Non-malignant Breast Epithelial Cells Research Articles

Exposure to the polyester PET precursor--terephthalic acid induces and perpetuates DNA damage-harboring non-malignant human breast cells.

Identification of early perturbations induced in cells from non-cancerous breast tissue is critical for understanding possible breast cancer risk from chemical exposure. We have demonstrated previously that exposure to the ubiquitous xenoestrogens, bisphenol A (BPA) and methyl paraben, promotes the hallmarks of cancer in non-malignant human high-risk donor breast epithelial cells (HRBECs) isolated from several donors. Here we show that terephthalic acid (TPA), a major chemical precursor of polyethylene terephthalate (PET) containers used for the storage of food and beverages, increased the ERα: ERβ ratio in multiple HRBEC samples, suggesting an estrogenic effect. Although, like BPA and methyl paraben, TPA also promoted resistance to tamoxifen-induced apoptosis, unlike these chemicals instead of inducing an increased S-phase fraction, TPA treatment arrested cell proliferation. DNA-PK, ATM and members of the MRN complex, known to be involved in DNA damage sensor and effector proteins, were elevated indicating induction of DNA strand breaks. Early DNA damage checkpoint response, mediated through p53/p21, led to G1 arrest in TPA-exposed cells. Removal of TPA from the growth medium resulted in the rapid induction of BCL2, increasing the ratio of anti-: pro-apoptotic proteins, together with overexpression of Cyclin A/CDK2 proteins. Consequently, despite elevated p53(pSer15) and H2AX(pSer139), indicating sustained DNA damage, TPA exposed cells resumed robust growth rates seen prior to TPA exposure. The propensity for the perpetuation of DNA aberrations that activate DNA damage pathways in non-malignant breast cells justifies careful consideration of human exposure to TPA, particularly at vulnerable life stages.

Effects of the exercise-inducible myokine irisin on malignant and non-malignant breast epithelial cell behavior in vitro.

Exercise has been shown to reduce risk and improve prognosis of several types of cancers. Irisin is a myokine linked to exercise and lean body mass, which is thought to favorably alter metabolism systemically, potentially providing benefit for metabolic disease (including cancer). We evaluated the effects of various concentrations of irisin (with and without post-translational modifications) on malignant and non-malignant breast epithelial cell number, migration and viability. Irisin significantly decreased cell number, migration and viability in malignant MDA-MB-231 cells, without affecting non-malignant MCF-10a cells. Moreover, irisin enhanced the cytotoxic effect of doxorubicin (Dox) when added to a wide spectrum of irisin concentrations in the malignant cell type (with simultaneous reduction in Dox uptake), which was not observed in non-malignant MCF-10a cells. Additionally, we found that irisin decreases malignant cell viability in part through stimulation of caspase activity leading to apoptotic death. Interestingly, we found that irisin suppresses NFκB activation, an opposite effect of other myokines such as tumor necrosis factor alpha (TNF-α). Our observations suggest that irisin may offer therapeutic benefits for breast cancer prevention and treatment possibly through an anti-inflammatory response, induction of apoptotic cell death, or through enhanced tumor sensitivity to common antineoplastic agents such as Dox.

Synergistic anticancer effects of a bioactive subfraction of Strobilanthes crispus and tamoxifen on MCF-7 and MDA-MB-231 human breast cancer cell lines.

BackgroundDevelopment of tumour resistance to chemotherapeutic drugs and concerns over their toxic effects has led to the increased use of medicinal herbs or natural products by cancer patients. Strobilanthes crispus is a traditional remedy for many ailments including cancer. Its purported anticancer effects have led to the commercialization of the plant leaves as medicinal herbal tea, although the scientific basis for its use has not been established. We previously reported that a bioactive subfraction of Strobilanthes crispus leaves (SCS) exhibit potent cytotoxic activity against human breast cancer cell lines. The current study investigates the effect of this subfraction on cell death activities induced by the antiestrogen drug, tamoxifen, in estrogen receptor-responsive and nonresponsive breast cancer cells.MethodsCytotoxic activity of SCS and tamoxifen in MCF-7 and MDA-MB-231 human breast cancer cells was determined using lactate dehydrogenase release assay and synergism was evaluated using the CalcuSyn software. Apoptosis was quantified by flow cytometry following Annexin V and propidium iodide staining. Cells were also stained with JC-1 dye to determine changes in the mitochondrial membrane potential. Fluorescence imaging using FAM-FLICA assay detects caspase-8 and caspase-9 activities. DNA damage in the non-malignant breast epithelial cell line, MCF-10A, was evaluated using Comet assay.ResultsThe combined SCS and tamoxifen treatment displayed strong synergistic inhibition of MCF-7 and MDA-MB-231 cell growth at low doses of the antiestrogen. SCS further promoted the tamoxifen-induced apoptosis that was associated with modulation of mitochondrial membrane potential and activation of caspase-8 and caspase-9, suggesting the involvement of intrinsic and extrinsic signaling pathways. Interestingly, the non-malignant MCF-10A cells displayed no cytotoxicity or DNA damage when treated with either SCS or SCS-tamoxifen combination.ConclusionsThe combined use of SCS and lower tamoxifen dose could potentially reduce the side effects/toxicity of the drug. However, further studies are needed to determine the effectiveness and safety of the combination treatment in vivo.

Breast carcinoma cells modulate the chemoattractive activity of human bone marrow-derived mesenchymal stromal cells by interfering with CXCL12

We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP-1) were cocultured with primary breast cancer cells, MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP-1 cells cultured with MCF-7 medium revealed CXCL12 (SDF-1) as one of the most significantly downregulated genes. Supernatant from both MCF-7 and MDA-MB231 reduced the CXCL12 promoter activity in SCP-1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans-well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR-23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony-forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches.

Ligand-dependent corepressor contributes to transcriptional repression by C2H2 zinc-finger transcription factor ZBRK1 through association with KRAB-associated protein-1

We identified a novel interaction between ligand-dependent corepressor (LCoR) and the corepressor KRAB-associated protein-1 (KAP-1). The two form a complex with C2H2 zinc-finger transcription factor ZBRK1 on an intronic binding site in the growth arrest and DNA-damage-inducible α (GADD45A) gene and a novel site in the fibroblast growth factor 2 (FGF2) gene. Chromatin at both sites is enriched for histone methyltransferase SETDB1 and histone 3 lysine 9 trimethylation, a repressive epigenetic mark. Depletion of ZBRK1, KAP-1 or LCoR led to elevated GADD45A and FGF2 expression in malignant and non-malignant breast epithelial cells, and caused apoptotic death. Loss of viability could be rescued by simultaneous knockdowns of FGF2 and transcriptional coregulators or by blocking FGF2 function. FGF2 was not concurrently expressed with any of the transcriptional coregulators in breast malignancies, suggesting an inverse correlation between their expression patterns. We propose that ZBRK1, KAP-1 and LCoR form a transcriptional complex that silences gene expression, in particular FGF2, which maintains breast cell viability. Given the broad expression patterns of both LCoR and KAP-1 during development and in the adult, this complex may have several regulatory functions that extend beyond cell survival, mediated by interactions with ZBRK1 or other C2H2 zinc-finger proteins.

Effects of Physiological Levels of the Green Tea Extract Epigallocatechin-3-Gallate on Breast Cancer Cells

Physiological concentrations of the green tea extract epigallocatechin-3-gallate (EGCG) caused growth inhibition in estrogen receptor α (ERα)-positive MCF7 cells that was associated with down-regulation of the ERα and reduced insulin-like growth factor binding protein-2 abundance and increased protein abundance of the tumor suppressor genes p53/p21. In contrast to MCF7 cells that have wt p53, EGCG alone did not change cell proliferation or death significantly in another ERα-positive cell line T47D that possesses mutant p53. EGCG increased ERα protein levels and as a consequence, the cells responded significantly better to an ERα antagonist tamoxifen (TAM) in the presence of EGCG. EGCG significantly increased cell death in an ERα-negative cell line, MDA-MB-231 that also possesses mutant p53. EGCG significantly increased the ERα and insulin-like growth factor-I receptor levels and thereby enhanced the sensitivities of the cells to TAM and a blocking antibody targeting the insulin-like growth factor-1 receptor (αIR3). In contrast to MCF7, T47D and MDA-MB-231 breast cancer cells that exhibited significant changes in key molecules involved in breast growth and survival upon treatment with physiological levels of EGCG, the growth, survival, and levels of these proteins in non-malignant breast epithelial cells, MCF10A cells, were not affected.

N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells.

BackgroundThe DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations.MethodsImmunoblot and comet assays were performed to detect DNA breaks and activation of the canonical checkpoint signalling kinases following NEU damage upto 2 hours. To investigate whether mismatch repair played a role in checkpoint activation, knock-down studies were performed while flow cytometry analysis was done to understand whether the activation of the checkpoint kinases was cell cycle phase specific. Finally, breast epithelial cells were grown as 3-dimensional spheroid cultures to study whether NEU can induce upregulation of vimentin as well as disrupt cell polarity of the breast acini, thus causing transformation of epithelial cells in culture.ResultsWe report a novel finding that NEU causes activation of major checkpoint signalling kinases, Chk1 and Chk2. This activation is temporally controlled with Chk2 activation preceding Chk1 phosphorylation, and absence of cross talk between the two parallel signalling pathways, ATM and ATR. Damage caused by NEU leads to the temporal formation of both double strand and single strand breaks. Activation of checkpoints following NEU damage is cell cycle phase dependent wherein Chk2 is primarily activated during G2-M phase whilst in S phase, there is immediate Chk1 phosphorylation and delayed Chk2 response. Surprisingly, the mismatch repair system does not play a role in checkpoint activation, at doses and duration of NEU used in the experiments. Interestingly, NEU caused disruption of the well-formed polarised spheroid archithecture and upregulation of vimentin in three-dimensional breast acini cultures of non-malignant breast epithelial cells upon NEU treatment indicating NEU to have the potential to cause early transformation in the cells.ConclusionNEU causes damage in mammalian cells in the form of double strand and single strand breaks that temporally activate the major checkpoint signalling kinases without the occurrence of cross-talk between the pathways. NEU also appear to cause transformation in three-dimensional spheroid cultures.

Suppressive role of miR-502-5p in breast cancer via downregulation of TRAF2

TRAF2 promotes cancer cell survival, proliferation and metastasis through the NF-κB pathway by directly interacting with various TNF recepors. However, the molecular mechanism of TRAF2 dysregulation in breast cancer remains to be elucidated. In the present study, miR-502-5p was predicted as a potential regulator of TRAF2. miR-502-5p was significantly downregulated in breast cancer tissues when compared to the level in paired normal breast tissues. The breast cancer cell lines including MCF-7 and MDA-MB-231 expressed a lower level of miR-502-5p when compared to the level in the non-malignant breast epithelial cell line MCF-10A. In vitro, miR-502-5p enhanced early apoptosis and inhibited proliferation of breast cancer cells. Luciferase reporter assay results showed that miR-502-5p could bind to the 3'-untranslated region of the TRAF2 gene, thus, exerting an inhibitory effect on TRAF2. Furthermore, silencing of TRAF2 exhibited effects similar to those of exogenous miR‑502-5p, while overexpression of TRAF2 partially abrogated miR-502-5p-mediated suppression in breast cancer cells. In conclusion, miR-502-5p may act as a tumor-suppressor gene by targeting oncogenic TRAF2 in breast cancer and, therefore, may be a potential diagnostic and anticancer therapeutic marker for breast cancer.

MiR-96 promotes tumor proliferation and invasion by targeting RECK in breast cancer

microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in diverse biological processes. The aim of the present study was to investigate the expression pattern of miR-96 in breast cancer and its biological role in tumor progression. The expression levels of miR-96 were analyzed in 38 breast cancer specimens and 6 breast cancer cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of miR-96 on proliferation was evaluated by MTT assays, and cell migration and invasion were evaluated by transwell assays in MDA-MB-231 human breast cancer cells. Luciferase reporter assays were performed to validate the regulation of a putative target of miR-96. The effects of modulating miR-96 on endogenous levels of this potential target were subsequently confirmed via qRT-PCR and western blot analysis. We found that expression of miR-96 was commonly upregulated in breast cancer cells and breast cancer specimens when compared with that in non-malignant breast epithelial cells and adjacent normal tissues. Ectopic expression of miR-96 promoted cellular proliferation, migration and invasion of breast cancer cells, whereas inhibition of miR-96 suppressed those functions. Luciferase assays revealed that miR-96 directly bound to the 3'-untranslated region (3'-UTR) of RECK. qRT-PCR and western blot analysis confirmed that miR-96 regulated the expression of RECK both at the mRNA and protein levels. Knockdown of RECK expression in MDA-MB-231 cells by siRNA significantly promoted cell proliferation, migration and invasion. Collectively, miR-96 was significantly upregulated in breast cancer. our data also delineate the molecular pathway by which miR-96 promotes breast cancer proliferation, migration and invasion. Our findings may have important implications for the treatment of breast cancer.

Abstract P6-03-01: Low dose exposure of non-malignant breast epithelial cells to the polyester and PET precursor, terephthalic acid, triggers broad-based dysfunctional changes

Abstract BACKGROUND: Terephthalic acid (TPA) is the raw material for the production of polyester fiber and polyethylene terephthalate (PET) or #1 plastic. With steadily increasing levels of TPA in our environment, concerns for public health are foreseeable. We report the role of low dose TPA exposure in the induction of well-accepted endpoints of cancer progression within non-cancerous breast cells. METHODS: In our previous work with bisphenol-A (BPA) and methylparaben (MP), we employed human, benign, high-risk donor breast epithelial cells (HRBECs) to study functional response to chemical exposure. To measure the effects of TPA, 3 independent spontaneously immortalized ER-positive HRBEC lines were treated with concentrations ranging from 100pM to 100μM for 7 days. Changes in the levels of key signaling proteins encoded by genes associated with estrogenic stimulation, cell cycle, DNA replication, DNA repair, and apoptosis, were analyzed by Western blots. Fluorescence-activated cell sorting (FACS) was used to quantitate alterations in cell proliferation and programmed cell death. TPA-induced DNA double strand breaks were visualized as nuclear foci of activated gH2AX, and p53 by immunofluorescence microscopy. RESULTS: TPA exposure was estrogenic in HRBECs as evident by ERα activation. Similar to our findings with BPA and MP, prior exposure to TPA at 1 or more concentrations in the low dose range enabled programmed cell death evasion in 3/3 immortalized HRBEC lines (p<0.01), further confirmed in finite-life HRBECs (p<0.001) from 3/3 independent donors. Otherwise, effects of TPA were distinct from those induced by BPA and MP. Instead of an increase in the S-phase fraction of the cell cycle, TPA treatment led to a G1-arrest. These G1-arrested cells accumulated the DNA repair proteins – DNA-PK, ATM (pSer1981), RAD50, MRE11, p53 (pSer15) and H2AX (pSer139), indicating the occurrence of DNA damage. TPA removal released HRBECs from G1-arrest and restored proliferation. However, due to the propensity for apoptosis evasion conferred by TPA, unrepaired cells displaying DNA repair signals persisted even in its absence. Taken together, these findings demonstrate perpetuation of cells with evidence of DNA damage from prior TPA exposure, and suggest a scenario whereby potential DNA aberrations incurred from exposure to this and other DNA damaging agents might be transmitted to the cell progeny. CONCLUSION: Supporting the novel observation of mTOR activation in HRBECs by BPA in our previous work, TPA-induced pathway perturbations demonstrate that the common practice of screening for potential breast carcinogens simply based on increased cell proliferation, presumably stimulated by the mitogenic activity of estrogen mimics, is an oversimplification of possible sources of risk to target cells within the breast. Using such limited tests, TPA would be deemed inactive – ostensibly a false negative result. Effects of environmental chemicals must therefore be evaluated by multiple endpoints in independent biological programs within responsive human cell types. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-03-01.

Borrelidin has limited anti‐cancer effects in bcl‐2 overexpressing breast cancer and leukemia cells and reveals toxicity in non‐malignant breast epithelial cells

Clinically effective anti-cancer drugs have to tread a narrow line between selective cytotoxicity on tumor cells and tolerable adverse effects against healthy tissues. This causes the failure of many potential cancer drugs in advanced clinical trials, hence signifying the importance of a comprehensive initial estimate of the cytotoxicity of prospective anti-cancer drugs in preclinical studies. In this study, the cytotoxicity of borrelidin, a macrolide antibiotic with a high cytotoxic selectivity for proliferating endothelial cells and leukemia cells, was tested on malignant and non-malignant breast cells. Highly metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-435) showed promising results and exhibited good sensitivity to borrelidin at low nanomolar concentrations, but borrelidin was cytotoxic to a non-malignant breast epithelial cell line (MCF10A) as well. Furthermore, although a high sensitivity of endothelial cells (human umbilical vein endothelial cells; HUVEC) and individual leukemia cell lines (Jurkat and IM9) to borrelidin was confirmed in this study, another leukemia cell line (HL60) and an immortalized endothelial cell line (EA.hy926) displayed a significantly decreased sensitivity. Reduced sensitivity to borrelidin was associated with elevated bcl-2 expression in these cell lines. In conclusion, the results presented show that borrelidin displays high and selective cytotoxicity against subgroups of cancer cells and endothelial cells, but, owing to its non-specific toxicity to non-malignant cells, its clinical application might be restricted because of likely adverse effects and limited efficacy in bcl2-overexpressing cancer cells.

NF-κB Regulates Radioresistance Mediated By β1-Integrin in Three-Dimensional Culture of Breast Cancer Cells

β1-integrin induction enhances breast cancer cell survival after exposure to ionizing radiation (IR), but the mechanisms of this effect remain unclear. Although NF-κB initiates prosurvival signaling pathways post-IR, the molecular function of NF-κB with other key elements in radioresistance, particularly with respect to extracellular matrix-induced signaling, is not known. We discovered a typical NF-κB-binding site in the β1-integrin promoter region, indicating a possible regulatory role for NF-κB. Using three-dimensional laminin-rich extracellular matrix (3D lrECM) culture, we show that NF-κB is required for β1-integrin transactivation in T4-2 breast cancer cells post-IR. Inhibition of NF-κB reduced clonogenic survival and induced apoptosis and cytostasis in formed tumor colonies. In addition, T4-2 tumors with inhibition of NF-κB activity exhibit decreased growth in athymic mice, which was further reduced by IR with downregulated β1-integrin expression. Direct interactions between β1-integrin and NF-κB p65 were induced in nonmalignant breast epithelial cells, but not in malignant cells, indicating context-specific regulation. As β1-integrin also activates NF-κB, our findings reveal a novel forward feedback pathway that could be targeted to enhance therapy.

Tumor necrosis factor alpha induces Warburg‐like metabolism and is reversed by anti‐inflammatory curcumin in breast epithelial cells

The reprogramming of cellular metabolism in cancer cells is a well-documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF-10a) and malignant (MCF-7) breast epithelial cells with low-level (10 ng/ml) tumor necrosis factor alpha (TNF-α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF-α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF-α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti-inflammatory agent curcumin in a dose-dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.

Targeting the Deregulated Spliceosome Core Machinery in Cancer Cells Triggers mTOR Blockade and Autophagy

The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology.

Abstract PD01-08: Differences in estrogen receptor signaling in non-malignant primary ER-positive breast epithelial cells and breast cancer.

Abstract Background: The estrogen receptor (ER) is expressed in ∼70% of sporadic breast cancer and activates genes driving cell proliferation and tumorigenesis. We have previously performed genome-wide analysis of ER binding sites in MCF-7 breast cancer cells, and identified distinct mechanisms of ER signaling. We have also previously used EpCAM and CD49f as markers to enrich for viable ER-positive (ER+) cells obtained from non malignant breast tissue. Here, we seek to elucidate differences in ER signaling between non-malignant and ER+ breast cancer cells. Methods: Primary breast epithelial cells were obtained from patients undergoing reduction mammoplasties and surgical excision of ER+ breast cancer. After dissociation of breast reductions into a single-cell suspension, ER+ mature luminal (ML; EpCAM+CD49f−) and luminal progenitor (LP; EpCAM+CD49f+) subpopulations were obtained by flow cytometry. Following estrogen stimulation, RNA was extracted for gene microarray analysis. ER chromatin immunoprecipitation and DNA sequencing (ChIP-seq) was performed. These results were compared to MCF-7 breast cancer cells. Results: Reduction mammoplasty and ER+ breast cancer tissues were analyzed, and compared to MCF-7 cells. Gene expression profiles were different between non-malignant tissue and ER+ breast cancer cells following estrogen stimulation, with a 2–3 fold higher number of ER regulated genes in ER+ breast cancer compared to ER+ non malignant cells, and few overlapping estrogen regulated genes. Genes that promotes cell cycling and cell proliferation were downregulated in non-malignant tissue, but were upregulated in breast cancer cells (P < 10–5). CYP1A1, a major estradiol metabolizing enzyme, was upregulated in normal cells but downregulated in ER+ breast cancer cells. Motif analysis of ER ChIP-seq data in normal and ER+ breast cancer tissues demonstrated an enrichment of ER motifs in the overlapping sites and an enrichment of FOXA1 motifs in ER+ breast cancer cells and TCF12 motifs in non-malignant ER+ epithelial cells. Conclusions: There are contrasting differences in ER signaling between normal mammary and breast cancer cells, with estrogen having anti-proliferative effects in normal luminal cells compared to pro-proliferative effects in breast cancer. ER ChIP-Seq has identified TCF12 as a major co-factor in non-malignant breast tissue whilst FOXA1 is a major co-factor in ER+ breast cancer. Our data provides evidence for key alterations in ER-signaling during tumorigenesis, and identifies potential mechanisms to target cancer specific ER signaling. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-08.

ATM Suppresses SATB1-Induced Malignant Progression in Breast Epithelial Cells

SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.

Bisphenol-A-induced inactivation of the p53 axis underlying deregulation of proliferation kinetics, and cell death in non-malignant human breast epithelial cells

Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway—accompanied by dose-dependent evasion of apoptosis and increased proliferation—in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21WAF1 and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ERα (Estrogen Receptor): ERβ ratio—an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures—another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth—a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.

P03-24 IGFBP-2 is a novel regulator of the estrogen receptor in breast epithelial cells

Background: IGFBP-2 is associated with breast cancer development and many studies have indicated a positive association between estrogen receptor (ERa) status and IGFBP-2 both in vitro and in vivo. It is well known that ERa can regulate expression of IGF family members including IGFBP-2. However, a role for IGFBP-2 in affecting ER levels has not been examined. Aims: To determine the effects of IGFBP-2 on ERa expression in breast epithelial cells. Methods: The relatively normal breast epithelial cells (MCF10A) and the ER-a positive breast cancer cells (T47D) were examined. MCF10A and T47D cells were treated with IGFBP-2 (0–600ng/ml and 0–100ng/ml respectively) in the presence or absence of a disintegrin, RGD (100ng/ml) an IGF-1R blocking antibody, aIR3 (1mg/ml), NBI-31772 peptide (1mM), that blocks the formation of IGF:IGFBP complexes, a PTEN inhibitor Dipotassium Bisperoxo(picolinato) oxovanadate (V) (bpV, 0.1mM) or an inhibitor of PI3K LY294002. IGFBP-2 silencing was achieved by using siRNA technology. Protein abundance of ER-a, IGFBP-2 and Sp1 was determined by western immunoblotting. Quantitative RT-PCR was used to examine mRNA expression. Association between Sp1 and the ERa gene was measured using a Chromatin Immunoprecipitation Assay (ChIP). Results: Adding exogenous IGFBP-2 to the T47D cells and the nonmalignant breast epithelial cells MCF10A induced an up-regulation of ER-a abundance, at both protein (3.5 and 2 fold increase respectively) and mRNA level. This effect of IGFBP-2 was blocked in the presence of a disintegrin in both cell types. With the cancer cells, the increase in ER-a required activation of the IGF-1R, a reduction in PTEN and activation of PI3K. Whereas in the normal cells, IGFBP-2 did not require activation of the IGF-1R and its effect on ER-a was independent of PI3K signalling. In both cell models, the up-regulation of ER-a was mediated by an increase in the association of Sp1 with the ER-a gene. Conclusion: The majority of newly diagnosed breast cancers are hormone-dependent with first line treatment being anti-estrogen therapy. These data suggest that IGFBP-2 could be an important regulator of ER-a and as such may be contributing to breast cancer development and progression. Acknowledgements: We thank the Breast Cancer Campaign for funding this work.

Hypoxic Conditions Induce a Cancer-Like Phenotype in Human Breast Epithelial Cells

IntroductionSolid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis.MethodsNormal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot.ResultsIn large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state.

The nucleoside antagonist cordycepin causes DNA double strand breaks in breast cancer cells

The fungal drug cordycepin (3-deoxyadenosine) is known to exert anti-tumor activities, preferentially by interfering with RNA synthesis. We have investigated the effect of cordycepin on human breast epithelial cell lines, ranging from non-malignant MCF10A cells to highly de-differentiated MDA-MB-435 cancer cells. Treatment of human breast cancer cells with cordycepin caused either apoptosis or persistent cell cycle arrest that was associated with reduced clonal growth of cordycepin-treated breast cancer cells. Highly de-differentiated breast cancer cell lines, such as MDA-MB-231 and MDA-MB-435, reacted more sensitive to cordycepin than less aggressive breast cancer cell lines (MCF7, T47D) or non-malignant breast epithelial cells (MCF10A), which poorly reacted to cordycepin. In cordycepin-sensitive breast cancer cells, a marked induction of the DNA damage response (DDR), including the phosphorylation of ATM, ATR, and histone γH2AX could be observed. These data indicate that cordycepin, which was believed to cause cancer cell death by inhibition of RNA synthesis, induces DNA double strand breaks in breast cancer cells. The genotoxic effect of cordycepin on breast cancer cells indicates a new mechanism of cordycepin-induced cancer cell death, and its activity against highly undifferentiated breast cancer cells provides a new perspective of how cordycepin may be used in the treatment of advanced breast cancer.