Identification Of Gli1 Binding Partners That Regulate Transactivation Or Cell Transformation

Activation of the GLI1 transcription factor and oncogene appears to play a role in the genesis of desmoplastic medulloblastomas, basal cell carcinomas, and some rhabdomyosarcomas. We identified interactions between GLI1 and proteins that enhance the transactivating or cell transforming function of GLI1. Such protein-protein interactions could potentially be targeted in the treatment of some GLI1-related malignancies. We focused on proteins interacting with GLI1 transactivation domains, believing these would be essential for transactivation and cell transformation. Previously, we identified an acidic alpha-helical transcription activation domain (TAD1; aa 1020-1091) at the carboxyl terminus of the GLI1 protein. We show that TAD1 physically interacts with the coactivator TAF9 in vitro by pull down assays and in rhabdomyosarcoma cells in vivo by proximity ligation assays and co-immunoprecipitation. Inhibition of this interaction reduces transactivation by GLI1 and the ability of GLI1 to transform RK3E cells. We identify a second GLI1 transactivation domain (TAD2; aa 635-740), which activates reporter gene transcription and accounts for the TAD1-independent cell transforming ability of GLI1. TAD2 contains conserved sequences among GLI1 family proteins, including a CBP binding domain identified in GLI3. However, consistent with in vitro data from others, we could not demonstrate interaction of TAD2 and CBP in rhabdomyosarcoma cells in vivo by proximity ligation assay. To identify TAD2 binding proteins, we performed yeast 2 hybrid screening and identified other transcription factors (Y-Box1, HSF2, RBMX2, and ZMYM2), cytoskeleton-associated proteins and membrane-associated proteins. Based on a previously described GLI1-p53 inhibitory loop that controls stem cell and tumor cell numbers, we demonstrate that p53 down-regulates TAD1 but not TAD2 function. Understanding the mechanism of transactivation and cell transformation by GLI1 can provide novel targeted approaches to inhibit the GLI1 transcription factor in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2200. doi:1538-7445.AM2012-2200