Estrogen-responsive Cell Line Research Articles

Differences in MCF-7 response to endocrine disruptors in waste, superficial, and treated water from Southern Brazil.

The aim of this study was to evaluate the effect of possible endocrine disruptors in surface and wastewater using a cell proliferation assay in an estrogen-responsive cell line (MCF-7). This study was conducted in the Sinos River (Brazil). The residual water was collected from a Pilot Treatment Plant (using Typha domingensis) and surface waters of the Luis Rau stream, the Sinos River, and the Water Treatment Station (WTS). After exposures (24-120h), a Sulforhodamine B assay was performed to determine the proliferation rate. The higher increase in proliferation rate was observed with the Luiz Rau stream and the sewage treated by macrophytes in a flotation filter. The results from WTS water remained with a proliferation rate similar to the negative control at all times, suggesting that the conventional treatment is partially effective for the withdrawal of endocrine-disrupting agents. The study demonstrated the efficiency of the MCF-7 line in assessing endocrine disruption caused by wastewater and surface water samples. Our results indicate that conventional water treatment can partially remove the polluting load of endocrine disruptors, minimizing their environmental and public health impacts. Besides, it demonstrates the need to expand sanitary services to improve the population's quality of life.

Using in vitro bioassays to guide the development of safer bio-based polymers for use in food packaging.

Petroleum-based polymers traditionally used for plastic packaging production have been shown to leach dangerous chemicals such as bisphenol-A (BPA). Bio-based polymers are potentially safer alternatives, and many can be sustainably sourced from waste streams in the food industry. This study assesses bio-based polymers undergoing food packaging development for migration of endocrine disrupting leachates at the level of estrogen, androgen and progestagen nuclear receptor transcriptional activity. Reporter gene assays were coupled with migration testing, performed using standardised test conditions for storage and temperature. Test samples include nine bio-based polymers and four inorganic waste additives mixed with a traditional petroleum-based polymer, polypropylene. Thermoplastic starch material, polybutylene succinate, polycaprolactone, polybutylene adipate terephthalate (PBAT), two polylactic acid (PLA)/PBAT blends, polyhydroxybutyrate (PHB) and eggshell/polypropylene (10:90) presented no significant reduction in metabolic activity or hormonal activity under any test condition. Polypropylene (PP) presented no hormonal activity. Metabolic activity was reduced in the estrogen responsive cell line after 10 days migration testing of eggshell/polypropylene (0.1:99.9) in MeOH at 40°C, and PP in MeOH and dH20. Estrogenic agonist activity was observed after 10 days in poultry litter ash/polypropylene (10:90) in MeOH at 20°C and 40°C, poultry feather based polymer in MeOH and dH2O at 40°C, and eggshell/polypropylene (40:60) and PLA in dH2O at 40°C. Activity was within a range of 0.26–0.50 ng 17β-estradiol equivalents per ml, equating to an estrogenic potency of 3–∼2800 times less than the estrogenic leachate BPA. Poultry litter ash/polypropylene (10:90) in MeOH for 10 days presented estrogenic activity at 20°C and 40°C within the above range and anti-androgenic activity at 40°C. Progestagenic activity was not observed for any of the compounds under any test condition. Interestingly, lower concentrations of eggshell or PP may eliminate eggshell estrogenicity and PP toxicity. Alternatively eggshell may bind and eliminate the toxic elements of PP. Similarly, PLA estrogenic activity was removed in both PLA/PBAT blends. This study demonstrates the benefits of bioassay guidance in the development of safer and sustainable packaging alternatives to petroleum-based plastics. Manipulating the types of additives and their formulations alongside toxicological testing may further improve safety aspects.

The aromatase inhibitor letrozole restores FADS2 function in ER+ MCF7 human breast cancer cells

PurposePlasticity in fatty acid metabolism is increasingly recognized as a major feature influencing cancer progression and efficacy of treatments. Estrogen receptor positive MCF7 human breast cancer cells have long been known to have no FADS2-mediated Δ6-desaturase activity. Our objective was to examine the effect of estrogen and the “antiestrogen” aromatase inhibitor letrozole, on Δ5- and Δ6-desaturase synthesized fatty acids in vitro. MethodsEicosa-11,14-dienoic acid (20:2n-6), a known substrate for both FADS1 and FADS2, was used as a sentinel of relative FADS2 and FADS1 activity. MCF7 cells and four additional estrogen responsive wild type cell lines (HepG2, SK-N-SH, Y79 and Caco2) were studied. FAME were quantified by GC-FID and structures identified by GCCACI-MS/MS. ResultsIn all five cell lines, estrogen caused a dose dependent decrease in sciadonic acid (5,11,14–20:3, ScA) via apparent inhibition of FADS1 activity, and had no effect on FADS2 catalyzed synthesis of dihomo-gamma linolenic acid (8,11,14–20:3; DGLA). In MCF7 cells, letrozole caused a dose dependent increase in FADS2-catalyzed DGLA synthesis, which plateaued in SK-N-SH cells. ConclusionLetrozole restores Δ6-desaturase mediated synthesis of the anti-inflammatory PGE1-precursor DGLA in vitro and is the first endocrine-active agent to have opposing effects on FADS1 and FADS2 catalyzed activities.

Isoflavonoid Profiling and Estrogen-Like Activity of Four Genista Species from the Greek Flora.

As part of our ongoing research on phytoestrogens, we investigated the phytochemical profile and estrogen-like activities of eight extracts from the aerial parts of four Genista species of Greek flora using estrogen-responsive cell lines. Ethyl acetate and methanolic extracts of G. acanthoclada, G. depressa, G. hassertiana, and G. millii were obtained with accelerated solvent extraction and their phytochemical profiles were compared using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (uHPLC-HRMS). Fourteen isoflavonoids, previously isolated from G. halacsyi, were used as reference standards for their identification in the extracts. Thirteen isoflavonoids were detected in both extracts of G. acanthoclada and G. hassertiana, while fewer and far fewer were detected in G. millii and G. depressa, respectively. The ethyl acetate extracts of G. hassertiana and G. acanthoclada displayed 2.45- and 1.79-fold higher, respectively, estrogen-like agonist activity in Ishikawa cells compared to MCF-7 cells at pharmacologically relevant concentrations. Both these extracts, but not that of G. depressa, contained mono- and di-O-β-d-glucosides of genistein as well as the aglycone, all three of which are known to display full estrogen-like activity at lower-than-micromolar concentrations. The possibility of using preparations rich in G. hassertiana and/or G. acanthoclada extracts as a potentially safer substitute for low-dose vaginal estrogen for menopausal symptoms is discussed.

Design, synthesis and anticancer/antiestrogenic activities of novel indole-benzimidazoles.

Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic effects. However, their structural similarities and molecular mechanisms shared with selective estrogen receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R1) and fifth (R2) positions of benzimidazole and indole groups, respectively. Subsequently, we performed 1H NMR, 13C NMR, and Mass spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor negative (ER-) cell line MDA-MB-231. R1:p-fluorobenzyl group members were selected as lead compounds for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling and gene enrichment analyses (GSEA) of the selected compounds (R1:p-fluorobenzyl: 48, 49, 50, 51; R1:3,4-difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-dependent manner.

Bioactivation of Quinolines in a Recombinant Estrogen Receptor Transactivation Assay Is Catalyzed by N-Methyltransferases.

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17β-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Abstract P5-04-26: Identification of preclinical mechanisms driving acquired resistance to endocrine therapy in estrogen-receptor positive (ER+) breast cancer cells

Abstract Estrogen Receptor positive (ER+) breast cancer accounts for the majority of breast cancer cases and standard of care for these tumors is treatment with endocrine therapy, including the blockade of estrogen production (i.e. aromatase inhibitors; AIs) as well as the use of antagonists of ER function, i.e. selective estrogen receptor modulators (SERMs, i.e. tamoxifen) and selective estrogen receptor degraders (SERDs, i.e. fulvestrant). Despite the initial dependency of ER+ breast tumors on estrogen and ER for their survival and proliferation, treatment in the metastatic setting invariably leads to therapeutic resistance. While mechanisms of resistance to AIs include mutations in the estrogen receptor gene ESR1, less is known about mechanisms of resistance to SERMs and SERDs, thus it is essential to further investigate the latter, in order to successfully treat relapsed patients. To pre-clinically model cell-autonomous acquired resistance to these agents, we used T47D, an ER+ and p53- estrogen-responsive cell line treated with increasing concentrations of the SERM/SERD hybrid (SSH) ER-targeting agent GDC-0810 over the period of several months during which individual clones with acquired resistance to GDC-0810 were selected. GDC-0810-resistant clones were cross-resistant to other endocrine agents, including SERMs (tamoxifen) and SERDs (fulvestrant), consistent with general loss of dependency on ER. Surprisingly, the cells also lost sensitivity to palbociclib, the latter likely linked to their loss of one copy of the retinoblastoma (Rb) tumor suppressor gene. Comprehensive genetic and phenotypic characterization of the resistant clones relative to the parental cells revealed multiple mutations and deletions in DNA repair and cell cycle genes, and associated defects in DNA repair and cell cycle checkpoints. Cell cycle, proteomic, and mRNA expression analysis of parental versus resistant clones at baseline and upon DNA damage, identified a distinct cell cycle profile in the GDC-0810-resistant clones, characterized by accumulation of cells in the mitotic phase. A broad chemical screen identified pharmacologic inhibitors of cell cycle regulators and chemotherapeutic drug classes that preferentially target the ER-independent, GDC-0810 resistant clones compared to the parental cells. Our work provides novel insights into mechanisms and biomarkers of acquired resistant to estrogen therapies in ER+ breast cancer and reveals the acquisition of actionable dependencies that may potentially be exploited in resistant tumors. Furthermore, our studies provide rationale for testing specific chemotherapy regimens upon endocrine resistance accompanied by cell cycle and DNA repair checkpoint dysfunction in ER+ breast cancer. Citation Format: Ong C, Daemen A, Merrick K, O'Brien T, Friedman L, Hatzivassiliou G. Identification of preclinical mechanisms driving acquired resistance to endocrine therapy in estrogen-receptor positive (ER+) breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-26.

Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation

Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L–P (1–5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity.

Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus × acerifolia buds

One flavonol glycoside, two O-isoprenylated flavonols, one α,α-dimethylallyl flavonol, one dihydrochalcone, two furanocoumarins and one terpenoid previously undescribed, along with 42 known compounds were isolated from the buds of two European Platanaceae, Platanus orientalis and Platanus × acerifolia. Their chemical structures were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR (COSY, NOESY, HSQC, and HMBC) experiments, as well as HRMS data. The estrogen-like and antiestrogen-like activity of dichloromethane and methanol extracts of P. orientalis and P. × acerifolia buds and isolated compounds was evaluated using estrogen-responsive cell lines. The potency of selected estrogen agonists to regulate gene expression through ERα and/or ERβ was compared with their in vitro osteoblastogenic activity. Kaempferol and 8-C-(1,1-dimethyl-2-propen-1-yl)-5,7-dihydroxyflavonol displayed osteoblastogenic as well as ERα-mediated estrogenic activity similar to estradiol.

Binary mixtures effects of food additives and cosmetic preservatives on an estrogen responsive cell line

Binary mixtures effects of food additives and cosmetic preservatives on an estrogen responsive cell line

Estrogenic activity of aromatic compounds present in mineral oil used in printing inks

Mineral oils (MO) used as ingredients for many printing inks are complex mixtures of hydrocarbons. Although migration of MO from paperboard food packaging made of recycled paper into food has been shown, the toxicological relevance of these findings is elusive. Therefore we investigated the estrogenic potential of mineral oil using different in vitro assays. Proliferative effects of MO in the estrogen responsive human breast cancer cell line MCF7 were assessed in the E-screen, and endogenous estrogen responsive gene expression was quantified by real-time PCR. Transactivation assays were performed in HeLa9903 containing the human estrogen receptor α (ERα) and an estrogen responsive element (ERE)-driven luciferase. MO primarily consist of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). These two fractions were separated by preparative HPLC and also assessed in the bioassays. MO in a thousand-fold dilution stimulated MCF7 proliferation but did not induce reporter gene expression in HeLa9903 cells. Furthermore, expression of the estrogen responsive genes PGR, TFF1 and GREB1 in MCF7 cells was induced after 24 h exposure to total MO. The estrogenic effects were derived from the MOAH fraction, as exposure of MCF7 cells to this fraction induced gene expression and increased proliferation. Estrogenic activity of 14 more tested MO correlated with the percentage of the MOAH fraction. In conclusion estrogenic action of aromatic substances derived from MO could be shown in vitro.

Single-Cell States in the Estrogen Response of Breast Cancer Cell Lines

Estrogen responsive breast cancer cell lines have been extensively studied to characterize transcriptional patterns in hormone-responsive tumors. Nevertheless, due to current technological limitations, genome-wide studies have typically been limited to population averaged data. Here we obtain, for the first time, a characterization at the single-cell level of the states and expression signatures of a hormone-starved MCF-7 cell system responding to estrogen. To do so, we employ a recently proposed model that allows for dissecting single-cell states from time-course microarray data. We show that within 32 hours following stimulation, MCF-7 cells traverse, most likely, six states, with a faster early response followed by a progressive deceleration. We also derive the genome-wide transcriptional profiles of such single-cell states and their functional characterization. Our results support a scenario where estrogen promotes cell cycle progression by controlling multiple, sequential regulatory steps, whose single-cell events are here identified.

Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.

Abstract P5-09-02: Cell culture models to study mechanisms for endocrine resistant breast cancer, new treatment options and new biomarkers

Abstract Introduction: Estrogen is the main driver of growth of the majority of breast cancers and many patients benefit from endocrine therapy. However, both innate and acquired resistance is a major problem. In order to study resistance mechanisms and to explore targeted treatment options for endocrine resistant breast cancer, we have developed in vitro cell culture models based on the estrogen receptor (ER) positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D. Methods: Panels of MCF-7 and T47D sublines with acquired resistant to tamoxifen, fulvestrant and aromatase inhibitors were established by selection of colonies surviving long term treatment. Long-term estrogen deprived (LTED) MCF-7/S9 cells were developed by step-wise serum reduction until transfer to serum-free medium. The endocrine resistant sublines were analysed with respect to expression and function of the ER and the HER system, with respect to response to 195 kinase inhibitors and to second-line endocrine therapy. Results: ER expression was reduced in tamoxifen and fulvestrant resistant MCF-7 sublines, unchanged in tamoxifen resistant T47D sublines, whereas fulvestrant resistant T47D sublines were ER negative. In general, ER was functional in the resistant MCF-7 cells. Both tamoxifen and fulvestrant resistant MCF-7 sublines expressed increased level of HER1, the activated form of HER3 and activated ERK1/2, whereas HER4 was severely decreased. In fulvestrant resistant T47D sublines, HER1 and HER4 expression were decreased whereas HER2 expression was highly upregulated. The kinase inhibitor screen disclosed HER receptors as common hits in the tamoxifen and fulvestrant resistant MCF-7 sublines. Regarding T47D, c-Src and Aurora were common hits for tamoxifen and fulvestrant resistant sublines. Tamoxifen resistant MCF-7 and T47D sublines were severely growth inhibited by ER knockdown with either siRNA or fulvestrant, but complete growth arrest required combined treatment with e.g ERK1/2 inhibitor. LTED MCF-7 cells were resistant to aromatase inhibitor treatment and sensitive to both tamoxifen and fulvestrant. Conclusions: In our large panels of MCF-7 and T47D derived endocrine resistant sublines, it was a general finding that ER is the main driver of growth of tamoxifen resistant tumor cells, and also of growth of LTED MCF-7 cells. The ER down modulator fulvestrant inhibited growth of tamoxifen resistant sublines but complete growth arrest required combined treatment with e.g. a kinase inhibitor targeting the activated growth promoting pathways in the resistant cells. In fulvestrant resistant sublines, ER played only a minor role in MCF-7 sublines and no role in T47D sublines. The HER system played a major role in fulvestrant resistant MCF-7 cells, whereas Aurora B and c-Src were new candidate targets for fulvestrant resistant T47D cells. These cell culture models reflect clinical findings and will be available for collaborative studies, e.g. to discover new predictive markers to select targeted therapy for resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-02.

Abstract P5-05-07: Promestriene effects on estrogen-sensitive breast cancer cell proliferation in vitro

Abstract Introduction: Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with potential as a topical treatment for vaginal atrophy caused by estrogen deprivation. Topical Promestriene is minimally absorbed with chronic use and has been reported to significantly improve the symptoms of vaginal atrophy. However, to be considered for use for these symptoms in estrogen responsive post-menopausal breast cancer survivors on an aromatase inhibitor, Promestriene would need to show none of the cyto-proliferative properties of estrogen. Methods: We conducted in vitro testing of estrogen responsive breast cancer cell line MCF-7, T-47D, and BT-474 for dose-dependent, estrogen-like functional responses to Promestriene in both estrogen-rich and estrogen-deprived conditions. As assay controls, cultures treated with Fulvestrant, Testosterone, Progesterone and Estradiol were analyzed in parallel. Analysis: Dose dependent Estrogen-like responses in the cell lines were measured as 1) proliferation using flow cytometric analysis of CSFE fluorescence, 2) estrogen receptor expression by flow cytometry, and 3) GREB 1 RNA expression using TaqMan real time PCR quantitative analysis. All cultures were grown to quiescence in RPMI medium supplemented with 10% fetal calf serum. To induce estrogen deprivation, half of the cultures were then held 3 days in media pre-treated and containing anti-estrogen specific antibodies to sequester any residual estrogen from the culture. Data for each culture treatment were analyzed using linear regression analysis and compared using multifactorial or pairwise analyses to determine the significance of estrogen-like responses relative to the estradiol and untreated controls. Results: In estrogen sufficient cultures, Promestriene exhibited no cell proliferative properties on MCF-7, BT-474, or T47-D tumor cells, even at very high concentrations. However, when estrogen in the media was sequestered and culture allowed to quiesce without estrogen receptor stimulation, low dose Promestriene effects on cell proliferation in MCF-7 estrogen responsive cells were not significantly different from low dose estradiol. Low dose Promestriene (2-10pg/ml), unlike comparable doses of estradiol, did not stimulate GREB1 expression in MCF-7 cells. In contrast, T-47D and BT-474 cell lines proliferated and increased GREB- expression in response Promestriene or Fulvestrant treatments. Conclusions: The potential estrogen-like properties of Promestriene to stimulate the growth of estrogen receptor responsive breast cancer cell lines, especially in estrogen-deprived conditions, suggest caution when prescribed for vaginal atrophy in post-menopausal breast cancer survivors on an aromatase inhibitor. Its ability to activate growth and gene expression in ER- breast cancer cells warrants further study. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-07.

Abstract 3687: Benzyl isothiocyanate downregulates Ronβ and sfRon to inhibit self-renewal of breast cancer stem cells.

Abstract Recent studies suggest that cancer stem cells play a major role not only in the initiation and maintenance of breast cancer but also in clinical response to therapy. Breast cancer stem cells (bCSC) are characterized by their ability to self-renew (asymmetrical division into a daughter bCSC and a progeny), form spheres under ultra-low attachment conditions in vitro, and express unique cell-surface markers (eg, CD44, ESA). Elimination of both therapy-resistant bCSC and therapy-sensitive epithelial tumor cells may be required for effective prevention of breast cancers. We have shown previously that dietary administration of benzyl isothiocyanate (BITC), a small molecule constituent of many edible cruciferous vegetables (eg, garden cress), significantly inhibits mammary cancer development in a transgenic mouse model (MMTV-neu) in association with apoptosis induction and tumor infiltration of T cells. The present study was undertaken to determine the effect of BITC on bCSC. Exposure of MCF-7 (an estrogen-responsive cell line) and SUM159 (a triple-negative cell line) human breast cancer cells to pharmacologic doses of BITC (0.5-2.5 μM) resulted in inhibition of mammosphere formation (first generation after BITC treatment and subsequent generations without BITC treatment), aldehyde dehydrogenase 1 (ALDH1) activity, and CD44high/CD24low/ESA+ population. Inhibition of bCSC self-renewal resulting from BITC exposure was accompanied by down-regulation of urokinase-type plasminogen activator (uPAR), Bmi-1, and Ron (Ronβ and sfRon) proteins in vitro and in MDA-MB-231 xenografts in vivo (uPAR and Bmi-1). Ectopic expression of uPAR alone was sufficient to drive stemness in MCF-7 and MDA-MB-468 cells. However, the BITC-mediated inhibition of mammosphere formation and ALDH1 activity was sustained even after stable overexpression of uPAR in MCF-7 or MDA-MB-468 cells. Similarly, Bmi-1 overexpression was largely dispensable for BITC-mediated suppression of bCSC. On the other hand, overexpression of Ron (full-length) as we as sfRon in MCF-7 cells conferred significant protection against BITC-mediated inhibition of bCSC self-renewal. Ectopic expression of sfRon, but not Ron, resulted in nearly 4-fold increase in ALDH1 activity compared with control cells. Novel conclusions of the present study are: (a) overexpression of sfRon is sufficient to drive stemness at least in MCF-7 cells (b) mammary cancer prevention by BITC likely involves removal of bCSC, and (c) BITC-mediated inhibition of bCSC is caused by suppression of Ron signaling. This study was supported by the grant CA129347-06 awarded by the National Cancer Institute. Citation Format: Su-Hyeong Kim, Anuradha Sehrawat, Shivendra V. Singh. Benzyl isothiocyanate downregulates Ronβ and sfRon to inhibit self-renewal of breast cancer stem 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 3687. doi:10.1158/1538-7445.AM2013-3687

Abstract 3682: Differential effect of withaferin A treatment on activation of Notch1 and Notch2 in human breast cancer cells.

Abstract Withaferin A (WA), a constituent of the Ayurvedic medicine plant Withania somnifera, exhibits in vivo efficacy against human breast cancer xenografts in athymic mice as well as spontaneous mammary cancer development in MMTV-neu transgenic mice. However, the mechanism underlying anticancer effect of WA is not fully understood. The present study was undertaken to determine the role of Notch family of transcription factors in anticancer effects of WA using human breast cancer cells as a model. Notch activation is linked to mammary carcinogenesis as evidenced by the following observations: (a) overexpression of activated Notch1 and Notch3 in transgenic mice blocks mammary gland development but induces tumorigenesis of the breast; (b) high level expression of Notch1 and its ligands is associated with poor outcome in breast cancer; (c) expression of Notch ligand Jagged1 is associated with recurrence in lymph node-negative breast cancer; and (d) Notch1 is involved in migration and invasion of human breast cancer cells. The present study reveals differential effect of WA treatment on activation of Notch1 versus Notch2 in human breast cancer cells. Exposure of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 (an estrogen-responsive cell line with wild-type p53) human breast cancer cells to pharmacological concentrations of WA (2 and 4 μM) resulted in cleavage (suggesting activation) of Notch2, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved (active) Notch1. The WA-mediated activation of Notch2 was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was statistically significantly augmented by knockdown of Notch2 using siRNA and shRNA. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. Moreover, withanone and withanolide A were practically inactive against MDA-MB-231 cell migration. The results of the present study suggest that anti-metastatic effect of WA may be augmented in the presence of Notch2 inhibitors. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Joomin Lee, Anuradha Sehrawat, Shivendra V. Singh. Differential effect of withaferin A treatment on activation of Notch1 and Notch2 in human 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 3682. doi:10.1158/1538-7445.AM2013-3682

Insulin-like Growth Factor-Dependent Proliferation and Survival of Triple-Negative Breast Cancer Cells: Implications for Therapy

Triple-negative breast cancers have a poor prognosis and are not amenable to endocrine- or HER2-targeted therapies. The prevailing view is that targeting the insulin-like growth factor (IGF) signal transduction pathway will not be beneficial for triple-negative breast cancers because their growth is not IGF-responsive. The present study investigates the importance of IGFs in the proliferation and survival of triple-negative breast cancer cells. Estrogen and progesterone receptors, HER2, type I IGF, and insulin receptors were measured by Western transfer analysis. The effects of IGF-1 on proliferation were assessed by DNA quantitation and on cell survival by poly (ADP-ribose) polymerase cleavage. The effect of IGF-1 on phosphorylation of the IGF receptors, Akt and mitogen-activated protein kinase, was measured by Western transfer analysis. Seven cell lines were identified as models of triple-negative breast cancer and shown to express IGF receptors at levels similar to those present in estrogen-responsive cell lines known to respond to IGFs. IGF-1 increased the proliferation and cell survival of all triple-negative cell lines. Proliferation was attenuated after reduction of type I IGF receptor expression. Cells that express higher levels of receptor were more sensitive to subnanomolar IGF-1 concentrations, but the magnitude of the effects was not correlated simply with the absolute amount or phosphorylation of the IGF receptors, Akt or mitogen-activated protein kinase. These results show that IGFs stimulate cell proliferation and promote cell survival in triple-negative breast cancer cells and warrant investigation of the IGF signal transduction pathway as a therapeutic target for the treatment of triple-negative breast cancer.

Abstract 3686: Activation of signal transducer and activator of transcription 3 fails to confer resistance against withaferin A-induced apoptosis in human breast cancer cells

Abstract We have shown previously that withaferin A (WA), a constituent of Indian Ayurvedic medicine plant Withania somnifera that has been used safely for thousands of years for treatment of different ailments, inhibits growth of cultured MDA-MB-231 (an estrogen receptor negative cell line with mutant p53) and MCF-7 (an estrogen-responsive cell line with wild-type p53) human breast cancer cells. Moreover, the WA administration significantly inhibits growth of MDA-MB-231 xenografts in female athymic mice without causing any side effects. The WA-mediated inhibition of breast cancer cell growth in culture and xenografts correlates with apoptosis induction. However, the mechanism underlying proapoptotic effect of WA is not fully understood. The present study was designed to study the role of signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor activated in many human malignancies including breast cancer, in proapoptotic response to WA using MDA-MB-231 and MCF-7 cells. Growth suppressive and proapoptotic concentrations of WA (2 and 4 μM) decreased constitutive (MDA-MB-231) or interleukin-6 (IL-6)-inducible (MDA-MB-231 and MCF-7) phosphorylation of STAT3 (Tyr705) and its upstream regulator Janus-activated kinase 2 (JAK2; Tyr1007/1008) in MDA-MB-231, which was accompanied by suppression of their protein levels especially at the higher concentration. Exposure of MDA-MB-231 or MCF-7 cells to WA also resulted in suppression of (a) transcriptional activity of STAT3 with or without IL-6 stimulation in both cells; (b) dimerization of STAT3 (MDA-MB-231); and (c) nuclear translocation of Tyr705 phosphorylated STAT3 in both cells. To our surprise, the IL-6-stimulation, either before or after WA treatment, did not have an appreciable effect on WA-mediated apoptosis in MDA-MB-231 or MCF-7 cell line. The IL-6-stimulated activation of STAT3 conferred only a modest protection against WA-mediated suppression of MDA-MB-231 cell invasion. General implication of these findings is that WA can trigger apoptosis and largely inhibit cell migration/invasion of breast cancer cells even after IL-6-induced activation of STAT3, which should be viewed as a therapeutic advantage for this agent. This investigation was supported by the USPHS grant CA142604-01, awarded by the National Cancer Institute. 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 3686. doi:10.1158/1538-7445.AM2011-3686

Abstract 860: Benzyl isothiocyanate inhibits oncogenic actions of leptin in human breast cancer cells by suppressing activation of signal transducer and activator of transcription 3

Abstract Obesity is a well-established risk factor for breast cancer progression and molecular effects of obesity are mediated by adipocytokine, leptin. Considering the important role of leptin in breast cancer growth and metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., gardencress), confers significant protection against mammary carcinogenesis in a transgenic mouse model (MMTV-neu). The BITC-mediated prevention of mammary cancer development in MMTV-neu mice correlated with reduced cell proliferation, increased apoptosis, and tumor infiltration of T cells. The present study provides first evidence for efficacy of BITC against oncogenic effects of letpin. BITC-treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 human breast cancer cells (an estrogen-responsive cell line with wild-type p53) at pharmacologic doses. Leptin-stimulated migration and invasion of these cells was also inhibited significantly in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC-treatment suppressed leptin-induced phosphorylation of signal transducer and activator of transcription 3 (Stat3) and cyclin D1 transactivation. BITC-treatment efficiently inhibited Stat3 recruitment to the cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a decrease in level of phospho-Stat3. These studies indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivastion. Taken together, these data show that BITC treatment can inhibit the oncogenic actions of leptin via blocking its downstream signaling molecule, Stat3. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels. This work was supported in part by the USPHS grants NCI-RO1 CA129347 (to SVS), NIDDK-K01DK076742 and R03DK089130 (to NKS), NCI-R01CA131294 and NCI-R21 CA155686 (to DS). 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 860. doi:10.1158/1538-7445.AM2011-860