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Breast Cancer Research

, 8:R63

First Online: 01 November 2006Received: 17 July 2006Revised: 04 September 2006Accepted: 01 November 2006


IntroductionNa-H exchanger regulatory factor 1 NHERF1, also known as EBP50 or NHERF is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity LOH at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified.

MethodsTo confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA shRNA to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses.

ResultsWe found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G1-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level.

ConclusionOur findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities.

AbbreviationsERoestrogen receptor

EREoestrogen response element


FACSfluorescence-activated cell sorting

FBSfoetal bovine serum

LOHloss of heterozygosity

MTT = 3-45-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide


NHE3Na-H exchanger isoform 3

NHERF1Na-H exchanger regulatory factor 1

PBSphosphate-buffered saline

PDGFRplatelet-derived growth factor receptor


PTENphosphatase and tensin homologue mutated in multiple advanced cancers 1

shRNAshort hairpin RNA

SYKspleen tyrosine kinase.

Electronic supplementary materialThe online version of this article doi:10.1186-bcr1616 contains supplementary material, which is available to authorized users.

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Autor: Yong Pan - Lei Wang - Jia Le Dai


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