Non-malignant Human Mammary Epithelial Cells Research Articles

Norepinephrine transporter expressed on mammary epithelial cells incorporates norepinephrine in milk into the cells

Mammary epithelial cells synthesize and secrete norepinephrine (NE) into breast milk to regulate β-casein expression through the adrenergic β2 receptor. We investigated the expression, localization, and roles of NE transporter (NET) in the mammary epithelium during lactation. mRNA and protein levels of NET were determined in primary normal human mammary epithelial cells (pHMECs) and non-malignant human mammary epithelial MCF-12A cells. In nursing CD1 mice, NET localized to the apical membranes of the mammary epithelium. The intracellular NE content of pHMECs incubated with NE increased. Although the β-casein concentration in milk was slightly higher at day 10 than at day 2 of lactation, the NE concentration and lactation-related proteins were only slightly changed on days 2–10. Restraint stress increased the NE concentration in milk from nursing mice and NET protein levels were significantly higher than in non-stressed nursing mice. NET is expressed on the apical membrane of mammary epithelial cells and incorporates NE in milk into cells, potentially regulating the NE concentration in milk.

Complementary actions of melatonin on angiogenic factors, the angiopoietin/Tie2 axis and VEGF, in co‑cultures of human endothelial and breast cancer cells.

Melatonin exerts oncostatic activity in breast cancer through antiangiogenic actions. There, the aim of the present study was to ascertain whether melatonin modulates, in a coordinated action, angiopoietin-1 (ANG-1), ANG-2, their cognate Tie2 receptor and VEGF in co-cultures of human endothelial cells (HUVECs) and breast cancer (MCF-7) cells. To accomplish this we used co-cultures of human breast cancer cells (MCF-7) or non-malignant human mammary epithelial cells (MCF‑10A) with endothelial cells (HUVECs). The presence of breast cancer cells increased HUVEC proliferation and 1mM melatonin prevented this effect. ANG-1, ANG-2 and VEGF levels in co-culture media and mRNA expression were upregulated and Tie2 mRNA expression was downregulated in the HUVECs and MCF-7. Melatonin (1mM) downregulated ANG-1, ANG-2 and VEGF levels in the co-culture media and mRNA expression in both types of cells and upregulated Tie2 mRNA expression in HUVECs. ANG-1, ANG-2, Tie2 and VEGF mRNA expression were not modified during HUVEC/MCF-10A co-culture. Estradiol (10nM) increased ANG-1, ANG-2 and VEGF mRNA expression in HUVECs and melatonin (1mM) counteracted this effect. We conclude that melatonin simultaneously coordinates downregulation of angiopoietins with a reduction in VEGF, which could be an effective therapeutic strategy for blocking tumor angiogenesis.

Abstract 405: A novel panel of 325 biomarkers is part of a large interconnected network representing multiple cell signaling pathways and allowing development of predictive tests for oncology drugs

Abstract Background: We developed an algorithm based on the gene expression of tumor biopsies to identify the best combination of biomarkers to reliably predict a patient’s response to relevant cancer treatments. This algorithm is derived from 325 genes whose expression showed significant changes during differentiation of non-malignant human mammary epithelial cells cultured in laminin-rich extracellular matrix. Of these 325 genes, 251 are novel and not present in 9 other cancer based gene expression panels such as FoundationOne or PAM50. These differentiated cells formed multicellular structures with defined lumens and tight junctions and with specific localizations of cadherin and integrins. In contrast, cells from breast cancers displayed a general loss of structure. Previous work showed that different sets of these 325 biomarkers accurately predicted overall breast cancer patients' survival or response to neoadjuvant therapy in multiple independent studies. Objective: Predict cell-signaling pathways, drug associations, and disease associations for the 325 biomarkers (BA325) in contrast to other cancer gene panels. Methods and Results: The Qiagen Ingenuity program was used to identify pathways and disease states containing significant overlap with BA325. Multiple cell signaling pathways including cell proliferation, migration, invasion, and metabolism were found in BA325, while most other cancer biomarker panels were highly concentrated in cell proliferation. Examples of significant pathway associations relevant for oncology drug discovery and targeted treatments include Cell Cycle Control of Chromosomal Replication (p=8.1E-14), Polo-like-kinase and HSP90 complex (p=6.3E-07), G2/M DNA Damage Checkpoint (p=6.4E-07), Integrin Signaling (p=3E-05), Integrin Linked Kinase Signaling (p=4.51E-05), BRCA1 DNA Damage Response (p=1.75E-04), Estrogen Mediated S-phase entry (p=4.24E-04), Regulation of Actin Based Motility by RHO (p=4.55E-04), Adherins Junction Remodeling (p=5.6E-04), Actin Cytoskeleton Signaling (p=7.25E-04), Mismatch Repair (p=1.6E-03), RAN Signaling (p=1.64E-03), Protein Ubiquitination (p=1.78E-03) and Cholesterol Biosynthesis (p=4.8E-03). This wide coverage of cellular signaling pathways provides an advantage in developing predictive tests for treatments targeting highly complex cell signaling networks. Surprisingly, even though the BA325 panel was obtained during non-malignant differentiation, the most significant disease state associated with these genes was mammary tumor, demonstrating the validity of this approach to probe breast cancer biology. Conclusion: This analysis demonstrates that the BA325 panel is useful both in understanding non-malignant mammary epithelial differentiation and breast cancer tumors. Citation Format: Edward C. Goodwin, Said Attiya, Marcia Fournier. A novel panel of 325 biomarkers is part of a large interconnected network representing multiple cell signaling pathways and allowing development of predictive tests for oncology drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 405. doi:10.1158/1538-7445.AM2017-405

Abstract P1-05-25: Patterns of translational dysregulation in models of estrogen receptor(+) and (-) breast cancer

Abstract Although our understanding of transcriptional dysregulation in cancer has expanded dramatically over the recent years, comparatively little is known about dysregulation in gene expression at the translational level. Yet, gene expression is extensively modulated at the level of translation. Leveraging the unbiased nature of next generation sequencing, we utilized the ribosome profiling strategy to characterize the translational profiles of a representative ER(+) (T47D) and ER(-) (SUM159) breast cancer cell line and compared them with the profiles of non-malignant human mammary epithelial cells (HMECs). The overarching hypothesis is that cancer cells differ from their non-malignant counterparts not only at the level of transcription but also at the level of translation and coordinate changes in transcription and translation lead to the phenotypic hallmarks of cancer. The results show that the median translational efficiency was higher and more variable in malignant cells as compared to HMECs (HMECs 1.09; T47D 1.6; SUM159 1.29. Variance of the log2 transformed values, 2.37; 3.92; 4.16, respectively). When cells were deprived of serum for 24 hours, translation was uniformly upregulated but this upregulation was more pronounced in malignant cells. A larger number of genes were differentially expressed at the translational as compared to the transcriptional level between non-malignant and malignant cells. There was an imperfect correlation between RNA levels and translational efficiency. In addition, individual transcripts were translated with variable efficiency between malignant and non-malignant cells suggesting that the translation of the same RNA sequence is modulated in a context-dependent manner. Although translation is globally upregulated in malignant cells, this upregulation is not uniform and the translational efficiency of multiple cancer-related mRNAs is not proportional to their abundance. For example, while the abundance of the CDK1 mRNA was not significantly different among the 3 cell lines, its translational efficiency was 1.5 and 2 times higher in the SUM159 and T47D cells, respectively. On the other hand, the mRNA of its cognate inhibitor CDKN1A was not only 4 times less abundant in the malignant cells but also half as efficiently translated as compared to HMECs. Ribosome profiling has consistently revealed translation outside of canonical protein-coding regions. While in HMECs only 5.3% of unique ribosome protected fragments aligned to non-protein encoding transcripts, the respective fractions were 8% for T47D and 19.2% for SUM159 cells and were consistently higher in malignant cells across all conditions tested. We found that the translation of RNAs previously thought to be noncoding, especially lincRNAs, was pervasively upregulated and to similar levels in T47D and SUM159 cells as compared to HMECs. Collectively, these results begin to unravel components of dysregulation in gene expression at the level of translation with important implications for the molecular pathophysiology of breast cancer and set the stage to advance such studies in primary human tumor samples. Citation Format: Vaklavas C, Myers RM, Grizzle WE, Blume SW. Patterns of translational dysregulation in models of estrogen receptor(+) and (-) breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-25.

Docosahexaenoic acid-acylated phloridzin, a novel polyphenol fatty acid ester derivative, is cytotoxic to breast cancer cells.

Docosahexaenoic acid-acylated phloridzin (PZ-DHA), a novel polyphenol fatty acid ester derivative, was synthesized through a regioselective acylation reaction with the aim of increasing the bioactivity of phloridzin (PZ) and docosahexaenoic acid (DHA). In this study, PZ-DHA's cytotoxic activity was explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA was selectively cytotoxic for mammary carcinoma (MDA-MB-231, MDA-MB-468, 4T1, MCF-7 and T-47D) cells compared to non-malignant human mammary epithelial cells (HMEC and MCF-10A) and fibroblasts by MTS assay and Annexin-V-FLUOS/propidium iodide staining. Flow cytometric analysis of Oregon Green 488- and Ki-67-stained MDA-MB-231 cells showed antiproliferative activity of PZ-DHA at a subcytotoxic concentration. PZ-DHA also arrested MDA-MB-231 cell division at the G2/M phase and down-regulated expression of cyclin B1 and cyclin-dependent kinase 1 (CDK1). PZ-DHA-induced apoptosis in MDA-MB-231 cells was confirmed by caspase 3/7 activation in a luminescence assay and DNA fragmentation by TUNEL staining. Moreover, MDA-MB-231 xenograft growth in non-obese diabetic severe combined immunodeficient mice was suppressed by intra-tumoral administration of PZ-DHA. This study shows that PZ-DHA is selectively cytotoxic to breast cancer cells in vitro and in vivo, suggesting that further investigations of PZ-DHA are warranted as a potential treatment for breast cancer.

WNT4 mediates the autocrine effects of growth hormone in mammary carcinoma cells.

The expression of Wingless and Int-related protein (Wnt) ligands is aberrantly high in human breast cancer. We report here that WNT4 is significantly upregulated at the mRNA and protein level in mammary carcinoma cells expressing autocrine human growth hormone (hGH). Depletion of WNT4 using small interfering (si) RNA markedly decreased the rate of human breast cancer cell proliferation induced by autocrine hGH. Forced expression of WNT4 in the nonmalignant human mammary epithelial cell line MCF-12A stimulated cell proliferation in low and normal serum conditions, enhanced cell survival and promoted anchorage-independent growth and colony formation in soft agar. The effects of sustained production of WNT4 were concomitant with upregulation of proliferative markers (c-Myc, Cyclin D1), the survival marker BCL-XL, the putative WNT4 receptor FZD6 and activation of ERK1 and STAT3. Forced expression of WNT4 resulted in phenotypic conversion of MCF-12A cells, such that they exhibited the molecular and morphological characteristics of mesenchymal cells with increased cell motility. WNT4 production resulted in increased mesenchymal and cytoskeletal remodeling markers, promoted actin cytoskeleton reorganization and led to dissolution of cell-cell contacts. In xenograft studies, tumors with autocrine hGH expressed higher levels of WNT4 and FZD6 when compared with control tumors. In addition, Oncomine data indicated that WNT4 expression is increased in neoplastic compared with normal human breast tissue. Accordingly, immunohistochemical detection of WNT4 in human breast cancer biopsies revealed higher expression in tumor tissue vs normal breast epithelium. WNT4 is thus an autocrine hGH-regulated gene involved in the growth and development of the tumorigenic phenotype.

Forkhead Box F1 promotes breast cancer cell migration by upregulating lysyl oxidase and suppressing Smad2/3 signaling.

BackgroundEpithelial-mesenchymal transition (EMT) increases cell migration and is implicated in cancer cell invasion and metastasis. We have previously described the involvement of the transcription factors, nuclear factor I-C2 (NFI-C2) and Forkhead box F1 (FoxF1), in the regulation of EMT and invasion during breast tumor progression. NFI-C2 counteracts these processes and FoxF1 is a directly repressed target of NFI-C2. FoxF1 induces EMT and invasiveness and enhances xenograft tumorigenicity in nude mice. Here we identify oppositely regulated targets of NFI-C2 and FoxF1 involved in these processes and further study a possible role for FoxF1 in tumorigenesis.MethodsWe used Affymetrix microarray to detect changes in the transcriptome of a mouse mammary epithelial cell line upon overexpression of NFI-C2 or FoxF1. To elucidate the effects and signaling events following FoxF1 overexpression we investigated in vitro invasion capacity and changes in transcription and protein expression resulting from RNAi and inhibitor treatment.ResultsThe extracellular matrix enzyme lysyl oxidase (LOX) was negatively regulated by NFI-C2 and positively regulated by FoxF1, and upregulation of LOX following FoxF1 overexpression in mouse mammary epithelial cells increased in vitro cell invasion. In the nuclei of FoxF1-overexpressing cells, the phosphorylation of Smad2 decreased, while that of p38 increased. Depletion of LOX by RNAi enhanced phosphorylation of Smad2 by a focal adhesion kinase (FAK)-dependent mechanism. In addition, induced expression of FoxF1 in a non-malignant human mammary epithelial cell line showed that the increase in LOX transcription and the suppression of Smad2 activity are early effects of FoxF1.ConclusionThese data show that FoxF1 enhances invasion in a LOX-dependent manner, is involved in the regulation of Smad2 signaling, and that FoxF1 overexpression ultimately leads to activation of p38 MAPK signaling. These findings provide new insights into the regulation of signaling pathways known to be important during breast tumor progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2196-2) contains supplementary material, which is available to authorized users.

Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis.

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.

Combinatorial DNA damage pairing model based on X-ray-induced foci predicts the dose and LET dependence of cell death in human breast cells.

In contrast to the classic view of static DNA double-strand breaks (DSBs) being repaired at the site of damage, we hypothesize that DSBs move and merge with each other over large distances (μm). As X-ray dose increases, the probability of having DSB clusters increases as does the probability of misrepair and cell death. Experimental work characterizing the X-ray dose dependence of radiation-induced foci (RIF) in nonmalignant human mammary epithelial cells (MCF10A) is used here to validate a DSB clustering model. We then use the principles of the local effect model (LEM) to predict the yield of DSBs at the submicron level. Two mechanisms for DSB clustering, namely random coalescence of DSBs versus active movement of DSBs into repair domains are compared and tested. Simulations that best predicted both RIF dose dependence and cell survival after X-ray irradiation favored the repair domain hypothesis, suggesting the nucleus is divided into an array of regularly spaced repair domains of ∼1.55 μm sides. Applying the same approach to high-linear energy transfer (LET) ion tracks, we are able to predict experimental RIF/μm along tracks with an overall relative error of 12%, for LET ranging between 30-350 keV/μm and for three different ions. Finally, cell death was predicted by assuming an exponential dependence on the total number of DSBs and of all possible combinations of paired DSBs within each simulated RIF. Relative biological effectiveness (RBE) predictions for cell survival of MCF10A exposed to high-LET showed an LET dependence that matches previous experimental results for similar cell types. Overall, this work suggests that microdosimetric properties of ion tracks at the submicron level are sufficient to explain both RIF data and survival curves for any LET, similarly to the LEM assumption. Conversely, high-LET death mechanism does not have to infer linear-quadratic dose formalism as done in the LEM. In addition, the size of repair domains derived in our model are based on experimental RIF and are three times larger than the hypothetical LEM voxel used to fit survival curves. Our model is therefore an alternative to previous approaches that provides a testable biological mechanism (i.e., RIF). In addition, we propose that DSB pairing will help develop more accurate alternatives to the linear cancer risk model (LNT) currently used for regulating exposure to very low levels of ionizing radiation.

Abstract P5-03-09: Phenotypic modulation of mammary stem cells by CAPE (caffeic acid phenethyl ester)

Abstract Background: Over 30% of cancer patients and up to 60% of breast cancer patients routinely use alternative formulations, commonly in combination with allopathic treatment, which may be linked with fewer adverse events. The naturopathic formulation propolis, and its active component Caffeic Acid Phenethyl Ester (CAPE), is a widely available honeybee product with an extended safety history credited with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE, inhibits growth of MCF-7 (ER+/PR+) and MDA-231 (TNBC) cells by cell cycle arrest and apoptosis; inhibits mdr-1 gene expression, NF-kB, and VEGF (1). In addition, CAPE inhibits the growth and tumorigenic potential of breast cancer stem cells derived from MDA-231 cells (2). We have demonstrated that CAPE, which is structurally similar to the hydroxamic acid class of HDAC inhibitors, mediates its effects on breast cancer through epigenetic modifications via histone deacetylase inhibition (unpublished data). We hypothesized that CAPE would inhibit mammary stem cells, which was tested in primary mouse mammary epithelial cells and a human breast epithelial cell line. Materials and Methods: Single mammary epithelial cells were isolated from Balb/c mice and cultured for 8 days in Ultra Low Attachment 96 well plates using mammosphere media containing 5%FBS and 5% Matrigel (3). The first generation of mammospheres were dissociated into single cells and recultured in the presence of CAPE for 8 days in secondary and tertiary passages. Alternatively, a Let7c-miRNA reporter expressed in MCF10a non-malignant human mammary epithelial cells were sorted to isolate Let7c-negative cells and placed in mammosphere culture and similarly treated with CAPE for 7 to 10 days. The mammospheres were stained with luminal marker K18 and basal marker K14 and examined using immunofluorescent microscopy. Results: CAPE treatment results in a dose dependent decrease in mammosphere forming efficiency (MFE) of mammospheres obtained from a primary culture of mammary gland derived epithelial cells as well as those derived from the MCF10A cell line. Furthermore, MFE of tertiary cultures is reduced even after CAPE is removed. Interestingly, mammospheres shifted from predominantly K14 to K18 positive in those mammospheres that formed at the lowest CAPE treatment concentration. Conclusion: CAPE inhibits mammosphere formation from normal mouse mammary gland and from a non-malignant human mammary cell line. The mechanism of CAPE effects may lie in its established role in inducing cell cycle arrest, apoptosis or via epigenetic mechanisms, among others. Interestingly, CAPE causes a shift from a basal to a luminal cell phenotype in mammospheres, suggesting an effect on lineage commitment. References 1) *Wu J, *Omene C, Karkaszka J, et al. CANCER LETTERS. 2011 Sep 1;308(1):43-53. Epub 2011 May 13. (* Share 1st authorship) 2) Omene C, Wu J, Frenkel K. INVESTIGATIONAL NEW DRUGS. 2012 Aug;30(4):1279-88. Epub 2011 May 3. 3) Guo W, Keckesova Z, Donaher JL, et al. CELL. 2012 Mar 2;148(5):1015-28. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-09.

Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis

Expression of the antioxidant enzyme EcSOD in normal human mammary epithelial cells was not recognized until recently. Although expression of EcSOD was not detectable in non-malignant human mammary epithelial cells (HMEC) cultured in conventional two-dimensional (2D) culture conditions, EcSOD protein expression was observed in normal human breast tissues, suggesting that the 2D-cultured condition induces a repressive status of EcSOD gene expression in HMEC. With the use of laminin-enriched extracellular matrix (lrECM), we were able to detect expression of EcSOD when HMEC formed polarized acinar structures in a 3D-culture condition. Repression of the EcSOD-gene expression was again seen when the HMEC acini were sub-cultured as a monolayer, implying that lrECM-induced acinar morphogenesis is essential in EcSOD-gene activation. We have further shown the involvement of DNA methylation in regulating EcSOD expression in HMEC under these cell culture conditions. EcSOD mRNA expression was strongly induced in the 2D-cultured HMEC after treatment with a DNA methyltransferase inhibitor. In addition, epigenetic analyses showed a decrease in the degree of CpG methylation in the EcSOD promoter in the 3D versus 2D-cultured HMEC. More importantly, >80% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA and protein expression levels compared with normal mammary tissues and there is an inverse correlation between the expression levels of EcSOD and the clinical stages of breast cancer. Combined bisulfite restriction analysis analysis of some of the tumors also revealed an association of DNA methylation with the loss of EcSOD expression in vivo. Furthermore, overexpression of EcSOD inhibited breast cancer metastasis in both the experimental lung metastasis model and the syngeneic mouse model. This study suggests that epigenetic silencing of EcSOD may contribute to mammary tumorigenesis and that restoring the extracellular superoxide scavenging activity could be an effective strategy for breast cancer treatment.

Epigenome remodelling in breast cancer: insights from an early in vitro model of carcinogenesis

Epigenetic gene regulation has influence over a diverse range of cellular functions, including the maintenance of pluripotency, differentiation, and cellular identity, and is deregulated in many diseases, including cancer. Whereas the involvement of epigenetic dysregulation in cancer is well documented, much of the mechanistic detail involved in triggering these changes remains unclear. In the current age of genomics, the development of new sequencing technologies has seen an influx of genomic and epigenomic data and drastic improvements in both resolution and coverage. Studies in cancer cell lines and clinical samples using next-generation sequencing are rapidly delivering spectacular insights into the nature of the cancer genome and epigenome. Despite these improvements in technology, the timing and relationship between genetic and epigenetic changes that occur during the process of carcinogenesis are still unclear. In particular, what changes to the epigenome are playing a driving role during carcinogenesis and what influence the temporal nature of these changes has on cancer progression are not known. Understanding the early epigenetic changes driving breast cancer has the exciting potential to provide a novel set of therapeutic targets or early-disease biomarkers or both. Therefore, it is important to find novel systems that permit the study of initial epigenetic events that potentially occur during the first stages of breast cancer. Non-malignant human mammary epithelial cells (HMECs) provide an exciting in vitro model of very early breast carcinogenesis. When grown in culture, HMECs are able to temporarily escape senescence and acquire a pre-malignant breast cancer-like phenotype (variant HMECs, or vHMECs). Cultured HMECs are composed mainly of cells from the basal breast epithelial layer. Therefore, vHMECs are considered to represent the basal-like subtype of breast cancer. The transition from HMECs to vHMECs in culture recapitulates the epigenomic phenomena that occur during the progression from normal breast to pre-malignancy. Therefore, the HMEC model system provides the unique opportunity to study the very earliest epigenomic aberrations occurring during breast carcinogenesis and can give insight into the sequence of epigenomic events that lead to breast malignancy. This review provides an overview of epigenomic research in breast cancer and discusses in detail the utility of the HMEC model system to discover early epigenomic changes involved in breast carcinogenesis.

Abstract 5160: Global metabolomic profiles distinguish malignant from non-malignant human mammary epithelial cell responses to endogenous p53 upregulation by MDM2 antagonism

Abstract Wildtype (wt) p53 can alter metabolic pathways and mitochondrial function but it is unclear whether these responses are similar in normal and malignant cells, where it is also hypothesized that p53wt can modulate the Warburg effect. Therefore, we used a small molecule inhibitor of MDM2, MI-63 (Ding et al., J. Med. Chem. 2006; Ascenta/Sanofi), to induce ∼5-fold intracellular increases in p53wt protein levels within 24 h of treating malignant (MCF-7) or non-malignant (MCF10A) human mammary epithelial cells, whereupon global biochemical changes were assayed in replicate (x6) extracts using various mass spectrometry platforms (GC/MS, LC/MS/MS; Metabolon, Durham, NC). Extracts from similarly treated p53mut malignant SKBr3 cells were also assessed. Normalized (to total protein) and imputed data from 310 known and detected biochemicals were compared for treatment effects (Wilcoxon rank sum test, p<0.05). Treatment significantly altered levels of 93 biochemicals (58 up, 35 down) in the malignant MCF-7 cells, 62 (39 up, 23 down) in the non-malignant MCF10A cells, and 24 (20 up, 4 down) in the malignant p53mut SKBr3 cells. Common to both MCF-7 and MCF10A, p53wt upregulation increased NAD+ and NADP+ levels, responses not seen in SKBr3 cells. Upregulation of p53wt selectively suppressed polyamines (spermidine, putrescine) in MCF10A, whose response to MI-63 is cytostatic unlike the apoptotic response of MCF-7 or treatment unaffected growth of SKBr3 cells. Relative to MCF10A, MCF-7 cells showed opposing energy-related biochemical responses in glucose, glucose-6-phophosphate, 3-phosphoglycerate and pyruvate pools, along with increases in the glutaminolysis product succinate, suggesting that p53wt upregulation enhances glycolytic and mitochondrial (TCA) production of pyruvate in malignant MCF-7 cells yet decreases glycolysis and pyruvate production in non-malignant MCF10A cells. This conclusion that p53wt upregulation increases glycolysis, pyruvate production and mitochondrial ATP synthesis in MCF-7 was confirmed using live cell extracellular flux analysis of oxygen consumption and acid production (Seahorse XF), which showed opposing treatment responses by MCF10A and no treatment effects in SKBr3. Altogether, these findings point to opposing metabolic and bioenergetic responses induced by p53wt upregulation in malignant and non-malignant mammary epithelial cells, consistent with p53wt induced reversal of the Warburg effect, and offering new insights into pathways associated with different therapeutic and cell fate responses induced by novel small molecule MDM2 antagonists like MI-63. 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 5160. doi:1538-7445.AM2012-5160

Abstract 2701: RORα suppresses breast tumor progression by inducing cell polarization and inhibiting cell invasion

Abstract Background: Disruption of tissue polarity is one of hallmarks of breast cancer development, which is associated with cell invasion and cancer dissemination. Therefore, identifying proteins and pathways that modulate cell polarity is crucial not only to advancing our knowledge of breast cancer, but also to discovering potential therapeutic targets. Methods: To identify potential regulators of tissue polarity, we analyzed over-represented DNA motifs in the promoter regions of the polarization genes that are significantly up-regulated genes in polarized human mammary epithelial cells in a physiologically relevant three-dimensional (3D) culture system. We assessed the protein levels of RORα by immunoblotting and immunohistochemistry in malignant and non-malignant human mammary epithelial cells as well as human breast cancer tissue array. To explore the functional significance of RORα down-regulation the malignance of breast cancer cells, we restored RORα expression in malignant T4-2 cells, MDA231 cells and silenced RORα expression in non-malignant S1 cells. The growth and malignant phenotypes of these cells were examined in 3D culture and xenograft model. Results: Results from promoter analysis suggest that RORα is a potential regulator of mammary tissue polarity and is inactivated in disorganized HMECs. Expression of RORα protein is significantly reduced in malignant breast cancer cell lines and this reduction is associated with disruption of acinar structure in 3D culture. Both mRNA and protein level of RORα were significantly reduced in human breast cancer tissues. Restoring RORα expression in T4-2 and MDA-MB 231 cells significantly suppressed proliferation, invasion and the malignant phenotypes in 3D culture. Silencing RORα expression in non-malignant S1 cells disrupts the polarized acinar morphogenesis. Restoring RORα expression in T4-2 and MDA-MB 231 cells also inhibits the tumor growth in nude mice. Moreover, patients with reduced expression of RORα in cancer tissue have significantly shorter survival. Conclusion: Together these results indicate that inactivation of RORα promotes the breast cancer development and progression by enhancing cell invasion and disturbing normal tissue architecture. RORα provide a potential therapeutic target for breast cancer. 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 2701. doi:1538-7445.AM2012-2701

RORα Suppresses Breast Tumor Invasion by Inducing SEMA3F Expression

Inactivation of tumor suppressors and inhibitory microenvironmental factors is necessary for breast cancer invasion; therefore, identifying those suppressors and factors is crucial not only to advancing our knowledge of breast cancer, but also to discovering potential therapeutic targets. By analyzing gene expression profiles of polarized and disorganized human mammary epithelial cells in a physiologically relevant three-dimensional (3D) culture system, we identified retinoid orphan nuclear receptor alpha (RORα) as a transcription regulator of semaphorin 3F (SEMA3F), a suppressive microenvironmental factor. We showed that expression of RORα was downregulated in human breast cancer tissue and cell lines, and that reduced mRNA levels of RORα and SEMA3F correlated with poor prognosis. Restoring RORα expression reprogrammed breast cancer cells to form noninvasiveness structures in 3D culture and inhibited tumor growth in nude mice, accompanied by enhanced SEMA3F expression. Inactivation of RORα in nonmalignant human mammary epithelial cells inhibited SEMA3F transcription and impaired polarized acinar morphogenesis. Using chromatin immunoprecipitation and luciferase reporter assays, we showed that transcription of SEMA3F is directly regulated by RORα. Knockdown of SEMA3F in RORα-expressing cancer cells rescued the aggressive 3D phenotypes and tumor invasion. These findings indicate that RORα is a potential tumor suppressor and inhibits tumor invasion by inducing suppressive cell microenvironment.

Abstract 5065: A 22-gene signature predicts response to chemotherapy in a broad range of breast cancer subtypes

Abstract Statement of Purpose: To test the utility of a novel, multifaceted 22-gene signature to predict response to chemotherapy in different molecular subtypes of breast cancer. Background: Only 15-25% of breast cancer patients from all histopathological subtypes currently respond to primary chemotherapy with a complete pathological response (pCR) and there is currently no test in widespread clinical use to predict whether a patient will respond. Here we present results describing a 22-gene signature that can identify breast cancer patients who will respond to taxol-combination chemotherapy in a wide range of molecular subtypes. The signature was identified using a biology-driven approach. We hypothesized that genes involved in normal human mammary acinar morphogenesis represent biomarkers capable of stratifying breast cancers. Methods: To determine whether genes with differential expression during acinar morphogenesis predict response to chemotherapy, we have analyzed gene expression microarray results. Three preexisting microarray datasets were used. 1) The dataset of Fournier et al (Cancer Res, 2006. 66:7095) obtained from a time course of two different non-malignant human mammary epithelial cell types grown in a laminin-rich extracellular matrix culture (lrECM). 2) The dataset of Hess et al (J Clin Oncol, 2006. 24:4236) including 133 patients with stage I-III breast cancer obtained before primary taxol-combination chemotherapy (TFAC). 3) The dataset of Popovici et al (Breast Cancer Res, 2010. 12:R5) including 278 patient samples that overlaps with the previous dataset. The assignment of molecular subtype of tumor samples was performed using the intrinsic gene set of 300 genes (Hu et al., BMC Genomics, 2006.7:96). Results: The majority of genes (12 out 22) down regulated upon growth arrest and polarity formation during acinar morphogenesis in lrECM were significantly over expressed in patients who responded to chemotherapy with pCR (p<0.05, Student's T-test and Fisher's Exact test). These 22 genes are referred to here as the 22-gene signature. To test this signature's ability to predict response in different molecular subtypes of breast cancer we applied logistic regression with three-fold cross validation. The 22-gene signature accurately predicted response in all samples with AUC 0.75 and in specific breast cancer molecular subtypes including ER-positive, ER-negative, luminal B, Her2-positive, and basal-like with average AUC 0.69. Conclusions: This study is the first example of a single gene signature to predict chemotherapy response in different molecular subtypes of breast cancer. Its predictive value in ER-negative and basal-like breast cancers is notable. Patients identified as non-responsive have the potential to benefit from adding an alternative treatment early in their care or from improved quality of life in forgoing an ineffective treatment. 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 5065. doi:10.1158/1538-7445.AM2011-5065

Abstract 3797: Wildtype p53 upregulation induces contrasting bioenergetic and metabolic responses in malignant and non-malignant mammary epithelial cells

Abstract In addition to its well established genome stabilizing and tumor suppressing role, p53 can also modulate cell metabolism and mitochondrial function. Small molecule MDM2 antagonists like nutlin-3 or MI-63 (Ascenta) are therapeutic prototypes that rapidly increase wildtype (wt) p53 levels in mammalian cells inducing apoptosis in cancer cells and growth arrest or senescence in non-malignant cells. We employed MI-63 (10 uM × 24h) to induce a near 10-fold increase in p53wt protein expression and compare its mitochondrial bioenergetic and metabolic consequences in malignant (MCF7) and non-malignant (MCF10A) human mammary epithelial cells, whose fates are differentially affected by MI-63. We utilized chromatin immunopreciptation-DNA sequencing (ChIP-seq; Illumina 3G sequencer) and expression microarrays (Affymetrix U133Av2) to identify genome-wide direct and indirect p53 target genes potentially affecting MCF7 mitochondrial structure and function. Within 24h of p53wt upregulation, 1903 genes were differentially expressed including 539 of the 2239 differentially bound direct p53 gene targets, leaving 1364 differentially expressed indirect p53 target genes. While p53 binding was not detected in the mitochondrial genome, GO analysis identified genes associated with the mitochondrial matrix (3 up, 28 down) and genes associated with the outer mitochondrial membrane (7 up, 10 down) as significantly enriched among the p53 regulated nuclear targets (EASE = 0.003, FDR p = 0.03 and EASE = 0.002, FDR p = 0.02 respectively). Real-time, kinetic measurement (Seahorse XF) of oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) was performed in adherent cultures of MCF7 and MCF10A cells after comparable 24 h MI-63 induction of p53wt. In contrast to the apparent inhibitory effect of p53wt induction on MCF7 transcription of mitochondrial matrix genes, basal and spare mitochondrial respiratory capacity were stimulated ∼2-fold. This p53wt induced increase in ATP supply and OCR was caused by slightly higher OXPHOS and maximal recruitment of glycolysis, assessed by ECAR and simulated by cell supplementation with pyruvate. Unlike this metabolic and bioenergetic response in the malignant MCF7 cells, p53wt upregulation in the non-malignant MCF10A cells not only failed to stimulate OCR and ECAR but significantly depressed glycolysis. While it is generally thought that the metabolic function of p53wt is to favor energy production by mitochondrial OXPHOS and resist the shift to glycolysis that is characteristically seen in cancer cells, our findings suggest that malignant and non-malignant epithelial cells may have different metabolic and bioenergetic responses to upregulated p53wt. Further studies are needed to determine if these different p53wt responses contribute to the different cell fate responses induced by MDM2 antagonists like MI-63. 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 3797. doi:10.1158/1538-7445.AM2011-3797

Quantitative Analysis of Three-Dimensional Human Mammary Epithelial Tissue Architecture Reveals a Role for Tenascin-C in Regulating c-Met Function

Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.

Estradiol decreases xanthine dehydrogenase enzyme activity and protein expression in non‐tumorigenic and malignant human mammary epithelial cells

The retinoic acid deficiency in breast tumour epithelial cells has been ascribed to an insufficient expression of either the enzyme(s) involved in its biosynthesis or the cellular retinol binding protein (CRBP) or both. In an attempt to define the mechanisms underpinning retinoic acid deficiency in these cell model systems, we have investigated the potential regulatory effect of oestrogen (17beta-estradiol) on one key player in retinoic acid biosynthesis, the xanthine dehydrogenase (XDH). This enzyme is consistently expressed and very active in non-malignant human mammary epithelial cells (HMEC), as opposed to tumour MDA-MB231 and MCF7 cells. In these latter two cell lines, as opposed to HMEC cells, we observe a residual ability of XDH to produce retinoic acid from retinaldehyde and the inability to use retinol, as a consequence of a deficit in CRBP. In addition, estradiol treatment of MDA-MB231 and MCF7 cells decreases protein expression and activity of the enzyme, with no modification of the mRNA transcript levels, eventually leading to deteriorate further retinoic acid production.

Interaction of E-cadherin and PTEN Regulates Morphogenesis and Growth Arrest in Human Mammary Epithelial Cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual-function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two nonmalignant human mammary epithelial cell lines that form polarized, growth-arrested structures (acini) when cultured in three-dimensional laminin-rich extracellular matrix gels (lrECM). As acini begin to form, PTEN accumulates both in the cytoplasm and at cell-cell contacts where it colocalizes with the E-cadherin/beta-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in Skbr-3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in three-dimensional lrECM, indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus seems to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.