Purpose:
Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of many different cell types. Escape from TGF-beta-induced growth inhibition is frequently observed in tumors. TGF-beta initiates its cellular responses by binding to and activating receptors with intrinsic serine/threonine kinase activity and their downstream effectors termed Smad proteins. Smads relay the TGF-beta signal from the cell membrane to the nucleus, where they affect the transcription of target genes. The specific aims of the proposed study are: (1) to identify interacting proteins for Smad3 and Smad4 through protein purification using Smad affinity columns, (2) to identify novel genes that perturb TGF-beta-induced growth arrest by performing genetic screens in mammalian cells, and (3) to determine the mechanism of action of the identified novel gene products in regulating TGF-beta/Smad signaling.
Plan of investigation:
Smad interacting proteins will be isolated through their specific interaction with recombinant (phosphorylated) Smad3 and Smad4 immobilized to a solid support, and additional purification by sodium dodecylsulfate polyacrylamide gels. Smad interacting proteins will be identified through peptide mass mapping and/or amino acid sequence analysis. The interaction will be analyzed in vivo by co-expression in mammalian cells, and their effect on Smad subcellular distribution, stability and transcriptional responses investigated. In addition, we will infect mink lung epithelial cells, that express the receptor for ecotropic Moloney retrovirus, with retroviral cDNA libraries. The cDNA library will be prepared from human cancer cell lines resistant to TGF-beta. The infected cells will be selected in hygromycin, and subsequently subjected to TGF-beta and clones that continue to grow isolated. The cDNAs will be recovered by Cre-mediated excision of integrated proviruses from the genome. In addition, we plan to use retroviral insertional mutagenesis in mink lung epithelial cells, and select for TGF-beta resistant clones. The (in)activated genes will be identified by cloning the flanking sequences. The level at which the components act in the TGF-beta signal transduction cascade will be elucidated, and their expression in TGF-beta resistant versus sensitive cells analyzed.
Significance of possible results / Relevance for cancer research:
Considering the pivotal role of Smad3 and Smad4 in TGF-beta signaling and their tumor suppressor function, the isolation of proteins that interact with these two proteins and regulate their activity, will yield valuable insights into these processes. Novel components may be identified, or our studies may reveal novel functions of already known proteins. The proposed genetic approaches have the potential to find critical components acting at any level in the TGF-beta signal transduction pathway, and may even function parallel to the established TGF-beta/Smad pathway.
TGF-beta receptors and Smads were found to be frequently mutated in particular human tumors, and this has been shown to correlate and contribute to an increased tumorigenicity. Mice deficient in Smad3 develop metastatic tumors in the colon. Elucidating the mechanisms that underlie TGF-beta/Smad signaling and resistance to TGF-beta-induced growth arrest through inactivation of TGF-beta signaling components or through overexpression of specific proto-oncogenes in tumor cells may allow for the development of novel strategies to treat cancer patients. |
