays an important role in breast cancer growth, it is also necessary for the differentiation of normal breast epithelial cells. How this functional conversion occurs, however, remains unknown. Based on a genome-wide sequencing study that identified mutations in several breast cancer genes, we examined some of the genes for mutations, expression levels, and functional effects on cell proliferation and tumorigenesis. We present the data for C1orf64 or ER-related factor (ERRF) from 31 cell lines and 367 primary breast cancer tumors. Whereas mutation of ERRF was infrequent (1 of 79 or 1.3%), its expression was up-regulated in breast cancer, and the up-regulation was more common in lower-stage tumors. In addition, increased ERRF expression was significantly associated with ER and/or progesterone receptor (PR) positivity, which was still valid in human epidermal growth factor receptor 2 (HER2)-negative tumors. In ER-positive tumors, ERRF expression was inversely correlated with HER2 status. Furthermore, higher ERRF protein expression was significantly associated with better disease-free survival and overall survival, particularly in ER- and/or PR-positive and HER2-negative tumors (luminal A subtype). Functionally, knockdown of ERRF in two ER-positive breast cancer cell lines, T-47D and MDA-MB-361, suppressed cell growth in vitro and tumorigenesis in xenograft models. These results suggest that ERRF plays a role in estrogen-ER-mediated growth of breast cancer cells and could, thus, be a potential therapeutic target.