Dissecting the transcriptional networks underlying breast cancer: NR4A1 reduces the migration of normal and breast cancer cell lines.
BACKGROUND
Breast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology.


METHODS
NR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting.


RESULTS
Breast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins.


CONCLUSIONS
NR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.
Alexopoulou
Annika N
AN
University of Oxford Branch, Ludwig Institute for Cancer Research, Old Road Campus, Off Roosevelt Drive, Oxford OX3 7DQ, UK. a.alexopoulou@prion.ucl.ac.uk
Leao
Maria
M
Caballero
Otavia L
OL
Da Silva
Leonard
L
Reid
Lynne
L
Lakhani
Sunil R
SR
Simpson
Andrew J
AJ
Marshall
John F
JF
Neville
A Munro
AM
Jat
Parmjit S
PS
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046888:Tissue Array Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D014157:Transcription Factors; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3