Abstract
IntroductionThe orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.ResultsFLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.ConclusionsOur data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.
Highlights
The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer
COUP-TFs are classified as orphan receptors, as they have no currently established physiological ligands, the crystal structure of the COUP-TFII LBD showed that its ‘‘auto-repressed conformation’’ was relieved by 9-cis and all-trans retinoic acids (9cRA and atRA) that bind the LBD with,17–26 mM affinity [4]
Identification of COUP-TFII-associated proteins C-terminal FLAG-tagged COUP-TFII was overexpressed in MCF-7 cells (,2-fold higher expression compared to COUP-TFII endogenous expression, Fig. S1 and S2) and interacting proteins were captured by immunoprecipitation (IP) with anti-FLAGaffinity gel (Fig. S2A)
Summary
The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. We reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. COUP-TFI (NR2F1) and COUP-TFII (NR2F2) are ‘orphan’ members of the steroid/nuclear receptor (NR) superfamily [1] COUP-TFs regulate gene transcription in a cell- and gene- specific manner. Gene knockout mice demonstrated that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) have distinct roles during embryogenesis, notably in the nervous and cardiovascular systems, respectively [2,3]. COUP-TFs are classified as orphan receptors, as they have no currently established physiological ligands, the crystal structure of the COUP-TFII LBD showed that its ‘‘auto-repressed conformation’’ was relieved by 9-cis and all-trans retinoic acids (9cRA and atRA) that bind the LBD with ,17–26 mM affinity [4]. Adjuvant endocrine therapies including the use of antiestrogens, e.g., tamoxifen (TAM), and aromatase inhibitors (AI), e.g., letrozole, are effective in reducing disease recurrence in many patients [9]
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