The constitutive activation of signal transducer and activator of transcription 3 (Stat3) is frequently detected in breast cancer tissues and cell lines. Stat3 has been classified as a proto-oncogene, because an activated form of Stat3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. Since Stat3 may play an important role in breast cancer, it is of interest to investigate the expression of phosphorylated Stat3, an activated form of Stat3, and its downstream mediators specifically in breast cancer, and to explore the possible mechanisms of Stat3 signaling pathway in oncogenesis of breast cancer. We analyzed Stat3 phosphorylation and expression of Stat3-regulated genes in breast cancer cell lines as well as invasive breast cancer tissues using tissue microarray slides. Our results showed that elevated levels of phosphorylation of Stat3 protein (Tyr705) were detected in 48 out of total 136 invasive breast tumors (35%) whereas normal breast tissues express much lower levels of Stat3 phosphorylation. The increased levels of Stat3 phosphorylation were associated with the metastasis in regional lymph nodes (P = 0.042) and the expression of progesterone receptor (P = 0.028) but not with distant metastasis, nor the expression of estrogen receptor. Our results also indicate that elevated levels of Stat3 phosphorylation were significantly associated with increased expression of potential downstream targets of Stat3 which include apoptosis inhibitors (Survivin, Mcl-1, HSP27, Adrenomedullin, and Bcl-xL), cell-cycle regulators (c-Fos, MEK5, and c-Myc), and inducer of tumor angiogenesis (VEGF, COX-2, MMP-2, MMP-10, and MMP-1) in invasive breast cancer tissues. Therefore, our findings suggest that constitutive Stat3 signaling may be one of the key upstream regulators to induce these downstream proteins, which may play important roles in Stat3-mediated oncogenesis in breast cancer. (c) 2005 Elsevier Inc. All rights reserved.