Estrogen receptors (ER) and nuclear factor-κB (NF-κB) are known to play important roles in breast cancer, but these factors are generally thought to repress each other's activity. However, we have recently found that ER and NF-κB can also act together in a positive manner to synergistically increase gene transcription. To examine the extent of cross-talk between ER and NF-κB, a microarray study was conducted in which MCF-7 breast cancer cells were treated with 17β-estradiol (E 2 ), tumor necrosis factor α (TNFα), or both. Followup studies with an ER antagonist and NF-κB inhibitors show that cross-talk between E 2 and TNFα is mediated by these two factors. We find that although transrepression between ER and NF-κB does occur, positive cross-talk is more prominent with three gene-specific patterns of regulation: (a) TNFα enhances E 2 action on ∼30% of E 2 -upregulated genes; (b) E 2 enhances TNFα activity on ∼15% of TNFα-upregulated genes; and (c) E 2 + TNFα causes a more than additive upregulation of ∼60 genes. Consistent with their prosurvival roles, ER and NF-κB and their target gene, BIRC3, are involved in protecting breast cancer cells against apoptosis. Furthermore, genes positively regulated by E 2 + TNFα are clinically relevant because they are enriched in luminal B breast tumors and their expression profiles can distinguish a cohort of patients with poor outcome following endocrine treatment. Taken together, our findings suggest that positive cross-talk between ER and NF-κB is more extensive than anticipated and that these factors may act together to promote survival of breast cancer cells and progression to a more aggressive phenotype. [Cancer Res 2009;69(23):8918-25]

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.

Constitutive activation of NFB in estrogen receptor (ER)-positive breast cancer is associated with tumor recurrence and development of anti-estrogen resistance. Furthermore, a gene expression signature containing common targets for ER and NFB has been identified and found to be associated with the more aggressive luminal B intrinsic subtype of ER-positive breast tumors. Here, we describe a novel mechanism by which ER and NFB cooperate to up-regulate expression of one important gene from this signature, ABCG2, which encodes a transporter protein associated with the development of drug-resistant breast cancer. We and others have confirmed that this gene is regulated primarily by estrogen in an ER-and estrogen response element (ERE)-dependent manner. We found that whereas proinflammatory cytokines have little effect on this gene in the absence of 17␤-estradiol, they can potentiate ER activity in an NFB-dependent manner. ER allows the NFB family member p65 to access a latent NFB response element located near the ERE in the gene promoter. NFB recruitment to the gene is, in turn, required to stabilize ER occupancy at the functional ERE. The result of this cooperative binding of ER and p65 at adjacent response elements leads to a major increase in both ABCG2 mRNA and protein expression. These findings indicate that estrogen and inflammatory factors can modify each other's activity through modulation of transcription factor accessibility and/or occupancy at adjacent response elements. This novel transcriptional mechanism could have important implications in breast cancer, where both inflammation and estrogen can promote cancer progression. The estrogen 17␤-estradiol (E 2 )2 is a steroid that plays an important role in reproductive tissues, as well as in the skeletal, cardiovascular, immune, and central nervous systems, by regulating a number of cellular processes, such as proliferation, differentiation, and survival. In the classical mechanism of estrogen action, E 2 binds to the estrogen receptor (ER) to promote receptor homodimerization. The ligand-bound receptor binds to cognate DNA sequences, called estrogen response elements (EREs), which leads to coregulator recruitment and target gene transcription. In addition to direct DNA binding, ER can also regulate gene transcription via protein-protein interaction with other DNA-binding transcription factors, such as the proteins of the AP-1 complex and Sp1 (1, 2).Estrogen can also modulate gene transcription by the proinflammatory transcription factor NFB, which, like ER, influences numerous cellular processes. In the classical NFB pathway, binding of proinflammatory cytokines to their receptors activates the IB kinase (IKK) complex, which phosphorylates the inhibitory protein IB, leading to its subsequent ubiquitination and proteasomal degradation. NFB family members p65 and p50 can then translocate to the nucleus and regulate transcription of a cohort of genes by binding to specific DNA elements called NFB response elements (NFBREs). Numerous studies in a variety of phy...

A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects.