STAT (Signal Transducers and Activators of Transcription) proteins are latent transcription factors that become activated by phosphorylation on a single tyrosine, typically in response to extracellular ligands. Ligand dependent activation of the STATs is often associated with differentiation and/or growth regulation while constitutive activation of STATs is often associated with growth dysregulation. In a growing number of human cancers (particularly breast cancer) and transformed cell lines, Stat3 is active as a persistently tyrosine phosphorylated DNA binding protein (constitutively activated). In a number of these cases, activated Stat3 is either required for transformation, enhances transformation or blocks apoptosis. We have engineered a constitutively dimerizeable Stat3, termed Stat3-C. This molecule is capable of driving transcription and induces cell transformation, thus qualifying Stat3 as a proto-oncogene. A number of Stat3 target genes have been identified which could participate in Stat3's role in cellular transformation. One potentially important Stat3 target genes includes CyclinD1, which is associated with breast cancer development. We have also developed an inducible system for expression of Stat3-C and observe over time inducible cellular transformation. Stat3 is constitutively activated in about6O percent of freshly isolated breast cancers, as determined by biochemical assays. We have developed an immunohistochemical technique to identify which cells contain constitutively active Stat3 protein. Preliminary evidence indicates that Ductal Carcinoma In Situ (DCIS) and some fraction of invasive cancers contain tyrosine phosphorylated Stat3 protein, suggesting that activated Stat3 may be an early indicator of the development of breast carcinoma. The purpose of this proposal is to 1) determine if Stat3-C can mediate transformation of Human Mammary Epithelial Cells (HMECs) as well as induce breast tumors in transgenic animals and to begin to understand the mechanisms by which constitutively activated Stat3 leads to cellular transformation; and 2) by immunohistochemistry determine the distribution of activated Stat3 in a variety of human cancerous breast tissue sections. We will then correlate the distribution of activated Stat3 with expression of other indicators of aggressiveness or differentiation. In addition, we will begin to characterize the potential regulators of Stat3 activation in these tissue specimens.
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