Identification and characterization of nucleolin as a COUP-TFII coactivator of retinoic acid receptor β transcription in breast cancer cells.
BACKGROUND
The 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.


RESULTS
FLAG-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.


CONCLUSIONS
Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.
Department of Biochemistry & Molecular Biology and Center for Genetics and Molecular Medicine, Louisville, Kentucky, United States of America.
Lacey M
LM
Litchfield
Krista A
KA
Riggs
Alyson M
AM
Hockenberry
Laura D
LD
Oliver
Katelyn G
KG
Barnhart
Jian
J
Cai
William M
WM
Pierce
Margarita M
MM
Ivanova
Paula J
PJ
Bates
Savitri N
SN
Appana
Susmita
S
Datta
Piotr
P
Kulesza
Jean
J
McBryan
Leonie S
LS
Young
Carolyn M
CM
Klinge
D000818:Animals; D052157:Aptamers, Nucleotide; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D051837:COUP Transcription Factor II / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D060787:Neoplasm Grading; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D046888:Tissue Array Analysis; D015533:Transcriptional Activation / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology; D000097796:Nucleolin
D052157:Aptamers, Nucleotide; D051837:COUP Transcription Factor II; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D000097796:Nucleolin; C513936:AGRO 100; C082925:retinoic acid receptor beta