Synergistic up-regulation of prostaglandin E synthase expression in breast cancer cells by 17beta-estradiol and proinflammatory cytokines

Abstract

Inflammatory mediators, such as cytokines and prostaglandins, play a fundamental role in estrogen-dependent breast cancer through their ability to up-regulate aromatase expression and subsequent local production of estrogens in the breast. To study the link between estrogens and inflammation further, we examined the regulation of prostaglandin E synthase (PTGES), a key enzyme in the production of prostaglandin E2. We found that 17beta-estradiol (E2) rapidly and robustly up-regulates PTGES mRNA and protein levels in estrogen receptor (ER)-positive breast cancer cells through ER recruitment to an essential estrogen response element located in the 5' flanking region of the PTGES gene. PTGES is also up-regulated by the proinflammatory cytokines TNFalpha or IL-1beta. Surprisingly, the combination of E2 and cytokines leads to a synergistic up-regulation of PTGES in an ER and nuclear factor-kappaB (NFkappaB)-dependent manner. This is in contrast to the mutual transrepression between ER and NFkappaB that has been well characterized in other cell types. Furthermore, we found enhanced recruitment of ERalpha as well as the NFkappaB family member, p65, to the PTGES estrogen response element by the combination of E2 and TNFalpha compared with either E2 or TNFalpha alone. The synergistic up-regulation of PTGES may result in enhanced prostaglandin E2 production, which in turn may further enhance aromatase expression and production of local estrogens. Our findings suggest that a finely tuned positive feedback mechanism between estrogens and inflammatory factors may exist in the breast and contribute to hormone-dependent breast cancer growth and progression.

Fig. 1

E2 up-regulates PTGES expression in ER-positive breast cancer cells. A, MCF-7 cells were treated for different lengths of time with 10 nm E2. PTGES mRNA was measured by QPCR, and fold change was calculated using the ΔΔcycle threshold method with 36B4 as an internal control. B, PTGES protein was examined by Western blotting after 24 h treatment of MCF-7 cells with E2. The amount of PTGES protein after treatment is indicated, relative to the internal control β-actin. C, E2 regulation of PTGES mRNA after 2 h treatment was examined by QPCR in ER-positive T47D cells and ER-negative MDA-MB-231 cells. D, PTGES mRNA levels were examined in MCF-7 cells pretreated for 2 h with the selective ER modulators (1 μm), Ral), 4-OHT, or ICI, followed by 2 h treatment with 10 nm E2.

Fig. 2

PTGES is regulated by E2 through an ERE located in the 5′ flanking region of the PTGES gene. A, A 2.9-kb fragment of the PTGES 5′ flanking region, containing an ERE at −2768, was cloned into the pGL4 luciferase reporter vector. The ERE was mutated (Mut-ERE) or a 400-kb region containing the ERE was deleted (2.5 kb PTGES). Reporter activity was measured in MCF-7 cells after transient cotransfection of PTGES reporter constructs and a Renilla control reporter and treatment with E2 for 4 h. B, ChIP assays were carried out in MCF-7 cells with an ERα-specific antibody or rabbit IgG after 45 min E2 treatment. QPCR was then carried out on immunoprecipitated chromatin for a region around the ERE in the PTGES gene, the ERE in the pS2 gene as a positive control, or a region of the PTGES gene located −3.9 kb upstream as a negative control. C, HeLa cells were transfected with the 2.9-kb PTGES reporter construct and an expression vector for wild-type ERα or ERα incapable of binding DNA, due to either a deletion (DBD-del) or mutation (DBD-mut) in the DBD. Reporter activity was measured after 16 h treatment with E2.

Fig. 3

Proinflammatory cytokines and E2 synergistically up-regulate PTGES mRNA levels. A, PTGES mRNA was examined by QPCR in MCF-7 cells that were treated with 10 nm E2 in combination with 10 ng/ml of the proinflammatory cytokines, TNFα, IL-1β, or IL-6. B, A time course was carried out with 10 nm E2, 10 ng/ml TNFα, or both. C, Cells were treated for 2 h with 10 nm E2 or 1 μm 4-OHT in the presence or absence of 10 ng/ml TNFα. PTGES mRNA levels were examined by QPCR. D, A dose response for E2 was carried out in the presence or absence of 10 ng/ml TNFα for 2 h. E, A dose response for TNFα was carried out in the presence or absence of 10 nm E2 for 2 h.

Fig. 4

ER and NFκB pathways are required for synergistic PTGES regulation. A, MCF-7 cells were pretreated for 2 h with 1 μm ICI or 1 μm of two different IKK inhibitors (IKK inhibitor VII, which inhibits both IKKα and IKKβ, and IKK-2 inhibitor VI, which specifically inhibits IKKβ; Calbiochem) and then treated for an additional 2 h with 10 nm E2 ± 10 ng/ml TNFα. B, Adenoviral delivery of GFP (control) or IκBα-DN was carried out 24 h before 2 h treatment with 10 nm E2 ± 10 ng/ml TNFα. PTGES mRNA levels were analyzed by QPCR.

Fig. 5

E2 and TNFα regulate PTGES through a primary transcriptional response. A, MCF-7 cells were transfected as described for Fig 2. Cells were treated with E2, TNFα, or both for 4 h, and reporter activity was measured using a dual-luciferase assay. B, MCF-7 cells were pretreated with 10 μg/ml of the protein synthesis inhibitor CHX or 1 μg/ml of the RNA synthesis inhibitor ActD followed by 2 h treatment with E2 ± TNFα. PTGES mRNA levels were examined by QPCR. Mut-ERE, Mutated ERE.

Fig. 6

E2 and TNFα enhance ER and p65 recruitment to the PTGES ERE. ChIP assays for ERα (A and B) or p65 (C) were carried out in MCF-7 cells treated for 45 min with E2, 4-OHT, and/or TNFα without pretreatment (A and C) or after 2 h pretreatment with the inhibitor of IKKβ (B). ERα and p65 recruitment to the PTGES and/or pS2 ERE were analyzed by QPCR.

Fig. 7

Potential positive feedback mechanisms in breast cancer between estrogens, inflammatory cytokines, and PGE2. PTGES is an important enzyme that acts downstream of COX-2 in the PGE2 biosynthesis pathway. Our findings suggest the presence of a positive feedback loop between estrogens and PGE2, whereby estrogens up-regulate PTGES expression and PGE2 up-regulates aromatase expression. Our findings also suggest that proinflammatory cytokines can positively reinforce this feedback loop by enhancing both the production and action of estrogens via increasing aromatase expression and influence of the NFκB pathway on ER recruitment and activity at the PTGES gene, resulting in a synergistic up-regulation of PTGES expression. PGH2, Prostaglandin H2.