The organochlorine o,p'-DDT plays a role in coactivator-mediated MAPK crosstalk in mcf-7 breast cancer cells.
OBJECTIVES: We and others have shown that DDT activates cell-signaling cascades, culminating in the activation of estrogen receptor-dependent and -independent gene expression. Here, we identify a mechanism by which DDT alters cellular signaling and gene expression, independent of the estrogen receptor.
METHODS: We performed quantitative polymerase chain reaction array analysis of gene expression in MCF-7 breast cancer cells using either estradiol ([E.sup.2]) or o,p'-DDT to identify distinct cellular gene expression responses. To elucidate the mechanisms by which DDT regulates cell signaling, we used molecular and pharmacological techniques.
RESULTS: [E.sup.2] and DDT treatment both altered the expression of many of the genes assayed, but up-regulation of vascular endothelial growth factor A (VEGFA) was observed only after DDT treatment, and this increase was not affected by the pure estrogen receptor [alpha] antagonist ICI 182780. Furthermore, DDT increased activation of the HIF-1 response element (HRE), a known enhancer of the VEGFA gene. This DDT-mediated increase in HRE activity was augmented by the coactivator CBP (CREB-binding protein) and was dependent on the p38 pathway.
CONCLUSIONS: DDT up-regulated the expression of several genes in MCF-7 breast cancer cells that were not altered by treatment with [E.sub.2], including VEGFA. We propose that this DDT-initiated, ER-independent stimulation of gene expression is due to DDT's ability to initiate crosstalk between MAPK (mitogen-activated protein kinase) signaling pathways and transcriptional coactivators.
KEY WORDS: breast cancer, CBP, coactivator, crosstalk, DDT, dichlorodiphenyltrichloroethane, endocrine-disrupting chemical, HIF-1[alpha], MAPK, organochlorine, p38 kinase, vascular endothelial growth factor. Environ Health Perspect 120:1291-1296 (2012). http://dx.doi.org/10.1289/ehp.1104296 [Online 18 May 2012]
Endocrine-disrupting chemicals (EDCs), such as polychlorinated biphenyls (PCBs), phthalates, phenolics, and other organochlorines, can affect the endocrine system by altering steroid receptor function, resulting in apparent estrogen-like activity and possible reproductive dysfunction (McLachlan 2001; McLachlan et al. 2006; Tilghman et al. 2010). The estrogen-like activity of the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) and its congeners was first shown > 50 years ago (Tullner 1961), yet the mechanism of action of DDT as a hormone remains an enigma (see McLachlan 2001 for review). Although its use has been restricted to use for mosquito control in developing countries with tropical climates, DDT remains active in the environment worldwide and bioaccumulates in the fat stores of animals and humans because of its lipophilic nature and chemical stability (Kelly et al. 2004). The DDT metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) continues to be detected in human serum with a high frequency at concentrations up to and exceeding 1,000 [micro]g/kg lipids (Cole et al. 2006). DDT and its metabolites have been associated with human diseases including type 2 diabetes (Codru et al. 2007; Rignell-Hydbom et al. 2007), testicular tumors (McGlynn et al. 2008), pancreatic cancer (Porta et al. 2008), endometrial cancer (Hardell et al. 2004), and breast cancer (Cocco et al. 2000; Rier and Foster 2002; Safe and Zacharewski 1997; Sasco 2003; Wolff et al. 1993), but mechanisms to explain these associations remain elusive.
DDT mimics the natural hormone estradiol ([E.sub.2]) and can bind to estrogen receptor [alpha] (ER[alpha]) (Ahlborg et al. 1995; Gulledge et al. 2001; Klotz et al. 1996; Kuiper et al. 1998). In addition, DDT exerts cellular effects independently of ER[alpha]. For example, we previously demonstrated that DDT and its active metabolites are capable of inducing AP-1 mediated transcription, in both ER[alpha]-positive and ER[alpha]-negative cells (Frigo et al. 2002). We have also shown that DDT activates transcription at multiple DNA response elements through p38-mediated phosphorylation and activation of the coactivators p300 (Bratton et al. 2009) and GRIP1 (Frigo et al. 2006). Using endometrial cells, we have shown that DDT can activate both the p38 and ERK1/2 (extracellular signal-regulated kinases 1/2) pathways, again independently of the ER (Frigo et al. 2004). Therefore, we hypothesized that treatment of MCF-7 breast cancer cells with DDT would result in an altered gene expression profile compared with cells treated with [E.sub.2], and that this altered phenotype could provide clues regarding the molecular mechanism of DDT's distinct effects on cell physiology.
Materials and Methods
Chemicals. We purchased o,p'-DDT, p,p'-DDT, o,p'- and p,p'-dichlorodiphenyl-dichloroethane (DDD), p,p'-dichlorodiphenyl acetic acid (DDA), and o,p'- and p,p'-DDE from AccuStandard (New Haven, CT); 17[beta]-estradiol ([E.sub.2]); all protease inhibitors; and porcine insulin from Sigma Chemical Company (St. Louis, MO); UO126 (an ERK inhibitor) from Promega (Madison, WI); SP600125 (a JNK inhibitor) from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA); and SB203580 (a p38[alpha]/[beta] inhibitor) from EMD Biosciences (Billerica, MA). Dulbecco's modified Eagle medium (DMEM), phenol-red free DMEM, fetal bovine serum (FBS), BME (basal medium Eagle) amino acids, MEM (minimum essential medium) amino acids, l-glutamine, penicillin, streptomycin, and sodium pyruvate were obtained from GibcoBRL (Gaitherburg, MD). We purchased charcoal-stripped FBS from HyClone (Logan, UT), Effectene from QIAGEN (Valencia, CA), and MPER (mammalian protein extraction reagent) from Pierce (Thermo Scientific, Rockford, IL).
Plasmids. Hypoxia-inducible factor 1 (HIF-1)-luciferase (HRE-luc) was donated by B.S. Beckman (Tulane University); CMV-GAL4 (negative control) was a gift from E. Flemington (Tulane University); and GAL4-CBP was donated by R. Goodman (Oregon Health Sciences University, Portland, OR). We purchased pFR-Luc [GAL4-luciferase (GAL4-luc) reporter] and pFC-MEK1 [CA-MKK1; constitutively active MAPK kinase (MKK) 1] from Stratagene (La Jolla, CA), and pcDNA3.1 from Invitrogen (Carlsbad, CA). pcDNA3-CA-MKK5 [CA-MKK5; constitutively active MAPK kinase (MKK) 5] and dominant-negative (DN) ERK2 (DN-ERK2) were gifts from J.-D. Lee (Scripps Research Institute, La Jolla, CA). pcDNA3-CA-MKK6 [CA-MKK6; constitutively active MAPK kinase (MKK) 6] and pcDNA3-CA-MKK7 [CA-MKK7; constitutively active MAPK kinase (MKK) 7] were gifts from J. Han (Scripps Research Institute). JNK1 and p38[alpha] MAPK DN mutants (DN-JNK1, DN-p38[alpha]) were provided by R. Davis (University of Massachusetts Medical School, Worcester, MA). GST (glutathione S-transferase) expression vector was purchased from Amersham Biosciences (Piscataway, NJ). pGEX-CBP1 (aa: 390-790) and pGEX-CBP3 (aa: 1990-2441) were gifts from R.G. Roeder (Rockefeller University, New York, NY). pGEX-CBP2 (aa:1680-1892) was generated by polymerase chain reaction (PCR) using HA-CBP (histone acetyltransferase-CREB-binding protein) full length (gift from R. Goodman, Oregon Health Sciences University) as a template. Resultant DNA was subcloned into the EcoR1/Sal1 site of pGEX-5X-1 (Amersham Pharmacia Biotech, Arlington Heights, IL).
Cell culture. ER-positive MCF-7 human breast carcinoma cells (Burow et al. 2000) and ER-negative human embryonic kidney (HEK) 293 cells (Kuiper et al. 1998) were maintained as previously described (Bratton et al. 2009; Rhodes et al. 2010). MCF-7 cells were grown for 48 hr in phenol red-free DMEM supplemented with 5% charcoalstripped FBS and supplements but without insulin (5% charcoal-stripped DMEM), as previously described (Burow et al. 1999). Fulvestrant resistant MCF-7F cells were grown as previously described (Fan et al. 2006).
Quantitative PCR (qPCR) array analysis. MCF-7 cells were seeded in 6-well plates, and drug treatment was initiated after 24 hr. Cells were lysed 48 hr later, and total RNA was harvested using the RNeasy Mini Kit (QIAGEN). We used the R[T.sup.2] First Strand cDNA kit (SABiosciences, Frederick, MD) to perform cDNA synthesis from total RNA according to the manufacturer's protocol. qPCR was then performed on a BioRad IQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using a 96-well R[T.sup.2] Profiler PCR Array (Breast Cancer and Estrogen Receptor Signaling PCR Array; PAHS-005; QIAGEN). Generation and analysis of cycle threshold (Ct) values were performed according to manufacturer's instructions for the array. Three independent arrays were run for each treatment; values are presented as fold change relative to several housekeeping genes (18S rRNA, HPRT1, RPL13A, GAPDH, and ACTB). qPCR of VEGFA mRNA was performed on samples of MCF-7 cells treated with either vehicle (i.e., DMSO), DDT, or DDT plus ICI 182780 (ICI) as previously described (Bratton et al. 2009). qPCR arrays of MCF-7F cells were run on samples isolated from three independent experiments using triplicate Breast Cancer and Estrogen Receptor Signaling PCR Arrays as previously described (Tilghman et al. 2012).
Luciferase assays. MCF-7 and HEK 293 cells were transfected as previously described (Bratton et al. 2010). A GAL4-luc reporter, along with an empty expression vector or a GAL4-CBP fusion, was transfected into HEK 293 cells. The cells were then treated with vehicle or different MAPK inhibitors for 1 hr, followed by addition of vehicle or 50 [micro]M o,p'-DDT for 18 hr. Luciferase activity was measured in 100 [micro]L of the lysed sample using a Berthold luminometer (Titertek Instruments Inc., Huntsville, AL) and 100 [micro]L Bright Glo luciferase assay reagent (Promega, Madison, WI).
GST-fusion protein purification and in vitro kinase assay. The GST and GST-CBP fusion proteins were generated as previously described (Bratton et al. 2009). Roughly, 3-5 [micro]g of eluted purified GST-fusion protein or 200 ng of purified mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (Upstate Biotechnology, Lake Placid, NY) was phosphorylated by activated p38[alpha] as previously described (Bratton et al. 2009). Samples were analyzed by 4-12% SDS-PAGE (Invitrogen), stained with coomassie blue to monitor expression, and subjected to autoradiography as described by Bratton et al. (2010).
DDT- and [E.sub.2]-induced gene expression. We used a qPCR-based human breast cancer pathway array to compare gene expression in MCF-7 breast cancer cells after treatment with vehicle, 1 nM [E.sub.2], or 10 [micro]M o,p'-DDT for 18 hr. [E.sub.2] and DDT both significantly altered the expression of 13 genes known to be involved in breast cancer signaling. Interestingly, several genes were differentially up-regulated by DDT compared with [E.sub.2], including Fas ligand (FASLG), integrin alpha 6 (ITGA6), and vascular endothelial growth factor A [VEGFA; an important factor in cellular angiogenic control mechanisms and differentiation (Zhang et al. 1995)] [Table 1; see also Supplemental Material, Table S2 (http://dx.doi.org/10.1289/ehp.1104296)]. To address whether the effect of DDT on VEGFA expression in MCF-7 cells is dependent on [E.sub.2] or ER[alpha], we assayed VEGFA expression by qPCR in MCF-7 cells incubated in the presence of the ER[alpha] inhibitor ICI. Because ICI had no effect on the DDT-mediated increase in VEGFA expression in MCF-7 cells, we concluded that the effect of DDT was ER[alpha] independent (Figure 1A). Consistent with this hypothesis, we observed a statistically significant increase in VEGFA expression in ER[alpha]-negative MCF-7F cells in response to DDT (Figure 1B; see also Supplemental Material, Table S1).
Table 1. qPCR array analysis of MCF-7 cells. Gene Description o,p'-DDT p-Value [E.sub.2] (DDT/veh) Bcl-2 B-cell 3.00 0.0011 2.65 CLL/lymphoma 2 CCNA1 Cyclin A1 1.97 0.0444 1.94 CTSD Cathepsin D 2.96 0.0228 2.64 FASLG Fas ligand 2.61 0.0156 0.98 FOSL1 FOS-like antigen 2.81 0.0002 2.72 1 HMGB1 High-mobility 1.70 0.0172 1.44 group box 1 IL6R Interleukin 6 2.11 0.0161 1.7 receptor ITGA6 Integrin, alpha 2.28 0.0376 1.47 6 NGFR Nerve growth 1.49 0.0486 1.33 factor receptor NME1 Non-metastatic 2.46 0.0006 2.96 cells 1 PGR Progesterone 229 0.0000 152 receptor SCGB1D2 Secretoglobin, 6.88 0.0035 2.43 family 1D, member 2 SERPINA3 Serpin peptidase 2.72 0.0139 2.62 inhibitor, clade a, member 3 SERPINB5 Serpin peptidase 4.70 0.0004 4.70 inhibitor, clade b, member 5 SLC7A5 Solute carrier 13.7 0.0002 11.62 family 7, member 5 STC2 Stanniocalcin 2 5.46 0.0001 3.94 TFF1 Trefoil factor 1 23.3 0.0000 28.93 VEGFA Vascular 1.97 0.0474 1.63 endothelial growth factor A Gene p-Value ([E.sub.2]/veh) Bcl-2 0.0006 CCNA1 0.0057 CTSD 0.0431 FASLG 0.9500 FOSL1 0.0000 HMGB1 0.0013 IL6R 0.0548 ITGA6 0.1152 NGFR 0.2321 NME1 0.0000 PGR 0.0000 SCGB1D2 0.0511 SERPINA3 0.0042 SERPINB5 0.0004 SLC7A5 0.0003 STC2 0.0000 TFF1 0.0000 VEGFA 0.1023 veh, vehicle. Significantly up-regulated genes are shown with their corresponding p-values (n = 3 separate arrays). Supplemental Material, Table S1. qPCR array of MCF-7F cells Gene Description Fold p value symbol Regulation (DDT/Veh) AR Androgen receptor -9.0317 0.440419 BAD BCL2-associated agonist of cell -15.366 0.208434 death BAG1 BCL2-associated athanogene -15.7251 0.253679 BCL2 B-cell CLL/lymphoma 2 -1.3582 0.03829 BCL2L2 BCL2-like 2 1.1961 0.052241 C3 Complement component 3 -1.1554 0.782079 CCNA1 Cyclin A1 1.0413 0.542735 CCNA2 Cyclin A2 -1.4557 0.011769 CCND1 Cyclin D1 -1.0058 0.972787 CCNE1 Cyclin E1 -1.21 0.23968 CD44 CD44 molecule (Indian blood 1.5422 0.002891 group) CDH1 Cadherin 1, type 1, E-cadherin -1.21 0.205846 (epithelial) CDKN1A Cyclin-dependent kinase inhibitor -2.5344 0.90007 1A (p21, Cip1) CDKN1B Cyclin-dependent kinase inhibitor -1.4557 0.00237 1B (p27, Kip1) CDKN2A Cyclin-dependent kinase inhibitor -1.5245 0.109123 2A (melanoma, p16, inhibits CDK4) CLDN7 Claudin 7 -1.5966 0.002555 CLU Clusterin -2.9113 0.021252 COL6A1 Collagen, type VI, alpha 1 1.0175 0.806653 CTNNB1 Catenin (cadherin-associated -1.5601 0.004348 protein), beta 1, 88kDa CTSB Cathepsin B -1.078 0.437803 CTSD Cathepsin D -1.21 0.063807 CYP19A1 Cytochrome P450, family 19, 1.2241 0.862951 subfamily A, polypeptide 1 DLC1 Deleted in liver cancer 1 -2.8448 0.010782 EGFR Epidermal growth factor receptor -1.5245 0.004899 ERBB2 V-erb-b2 erythroblastic leukemia -9.9062 0.070662 viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) ESR1 Estrogen receptor 1 -9.0317 0.122889 ESR2 Estrogen receptor 2 (ER beta) -1.2672 0.401208 FAS Fas (TNF receptor superfamily, -1.129 0.09034 member 6) FASLG Fas ligand (TNF superfamily, -1.6339 0.066675 member 6) FGF1 Fibroblast growth factor1 1.4061 0.291349 (acidic) FLRT1 Fibronectin leucine rich -1.0058 0.986079 transmembrane protein 1 FOSL1 FOS-like antigen 1 1.4726 0.009182 GABRP Gamma-aminobutyric acid (GABA) A -3.1932 0.003233 receptor, pi GATA3 GATA binding protein 3 -1.4557 0.00488 GNAS GNAS complex locus -3.1932 0.002396 GSN Gelsolin -1.078 0.416832 HMGB1 High mobility group box 1 2.7479 0.7239 HSPB1 Heat shock 27kDa protein 1 -68.9909 0.416565 ID2 Inhibitor of DNA binding 2, -1.3272 0.077236 dominant negative helix-loop-helix protein IGFBP2 Insulin-like growth factor -1.7921 0.016659 binding protein 2, 36kDa IL2RA Interleukin 2 receptor, alpha 1.116 0.50141 IL6 Interleukin 6 (interferon, beta 1.374 0.053573 2) IL6R Interleukin 6 receptor 1.4726 0.041435 IL6ST Interleukin 6 signal transducer -1.129 0.097104 (gp130, oncostatin M receptor) ITGA6 Integrin, alpha 6 1.4061 0.000454 ITGB4 Integrin, beta 4 -1.834 0.004426 JUN Jun proto-oncogene -1.4557 0.056947 KIT V-kit Hardy-Zuckerman 4 feline -1.6339 0.066675 sarcoma viral oncogene homolog KLF5 Kruppel-like factor 5 1.0413 0.840779 (intestinal) KLK5 Kallikrein-related peptidase 5 -1.6339 0.002613 KRT18 Keratin 18 -1.6339 0.000497 KRT19 Keratin 19 -1.1554 0.082243 MAP2K7 Mitogen-activated protein kinase -1.1824 0.380596 kinase 7 MKI67 Antigen identified by monoclonal -39.6248 0.06399 antibody Ki- 67 MT3 Metallothionein 3 -1.6339 0.066675 MUC1 Mucin 1, cell surface associated -1.4897 0.014666 NFYB Nuclear transcription factor Y, -1.3272 0.000844 beta NGF Nerve growth factor (beta -1.6339 0.066675 polypeptide) NGFR Nerve growth factor receptor -2.7798 0.000196 NME1 Non-metastatic cells 1, protein 1.0905 0.235201 (NM23A) expressed in PAPPA Pregnancy-associated plasma -1.6339 0.066675 protein A, pappalysin 1 PGR Progesterone receptor -1.6339 0.066675 PLAU Plasminogen activator, urokinase -1.129 0.425072 PTEN Phosphatase and tensin homolog -1.0534 0.264509 PTGS2 Prostaglandin-endoperoxide -1.6339 0.066675 synthase 2 (prostaglandin G/H synthase and cyclooxygenase) RAC2 Ras-related C3 botulinum toxin -1.6339 0.066675 substrate 2 (rho family, small GTP binding protein Rac2) RPL27 Ribosomal protein L27 1.2527 0.067928 SCGB1D2 Secretoglobin, family 1D, member -1.078 0.618955 2 SCGB2A1 Secretoglobin, family 2A, member 1.6915 0.113076 1 SERPINA3 Serpin peptidase inhibitor, clade 1.3119 0.061166 A (alpha-1 antiproteinase, antitrypsin), member 3 SERPINB5 Serpin peptidase inhibitor, clade 1.2241 0.157399 B (ovalbumin), member 5 SERPINE1 Serpin peptidase inhibitor, clade 1.2819 0.040936 E (nexin, plasminogen activator inhibitor type 1), member 1 SLC7A5 Solute carrier family 7 (amino 1.2819 0.191025 acid transporter light chain, L system), member 5 SPRR1B Small proline-rich protein 1B -4.9531 0.730958 STC2 Stanniocalcin 2 1.8129 0.001576 TFF1 Trefoil factor 1 1.4389 0.010301 TGFA Transforming growth factor, -1.1554 0.340467 alpha THBS1 Thrombospondin 1 -1.1554 0.122357 THBS2 Thrombospondin 2 -1.3272 0.025363 TIE1 Tyrosine kinase with -1.5245 0.089397 immunoglobulin-like and EGF-like domains 1 TNFAIP2 Tumor necrosis factor, -1.5966 0.018487 alpha-induced protein 2 TOP2A Topoisomerase (DNA) II alpha -1.4224 0.00628 170kDa TP53 Tumor protein p53 -1.3899 0.058272 VEGFA Vascular endothelial growth 1.2241 0.041647 factor A B2M Beta-2-microglobulin 127.2628 0.667939 Supplemental Material, Table S1. qPCR array analysis of MCF-7F cells. qPCR arrays of MCF-7F cells treated with either vehicle or 10[micro]M DDT were run on samples isolated from three independent experiments using triplicate Breast Cancer & Estrogen Signaling PCR Arrays. Supplemental Material, Table S2. qPCR array of MCF-7 cells. Gene Description Fold p symbol Regulation value (DDT/Veh) AR Androgen receptor 0.73 0.0285 BAD BCL2-associated agonist of 1.06 0.8421 cell death BAG1 BCL2-associated athanogene 0.59 0.1056 BCL2 B-cell CLL/lymphoma 2 3.00 0.0011 BCL2L2 BCL2-like 2 1.22 0.4896 C3 Complement component 3 0.81 0.6753 CCNA1 Cyclin A1 1.97 0.0444 CCNA2 Cyclin A2 1.28 0.1708 CCND1 Cyclin D1 2.28 0.0656 CCNE1 Cyclin E1 0.81 0.2212 CD44 CD44 molecule (Indian blood 1.57 0.0736 group) CDH1 Cadherin 1, type 1, 0.76 0.0010 E-cadherin (epithelial) CDKN1A Cyclin-dependent kinase 0.69 0.2083 inhibitor 1A (p21, Cip1) CDKN1B Cyclin-dependent kinase 1.01 0.9595 inhibitor 1B (p27, Kip1) CDKN2A Cyclin-dependent kinase 1.51 0.6186 inhibitor 2A (melanoma, p16, inhibits CDK4) CLDN7 Claudin 7 1.23 0.4211 CLU Clusterin 0.21 0.0001 COL6A1 Collagen, type VI, alpha 1 0.68 0.0833 CTNNB1 Catenin 0.80 0.3459 (cadherin-associated protein), beta 1, 88kDa CTSB Cathepsin B 0.63 0.1371 CTSD Cathepsin D 2.96 0.0228 CYP19A1 Cytochrome P450, family 19, 1.59 0.3552 subfamily A, polypeptide 1 DLC1 Deleted in liver cancer 1 0.85 0.4211 EGFR Epidermal growth factor 0.46 0.0146 receptor ERBB2 V-erb-b2 erythroblastic 0.46 0.0294 leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) ESR1 Estrogen receptor 1 0.71 0.2677 ESR2 Estrogen receptor 2 (ER 0.87 0.4867 beta) FAS Fas (TNF receptor 1.35 0.1461 superfamily, member 6) FASLG Fas ligand (TNF 2.61 0.0156 superfamily, member 6) FGF1 Fibroblast growth factor 1 0.64 0.0916 (acidic) FLRT1 Fibronectin leucine rich 1.38 0.5102 transmembrane protein 1 FOSL1 FOS-like antigen 1 2.81 0.0002 GABRP Gamma-aminobutyric acid 0.16 0.0152 (GABA) A receptor, pi GATA3 GATA binding protein 3 1.01 0.9697 GNAS GNAS complex locus 0.61 0.1166 GSN Gelsolin 0.36 0.0008 HMGB1 High mobility group box 1 1.70 0.0172 HSPB1 Heat shock 27kDa protein 1 0.87 0.4067 ID2 Inhibitor of DNA binding 2, 0.87 0.5347 dominant negative helix-loop-helix protein IGFBP2 Insulin-like growth factor 0.80 0.6142 binding protein 2, 36kDa IL2RA Interleukin 2 receptor, 0.79 0.4674 alpha IL6 Interleukin 6 (interferon, 0.54 0.0456 beta 2) IL6R Interleukin 6 receptor 2.11 0.0161 IL6ST Interleukin 6 signal 1.06 0.7558 transducer (gp130, oncostatin M receptor) ITGA6 Integrin, alpha 6 2.28 0.0376 ITGB4 Integrin, beta 4 0.52 0.0346 JUN Jun proto-oncogene 1.23 0.6386 KIT V-kit Hardy-Zuckerman 4 0.73 0.4273 feline sarcoma viral oncogene homolog KLF5 Kruppel-like factor 5 0.58 0.0120 (intestinal) KLK5 Kallikrein-related 0.60 0.4859 peptidase 5 KRT18 Keratin 18 0.44 0.0176 KRT19 Keratin 19 0.98 0.8705 MAP2K7 Mitogen-activated protein 1.43 0.3934 kinase kinase 7 MKI67 Antigen identified by 1.09 0.7367 monoclonal antibody Ki-67 MT3 Metallothionein 3 1.16 0.7076 MUC1 Mucin 1, cell surface 0.58 0.0901 associated NFYB Nuclear transcription 0.77 0.2585 factor Y, beta NGF Nerve growth factor (beta 0.63 0.1830 polypeptide) NGFR Nerve growth factor 1.49 0.0486 receptor NME1 Non-metastatic cells 1, 2.46 0.0006 protein (NM23A) expressed in PAPPA Pregnancy-associated plasma 0.56 0.0647 protein A, pappalysin 1 PGR Progesterone receptor 229.01 0.0000 PLAU Plasminogen activator, 0.32 0.0017 urokinase PTEN Phosphatase and tensin 0.86 0.2443 homolog PTGS2 Prostaglandin-endoperoxide 2.38 0.4210 synthase 2 (prostaglandin G/H synthase and cyclooxygenase) RAC2 Ras-related C3 botulinum 3.21 0.1364 toxin substrate 2 (rho family, small GTP binding protein Rac2) RPL27 Ribosomal protein L27 1.06 0.8228 SCGB1D2 Secretoglobin, family 1D, 6.88 0.0035 member 2 SCGB2A1 Secretoglobin, family 2A, 1.36 0.3466 member 1 SERPINA3 Serpin peptidase inhibitor, 2.72 0.0139 clade A (alpha- 1 antiproteinase, antitrypsin), member 3 SERPINB5 Serpin peptidase inhibitor, 4.70 0.0004 clade B (ovalbumin), member 5 SERPINE1 Serpin peptidase inhibitor, 0.35 0.0181 clade E (nexin, plasminogen activator inhibitor type 1), member 1 SLC7A5 Solute carrier family 7 13.72 0.0002 (amino acid transporter light chain, L system), member 5 SPRR1B Small proline-rich protein 1.24 0.6745 1B STC2 Stanniocalcin 2 5.46 0.0001 TFF1 Trefoil factor 1 23.30 0.0000 TGFA Transforming growth factor, 1.09 0.6578 alpha THBS1 Thrombospondin 1 0.62 0.0277 THBS2 Thrombospondin 2 0.78 0.2588 TIE1 Tyrosine kinase with 0.67 0.2734 immunoglobulin-like and EGF-like domains 1 TNFAIP2 Tumor necrosis factor, 0.26 0.0021 alpha-induced protein 2 TOP2A Topoisomerase (DNA) II 0.97 0.9093 alpha 170kDa TP53 Tumor protein p53 0.90 0.5926 VEGFA Vascular endothelial growth 1.97 0.0474 factor A B2M Beta-2-microglobulin 1.02 0.8612 Gene Fold p symbol Regulation value ([E.sub.2]/V eh) AR 0.70 0.0261 BAD 0.79 0.4956 BAG1 0.52 0.0207 BCL2 2.65 0.0006 BCL2L2 1.10 0.7312 C3 0.55 0.1100 CCNA1 1.94 0.0057 CCNA2 1.41 0.0085 CCND1 1.16 0.7079 CCNE1 0.81 0.2008 CD44 1.55 0.0529 CDH1 0.78 0.0179 CDKN1A 0.91 0.6773 CDKN1B 0.88 0.6278 CDKN2A 0.82 0.4367 CLDN7 0.97 0.9236 CLU 0.11 0.0000 COL6A1 0.46 0.0008 CTNNB1 0.70 0.1403 CTSB 0.60 0.0041 CTSD 2.64 0.0431 CYP19A1 1.15 0.7322 DLC1 0.62 0.0170 EGFR 0.45 0.0060 ERBB2 0.33 0.0061 ESR1 0.49 0.0406 ESR2 0.68 0.0994 FAS 1.15 0.4104 FASLG 0.98 0.9500 FGF1 0.40 0.0032 FLRT1 0.94 0.8911 FOSL1 2.72 0.0000 GABRP 0.10 0.0001 GATA3 0.79 0.4808 GNAS 0.56 0.0360 GSN 0.38 0.0003 HMGB1 1.44 0.0013 HSPB1 0.99 0.9174 ID2 0.79 0.1426 IGFBP2 0.78 0.5921 IL2RA 1.01 0.9635 IL6 0.72 0.1373 IL6R 1.70 0.0548 IL6ST 0.83 0.3089 ITGA6 1.47 0.1152 ITGB4 0.45 0.0138 JUN 1.02 0.9488 KIT 0.82 0.4367 KLF5 0.46 0.0012 KLK5 0.83 0.6677 KRT18 0.42 0.0027 KRT19 1.19 0.2614 MAP2K7 0.78 0.5801 MKI67 1.07 0.7722 MT3 0.91 0.7190 MUC1 0.44 0.0202 NFYB 0.69 0.0027 NGF 0.82 0.4367 NGFR 1.33 0.2321 NME1 2.96 0.0000 PAPPA 0.72 0.1744 PGR 152.01 0.0000 PLAU 0.29 0.0003 PTEN 0.86 0.1711 PTGS2 0.82 0.4367 RAC2 1.55 0.1196 RPL27 1.22 0.3457 SCGB1D2 2.43 0.0511 SCGB2A1 1.05 0.8463 SERPINA3 2.62 0.0042 SERPINB5 4.70 0.0004 SERPINE1 0.42 0.0139 SLC7A5 11.62 0.0003 SPRR1B 1.75 0.0664 STC2 3.94 0.0000 TFF1 28.93 0.0000 TGFA 0.94 0.7549 THBS1 0.80 0.1801 THBS2 0.61 0.0165 TIE1 0.82 0.4367 TNFAIP2 0.23 0.0007 TOP2A 0.91 0.6314 TP53 0.88 0.5090 VEGFA 1.63 0.1023 B2M 0.79 0.0374 Supplemental Material, Table S2. qPCR array analysis of MCF-7 cells. qPCR arrays of MCF-7 cells treated with either vehicle, 10[micro]M DDT, or 1 nM [E.sub.2] were run on samples isolated from three independent experiments using triplicate Breast Cancer & Estrogen Signaling PCR Arrays.
DDT and its metabolites activate the HIF-1 response element (HRE). The VEGFA gene contains an estrogen responsive element (Kazi et al. 2005; Stoner et al. 2000, 2004) and is regulated by estrogens in mammary and uterine cells (Hyder et al. 1996; Nakamura et al. 1996, 1999). However, VEGFA expression is down-regulated by [E.sub.2] in human breast cancer cells (Hyder et al. 1998). We previously showed that DDT stimulated transcription in ER[alpha]-negative human embryonic kidney cells by activating the HRE (Bratton et al. 2009). Because VEGFA contains an HRE within its promoter (Liu et al. 1995), we tested the effects of DDT and DDT metabolites on transcription of an HRE-luc reporter construct in MCF-7 breast cancer cells. Transcription was more than doubled in response to 10 [micro]M o,p'-DDT (Figure 2A). HRE activity also increased significantly in response to the active metabolites p,p'-DDT, p,p'-DDD, o,p'-DDE, and p,p'-DDE, but not in response to the inactive metabolite p,p'-DDA (Figure 2A). [E.sub.2] also activated the HRE-luc reporter in MCF-7 cells, but this effect was blocked by ICI (Figure 2B). This suggests that [E.sub.2] can activate HREs; this is not surprising considering the general nature of the HRE reporter and the possibility that HREs are located within genes mediated by [ER[alpha]-E.sub.2]. Our cumulative results suggest that DDT alters VEGFA expression in MCF-7 cells in part by activating an HRE within the VEGFA promoter, in a manner independent of the ER[alpha] or [E.sub.2]. However, the fact that [E.sub.2] stimulates an HRE reporter in MCF-7 cells leaves open the possibility that the DDT effect on VEGFA expression could be mediated, at least in part, through the ER[alpha]-[E.sub.2] pathway.
DDT potentiates CBP-induced transcriptional activation of the HRE. CBP is a general transcriptional coactivator that functions to regulate gene expression through interaction with various transcription factors, including CREB (Giordano and Avantaggiati 1999), Elk 1 (Janknecht and Nordheim 1996), c-Jun (Giordano and Avantaggiati 1999), and TBP (TATA box binding protein) (Goodman and Smolik 2000). Based on previously published data showing a direct interaction between HIF-1 and CBP (Dames et al. 2002), we hypothesized that DDT activation of CBP may potentiate the activation of HRE-mediated transcription. HRE-luc activity was unchanged in MCF-7 cells transfected with CBP, but activity increased approximately 3.3 times following the addition of 10 [micro]M DDT to CBP-transfected cells, compared with only a 2 x increase in cells transfected with an empty vector (p < 0.001 for CBP-positive versus CBP-negative cells) (Figure 2C). Other DDT metabolites also enhanced activation of the HRE-luc construct in cells expressing CBP, with the exception of the negative metabolite control p,p-DDA (Figure 2D).
DDT and its active metabolites potentiate CBP activity. HIF-1 forms a complex with CBP that increases CBP's transactivation potential (Arany et al. 1996; Dames et al. 2002; Ema et al. 1999). We tested effects of DDT on CBP activity using a mammalian one-hybrid assay in which the full-length CBP is tethered to GAL4-DBD in conjunction with a GAL4 responsive luciferase reporter. Because our results suggested that the effect of DDT on VEGFA expression was ER[alpha]-independent, we used ER[alpha]-negative HEK 293 cells for this and subsequent experiments. The active DDT metabolites o,p'-DDT, p,p'-DDT, and o,p'-DDD potentiated CBP transactivation in a dose-dependent manner, whereas the inactive DDT metabolite, p,p'-DDA, had no effect (Figure 3A).
DDT activation of CBP is dependent on the p38[alpha] MAPK pathway. We previously demonstrated that AP-1 stimulation by DDT is dependent upon the p38[alpha] MAPK cascade (Frigo et al. 2004). Therefore, we tested the role of individual MAPK signaling pathways on DDT's activation of CBP. HEK 293 cells were transfected with GAL4-CBP and either empty vector or vectors overexpressing constitutively active MKK1, MKK5, MKK6, or MKK7 mutants that selectively activate ERK1/2, ERK5, p38[alpha], and JNK (respectively). MKK6, and to a lesser extent MKK1, potentiated CBP activity (Figure 3B). We next tested whether p38[alpha] was necessary for DDT-induced activation of CBP in HEK 293 cells transfected with GAL4-CBP (a GAL4-luc reporter) and increasing concentrations of DN-p38[alpha], DN-ERK1/2, or DN-JNK1 in the presence of 50 [micro]M o,p'-DDT. DDT-mediated activation of CBP was significantly inhibited in the absence of p38[alpha]-DN expression and to a lesser extent by ERK1/2-DN (Figure 3C). To confirm our molecular findings, we blocked DDT-induced coactivator activity with pharmacological inhibitors of the MAPK pathways. A GAL4-luc reporter, along with an empty expression vector or a GAL4-CBP fusion, was transfected into HEK 293 cells. The cells were then treated with vehicle or different MAPK inhibitors for 1 hr, followed by addition of vehicle or 50 [micro]M o,p'-DDT for 18 hr. The p38[alpha]/[beta] inhibitor SB203580 significantly blocked (p < 0.01) o,p'-DDT induction of CBP activity (Figure 3D), whereas neither the ERK inhibitor UO126 nor the JNK inhibitor SP600125 had a significant effect (Figure 3D). Collectively, these data confirm that DDT activates the transcriptional coactivator CBP via the p38 MAPK pathway.
DDT induces the p38[alpha]-mediated phosphorylation and transcriptional activation of CBP. Various kinases have been shown to potentiate CBP by phosphorylation (Ait-Si-Ali et al. 1999; Constantinescu et al. 2004). We hypothesized that p38B MAPK directly phosphorylates CBP, leading to its potentiation. To test this, we bacterially expressed recombinant CBP fused to GST for purification (Figure 4A) and subjected the purified proteins to an in vitro kinase assay in the presence of [.sup.32]P (phosphorus-32) and activated p38[alpha] MAPK. The C-terminal fragments of CBP containing amino acids 1680-1892 and, to a lesser extent, 1990-2441 were phosphorylated by activated p38[alpha], whereas the N-terminal fragment (amino acids 390-790) was not (Figure 4B). Activation of the C-terminal of CBP by DDT was tested using a deletion mutant of CBP containing amino acids 1300-2441 in a GAL4 fusion vector (Figure 5A). We over-expressed either the empty vector or a constitutively active MKK6 mutant in HEK 293 cells, in the presence or absence of o,p'-DDT. MKK6 activated the C-terminal of CBP in the absence of o,p'-DDT, but CBP activity was further augmented with the addition of 50 [micro]M o,p'-DDT (Figure 5B). Taken together, these results suggest that DDT augments p38 activity, which in turn phosphorylates CBP within its C-terminal, resulting in increased CBP transcriptional activation.
Although estrogenic activity of DDT has been reported (Ahlborg et al. 1995; Gulledge et al. 2001; Klotz et al. 1996; Kuiper et al. 1998), the mechanism underlying the hormone activity of the organochlorine pesticide remains unclear. We have previously shown that DDT and its metabolites activate transcription factors such as AP-1 independently of ER[alpha] (Bratton et al. 2009; Frigo et al. 2004). In the present study, we further investigated the molecular differences in hormone action between DDT and [E.sub.2] and characterized the qualities of DDT compared with other compounds that display estrogen-like properties. Although DDT and [E.sub.2] both stimulated the transcription of a subset of ER[alpha]-regulated genes, including Bcl-2, PgR, and trefoil factor 1 (TFF1), DDT also up-regulated genes that were not affected by [E.sub.2], including FASLG, ITGA6, and VEGFA (Table 1).
Differential gene expression induced by "estrogenic" environmental contaminants has been reported. For example, Goodson and colleagues treated nonmalignant high-risk donor breast epithelial cells (HRBECs) with [E.sub.2] and BPA; using global gene expression analysis, they determined that BPA produced a distinct gene expression pattern compared with [E.sub.2] (Dairkee et al. 2008; Goodson et al. 2011). Han et al. (2010) recently reported that DDT up-regulated aromatase gene expression in MCF-7 cells independently of ER function. Results of the present study also suggest that DDT is capable of altering gene expression in breast cancer cells in a manner different from that of [E.sub.2].
Our gene expression analysis revealed that DDT up-regulated VEGFA, an important factor in angiogenic cell response and regulation, as well as cell differentiation (Zhang et al. 1995). DDT increased VEGFA expression in MCF-7 cells, even in the presence of the pure antiestrogen ICI, suggesting that the DDT effect is ER[alpha] independent. In addition, DDT increased VEGFA expression in the ER[alpha]-negative MCF-7F cell line. Although crosstalk can occur between DDT signaling estrogen response elements, as previously shown (Bratton et al. 2009), the results presented here strongly suggest that DDT-altered VEGFA expression in MCF-7 breast cancer cells is ER[alpha] independent.
Our results also suggest that DDT and its metabolites potentiate the activity of HIF-1[alpha], which is known to bind the VEGFA promoter (Liu et al. 1995). However, because [E.sub.2] activated the HRE reporter, ER[alpha]-independent effects of [E.sub.2] on HRE activation and VEGFA expression remain a possibility. We have previously shown that DDT can regulate gene expression through the phosphorylation of coregulatory proteins such as SRC-2/GRIP1 (glucocorticoid receptor-interacting protein 1, steroid receptor coactivator-2), a member of the NCoA family of coregualtors (Frigo et al. 2006). Here, we demonstrated that active DDT compounds increased CBP activity and CBP-mediated transactivation of an HRE-linked reporter gene. DDT concentrations used in our experiments (10-50 [micro]M) may appear high, but DDT metabolite levels > 20 ng/mL in blood (equivalent to 63 [micro]M) have been reported (Longnecker et al. 2002; Lopez-Carrillo et al. 2001; Martin et al. 2002), as well as levels > 4 mM in soils throughout North America (Aigner et al. 1998; Falconer et al. 1997; U.S. Geological Survey 2001). These results, taken together, support a role for DDT in activation of the CBP-HIF-1 complex and suggest a mechanism by which DDT increases VEGFA expression.
We used both molecular and pharmacological tools to investigate the role of MAPK pathways in the DDT-CBP-HIF-1 signaling cascade. We showed that activation of the p38 pathway potentited CBP activity and that DDT's effect on CBP activation was inhibited by blocking p38[alpha]. Finally, we showed that p38[alpha] directly phosphorylated the C-terminal of CBP, and that p38 activated CBP via its C-terminal region. These data, in conjunction with published reports of a direct interaction between the coactivator CBP and HIF-1[alpha] (Dames et al. 2002) suggest a mechanism for the expression of VEGFA in MCF-7 cells following DDT exposure: DDT activates p38, which leads to phosphorylation of CBP and enhanced binding to HIF-1[alpha]; the resulting HIF-1[alpha]-CBP complex binds to VEGFA promoter, increasing its transcription (Figure 6).
Overall, our data demonstrate a link between organochlorine-mediated cell signaling through a MAPK pathway and the direct phosphorylation and regulation of coactivator function. These data suggest that coactivator phosphorylation might serve as a cellular sensor of environmental stress and lead to the modulation of key sets of adaptive genes. Moreover, these results suggest a possible mechanism by which environmental compounds may exert more, or less, [E.sub.2]-like potency than their ER[alpha] affinity implies.
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Melyssa R. Bratton, (1), (2) Daniel E. Frigo, (3) H. Chris Segar, (4) Kenneth P. Nephew, (5) John A. McLachlan, (1), (2) Thomas E. Wiese, (6) and Matthew E. Burow (2), (4)
(1) Department of Pharmacology, and (2) Center for Bioenvironmental Research, Tulane University, New Orleans, Louisiana, USA; (3) Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA; (4) Department of Medicine, Section of Hematology and Medical Oncology, Tulane University Health Sciences Center, New Orleans, Lousiana, USA; (5) Department of Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana, USA; (6) Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, Louisiana, USA
Address correspondence to M.E. Burow, Tulane University School of Medicine, Section of Hematology and Medical Oncology, 1430 Tulane Ave., Box SL-78, New Orleans, LA 70112 USA. Telephone: (504) 988-6688. Fax: (504) 988-5483. E-mail: firstname.lastname@example.org
Supplemental Material is available online (http://dx.doi.org/10.1289/ehp.1104296).
This work was supported by the Office of Naval Research (N00014-11-1-0177 to J.A.M. and M.E.B.), the National Institutes of Health (DK059389 to M.E.B., 5G12RR026260-02 to T.E.W., K01DK084205 to D.E.F., and National Cancer Institute U54 CA113001-07 to K.P.N.), and the Department of Defense (W81XWH-04-1-0557 BC030300 to T.E.W.).
The authors declare they have no actual or potential competing financial interests.
Received 3 August 2011; accepted 18 May 2012.
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|Author:||Bratton, Melyssa R.; Frigo, Daniel E.; Segar, H. Chris; Nephew, Kenneth P.; McLachlan, John A.; Wies|
|Publication:||Environmental Health Perspectives|
|Date:||Sep 1, 2012|
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