A study of the signaling molecules implicated in heregulin-mediated mammary cell differentiation in a breast carcinoma cell line

Abstract

A complete understanding of mammary cell differentiation is required in order to fully understand the molecular basis of breast cancer. Members of the ErbB receptor tyrosine kinase family have been implicated in the etiology and progression of many types of human cancers, including breast cancer. Heregulin (HRG), a ligand for the ErbB3 and ErbB4 receptors, plays a key role in mammary gland development. During pregnancy, HRG-mediated ErbB receptor signaling is responsible for the formation of lobuloalveoli followed by terminal mammary cell differentiation. In some mammary carcinoma cell lines, such as the AU565 cell line, HRG has been shown to induce cellular differentiation. In order to further characterize HRG-ErbB3 signaling, our laboratory previously used a yeast two-hybrid interaction screen to isolate ErbB3 binding proteins. EBP1(ErbB3 Binding Protein 1) was identified and has been demonstrated to dissociate from the ErbB3 receptor upon HRG stimulation. We further characterized this signaling protein by generating a polyclonal antibody against GST-EBP1. Protein and mRNA expression profiles were examined in a variety of cell lines. EBP1 expression was ubiquituous, and not limited to ErbB3-expressing cell lines. We also determined the biological effects of transfecting ebp1 cDNA into breast carcinoma cells. The EBP1 protein is basally phosphorylated in vivo on serine and threonine residues and this phosphorylation is enhanced after thirty minutes of heregulin stimulation. Both serine and threonine residues of GST-EBP1 fusion protein are phosphorylated by PKC in vitro. In vivo, we demonstrated that whereas HRG-induced phosphorylation of EBP1 occurs predominantly in a PKC-independent manner, basal EBP1 phosphorylation is dependent upon the activation of PKC. In conclusion, sustained activation of the ERK signaling pathway is essential for HRG-induced differentiation of the AU565 mammary carcinoma cell line. The ErbB3 binding phospho-protein EBP1 has also been implicated as a potential signaling molecule involved in this process. (Abstract shortened by UMI.)