Breast Cancer-derived MCF-7 Cells Research Articles

The SP/NK1R system promotes the proliferation of breast cancer cells through NF-κB-mediated inflammatory responses.

Numerous molecules have been introduced to participate in the formation of breast cancer, the most common malignancy in women. Among them, neuropeptide substance P (SP) and its related receptor neurokinin-1 receptor (NK1R) have attracted unprecedented attention in tumorigenesis processes. In this study, we investigated the effect of the SP/NK1R pathway on the induction of oxidative stress in breast cancer and examine the therapeutic potential of NK1R inhibition in this malignancy. MCF-7 cells were treated with varying concentrations of SP and aprepitant, an FDA-approved NK1R antagonist, either as a single drug or in a combined modality. Resazurin assay was used to evaluate the anti-cancer ability of aprepitant. The alteration in the intracellular levels of reactive oxygen species (ROS) and gene expression were determined using ROS assay and the qRT-PCR analysis, respectively. The stimulation of the SP/NK1R axis in the MCF-7 cells was coupled with the accumulation of ROS as well as upregulation of NF-κB and its related pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and IL-6. In contrast, the suppression of NK1R by aprepitant halted the viability of MCF-7 cells, at least partly due to p53-mediated upregulation of p21. Moreover, aprepitant attenuated the oncogenic properties of SP by preventing the oxidative property of this neuropeptide. Overall, our results suggest that the SP/NK1R pathway might play a critical role in breast cancer pathogenesis, probably through inducing ROS/NF-κB-mediated inflammatory responses. Moreover, it seems that blockage of the axis has promising therapeutic value against breast cancer cells. Schematic representation proposed for the plausible mechanism by which the stimulation of the SP/NK1R might induce oxidative stress in breast cancer-derived MCF-7 cells. Once SP interacts with NK1R, this signaling axis could disturb the balance between the expression of p53 and NF-κB, an event that leads to the accumulation of ROS within MCF-7 cells. The produced ROS, in turn, elevates the expression of pro-inflammatory cytokines (TNF-α and IL-6) and downregulates the expression of p21. On the other hand, aprepitant, an antagonist of NK1R, could reduce the survival of proliferative capacity of MCF-7 cells by decreasing the intracellular levels of ROS and p53-mediated up-regulation of p21. Along with the effect on p53, aprepitant could also reduce the expression of NF-κB and its related pro-inflammatory cytokines.

Microdeletion at ESR1 Intron 6 (DEL_6_75504) Is a Susceptibility Factor for Cryptorchidism and Hypospadias.

We have previously reported that a specific "AGATC" haplotype in a > 34 kb tight linkage disequilibrium (LD) block within ESR1 is strongly associated with cryptorchidism and hypospadias in Japanese boys. However, a true susceptibility factor linked to the "AGATC" haplotype remains to be identified. We performed various molecular studies in hitherto unreported 230 Italian boys (80 with cryptorchidism and 150 with normal genitalia) and previously reported and newly recruited 415 Japanese boys (149 with cryptorchidism, 141 with hypospadias, and 125 with normal genitalia). We also performed ESR1 expression analyses using breast-cancer derived MCF-7 cells. Haplotype analysis revealed the LD block and positive association of the "AGATC" haplotype with cryptorchidism in Italian boys. Whole genome sequencing identified an identical 2,249 bp microdeletion (ΔESR1) generated by a microhomology-mediated replication error in both Japanese and Italian boys with the specific haplotype. ΔESR1 was found to be strongly associated with cryptorchidism and hypospadias by Cochran-Armitage trend test, and was revealed to show nearly absolute LD with the "AGATC" haplotype. ESR1 expression was upregulated in MCF-7 cells with a homozygous deletion encompassing ΔESR1 and those with a homozygous deletion involving a CTCF-binding site within ΔESR1. The results reveal that ΔESR1, which has been registered as "DEL_6_75504" in "gnomAD SVs v2.1", is the true susceptibility factor for cryptorchidism and hypospadias. It appears that ΔESR1 was produced in a single ancestral founder of modern humans and has been maintained within the genome of multiple ethnic groups by selection.

Development of Antibody-like Proteins Targeting the Oncogenic Ser/Thr Protein Phosphatase PPM1D

PPM1D, a protein Ser/Thr phosphatase, is overexpressed in various cancers and functions as an oncogenic protein by inactivating the p53 pathway. Therefore, molecules that bind PPM1D are expected to be useful anti-cancer agents. In this study, we constructed a phage display library based on the antibody-like small molecule protein adnectin and screened for PPM1D-specific binding molecules. We identified two adnectins, PMDB-1 and PMD-24, that bind PPM1D specific B-loop and PPM1D430 as targets, respectively. Specificity analyses of these recombinant proteins using other Ser/Thr protein phosphatases showed that these molecules bind to only PPM1D. Expression of PMDB-1 in breast cancer-derived MCF-7 cells overexpressing endogenous PPM1D stabilized p53, indicating that PMDB-1 functions as an inhibitor of PPM1D. Furthermore, MTT assay exhibited that MCF-7 cells expressing PMDB-1 showed inhibition of cell proliferation. These data suggest that the adnectin PMDB-1 identified in this study can be used as a lead compound for anti-cancer drugs targeting intracellular PPM1D.

GPR91 Receptor Mediates Protection against Doxorubicin-Induced Cardiotoxicity without Altering Its Anticancer Efficacy. An In Vitro Study on H9C2 Cardiomyoblasts and Breast Cancer-Derived MCF-7 Cells.

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte’s death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by 1H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis-epoxysuccinate (cis-ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis-ES either alone or in combination with DOX in H9C2 cells. A 1H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis-ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis-ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.

Alternatively Constructed Estrogen Receptor Alpha-Driven Super-Enhancers Result in Similar Gene Expression in Breast and Endometrial Cell Lines.

Super-enhancers (SEs) are clusters of highly active enhancers, regulating cell type-specific and disease-related genes, including oncogenes. The individual regulatory regions within SEs might be simultaneously bound by different transcription factors (TFs) and co-regulators, which together establish a chromatin environment conducting to effective transcription. While cells with distinct TF profiles can have different functions, how different cells control overlapping genetic programs remains a question. In this paper, we show that the construction of estrogen receptor alpha-driven SEs is tissue-specific, both collaborating TFs and the active SE components greatly differ between human breast cancer-derived MCF-7 and endometrial cancer-derived Ishikawa cells; nonetheless, SEs common to both cell lines have similar transcriptional outputs. These results delineate that despite the existence of a combinatorial code allowing alternative SE construction, a single master regulator might be able to determine the overall activity of SEs.

Metformin inhibits human breast cancer cell growth by promoting apoptosis via a ROS-independent pathway involving mitochondrial dysfunction: pivotal role of superoxide dismutase (SOD).

Despite a growing body of evidence indicating a potential efficacy of the anti-diabetic metformin as anti-cancer agent, the exact mechanism underlying this efficacy has remained largely unknown. Here, we aimed at assessing putative mechanisms associated with the ability of metformin to reduce the proliferation and migration of breast cancer cells. A battery of in vitro assays including MTT, colony formation, NBT and scratch wound healing assays were performed to assess the viability, proliferation, anti-oxidative potential and migration of breast cancer-derived MCF-7, MDA-MB-231 and T47D cells, respectively. Reactive oxygen species (ROS) assays along with fluorescence microscopy were used to assess apoptotic parameters. Quantification of SOD, Bcl-2, Bax, MMPs, miR-21 and miR-155 expression was performed using qRT-PCR. We found that metformin inhibited the growth, proliferation and clonogenic potential of the breast cancer-derived cells tested. ROS levels were found to be significantly reduced by metformin and, concomitantly, superoxide dismutase (SOD) isoforms were found to be upregulated. Mitochondrial dysfunction was observed in metformin treated cells, indicating apoptosis. In metastatic MDA-MB-231 cells, migration was found to be suppressed by metformin through deregulation of the matrix metalloproteinases MMP-2 and MMP-9. The oncogenic microRNAs miR-21 and miR-155 were found to be downregulated by metformin, which may be correlated with the suppression of cell proliferation and/or migration. Our data indicate that metformin may play a pivotal role in modulating the anti-oxidant system, including the SOD machinery, in breast cancer-derived cells. Our observations were validated by in silico analyses, indicating a close interaction between SOD and metformin. We also found that metformin may inhibit breast cancer-derived cell proliferation through apoptosis induction via the mitochondrial pathway. Finally, we found that metformin may modulate the pro-apoptotic Bax, anti-apoptotic Bcl-2, MMP-2, MMP-9, miR-21 and miR-155 expression levels. These findings may be instrumental for the clinical management and/or (targeted) treatment of breast cancer.

Leptin promotes the migration and invasion of breast cancer cells by upregulating ACAT2.

Previously, it has been shown that obesity may be considered as a risk factor for breast cancer in postmenopausal women. Leptin, a hormone whose level is elevated in obesity, has been suggested to be involved in the development of breast cancer, and univariate survival analyses have shown that over-expression of ACAT2, an enzyme that is involved in the production of cholesteryl esters, may be associated with a poor prognosis. Here, we aimed to investigate the effect of leptin on the proliferation, migration and invasion of breast cancer cells, as well as to elucidate its underlying mode of action. Gene expression changes in leptin treated breast cancer-derived MCF-7, T47D and BT474 cells were assessed using PCR array, qRT-PCR and Western blot analyses. The expression patterns of Ob-R (leptin receptor) and ACAT2 in breast cancer cells and primary breast cancer tissue samples were analyzed using immunofluorescence and immunohistochemistry, respectively. Leptin-induced proliferation of breast cancer cells was assessed using a CCK8 assay, and scratch wound and Transwell assays were used to assess breast cancer cell invasion and migration. We found that, among the genes tested, ACAT2 expression exhibited the most significant changes in the leptin treated cells. In addition, we found that inhibition of ACAT2 expression using pyripyropene A (PPPA) or siRNA-mediated gene silencing significantly decreased leptin-induced proliferation, migration and invasion of MCF-7 and T47D cells. Subsequent Western blot analyses strongly indicated that the PI3K/AKT/SREBP2 signaling pathway was involved in leptin-induced ACAT2 upregulation in both MCF-7 and T47D cells. Finally, through the analysis of primary breast cancer tissue samples we found that ACAT2 may affect cancer progression through activation ofthe Ob-R. Our data indicate that leptin may enhance the proliferation, migration and invasion of breast cancer cells via ACAT2 up-regulation through the PI3K/AKT/SREBP2 signaling pathway. Therefore, the leptin/ACAT2 axis may represent an attractive therapeutic target for breast cancer, particularly in postmenopausal and/or obese women.

TRIM28 interacts with EZH2 and SWI/SNF to activate genes that promote mammosphere formation.

Histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2) is generally associated with H3K27 methylation and gene silencing, as a member of the polycomb repressor 2 (PRC2) complex. Immunoprecipitation and mass spectrometry of the EZH2-protein interactome in estrogen receptor positive, breast cancer-derived MCF7 cells revealed EZH2 interactions with subunits of chromatin remodeler SWI/SNF complex and TRIM28, which formed a complex with EZH2 distinct from PRC2. Unexpectedly, transcriptome profiling showed that EZH2 primarily activates, rather than represses, transcription in MCF7 cells and with TRIM28 co-regulates a set of genes associated with stem cell maintenance and poor survival of breast cancer patients. TRIM28 depletion repressed EZH2 recruitment to chromatin and expression of this gene set, in parallel with decreased CD44hi/CD24lo mammosphere formation. Mammosphere formation, inhibited by EZH2 depletion, was rescued by ectopic expression of EZH2 but not by TRIM28 expression or by EZH2 mutated at the region (pre-SET domain) of TRIM28 interaction. These results support PRC2-independent functions of EZH2 and TRIM28 in activation of gene expression that promotes mammary stem cell enrichment and maintenance.

PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells.

Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.

11-Ketotestosterone Is a Major Androgen Produced in Human Gonads

11-ketotestosterone (11-KT) is a novel class of active androgen. However, the detail of its synthesis remains unknown for humans. The objective of this study was to clarify the production and properties of 11-KT in human. Design, Participants, and Methods: Expression of cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 (key enzymes involved in the synthesis of 11-KT) were investigated in human gonads. The production of 11-KT was investigated in Leydig cells. Plasma concentrations of testosterone and 11-KT were measured in 10 women and 10 men of reproductive age. Investigation of its properties was performed using breast cancer-derived MCF-7 cells. Cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 were detected in Leydig cells and theca cells. Leydig cells produced 11-KT, and relatively high levels of plasma 11-KT were measured in both men and women. There was no sexual dimorphism in the plasma levels of 11-KT, even though testosterone levels were more than 20 times higher in men than in women. It is noteworthy that the levels of testosterone and 11-KT were similar in women. In a luciferase reporter system, 11-KT activated human androgen receptor-mediated transactivation. Conversely, 11-KT did not activate estrogen receptor-mediated transactivation in aromatase-expressed MCF-7 cells, whereas testosterone did following conversion to estrogen. 11-KT did not affect the estrogen/estrogen receptor -mediated cell proliferation of MCF-7 cells. Furthermore, it significantly inhibited cell proliferation when androgen receptor was transfected into MCF-7 cells. The current study indicates that 11-KT is produced in the gonads and represents a major androgen in human. It can potentially serve as a nonaromatizable androgen.

O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.

Abstract 746: Functional crosstalk between the p53 and NF-kB transcription factors.

Abstract The tumor suppressor p53 and NF-kB are sequence-specific transcription factors (TFs) playing crucial roles in controlling cell proliferation and cell survival with an undeniable impact on cancer progression. In response to cellular stresses they induce the expression of a large variety of genes. Synergistic or antagonistic interactions between the p53 and NF-kB proteins may modulate the expression of common targets and potentially lead to a modified cellular response. To specifically investigate cell-autonomous, cooperative interactions between NF-kB and p53 on gene expression changes, we performed a genome-wide transcriptome analysis in the breast-cancer derived MCF-7 cells following single or combinatorial treatments with the chemotherapeutic drug doxorubicin (DXR) and the NF-kB inducer TNF-alpha (TNF). The microarray results revealed 720 upregulated (log2FC>1.5) and 926 repressed genes for the DXR+TNF treatment (log2FC 0.1} and 188 repressed genes {log2[FCDXR+TNF] - (log2[FCDXR] + log2[FCTNF]) < -0.1}. Notably, gene ontology analysis applied to these gene groups indicated an apparent enrichment for epithelial mesenchymal transition and cell migration functions. We focused the validation experiments on 15 up-regulated genes to investigate the direct involvement of p53 and NF-kB on their expression using RT-qPCR assays coupled to p53 activation by DXR, 5-fluorouracil or Nutlin, p53 RNAi and/or NF-kB chemical inhibition. In silico analysis of the potential target genes’ promoter sequence identified both p53 and NF-kB responsive regions where cis-mediated interactions of these TFs may occur, a result that has been followed up by mining of available ChIP-seq data. Among the 15 chosen targets, we uncovered five (LAMP3, ETV7, UNC5B, NTN1 and PLK3) that were induced synergistically after DXR + TNF treatment in MCF-7 breast cancer cells. Over-expression of LAMP3 has already been associated with an increased metastatic potential for particular cancers, and ETV7 can act as a regulator of cell proliferation. To study the migratory phenotype associated with the DXR+TNF combinatorial treatment, we performed migration assays using three different approaches: trans-well migration assay, Xcelligence Migration Assay and wound healing. The results consistently showed an increase in migration potential upon DXR+TNF compared to mock or to single treatments. We propose that the functional crosstalk between the p53 and NF-kB TFs can lead to the activation of specific gene expression programs that may impact on cancer phenotypes. Given that most tumors carry an activated NF-kB pathway, and a significant proportion of breast cancers, particularly the luminal subtype, maintains wild type p53, the uncovered crosstalk could potentially modify the efficiency of cancer therapy. Citation Format: Alessandra Bisio, Judit Zámborszky, Sara Zaccara, Mattia Lion, Toma Tebaldi, Yari Ciribilli, Alberto Inga. Functional crosstalk between the p53 and NF-kB transcription factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2013-746

Abstract 4199: CDKN2A/p16INK4a 5′UTR variants in melanoma predisposition: Lost in translation, somewhere

Abstract The CDKN2A gene is the most common high penetrance susceptibility gene identified to date in melanoma families. While functional tests for determining the pathogenicity of missense germline mutations in the CDKN2A coding region have been developed, rare polymorphisms or sequence variants at the CDKN2A/ p16INK4a 5′UTR, encountered during routine screening, are usually defined as variants with unknown significance after determining their frequency in control population and the cosegregation analysis in the family, when possible. We recently developed reporter assays to study a panel of p16INK4a 5′UTR variants identified as heterozygous changes in patients from a hospital-based series of melanoma cases (c.-21C>T; c.-25C>T&c.-180G>A; c.-56G>T; c.-67G>C). Monocistronic as well as bicistronic luciferase-based reporter vectors were developed and used to test wild type and variant p16INK4a 5′UTR activity upon transient transfection in melanoma-derived cells (WM266-4, G361 and SK-Mel-5) and in the breast cancer-derived MCF7 cells. Results revealed that the c.-21C>T variant had a strong negative impact on the reporter activity, similar to that of the known melanoma-predisposing mutation c.-34G>T, included as a control. The variants at –56 and at –25&-180 exhibited a milder impact, while results with c.-67G>C were dependent on the type of reporter vector. Quantification of the luciferase mRNA conducted in parallel with the luciferase assay indicated that the impact of the variants was mainly post-transcriptional. We also applied a polysomal profiling technique to measure allelic imbalance starting from heterozygous patient-derived cell lines and found that the c.-21C>T variant but also c.-56T>G and c.-67G>C exhibited lower association with the polysomes suggestive of reduced mRNA translation efficiency. A panel of eleven additional germline variants in the 5′UTR of p16INK4a is being investigated. In particular we are focusing on the functional interactions between wild type and variant p16INK4a 5′- and 3′-UTR sequences and on the impact the variants can have on the targeting of the p16INK4a mRNA by microRNAs (including mir-346, -636, -639, -935) or RNA binding proteins (including YB-1). 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 4199. doi:1538-7445.AM2012-4199

Meta-analysis of estrogen response in MCF-7 distinguishes early target genes involved in signaling and cell proliferation from later target genes involved in cell cycle and DNA repair

BackgroundMany studies have been published outlining the global effects of 17β-estradiol (E2) on gene expression in human epithelial breast cancer derived MCF-7 cells. These studies show large variation in results, reporting between ~100 and ~1500 genes regulated by E2, with poor overlap.ResultsWe performed a meta-analysis of these expression studies, using the Rank product method to obtain a more accurate and stable list of the differentially expressed genes, and of pathways regulated by E2. We analyzed 9 time-series data sets, concentrating on response at 3-4 hrs (early) and at 24 hrs (late). We found >1000 statistically significant probe sets after correction for multiple testing at 3-4 hrs, and >2000 significant probe sets at 24 hrs. Differentially expressed genes were examined by pathway analysis. This revealed 15 early response pathways, mostly related to cell signaling and proliferation, and 20 late response pathways, mostly related to breast cancer, cell division, DNA repair and recombination.ConclusionsOur results confirm that meta-analysis identified more differentially expressed genes than the individual studies, and that these genes act together in networks. These results provide new insight into E2 regulated mechanisms, especially in the context of breast cancer.

Uptake transporter organic anion transporting polypeptide 1B3 contributes to the growth of estrogen-dependent breast cancer

Estrone-3-sulfate is one of the most abundant estrogen precursors in postmenopausal women. We previously showed that estrone-3-sulfate transporters are present in human breast cancer-derived MCF-7 cells (J. Pharmacol. Exp. Ther. 311 (2004) 1032–1037) and that inhibition of estrone-3-sulfate uptake resulted in the suppression of cell growth (Pharm. Res. 22 (2005) 1634–1641); therefore, estrone-3-sulfate transporter should be a novel target for therapy of hormone-dependent breast cancers. The purpose of the present study is to identify the transporter(s) responsible for the uptake of estrone-3-sulfate in breast cancer cells. We obtained two subclones of MCF-7 cells with different estrone-3-sulfate uptake activities and searched for differentially expressed transporter genes by means of DNA microarray analysis. Among several candidate transporters identified, OATP1B3 was further evaluated, since the uptake characteristics of estrone-3-sulfate by MCF-7 cells seemed consistent with the transport properties of OATP1B3. The contribution of OATP1B3 to estrone-3-sulfate uptake by MCF-7 cells was examined by the relative activity factor (RAF) method, and was calculated to amount to 6%. This result suggests that OATP1B3 is one of the transporters contributing to the supply of the estrogen precursor estrone-3-sulfate to estrogen-dependent breast cancer cells.

The Coordinated P53 and Estrogen Receptor Cis-Regulation at an FLT1 Promoter SNP Is Specific to Genotoxic Stress and Estrogenic Compound

BackgroundRecently, we established that a C>T single nucleotide polymorphism (SNP) in the promoter of the VEGF receptor FLT1 gene generates a ½ site p53 response element (RE-T) that results in p53 responsiveness of the promoter. The transcriptional control required an estrogen receptor (ER) ½ site response element (ERE1) 225 nt upstream to the RE-T.Methodology/Principal FindingsHere we report the identification of a second ER ½ site (ERE2) located 145 bp downstream of the RE-T and establish that both EREs can impact p53-mediated transactivation of FLT1-T in a manner that is cell type and ER level dependent. Gene reporter assays and ChIP experiments conducted in the breast cancer-derived MCF7 cells revealed that the ERE2 site was sufficient for p53-mediated ERα recruitment and transactivation of the FLT1-T promoter/reporter construct. Surprisingly, unlike the case for other p53 target promoters, p53-mediated transactivation of FLT1-T constructs or expression of the endogenous FLT1 gene, as well as binding of p53 and ER at the promoter constructs, was inducible by doxorubicin but not by 5-fluorouracil. Furthermore, ER activity at FLT1-T was differentially affected by ER ligands, compared to a control TFF1/pS2 ER target promoter. The p53-related transcription factors (TFs) p73 and p63 had no effect on FLT1 transactivation.Conclusions/SignificanceWe establish a new dimension to the p53 master regulatory network where p53-mediated transcription from a ½ site RE can be determined by ER binding at one or more cis-acting EREs in manner that is dependent on level of ER protein, the type of ER ligand and the specific p53-inducing agent.

Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Functional analysis of CDKN2A/p16INK4a 5′-UTR variants predisposing to melanoma

Germline CDKN2A mutations are observed in 20-50% of melanoma-prone families. We identified melanoma patients that were heterozygous for non-coding germline variants in the 5'-UTR of CDKN2A (c.-21C > T; c.-25C > T&c.-180G > A; c.-56G > T; c.-67G > C) and examined their impact on the p16(INK4a) 5'-UTR activity using two luciferase-based reporter vectors that differ in basal transcription level and that were transfected into the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells. The wild-type 5'-UTR sequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G > T), was included as controls. Results revealed that the variants at -21 and -34 severely reduced the reporter activity. The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C were dependent on the plasmid type. Quantification of the luciferase mRNA indicated that the effects of the variants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmed that c.-21C > T and c.-34G > T had a severe negative impact in both cell lines. We also applied a polysomal profiling technique to samples heterozygous for the 5'-UTR variants, including patient-derived lymphoblasts. Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > C variants also reduced mRNA translation efficiency. Overall, our results suggest that the c.-21C > T sequence variant is a melanoma-predisposing mutation. The c.-25C > T&c.-180G > A and particularly the c.-56G > T variants showed a range of intermediate functional defects in the different assays, and were not observed in the control population. We propose that these variants should be considered as potential mutations.

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.

Effect of oil-in-water lipid emulsions prepared with fish oil or soybean oil on the growth of MCF-7 cells and HepG2 cells

The growth of human breast cancer-derived MCF-7 cells was affected by oil-in-water lipid emulsions prepared with fish oil (FO) rich in n-3 fatty acids (FAs) and egg-yolk phosphatides (EYP) (FO-emulsions), but not by lipid emulsions prepared with soybean oil (SO) and EYP (SO-emulsions). On the other hand, the growth of human hepatocarcinoma HepG2 cells was affected by neither SO-emulsions nor FO-emulsions. The growth inhibition of MCF-7 cells in the presence of FO-emulsions was not affected by trolox, but was inhibited by alpha-lipoic acid, and was even potentiated by ebselen, which works as an antioxidant as well as a lipoxygenase inhibitor. Since prostaglandin E(3), generated from n-3 FAs by cyclooxygenases, has a suppressive effect on tumour cell growth, and increases when lipoxygenases are inhibited, these findings suggest that lipid emulsions incorporating triglycerides of n-3 FAs might be effective in suppressing the growth of MCF-7 cells, possibly via oxidative stress and through eicosanoid production with anti-proliferating activity against cancer cells.