Primary Human Mammary Epithelial Cells Research Articles

Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (–ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore.

RelA-Induced Interferon Response Negatively Regulates Proliferation.

Both oncogenic and tumor-suppressor activities are attributed to the Nuclear Factor kappa B (NF-kB) pathway. Moreover, NF-kB may positively or negatively regulate proliferation. The molecular determinants of these opposing roles of NF-kB are unclear. Using primary human mammary epithelial cells (HMEC) as a model, we show that increased RelA levels and consequent increase in basal transcriptional activity of RelA induces IRF1, a target gene. Induced IRF1 upregulates STAT1 and IRF7, and in consort, these factors induce the expression of interferon response genes. Activation of the interferon pathway down-regulates CDK4 and up-regulates p27 resulting in Rb hypo-phosphorylation and cell cycle arrest. Stimulation of HMEC with IFN-γ elicits similar phenotypic and molecular changes suggesting that basal activity of RelA and IFN-γ converge on IRF1 to regulate proliferation. The anti-proliferative RelA-IRF1-CDK4 signaling axis is retained in ER+/HER2- breast tumors analyzed by The Cancer Genome Atlas (TCGA). Using immuno-histochemical analysis of breast tumors, we confirm the negative correlation between RelA levels and proliferation rate in ER+/HER2- breast tumors. These findings attribute an anti-proliferative tumor-suppressor role to basal RelA activity. Inactivation of Rb, down-regulation of RelA or IRF1, or upregulation of CDK4 or IRF2 rescues the RelA-IRF1-CDK4 induced proliferation arrest in HMEC and are points of disruption in aggressive tumors. Activity of the RelA-IRF1-CDK4 axis may explain favorable response to CDK4/6 inhibition observed in patients with ER+ Rb competent tumors.

Abstract 1071: Age-dependent DNA methylation in normal breast epithelium and breast cancer

Abstract Breast cancer risk factors include age, genetic alterations, weight, diet and others. More recently accumulating evidence suggests that epigenetic alterations are frequent and play important role in breast cancer development and progression. Epigenetic alterations include changes in DNA methylation, histone modifications and miRNA expression in the absence of changes in DNA sequence. The epigenetic changes in cancer include global loss of DNA methylation that might lead to genomic instability and gain of methylation at many promoters of tumor suppressor genes that leads to gene silencing. Interestingly, work from our own laboratory and others have established that in many different tissues, DNA methylation changes due to age have similar patterns as to changes that occur in cancer. Therefore, the goal of this study is to identify and characterize age-dependent DNA methylation changes in normal breast epithelium and their contribution to breast cancer development. To differentiate between age-dependent and independent DNA methylation changes, we utilized DNA extracted from 27 primary human mammary epithelial cells (age range 33-82 years old) derived from adjacent or contralateral normal mammary tissue of breast cancer patients. We measured DNA methylation using a highly sensitive methodology developed in our laboratory called Digital Restriction Enzyme Analysis of Methylation (DREAM). We also studied DNA methylation in six different breast cancer cell lines by DREAM. We also used publically available 450K TCGA data for 685 breast tumor and 98 normal samples. Using bioinformatic approach, we have defined age dependent (Spearman R > |0.38|, p <0.05) and independent (Spearman R < |0.37|, p>0.05) sites that exhibit DNA methylation changes in normal breast epithelium. Interestingly, we found that the age-related genes are more seeded (exhibit low level of methylation) in their promoter CpG islands and are enriched for polycomb group target genes compared to the non-age related genes (p value = 0.032). Furthermore, these age-related genes (for example HOXD9, TDRD10, MYOD1, DPYS, GDA, GIPC2, FAM162B, LAMA1, PKDREJ, etc) have increased DNA methylation in tumors compared to the normal samples in TCGA with a methylation difference of up to 50%. These changes are significantly different compared to not-age related genes or random genes (p<0.0001). The age-related genes that gained DNA methylation in tumors in TCGA dataset, were also hypermethylated in breast cancer cell lines compared to immortalized human mammary epithelial cells. Thirty one percent of the common genes between TCGA data set and breast cancer cell lines, showed decreased mRNA expression in RNA-seq data available through the TCGA data portal. Therefore, in this study we have characterized age-dependent epigenetic changes in normal breast epithelium as potential breast cancer risk associated alterations. Citation Format: Shoghag B. Panjarian, Carolyn Slater, Jozef Madzo, Jaroslav Jelinek, Xiaowei Chen, Jean-Pierre Issa. Age-dependent DNA methylation in normal breast epithelium and breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2015-1071

Abstract 3488: BPM 31510 has broad utility as both a single agent and in combination with standard-of-care chemotherapy in triple-negative breast cancer

Abstract Deregulated metabolism is recognized as a ‘hallmark of cancer’ although our understanding of the interplay between metabolic pathway activity and responses to standard-of-care chemotherapy remains limited. In vitro studies have demonstrated that BPM 31510, a Coenzyme Q10-containing proprietary formulation, alters both glycolysis and mitochondrial OXPHOS in cancer cells. Building on the clinical observation that triple-negative breast cancer (TNBC) is distinctly glycolytic compared to other breast sub-types, we examined the effects of BPM 31510 alone and in combination with standard-of-care chemotherapy in a murine xenograft model of TNBC (MDA-MB231). Tumor-bearing mice were treated with cycles of the standard ‘TAC regimen’ (5mg/kg docetaxel and 1 mg/kg doxorubicin for 3 days followed by 35 mg/kg cyclophosphamide) with or without BPM 31510 every 3 weeks. As expected, chemotherapy treatment extended survival. Notably, BPM 31510 was more effective as a single agent compared to traditional chemotherapy and extended survival by more than 6 weeks. The combination of BPM 31510 with chemotherapy further enhanced survival over either treatment regimen alone, and this was associated with attenuated tumor growth over the duration of the study. Analysis of a subset of tumors after 2 cycles of treatment confirmed a decrease in tumor weight in all treatment conditions compared to control and a significant synergistic effect of combined therapy (p = 0.02). Importantly, changes were not observed in the weight of other organs from the same mice, suggesting lack of systemic toxicity. Evidence of decreased proliferation (Ki67 staining) and enhanced caspase activity were also observed in tumors by immunohistochemical analysis. Assessment of the BPM 31510 IC50 in vitro using a panel of primary human mammary epithelial cells (HMEC), non-tumorigenic (MCF12A), and breast cancer cell lines demonstrated selective cytotoxicity in cancer cells compared to primary and non-tumorigenic cells. Moreover, MDA-MB231 was the most sensitive to BPM 31510-induced cell death in the breast cancer cell panel that was representative of multiple breast tumor sub-types. Taken together, these data demonstrate that the metabolic modulating agent BPM 31510 has broad utility as both a single agent and in combination with standard-of-care in breast cancer including distinctly glycolytic TNBC for which prognosis is poor and few therapeutic options exist. Citation Format: Niven R. Narain, Anne R. Diers, Arleide Lee, Rakibou Ouro-Djobo, Vivek K. Vishnudas, Ely Benaim, Joaquin J. Jimenez, Rangaprasad Sarangarajan. BPM 31510 has broad utility as both a single agent and in combination with standard-of-care chemotherapy in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3488. doi:10.1158/1538-7445.AM2015-3488

Abstract 2027: MECP2 is a frequently amplified oncogene with an unusual epigenetic mechanism of action

Abstract Introduction: We demonstrate that MECP2 is an oncogene, characterize the extent of MECP2 involvement in human cancers, and explore its mechanism of action. Experimental Procedures: To identify new oncogenes that drive cancer development, we conducted an unbiased genome-scale screen for genes that can substitute for activated RAS in oncogenic transformation and explored one screen hit in detail. Results: One of the new potential oncogenes identified in this screen, Methyl CpG Binding Protein 2 (MECP2), has no previously described role in malignancy and is amplified across 18% of all human cancers in the TCGA collection. MECP2 is an X-linked gene known to bind methylated cytosines, and can act as a transcriptional repressor in this context. Recent studies show that it also acts as a transcriptional activator, likely through binding to another epigenetic modification of DNA, 5-hydroxymethylcytosine (5hmC). MECP2 was as potent as activated RAS in conferring anchorage independent growth upon primary human mammary epithelial cells (HMECs) previously transduced with the SV40 early region and hTERT (N−RAS HMECs). MECP2 partially rescued the growth inhibition of RAS-addicted human cancer cell lines after the shRNA-mediated suppression of RAS. MECP2 expresses two spliced isoforms; experiments showed the short isoform activated the MAPK pathway, while both isoforms activated the PI3K pathway. Neither isoform alone conferred growth of N−RAS HMECs as xenografts in nude mice; however, both isoforms together allowed tumor growth. The cancer-promoting activities of MECP2 absolutely required its DNA-binding activity. A mutant allele of MECP2, R133C, is known to have defective 5hmC binding but largely preserved 5-methylcytosine binding; interestingly, this mutation was 10% as active as wt MECP2 in conferring anchorage independent growth. We searched for genes whose expression is modulated by MECP2 that might be important for transformation; the kinase PAK3 was found to be required for the ability of MECP2 to confer anchorage independent growth, and was itself able to confer anchorage independent growth upon N−RAS HMECs. A number of human cancer cell lines have amplified, overexpressed MECP2 and showed significant growth inhibition after shRNA-mediated downregulation of MECP2 (MECP2 addiction). N−RAS HMECs transformed with MECP2 were an order of magnitude more sensitive to the DNA methylation inhibitor 5-azacytidine than isogenic cells transformed by activated RAS, or isogenic cells without an additional transforming gene. Conclusion: MECP2 is a commonly amplified and overexpressed oncogene whose two splicing isoforms together recapitulate the major oncogenic activities of activated RAS. Because MECP2 requires DNA binding to methylated or hydroxymethylated cytosines for its tumor-promoting activities, DNA methylation inhibition with FDA-approved drugs may be therapeutic for tumors overexpressing MECP2. Citation Format: Manish Neupane, Allison P. Clark, Nicolai J. Birkbak, Aron Eklund, Steven E. Schumacher, Rameen Beroukhim, Zoltan Szallasi, Marc Vidal, David E. Hill, Daniel P. Silver. MECP2 is a frequently amplified oncogene with an unusual epigenetic mechanism of action. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2015-2027

Characterization of CM572, a Selective Irreversible Partial Agonist of the Sigma-2 Receptor with Antitumor Activity.

The sigma-2 receptors are promising therapeutic targets because of their significant upregulation in tumor cells compared with normal tissue. Here, we characterize CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one] (sigma-1 Ki ≥ 10 µM, sigma-2 Ki = 14.6 ± 6.9 nM), a novel isothiocyanate derivative of the putative sigma-2 antagonist, SN79 [6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one]. CM572 bound irreversibly to sigma-2 receptors by virtue of the isothiocyanate moiety but not to sigma-1. Studies in human SK-N-SH neuroblastoma cells revealed that CM572 induced an immediate dose-dependent increase in cytosolic calcium concentration. A 24-hour treatment of SK-N-SH cells with CM572 induced dose-dependent cell death, with an EC50 = 7.6 ± 1.7 µM. This effect was sustained over 24 hours even after a 60-minute pretreatment with CM572, followed by extensive washing to remove ligand, indicating an irreversible effect consistent with the irreversible binding data. Western blot analysis revealed that CM572 also induced cleavage activation of proapoptotic BH3-interacting domain death agonist. These data suggest irreversible agonist-like activity. Low concentrations of CM572 that were minimally effective were able to attenuate significantly the calcium signal and cell death induced by the sigma-2 agonist CB-64D [(+)-1R,5R-(E)-8-benzylidene-5-(3-hydroxyphenyl)-2-methylmorphan-7-one]. CM572 was also cytotoxic against PANC-1 pancreatic and MCF-7 breast cancer cell lines. The cytotoxic activity of CM572 was selective for cancer cells over normal cells, being much less potent against primary human melanocytes and human mammary epithelial cells. Taken together, these data show that CM572 is a selective, irreversible sigma-2 receptor partial agonist. This novel irreversible ligand may further our understanding of the endogenous role of this receptor, in addition to having potential use in targeted cancer diagnosis and therapy.

Abstract P4-05-04: MECP2 is a frequently amplified oncogene and potential therapeutic target in TNBC

Abstract Background: To identify new oncogenes that drive cancer development, we conducted an unbiased genome-scale screen for genes that can substitute for activated RAS in oncogenic transformation. We focused attention on one of the new potential oncogenes identified in this screen, Methyl CpG Binding Protein 2 (MECP2), which has no previously described role in malignancy and is amplified across ∼20% of all human cancers, and ∼30% of Triple-Negative Breast Cancer (TNBC). MECP2 is an X-linked gene known to bind methylated cytosines, and can act as a transcriptional repressor in this context. Recent studies show that it also acts as a transcriptional activator, likely through binding to another epigenetic modification of DNA, 5-hydroxymethylcytosine (5hmC). Results: MECP2 is as potent as activated RAS in conferring anchorage independent growth upon primary human mammary epithelial cells (hMECs) previously transduced with the SV40 early region and hTERT (N−RAS hMECs). MECP2 partially rescues the growth inhibition of RAS-addicted human cancer cell lines after the shRNA-mediated suppression of RAS. MECP2 expresses two spliced isoforms; experiments showed the short isoform activates the MAPK pathway, while the long isoform activates the PI3K pathway. Neither isoform alone can cause growth of N−RAS hMECs as a xenograft in nude mice; together, they can. The transformation and growth factor pathway induction activities of MECP2 absolutely require its DNA-binding activity. A number of TNBC cell lines have amplified, overexpressed MECP2, and of the first 13 TNBC patient-derived xenografts examined, 4 have MECP2 overexpression. Several TNBC cell lines that have amplified, overexpressed MECP2 show significant growth inhibition after shRNA-mediated downregulation of MECP2 (MECP2 addiction), while a breast cancer cell line without MECP2 overexpression showed no such inhibition. N−RAS hMECs transformed with MECP2 are an order of magnitude more sensitive to either of the DNA methylation inhibitors 5-azacytidine or decitabine than isogenic cells transformed by activated RAS, or isogenic cells without an additional transforming gene. Further, we find that combined treatment with the DNA methylation inhibitor 5-azacytidine and the HDAC inhibitor Trichostatin A is synergistic in our hMEC experimental system. Conclusion: MECP2 is a commonly amplified and overexpressed oncogene whose two splicing isoforms together recapitulate the major oncogenic functions of activated RAS. Because MECP2 requires DNA binding to methylated or hydroxymethylated cytosines for its tumor-promoting activities, DNA methylation inhibition with FDA-approved drugs may be therapeutic for tumors overexpressing MECP2. Citation Format: Daniel P Silver, Manish Neupane, Allison P Clark, Marc Vidal, David E Hill. MECP2 is a frequently amplified oncogene and potential therapeutic target in TNBC [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-04.

A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation.

IntroductionThe extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes.MethodsWe developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance.ResultsWe identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein–coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype–independent overall survival differences.ConclusionDiverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0510-y) contains supplementary material, which is available to authorized users.

Quantification of regenerative potential in primary human mammary epithelial cells.

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

C-MYC is a radiosensitive locus in human breast cells.

Ionising radiation is a potent human carcinogen. Epidemiological studies have shown that adolescent and young women are at increased risk of developing breast cancer following exposure to ionising radiation compared with older women, and that risk is dose-dependent. Although it is well understood which individuals are at risk of radiation-induced breast carcinogenesis, the molecular genetic mechanisms that underlie cell transformation are less clear. To identify genetic alterations potentially responsible for driving radiogenic breast transformation, we exposed the human breast epithelial cell line MCF-10A to fractionated doses of X-rays and examined the copy number and cytogenetic alterations. We identified numerous alterations of c-MYC that included high-level focal amplification associated with increased protein expression. c-MYC amplification was also observed in primary human mammary epithelial cells following exposure to radiation. We also demonstrate that the frequency and magnitude of c-MYC amplification and c-MYC protein expression is significantly higher in breast cancer with antecedent radiation exposure compared with breast cancer without a radiation aetiology. Our data also demonstrate extensive intratumor heterogeneity with respect to c-MYC copy number in radiogenic breast cancer, suggesting continuous evolution at this locus during disease development and progression. Taken together, these data identify c-MYC as a radiosensitive locus, implicating this oncogenic transcription factor in the aetiology of radiogenic breast cancer.

Human breast progenitor cell numbers are regulated by WNT and TBX3.

BackgroundAlthough human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties.MethodsTo examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone.ResultsGrowth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response.ConclusionsWe identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine WNT signaling is stimulated by estrogen and progesterone, while autocrine WNT signaling is induced by the embryonic T-box transcription factor TBX3.

Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: role of key tyrosines and lysines.

Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.

Abstract 4333: Metabolite profiling reveals druggable metabolic distinctions between basal-like and non-basal-like triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that represents about 15-20% of all breast cancers. Because TNBC tumors do not express the estrogen or progesterone receptor and lack HER2 amplification, the disease is not responsive to current targeted therapies. The development of therapeutic approaches specific for TNBC is hindered by genetic heterogeneity, and significant efforts are being made to subtype the disease. To this end, we performed metabolite profiling (metabolomics) to characterize metabolic fingerprints within TNBC in order to define metabolic subtypes, and identify molecular drivers for the development of targeted therapies. We profiled twelve well-characterized TNBC-derived cell lines as well as a non-transformed, immortalized breast cell line and two primary human mammary epithelial cell lines. Those cancer cell lines recapitulate all 7 genetic subtypes of TNBC which were proposed recently based on mRNA gene expression profiles (1). Our approaches used and data generated have implications for drug target discovery. Hierarchical clustering based on high quality intracellular metabolites clearly and reproducibly segregated the TNBC cell lines from the non-transformed lines. Alterations in energy utilization, lipid metabolism, and other pathways of importance to highly proliferative cells differed significantly from the control cell line MCF-10A. In addition, TNBC cell lines segregated into two discrete groups, suggesting the existence of two major metabolic subtypes of TNBC, which correlated with basal-like vs. non-basal-like gene expression. Metabolites like glutamate and glutamine, serine, glycine, trans-4-hydroxyproline, 5-oxoproline, several complex lipids (phosphatidylcholines and sphingomyelins), myo-inositol, polyamines spermidine and putrescine represented metabolites differing significantly between TNBC metabolic subtypes. Ongoing studies are evaluating whether these differences represent dependencies with therapeutic relevance. Metabolite profiling was also used to identify potential metabolic liabilities generated by treatment with clinical kinase inhibitors. Response of the metabolome to treatment with rapamycin, sorafenib, imatinib, and lapatinib in four genetically diverse TNBC cell lines and MCF-10A control cell lines revealed specific drug-induced metabolic alterations. Co-targeting kinases and metabolic targets may offer an approach to synthetic lethality with a reduced likelihood for the development of drug resistance. (1) (Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121) Citation Format: Alexander Beatty, Lauren Fink, Ulrike Rennefahrt, Alexander Strigun, Erik Peter, Hajo Schiewe, Regina Reszka, Jeffrey R. Peterson. Metabolite profiling reveals druggable metabolic distinctions between basal-like and non-basal-like triple-negative breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4333. doi:10.1158/1538-7445.AM2014-4333

Abstract 1112: Role of osteoprotegerin in inflammatory and invasive breast cancer

Abstract Inflammatory breast cancer (IBC) is a highly aggressive and angioinvasive form of breast cancer associated with a high incidence of early nodal and systemic metastasis. A proactive, complex and dynamic tumor microenvironment of IBC adds to the grim scenario of the disease by accumulating inflammatory and angiogenic growth factors and creating a niche for the growth and metastasis of tumor cells. The secretions from the primary inflammatory ductal carcinoma of the breast (SUM149PT), and highly invasive ductal carcinoma cells (SUM1315MO2) secrete unusually high levels of osteoprotegerin (OPG) as compared to those obtained from the primary human mammary epithelial cells (HMEC). OPG, which plays a pivotal role in bone remodeling by regulating osteoclast formation, is a multifaceted molecule, and has been found to be involved in multiple myeloma and prostate cancers. This prompted us to examine the role of OPG in the aggressive breast cancers, which has not been deciphered till date. For a better understanding of the OPG functions, apart from using adherent cells, we used in vitro ‘sphere culture’ as our working model. We studied the functional significance of OPG in aggressive breast cancer biology by utilizing well standardized techniques such as OPG staining (immunohistochemistry, immunofluorescence), OPG secretion (ELISA), angiogenesis (in vitro tube formation), proliferation (MTT), and cell cycle/aneuploidy analysis (PI staining). We demonstrated that (1) OPG secretion as well as protein level is upregulated in IBC (SUM149PT) and highly Invasive ductal carcinoma cells (SUM1315MO2), and their spheres compared to those of normal human mammary epithelial (HMEC); (2) OPG stained specifically in patient breast cancer tissue samples when compared to the uninvolved tissue from the same patient; (3) OPG rich secretions from IBC and highly invasive breast cancer were highly angiogenic; (4) OPG enriched culture medium induced proliferation of the control HMEC spheres; (5) the presence of OPG triggered the onset of aneuploidy in the control HMEC adherent cells as well as spheres. The functional studies showing the biological significance of OPG (i,e its effect on proliferation, angiogenesis, and aneuploidy) were confirmed using recombinant human OPG and OPG rich/depleted medium obtained from breast cancer cells. Overall our studies highlight the link between OPG and aneuploidy in aggressive breast cancer cells, and opens up avenues for new therapeutic targets for treating IBC. Citation Format: Sudeshna Goswami, NEELAM SHARMA-WALIA. Role of osteoprotegerin in inflammatory and invasive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1112. doi:10.1158/1538-7445.AM2014-1112

Abstract 242: Dietary PUFA's alter the visceral adipose secretome, which modulates mammary stem cell self-renewal

Abstract Rationale: Dietary PUFA's alter the expression and secretion of a variety of adipokines. Diets rich in omega-6 fatty acids shift the leptin:adiponectin ratio in favor of leptin, whereas diets rich in omega-3 fatty acids have the opposite effect. We tested the hypothesis that different ratios of adipose-derived factors differentially drive or inhibit normal mammary stem cell self-renewal. Methods: F344 rats were fed an obesogenic diet supplemented with arachidonic acid (AA) and eicosapentanoic acid (EPA) in either a 1:0 or 1:0.4 ratio. At 20 weeks, the animals were sacrificed and the visceral adipose explants were maintained in culture for up to 10 days. Conditioned media was collected at 48 hours. Adipokine concentrations were measured with the Proteome Profiler Adipokine Array and normalized to a leptin ELISA. Primary human mammary epithelial cells and the MCF10A breast cell line were expanded in a non-adherent sphere-forming assay as a measure of stem cell self-renewal. Cell suspensions were exposed to adipose conditioned media (1 mg/ml total protein concentration). After 10 days, spheres were counted and sphere size was measured using ImageJ. Results: Adipose explants were maintained in culture for up to 10 days with >99% viability. The secretome from the rats fed omega-6 PUFA's was enriched for leptin, resistin, serpin E1, and lipocalin-2. The omega-3 rich diet led to enrichment for adiponectin, pentraxin, and RBP4 in the adipose secretome. The adipose conditioned media from the omega-6 fed rats increased mammary stem cell self-renewal by 190%, whereas that from the rats fed omega-3 PUFA's attenuated this effect. Conclusion: The n3:n6 dietary ratio promotes a shift in the ratios of multiple secreted adipokines. These ratios have a differential effect on mammary stem cell self-renewal. A diet rich in omega-6 PUFA's leads to increased stem cell self-renewal, whereas a diet supplemented with EPA attenuated this effect. Further studies will clarify the role of specific adipokines on mammary stem cell self-renewal. Selected adipokine levels in adipose conditioned mediaOmega-3 rich dietOmega-6 rich dietn6:n3 dietary ratio1:0.41:0.0Adiponectin1864.7778.3Leptin181.2208.7Lipocalin-244.1323.1Pentraxin-277.00.8Pentraxin-319.29.3Pref-122.98.8Resistin737.01372.3Serpin E125.877.4RBP41136.0318.1 Citation Format: Ray Esper, Michael Dame, Djuric Zora, William Smith, Wicha Max, Dean Brenner. Dietary PUFA's alter the visceral adipose secretome, which modulates mammary stem cell self-renewal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 242. doi:10.1158/1538-7445.AM2014-242

Abstract 3037: Adipokines modulate human mammary stem cell self-renewal through mTOR

Abstract Rationale: Excess visceral adiposity is associated with an increased incidence of a variety of human ailments, including cancer. We tested the hypothesis that adipose-derived factors drive normal mammary stem cell self-renewal. Methods: Visceral adipose tissue explants from obese F344 rats and cultured mouse 3T3-L1 adipocytes were grown with serum-free media for 48 hours. Primary human mammary epithelial cells, MCF10A, SUM149 and MCF7 cells were exposed to rhleptin (500 ng/ml), or adipocyte conditioned media and expanded in a non-adherent sphere forming assay for 10 days. Spheres were counted and size was quantified in Image J. Adipokine concentrations were assayed with the Proteome Profiler Adipokine Array. Other cultures were pre-treated with mTOR inhibitors rapamycin (100 nM) or everolimus (100 nM) for 2 hours prior to exposure to rhleptin or adipocyte conditioned media for 2 hours. S6 protein phosphorylation was quantified from Western blots. Results: Data shown in Table 1 suggest that conditioned media from adipose tissue and from 3T3-L1 adipocytes increase human mammary stem cell self-renewal by 103% and 189%, respectively, whereas rhleptin increases stem cell self-renewal by 37%. Blockade of leptin activity with neutralizing antibodies or chimeric soluble receptors reduced the adipocyte conditioned media's stimulation of mammary epithelial cell self-renewal by 10-15%. Media conditioned by the mouse 3T3-L1 adipocytes or primary rat visceral adipose explants increased pS6 protein by 170% in MCF10A cells. mTOR inhibitors reduced rhleptin and adipocyte conditioned media stimulation of self-renewal in MCF10A and normal human mammary stem cells by 78-85%. Conclusion: Adipocyte secreted factors and rhleptin promote increased mammary epithelial cell self-renewal through a process involving the activation of mTOR. Leptin contributes to a small fraction of the activity in adipocyte conditioned media, and is sufficient but not required for mammary stem cell self-renewal. Change in stem cell self-renewalrhLeptin3T3-L1 Adipocyte Conditioned MediaRat Adipose Tissue Conditioned MediaBaseline # Spheres% Change (± SEM)P Value% Change (± SEM)P Value% Change (± SEM)P ValuePrimary human organoids19737 ± 7.005104 ± 20.006190 ± 26.001MCF-10A cells28834 ± 16.043320 ± 32.002389 ± 27.002MCF-7 cells57638 ± 9.007142 ± 15.001262 ± 40.006SUM149 cells95435 ± 6.09174 ± 20.012167 ± 21.003 Citation Format: Ray Esper, Dame Michael, Zora Djuric, William Smith, Max Wicha, Dean Brenner. Adipokines modulate human mammary stem cell self-renewal through mTOR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2014-3037

Abstract 3403: Circadian fluctuations in “housekeeping” gene expression measured by RNA-seq

Abstract A housekeeping gene is described as a gene that is stably expressed across tissues under normal conditions. These genes are extensively used in gene expression studies, particularly those involving quantitative real-time PCR, as a means to control for overall RNA abundance. Previous studies have reported extensive circadian variation at the transcriptional level, with as many as 10% of all expressed transcripts cycling over the course of a day. To determine the influence of circadian fluctuation on housekeeping gene stability, and the corresponding implications for estimates of gene expression, we evaluated time-course RNA-seq data from primary human mammary epithelial cells. These data demonstrate substantial time-dependent variability in the expression of several commonly used housekeeping genes. Of the 12 housekeeping genes evaluated, 8 had greater than 15% coefficient of variation (CV) with b-actin and GAPDH having CVs of 33.9% and 36.9%, respectively. In order to assess the impact of housekeeping gene variability on expression estimates of target genes which may use housekeeping transcript levels for the purposes of normalization, we stratified the 12 housekeeping genes into three categories: high variability, moderate variability, and low variability, and analyzed the effect of time-dependent fluctuations in several clinically-relevant targets, including ERBB2 and the genes used in a breast cancer panel to predict likelihood of recurrence. ERBB2, a proto-oncogene, is over-expressed in 15-30% of breast tumors and is associated with increased disease recurrence and a poor prognosis. Evaluating ERBB2 expression in our RNA-Seq data, which is normalized internally using both transcript length and total reads, we find that expression of ERBB2 is relatively stable over time (average FPKM = 23.28, SD= 2.91). However, when expression is normalized to genes in the high variability housekeeping set, expression fluctuates by as much as 50% above or below the mean. In contrast, normalization to genes in the low variability set provides an accurate depiction of expression across all time points. We also evaluated all genes in a commonly used clinical assay for predicting recurrence in newly diagnosed, early stage breast cancer patients. We find that a majority of the 16 genes included in the assay vary considerably over time when normalized to the 5 housekeeping genes identified by the assay developer, which could have important implications for result interpretation. Our RNA-Seq time-course data suggests that there is circadian variability in housekeeping gene expression and that it is important to consider circadian fluctuations during the experimental design phase in order to identify appropriate genes for RNA content normalization. Citation Format: Tracy L. Peters, Elizabeth J. Ferree, Yaou Sheng, Aaron E. Hoffman. Circadian fluctuations in “housekeeping” gene expression measured by RNA-seq. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3403. doi:10.1158/1538-7445.AM2014-3403

Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene.

quinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58-836 for CYP1A1, 336-5587 for CYP1B1 and 5943-40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251-13234 for CYP1A1, 4133-57078 for CYP1B1 and 4456-55887 for NQO1. There were 3.5 (mean, range 0.2-15.8) BPdG adducts/10(8) nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO1 protein, or NQO1 enzyme activity.

Aldehyde dehydrogenase and estrogen receptor define a hierarchy of cellular differentiation in the normal human mammary epithelium.

IntroductionAlthough estrogen and progesterone play a key role in normal mammary development and in breast cancer, the potential for proliferation and lineage differentiation as well as origin of cells that express the estrogen receptor (ER) in normal breast epithelium are not known. Some evidence suggests that normal human mammary stem/progenitor cells are ER–, but the identity of these cells and the cellular hierarchy of breast epithelium are still subjects of controversy. It is likely that elucidation of these aspects will bring insight into the cellular origin of breast cancer subtypes.MethodsWe used fluorescence-activated cell sorting of primary human mammary epithelial cells along with in vitro and in vivo functional assays to examine the hierarchic relation between cells with aldehyde dehydrogenase enzymatic activity (ALDH+ cells) and ER+ cells in the normal human breast epithelium. We assessed the proliferation and lineage differentiation potential of these cells in vitro and in vivo. A gene reporter assay was used to separate live ER+ and ER– mammary epithelial cells. With shRNA-mediated knockdown, we investigated the role of ALDH isoforms in the functionality of mammary epithelial progenitor cells.ResultsWe describe a cellular hierarchy in the normal human mammary gland in which ER–/ALDH+ cells with functional properties of stem/progenitor cells generate ER+ progenitor cells, which in turn give rise to cells of luminal lineage. We show that the ALDH1A1 isoform, through its function in the retinoic acid metabolism, affects the proliferation and/or early differentiation of stem/progenitor cells and is important for branching morphogenesis.ConclusionsThis study presents direct evidence that ER+ cells are generated by ER–/ALDH+ stem/progenitor cells. We also show that ER+ cells are able to generate cell progeny of luminal lineage in vitro and in vivo. Loss of ALDH1A1 function impairs this process, as well as branching morphogenesis and clonogenicity in suspension culture. This latter effect is reversed by treatment with retinoic acid.

Interactions between exosomes from breast cancer cells and primary mammary epithelial cells leads to generation of reactive oxygen species which induce DNA damage response, stabilization of p53 and autophagy in epithelial cells.

Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.