ABSTRACT
After the publication of this work [1] errors were noticed in the total protein loading controls for Figs. 1C, 2B, 3B and 4B.
ABSTRACT
Survivin has emerged as a unique regulator of cell death through its response to growth factors, such as basic fibroblast growth factor (bFGF), which we have previously shown to be mitogen-activated protein kinase (MAPK) dependent. The transcriptional complex myc/max is an oncogene that lies downstream of the MAPK pathway, suggesting a possible role in survivin's regulation. In this study, we investigated the ability of bFGF to induce signalling of the MAPK effector transcription factor c-myc in human breast cancer. Treatment of SK-BR-3 breast cancer cell line with growth factor induced survivin expression and recruitment of c-myc to its response element in the promoter region of the target gene survivin as demonstrated by electromobility shift analysis and chromatin immunoprecipitation assays. The promoter region of survivin was assessed using bioinformatic techniques and DNA footprinting. Overexpression of c-myc increased survivin protein expression. This effect was eliminated when siRNA against c-myc was transfected into the cells. c-Myc drove transcriptional activity of survivin when transfected into SK-BR-3 cells with a luciferase reporter vector harbouring the c-myc response element specific for survivin. Using confocal fluorescent microscopy, myc was located to the nucleus of breast tumour epithelial cells and was found to be significantly associated with survivin (P < 0.0001). These data provide evidence that growth factors can signal through the transcription factor c-myc in human breast cancer. They also indicate a role for c-myc in the transcriptional regulation of survivin in breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Base Sequence , Breast Neoplasms/genetics , Cell Line, Tumor , Humans , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins/genetics , Molecular Sequence Data , Neoplasm Proteins/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/genetics , Survivin , Transcription, Genetic/genetics , Up-RegulationABSTRACT
INTRODUCTION: Oestrogens can mediate some of their cell survival properties through a nongenomic mechanism that involves the mitogen-activated protein kinase (MAPK) pathway. The mechanism of this rapid signalling and its dependence on a membrane bound oestrogen receptor (ER), however, remains controversial. The role of G-protein-coupled receptor and epidermal growth factor (EGF) receptor in an ER-independent signalling pathway modulated by oestrogen was investigated. METHODS: ER-positive and ER-negative breast cancer cell lines (MCF-7 and SKBR3) and primary breast cancer cell cultures were used in this study. Cell proliferation was assessed using standard MTT assays. Protein and cAMP levels were detected by Western blotting and ELISA, respectively. Antigen localization was performed by immunocytochemistry, immunohistochemistry and immunofluorescence. Protein knockdown was achieved using small interfering RNA technologies. RESULTS: EGF and oestrogen, alone and in combination, induced cell proliferation and phosphorylation of MAPK proteins Raf and ERK (extracellular signal regulated kinase)1/2 in both ER-negative SKBR3 and ER-positive MCF-7 human breast cancer cell lines. Increased Raf phosphorylation was also observed in primary human breast cultures derived from ER-positive and ER-negative breast tumours. Oestrogen induced an increase in intracellular cAMP in ER-negative SKBR3 human breast cancer cells. Oestrogen-mediated cell growth and phosphorylation of MAPK was modified by the EGF receptor antagonist AG1478, the G-protein antagonist pertussis toxin, and the angiotensin II receptor antagonist saralasin. Knockdown of angiotensin II type 1 receptor (AT1) protein expression with small interfering RNA attenuated oestrogen-induced Raf phosphorylation in ER-negative cells. AT1 receptor was found to be expressed in the cell membrane of breast tumour epithelial cells. CONCLUSION: These findings provide evidence that, in breast cancer cells, oestrogen can signal through AT1 to activate early cell survival mechanisms in an ER-independent manner.
