| Each year more than one million new cases of breast cancer are diagnosed worldwide and an estimated 370,000 women die from breast cancer. Although the vast majority of fatal breast cancer cases involve metastatic spread of the primary tumor, the formation of metastases is still a poorly understood, complex process. Identifying the early molecular changes that facilitate metastasis of breast cancer will lead to new molecular targets for prevention of metastases and improved therapies.; Intriguing data from the mentor's laboratory show loss of activation of the transcription factor Stat5 during breast cancer progression and that tumors without active Stat5 have higher histological grade, increased mitotic rate, and unfavorable prognosis. Furthermore, data developed in the preparation of this dissertation indicate a substantial growth-inhibitory and pro-differentiation role for Stat5 in mammary epithelial cells. Based on these and other recent observations, we postulate that loss of Stat5 activation in breast cancer represents a progression event that leads to dedifferentiation and increased risk of metastatic invasion. Therefore, a critical analysis of the role of Staff in human breast cancer is warranted, including systematic efforts to identify genes directly controlled by Stat5.; The recent completion of the human genome sequence presents new opportunities for global identification of Stat5 target genes. Work performed in the preparation of this dissertation has established new methodology to capture, clone, sequence, and validate physiological Stat5 DNA-binding sites in a genome-wide manner. The method can also be used to determine whether Stat5 interacts with a known Stat5-responsive promoter within a given experimental context when coupled with PCR amplification of the target DNA. Using this methodology, we have demonstrated that glucocorticoids markedly alter the pattern of chromatin access for Stat5 binding in breast cancer cells. Furthermore, we also report novel methodology to specifically identify transcripts directly regulated by Stat5, based on dominant-negative, differential suppression of Stat5 regulated transcripts and large-scale gene chip analysis. As a result of this work, rapid progress in genome-wide identification of Stat5 target genes is now possible, as well as molecular mapping of the regulatory role of Stat5 in breast cancer. |
