Abstract 2118: Phenotypic diversity of disease-associated transforming growth factor-β (TGF-β) type I receptor gene (TGFBR1) mutants
Authors: Rosman Diana S. , Bromberg Yana , Weinstein Aliyah , Reiss Michael 
Published: July 2012

Abstract
Transforming Growth Factor-β (TGF-β) inhibits cell growth and drives of epithelial-to-mesenchymal transition, invasion and migration. Somatic mutations in the TGFBR1 and -2 genes have been found in cancer (n=9), while germline mutations are the proximal cause of the Loeys-Dietz aortic aneurysm syndrome (LDS)(n=42). Each of the 40 missense mutations associated with disease affect the protein kinase domain. The majority of mutations affect buried residues, where biochemically non-conservative substitutions may disrupt protein folding. This is in sharp contrast with disease-associated mutations in TGFBR2, all of which affect solvent accessible residues and likely alter protein-protein interactions. We performed an in silico analysis of TGFBR1 mutants using SNAP (Screening for Non-Acceptable Polymorphisms), a sequence-based method to predict the functional effects of single amino acid substitutions. First, the means of SNAP scores for all possible non-conservative substitutions at each residue were used to predict functionally important domains of the wild type protein. All of the disease-associated TGFBR1 mutants was located within such domain. Secondly, 35 (92%) of the 38 disease-associated TGFBR1 missense mutations were correctly classified as non-neutral. In order to validate these predictions experimentally, we have constructed a library of HA-tagged mutants in a pcDNA3 backbone. Thus far, six of these (T200I, K223E, A230T, K232E, N267H, and S387Y) have been characterized in detail. The known kinase inactive K232R mutant was included as negative control. To determine the effect of these mutations on TGF-β-regulated gene transcription, we carried out transient transfection assays using the Smad-responsive SBE4-Luc reporter in both wild type (Mv1Lu) and TGFBR1- deficient (R1B) mink lung epithelial cells. In R1B cells, the T200I, K232E, and K232R mutants were inactive, while A230T, K223E, N267H, and S387Y restored TGF-β-induced SBE4-Luc responses. Interestingly, the K223E mutation constitutively activates SBE4-Luc signaling in the absence of TGF-β, while the response of the S387Y mutant to TGF-β is attenuated. As expected, T200I, K232E and K232R were unable to phosphorylate Smads 2 and -3, while S387Y, K223E, and A230T had retained their kinase activity. Interestingly, the ability of the N267H mutant to activate pSmad2 is attenuated compared to wild type TGFBR1. Additionally, A230T is able to induce pSmad2 in the presence of the kinase inhibitor, SD-093, indicating that this mutant kinase is constitutively active. In conclusion, disease-associated TGFBR1 gene mutants display considerable phenotypic diversity, ranging from complete loss of function to constitutive activation. These findings have important implications for the selection of patients with cancer or LDS for treatment with TGFBR1 selective kinase inhibitors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2118. doi:10.1158/1538-7445.AM2011-2118