) Breast cancer is the second leading cause of cancer death among women in the United States. One major cause of mortality is the formation of metastases, the spread of cancer cells to distant areas of the body by way of the lymph system or the bloodstream. Breast tumor cells metastasize to axillary lymph nodes, the skeleton, and other parts of the body. The inhibition of metastasis is an important goal for therapeutic intervention. One important regulator of the metastatic potential of tumor cells is the transforming growth factor-beta (TGF-beta). TGF-beta has been suggested to play a dual role in the development and progression of tumors of epithelial origin. TGF-beta can function as a tumor suppressor in the early stages and as a tumor promoter in the later stages of the disease. This apparent switch of the role of TGF-beta is reflected in changes of tumor cell responsiveness. The molecular basis for the altered responsiveness is not well understood, but it has been suggested that a cooperation between TGF-beta and Ras signaling pathways is required. A major role of the Smad proteins in mediating the growth inhibitory response of normal epithelial cells to TGF-beta has been well established. However, TGF-beta signaling events leading to epithelial-mesenchymal transition (EMT) and the acquisition of increased tumorigenic properties of transformed epithelial cells are currently unknown.
The specific aims of the proposed project are (a) to determine the contribution of Smad-independent signaling to TGF-beta-mediated EMT of breast cancer cells, and (b) to identify novel signaling molecules interacting with TGF-beta receptor complexes in invasive breast cancer cells. Structure-function analysis of identified molecules will be carried out with the aim of delineating specific structural motifs which could serve as potential targets for drug development. Technically the project will involve the establishment of stable cell lines expressing dominant-negative constructs which specifically inhibit TGF- beta/Smad signaling. These cell lines will be used to determine the contribution of Smad signaling to the described TGF-beta effects on breast tumor cells. Another cell line expressing a kinase-inactive TGF-beta type I receptor will be established and used to isolate biochemically polypeptides which associate with the receptor complexes in tumor cells. The involvement of identified polypeptides in TGF-beta mediated EMT and gain of tumorigenic properties will be studied in both in vitro and in vivo assays.
