Novel Inhibitors of FGF Signal Transduction in Breast Cancer: Targeting the FGFR Adapter Protein SNT-1
Annual rept. 1 Mar 2000-1 Mar 2001
SOUTHERN RESEARCH INST BIRMINGHAM AL
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An enhanced growth of FGF treated estrogen receptor positive ER breast cancer cells in medium containing an antiestrogen ICI 182,780 suggests that FGF signaling completely bypasses a requirement for ER-alpha activation for growth. A lipid-anchored docking protein SNT-1 links FGFR molecules with the RasMAP kinase signaling pathway. The main objective of this work is the validation of SNT-1 as a useful target for drug development. For this purpose we cloned a phosphotyrosine binding domain PTB of SNT-1 in an expression vector and stably transfected a human ER breast cancer cells to study the possible dominant negative effect of the PTB domain on FGF dependent growth phenotype. Using semiquantitative RT PCR we demonstrated different levels of mRNA expression in different transfected clones and studied the FGF-dependent growth of the clones expressing higher levels of the PTB domain. We developed a colony forming test in FGF-dependent growth conditions to study a possible dominant negative effect of the SNT-l PTB domain. A mammalian two-hybrid system which we developed lacked the sensitivity for fine mapping of the SNT-l PTB domain interaction with the FGFR juxtamembrane domain. We overexpressed the PTB domain of SNT-l in E.coli and purified a protein to be used in the Biacore system and for crystallization experiments to study the PTB domain structure and its interaction with FGFR1,2,3,4 peptides.
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