Cellular Location and Expression Pattern of NHERF1 in Human Breast Cancer and the Impact of NHERF1 on the Growth of Breast Cancer Cells.

Abstract

Abstract BACKGROUND: The Na+/H+ exchange regulatory factor 1 (NHERF1) is an adapter protein that, when present in the cytoplasmic region, regulates trafficking and signaling of several G protein-coupled receptors (GPCRs). However, it has been recently suggested that, when located in the nucleus of cells, NHERF1 may have an oncogenic role in cancer. Recent work has shown that NHERF1 may be involved in the progression of certain solid tumours including breast cancer. The aim of the current study were: first to evaluate the spatial and cellular location of the NHEREF1 protein in mammary tissues, mammary epithelial and cancer cells, second to determine the expression pattern of the NHERF1 transcript in human breast cancer and deduce a possible association with clinical and pathological factors and, finally, to investigate the cellular impact of NHERF1 expression on breast cancer cells.MATERIALS AND METHODS: Expression of NHERF1 was examined in a cohort of breast tissue samples. The protein levels and distributions were assessed using immunohistochemical staining (IHC) and imaging analysis tools. The distribution of the NHERF1 protein in nucleus and cytoplasm was calculated using the protein staining intensity ratio between the two compartments. The transcript level was determined using quantitative real time-PCR. Constructed ribozyme transgenes were used to knock-down NHERF1 in MCF-7 cells, and the effect this had on in vitro cell growth was examined using in vitro methods.RESULTS: NHERF1 protein staining was seen in both normal epithelial cells and cancer cells in tissues. However, the staining pattern in cancer cells and normal epithelial cells was different. The protein was seen at a higher level in the nucleaus of cancer cells, as shown by a higher nuclear/cytoplasmic ratio of NHERF1 staining in breast cancer cells when compared with that in normal mammary epithelial cells (p=0.038). NHERF1 expression was increased in high grade tumours compared with low grade tumours (p=0.0016, grade-3 vs grade-1). Quantitative analysis of the NHERF1 transcript revealed a higher level expression in samples from patients with poor prognosis and that this was linked to the long term survival: mean survival for patients with high NHERF1 was 102 (55-148.8, 95%CI) months compared with 136 (126.6-145.9) months for patients with low NHERF1 transcripts. Using human breast cancer cell line, MCF7, we created NHERF1 knockdown subline. Loss of NHERF1 in the MCF-7 subline resulted in an increase in the growth rate, in vitro.CONCLUSION: This study shows that NHERF1 influences the growth of breast cancer cells. However, the effect of NHERF1 is dependent upon the levels of expression of NHERF1 in breast cancer tissues and most importantly determined by the cellular location of this protein in cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3155.