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The organochlorine o,p'-DDT plays a role in coactivator-mediated MAPK crosstalk in mcf-7 breast cancer cells.

BACKGROUND: The organochlorine dichlorodiphenyltrichloroethane (DDT), a known estrogen mimic and endocrine disruptor, has been linked to animal and human disorders. However, the detailed mechanism(s) by which DDT aFFects cellular physiology remains incompletely defined.

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).

Results

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.

Discussion

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).

Conclusions

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: mburow@tulane.edu

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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Title Annotation:Research
Author:Bratton, Melyssa R.; Frigo, Daniel E.; Segar, H. Chris; Nephew, Kenneth P.; McLachlan, John A.; Wies
Publication:Environmental Health Perspectives
Article Type:Report
Date:Sep 1, 2012
Words:8074
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