We have recently shown that an amphiregulin-mediated autocrine loop is responsible for growth factor–independent proliferation, motility, and invasive capacity of some aggressive breast cancer cells, such as the SUM149 breast cancer cell line. In the present study, we investigated the mechanisms by which amphiregulin activation of the epidermal growth factor receptor (EGFR) regulates these altered phenotypes. Bioinformatic analysis of gene expression networks regulated by amphiregulin implicated interleukin-1α (IL-1α) and IL-1β as key mediators of amphiregulin's biological effects. The bioinformatic data were validated in experiments which showed that amphiregulin, but not epidermal growth factor, results in transcriptional up-regulation of IL-1α and IL-1β. Both IL-1α and IL-1β are synthesized and secreted by SUM149 breast cancer cells, as well as MCF10A cells engineered to express amphiregulin or MCF10A cells cultured in the presence of amphiregulin. Furthermore, EGFR, activated by amphiregulin but not epidermal growth factor, results in the prompt activation of the transcription factor nuclear factor–κB (NF-κB), which is required for transcriptional activation of IL-1. Once synthesized and secreted from the cells, IL-1 further activates NF-κB, and inhibition of IL-1 with the IL-1 receptor antagonist results in loss of NF-κB DNA binding activity and inhibition of cell proliferation. However, SUM149 cells can proliferate in the presence of IL-1 when EGFR activity is inhibited. Thus, in aggressive breast cancer cells, such as the SUM149 cells, or in normal human mammary epithelial cells growing in the presence of amphiregulin, EGFR signaling is integrated with NF-κB activation and IL-1 synthesis, which cooperate to regulate the growth and invasive capacity of the cells. (Mol Cancer Res 2007;5(8):847–62)