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

Abstract

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 (E₂) 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: E₂ 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 α 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 E₂, 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.

Figure 1

VEGFA expression in ERα-positive MCF‑7 cells incubated for 18 hr with vehicle, 10 μM DDT, or DDT + ICI (100 nM) (A) and ERα-negative MCF‑7F cells incubated for 18 hr with vehicle or 10 μM DDT (B). qPCR results are presented as fold change relative to housekeeping genes. *p < 0.05 compared with vehicle control (n = 3).

Figure 2

Organochlorines augment CBP activation of transcription from an HRE. (A) MCF‑7 cells transfected with an HRE-luc reporter and incubated with DDT metabolites (10 μM; n = 3). (B) MCF‑7 cells transfected with an HRE as in A, followed by incubation with vehicle, E₂, or E₂ + ICI (n = 3). (C) MCF‑7 cells transfected with an empty vector or a CBP expression vector plus an HRE-containing luciferase reporter and incubated overnight with vehicle or 10 μM o,p’-DDT (n = 3). (D) MCF‑7 cells treated as in C with metabolites 10 μM DDT (n = 4–6). Luminescence values are shown as the mean ± SE percentage of vehicle control, with the vehicle control set to 100%. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with vehicle control (A and B), the metabolite without CBP (D), or as indicated (C).

Figure 3

DDT and its metabolites stimulate the coactivator CBP through activation of the p38 MAPK pathway. (A) ER-negative HEK 293 cells transfected with GAL4-CBP and a GAL4-luc reporter and incubated overnight with organochlorines; n = 4. (B) HEK 293 cells transfected overnight with GAL4‑CBP, a GAL4-luc reporter, and either an empty vector or a vector expressing constitutively active MKK1 (ERK1/2), MKK5 (ERK5), MKK6 (p38), or MKK7 (JNK); n = 3. (C) HEK 293 cells transfected with GAL4-CBP, GAL4-luc, and increasing amounts of dominant negative (DN) mutants and then treated with 50 μM o,p’‑DDT for 24 hr; n = 4–6. (D) HEK 293 cells transfected with either empty vector or GAL4-CBP and GAL4-luc; after 6 hr, MAPK inhibitors were added (1 μM UO126, 1 μM SP600125, 6 μM SB203580), followed 1 hr later by 50 μM o,p’‑DDT for 18 hr (n = 4). Values are mean ± SE luciferase activity, with control values set to 100%. *p < 0.05, **p < 0.01, and ^#p < 0.001, compared with vehicle control (A) or empty vector control (B,C,D).

Figure 4

Activated p38α phosphorylates CBP in vitro. (A) Schematic of GST fusion proteins used for the in vitro kinase assays; aa, amino acids. (B) GST fusion proteins were purified and standardized according to protein concentration. Purified activated p38α was used to phosphorylate GST-CBP fragments in the presence of ³²P-ATP followed by SDS‑PAGE and coomassie staining (left). Gels were dried and autoradiographed (right). GST-MAPKAPK2 was used as a positive control for p38α phosphorylation. The white arrow indicates phosphorylated GST-MAPKAPK2, and black arrows indicate phosphorylated GST-CBP fragments. Similar results were obtained in three independent experiments.

Figure 5

DDT and p38α target the C‑terminal of CBP. (A) Schematic of GAL4-CBP fusion constructs used for mammalian one-hybrid analysis; aa, amino acids. (B) HEK 293 cells transfected for 6 hr with full-length GAL4-CBP (FL) or the C‑terminal (C‑term) fragment GAL4-CBP (aa 1300-2441) plus GAL4‑luc. Some cells were also transfected with the CA-MKK6 mutant; other cells were transfected with MKK6-CA and incubated overnight with 50 μM o,p’‑DDT. Values represent the percent change (mean ± SE; n = 4) in CBP activity, with the vehicle set to 100%. **p < 0.01, and ^#p < 0.001 compared with vehicle control.

Figure 6

Proposed mechanism of organochlorine-mediated up‑regulation of VEGFA expression. We propose a mechanism whereby o,p’-DDT stimulates phosphorylation of p38 kinase, which in turn phosphorylates CBP. The activated CBP binds HIF-1, and this complex binds to the VEGFA promoter at the HRE, thereby increasing transcription of VEGFA.