Stimulatory Cross-talk between NFAT3 and Estrogen Receptor in Breast Cancer Cells

Abstract

Estrogen receptors (ER␣ and ER␤) are ligand-regulated transcription factors that play critical roles in the development and progression of breast cancer by regulating target genes involved in cellular proliferation. The transcriptional activity of ER␣ and ER␤ is known to be modulated by cofactor proteins. We used a yeast twohybrid system and identified NFAT3 as a novel ER␤-binding protein. NFAT3 interacted with ER␣ and ER␤ both in vitro and in mammalian cells in a ligand-independent fashion. NFAT3 bound specifically to the ER␤ region containing the activation function-1 domain, a ligand-independent transactivation domain. Overexpression of NFAT3 enhanced both ER␣ and ER␤ transcriptional activities in a ligand-independent manner and up-regulated downstream estrogen-responsive genes including pS2 and cathepsin D. Reduction of endogenous NFAT3 with NFAT3 small interfering RNA or overexpression of NFAT3 deletion mutants that lack the ER-binding sites reduced the NFAT3 coactivation of ER␣ and ER␤. NFAT3 increased binding of ER␣ to the estrogen-responsive element and was recruited to endogenous estrogen-responsive promoters. NFAT3 was expressed differentially in many breast cancer cell lines and overexpressed in a subset of breast cancer patients. Knockdown of endogenous NFAT3 reduced the growth of human breast cancer ZR75-1 cells in a ligand-independent manner. Taken together, these results suggest that NFAT3 may play important roles in ER signaling and represent a novel target for breast cancer therapy.Estrogen receptors (ER␣ and ER␤) 3 are members of the steroid hormone superfamily of nuclear receptors that act as ligand-activated transcription factors (1-3). Both of the two receptors regulate gene transcription either by binding directly to estrogen-responsive elements (ERE) located within the promoter regions of target genes or interacting with other transcription factors such as AP1 and SP1 (4, 5). ER␣ and ER␤ share structural similarity characterized by several functional domains. Two distinct activation function (AF) domains contribute to the transcriptional activity of the two receptors. The first activation function AF-1, a ligand-independent transactivation domain, is located at the N terminus, whereas the second, ligand-dependent activation function, AF-2, is located at the C terminus, overlapping the ligandbinding domain. The AF-1 activity of ER␤ is weak, compared with that of ER␣, whereas their AF-2 activities are similar (6). In most cases, the AF-1 and AF-2 domains interact functionally to enhance transcription in a cooperative manner. The DNA-binding domain (DBD) of the two receptors is centrally located. ER␣ and ER␤ possess similar binding affinities for estrogen and their cognate DNA binding site, which is probably caused by the high degree of sequence homology they share in their ligand and DNA binding domains (7). The ligand binding domain shows 58% homology between ER␣ and ER␤. The DNA binding domain is identical between the two receptors except for three amino acids. However, the N terminus co...

NFAT3 transcription factor inhibits breast cancer cell motility by targeting the Lipocalin 2 gene

“…Annotations: ERA (immunohistochemistry); p, positive; n, negative; 2-25 refers to the earlier reported patients' references (Bertheau et al, 2007). NFAT3/LCN2 axis inhibits breast carcinoma motility M Fougère et al However, we could not confirm (data not shown) the reports indicating that NFAT3 can associate with ERA and did not see any effects of NFAT3 on ERA transcriptional activity (Zhang et al, 2005;Qin et al, 2008). Interestingly, although both ERA þ cell migration and invasion were inhibited by LCN2 downregulation, the later was not enough to inhibit migration in ERAÀ cells, suggesting that cooperation of other critical genes was then required.…”

“…Moreover, when ERA þ breast carcinoma cell lines are re-implanted in mice, tumors arise only in the presence of estrogen and they are poorly metastatic compared with the cells that do NFAT3/LCN2 axis inhibits breast carcinoma motility M Fougère et al not express ERA (Price et al, 1990). Indeed, some studies have reported that NFAT3 is specifically present in breast cancer patients who express ERA (Gruvberger et al, 2001;Zhang et al, 2005). In this report, we unravel a new regulatory circuit, wherein NFAT3 is an anti-motility factor that acts by blocking LCN2 expression in ERA þ breast cancer.…”

The Hepatitis B Virus X Protein Disrupts Innate Immunity by Downregulating Mitochondrial Antiviral Signaling Protein

“…Immunohistochemistry was performed as previously described (35). Rabbit anti-MAVS (Abcam) were used as primary Abs.…”

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

“…The expression of FBXO44 and BRCA1 in the clinical samples was detected by immunohistochemistry as described previously (29). Briefly, the antigens were retrieved by autoclaving the sections in citric acids buffer and incubated with rabbit anti-FBXO44 antibody (Sigma) or mouse anti-BRCA1 antibody (Calbiochem) at a dilution of 1/100 (anti-FBXO44) or 1/50 (anti-BRCA1) for 1h at room temperature.…”