AbstractHeregulin (HRG) and type I receptor tyrosine kinase (RTK) expression was investigated in the highly invasive and metastatic LM3 cell line, our previously described model of metastasis for mammary cancer (Bal de Kier Joffe et al. [1986] Invasion Metastasis 6:302–12; Urtreger et al. [1997] Int J Oncol 11:489–96). Although LM3 cells do not express HRG, they exhibit high levels of ErbB‐2 and ErbB‐3 as well as moderate expression of ErbB‐4. Addition of exogenous HRGβ1 resulted in inhibition of both proliferation and migration of LM3 cells. HRGβ1 was also able to decrease the activity of urokinase‐type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP‐9), 2 key enzymes in the invasion and metastatic cascade. HRGβ1 treatment of LM3 cells induced tyrosine phosphorylation of ErbB‐2, ErbB‐3 and ErbB‐4 as well as the formation of ErbB‐2/ErbB‐3 and ErbB‐2/ErbB‐4 heterodimers. Assessment of the signaling pathways involved in HRGβ1 action indicated that the addition of HRGβ1 to LM3 cells resulted in activation of phosphatidylinositol 3‐ kinase (PI‐3K) and in strong induction of the association of the p85 subunit of PI‐3K with ErbB‐3. HRGβ1 also caused the rapid activation of ERK1/ERK2 and Stat3 and Stat5 (signal transducers and activators of transcription [STAT]). This is the first demonstration of the ability of HRGβ1 to activate STATs in mammary tumor cells. Blockage of PI‐3K activity with its chemical inhibitor wortmannin, or of MEK1/ERKs activity with PD98059, resulted in suppression of the ability of HRGβ1 to inhibit LM3 cell growth. Notwithstanding the suppression of these 2 signaling pathways, HRGβ1 still proved capable of inhibiting uPA activity. Therefore, our results provide evidence that signaling pathways involved in HRGβ1‐induced proliferation appear to be distinct from those involved in HRGβ1 regulation of uPA, a protease that plays a pivotal role in invasion and metastasis. © 2002 Wiley‐Liss, Inc.