AbstractThe inappropriate activation of one or more members of the ErbB family of receptor tyrosine kinases [ErbB‐1 (EGFR), ErbB‐2, ErbB‐3, ErbB‐4] has been linked with oncogenesis. ErbB‐2 is frequently coexpressed with ErbB‐3 in breast cancer cells and in the presence of the ligand heregulin (HRG) the ErbB‐2/ErbB‐3 receptors form a signaling heterodimer that can affect cell proliferation and apoptosis. The major goal of the present study was to determine whether endogenous HRG causes autocrine/paracrine activation of ErbB‐2/ErbB‐3 and contributes to the proliferation of mammary epithelial tumor cells. Tyrosine‐phosphorylated (activated) ErbB‐2 and ErbB‐3 receptors were detected in the majority of extracts from tumors that had formed spontaneously or as a result of oncogene expression. HRG‐1 transcripts and protein were found in the epithelial cells of most of these mouse mammary tumors. Various mouse mammary cell lines also contained activated ErbB‐2/ErbB‐3 and HRG transcripts. A ∼50 kDa C‐terminal fragment of pro‐HRG was detected, which indicates that the HRG‐1 precursor is readily processed by these cells. It is likely that the secreted mature HRG activated the ErbB‐2/3 receptors. Addition of an antiserum against HRG to the mammary epithelial tumor cell line TM‐6 reduced ErbB‐3 Tyr‐phosphorylation. Treatment with HRG‐1 siRNA oligonucleotides or infection with a retroviral construct to stably express HRG siRNA effectively reduced HRG protein levels, ErbB‐2/ErbB‐3 activation, and the rate of proliferation, which could be reversed by the addition of HRG. The cumulative findings from these experiments show that coexpression of the HRG ligand contributes to activation of ErbB‐2/Erb‐3 in mouse mammary tumor cells in an autocrine or paracrine fashion. Published 2006 Wiley‐Liss, Inc.