Stat5a IS TYROSINE PHOSPHORYLATED AND NUCLEAR LOCALIZED IN A HIGH PROPORTION OF HUMAN BREAST CANCERS Ion COTARLA 1,2 , Shuxun REN 1 , Ying ZHANG 3 , Edmund GEHAN 3 , Baljit SINGH 4 and Priscilla A. FURTH 1,2 * 1 Department of Oncology, Georgetown University, Washington, DC, USA 2 Graduate Program in Tumor Biology, Georgetown University, Washington, DC, USA 3 Biostatistics Shared Resource, Lombardi Cancer Center, Washington, DC 4 Department of Pathology, Georgetown University, Washington, DC, USA Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that are acti- vated and translocated into the nucleus after phosphoryla- tion at a conserved tyrosine residue. Mouse model studies have demonstrated that activated Stat5a acts as a critical survival factor for normal, preneoplastic and malignant mammary epithelial cells. Very limited information is avail- able, however, on the expression, tyrosine phosphorylation status and nuclear localization of Stat5a in human breast cancers. In our study, the pattern of Stat5a cellular localiza- tion was analyzed by immunohistochemistry in a series of 83 randomly selected primary human breast adenocarcinomas. Immunoprecipitation/Western blotting and immunohisto- chemistry assays employing different phospho-specific anti- bodies verified Stat5a tyrosine phosphorylation status. Stat5a was nuclear localized and tyrosine phosphorylated in 59 of 78 (76%) breast cancers examined; 38 of 78 (49%) demonstrated Stat5a nuclear localization in more than 25% of the breast cancer cells within the adenocarcinomas. Nu- clear localized Stat5a was associated positively with in- creased levels of histologic differentiation (p  0.03). A sta- tistically significant positive association with p27 nuclear localization also was identified (p  0.05). No relationship was found between nuclear localized Stat5a and menopausal status, tumor size, ploidy, percentage of cells in S-phase, lymph node metastases, ER, ErbB2, nuclear localized p21 or nuclear localized Stat5b/Stat3. As its role in human breast cancer progression and response to therapy is defined, Stat5a could become a new molecular target for breast can- cer therapy. © 2003 Wiley-Liss, Inc. Key words: Stat5a; breast cancer; differentiation; p27; Stat5b; Stat3; nuclear localization Discovered originally as a mammary gland factor (MGF), 1 signal transducer and activator of transcription (Stat) 5a plays the most prominent role of all STAT family members during mouse mammary gland development. 2 Activated Stat5a is required for lactation and contributes to the survival and terminal differentia- tion of mammary epithelial cells during pregnancy and lacta- tion. 3 Basal activation of Stat5a in virgin mice and in normal non-pregnant human breast tissues may contribute to early differ- entiation and survival of mammary epithelial cells outside of pregnancy. 4 Like other family members, Stat5a is activated through phos- phorylation at an invariant tyrosine residue in the cytoplasm and then translocated into the nucleus where it can act as a transcrip- tion factor. 5–7 The cytokine-inducible SH2 protein (CIS)/suppres- sor of cytokine signaling (SOCS) family in concert with protein inhibitor of activated Stat (PIAS) family members and various protein tyrosine phosphatases negatively regulate the activity of STATs. 8,9 Serine phosphorylation of STATs contributes to the fine modulation of their transcriptional activity. 7,9,10 In normal mam- mary epithelial cells, the activation of both Stat5a and its closely related family member arising from gene duplication, Stat5b, 11 is governed by the prolactin-signaling pathway. 9,12 Activated Stat5a/b also act to promote the survival and differentiation of hematopoietic progenitor cells of both myeloid and lymphoid lineages. 13–16 Aberrant activation of Stat5 through BCR-ABL has been identified as a key oncogenic event in chronic myelogenous leukemia 17,18 and impairs the apoptotic response to chemothera- peutic agents and DNA damage in various hematopoietic cancer cell lines. 19 –21 Stat3 is another family member located on the same chromo- some 22 that is expressed and activated in human breast cancers and breast cancer cell lines. 7,23–25 Interestingly, activated Stat3 induces physiological apoptosis of mammary epithelial cells in the mouse mammary gland during involution, 26,27 but constitutive activation of Stat3 promotes cell survival and growth in breast cancer cell lines. 7,28 Nevertheless, activated Stat3 has been correlated recently with better prognosis in node-negative breast cancers. 25 Experiments in mouse models of mammary cancer progression have demonstrated that activated Stat5a is an important survival factor for both preneoplastic and malignant mammary epithelial cells, which promotes cancer progression. 29,30 Only limited infor- mation is available, however, on the expression, tyrosine phos- phorylation status and nuclear localization of Stat5a in primary human breast cancers. No immunohistochemical studies of Stat5a in breast cancer have been reported to date. One group of inves- tigators was able to detect by electrophoretic mobility shift assays (EMSAs) high Stat5a DNA binding activity only in 1 of 63 human breast cancer samples, but the authors comment that the DNA binding activity was either low or below the limit of the sensitivity of their detection method in the other tumor samples. They were not able to detect tyrosine phosphorylation of Stat5 by Western blotting. 24 In contrast, activation of Stat5a/b by both prolactin and epidermal growth factor (EGF)-related pathways clearly can be Abbreviations: CIS, cytokine-inducible SH2 protein; EGF, epidermal growth factor; EMSAs, electrophoretic mobility shift assays; IHC, immu- nohistochemistry; IP, immunoprecipitation; Jak, Janus kinase; MGF, mam- mary gland factor; PIAS, protein inhibitor of activated Stat; SOCS, sup- pressor of cytokine signaling; STAT, signal transducer and activator of transcription; WB, Western blot. Grant sponsor: US Army Medical Research; Grant number: DAMD17- 01-1-0310, DAMD 17-03-1-0185; Grant sponsor: NCI; Grant number: P30-CA51008. Dr.Ren’s current address is: Department of Anesthesiology, University of California at Los Angeles, Los Angeles, CA. The first two authors contributed equally to this paper. *Correspondence to: Lombardi Cancer Center, Georgetown University, New Research Building, Room E518, 3970 Reservoir Rd. NW, Washing- ton, DC 20057-1469. Fax: +1-202-6877505. E-mail: paf3@georgetown.edu Received 3 June 2003; Revised 22 July 2003; Accepted 9 September 2003 DOI 10.1002/ijc.11619 Int. J. Cancer: 108, 665– 671 (2004) © 2003 Wiley-Liss, Inc. Publication of the International Union Against Cancer