Breast cancer is the most common malignancy diagnosed among women in western countries and is the leading cause of death from disease in women aged 25-54. Mortality from breast cancer nearly always results from metastatic disease, rather than from growth of the primary tumor. Consequently, a better understanding of the regulatory molecules and biological processes that drive metastasis is essential for developing more effective cancer therapies. The goal of this project is to identify the roles that a novel serine/threonine kinase, Hunk, plays during breast cancer metastasis, and to thereby facilitate the development of innovative approaches to treat this disease. ? ? Hunk is a SNF1-AMPK-related protein kinase that was identified based upon its dynamic expression during mammary gland development, its upregulation in murine mammary tumors and its association with pathologic hallmarks of aggressive human breast cancer. To investigate the physiological roles of this enzyme, mice bearing a targeted deletion in the Hunk locus were generated. Analysis of these mice has revealed that while Hunk is dispensable for normal development, it is required for the metastasis of mycinduced mammary tumors in a manner that depends upon its kinase activity. However, the mechanisms by which Hunk contributes to metastasis are unknown, as are the pathways that regulate Hunk. ? ? In this application, I will use both in vitro and in vivo approaches to identify the cellular and molecular bases for Hunk-dependent metastasis. In the first aim of this proposal, human tumor cell lines will be utilized to determine Hunk's contribution to their metastatic behavior. Assays will include a structure-function analysis of Hunk domains and the identification and evaluation of Hunk target genes. In the second aim, the regulation of Hunk activity will be explored by examining its phosphorylation status, protein stability, and potential protein interaction partners. Finally, constitutive and conditional Hunk-deficient mice will be employed to determine precisely where in the metastatic cascade Hunk is required and to assess Hunk's contributions to the maintenance of both tumor cell metastatic potential and metastases. Together, these experiments will provide mechanistic insight into the roles that Hunk plays in mammary tumor progression with direct relevance to improving anti-neoplastic therapy. ? ? The studies outlined in this proposal will allow me to learn cutting-edge techniques at one of the country's top institutes, under the supervision of a recognized expert in the field of breast cancer research. The practical as well as intangible skills gained during this training period will provide me with the tools necessary to establish an independent research program bridging the fields of developmental and cancer biology. ? ? ?