Abstract 5053: Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line.

Abstract

Abstract Medulloblastoma is one of the most frequent and aggressive tumors of childhood. The Sonic hedgehog (Shh) pathway, related to human development, is altered in most medulloblastomas: genes like Ptch, Smo, or Sufu suffer mutations in 15% to 25% of these tumors. We tested Shh inhibition in the Daoy medulloblastoma cell line by two methods: a molecular one (direct Gli1 siRNA inhibition); and a pharmacological inhibition of Smo, upstream of Gli1, by cyclopamine. Afterwards, a comparison of cellular and molecular responses was done. We proved that MTT cell viability, and cell migration assessed by the scratching assay decreased after Shh inhibition. Furthermore, colony formation assay in culture decreased by 70%, and colony formation assay in soft agar decreased up to 90% when Shh inhibition was applied. As a whole, Shh inhibition conferred a less in vitro tumorigenic status to Daoy cells. Moreover, we assessed the expression of different Gli1 target genes and other genes, before and after Shh inhibition, and found that Shh shows a crosstalk with oncogenes and tumor suppressor genes that have been described in numerous tumors. Therefore, we found downregulation of Ptch1, Cyclin D2, Plakoglobin, Nkx2.2, Bmi1, Smo and N-myc after Shh inhibition. Sufu and Gli3 showed parallel results, where Gli1 siRNA did neither decrease nor increase expression of both genes, whereas cyclopamine reduced them in 15-25%. Pax6 mRNA levels were upregulated by either Gli1 siRNA or cyclopamine. Finally, Notch1 was upregulated after inhibition of Shh, while Notch2 showed contrasting results, as siRNA inhibition decreased its expression, while cyclopamine increased it. All these experiments give an overview of the Shh pathway in medulloblastoma, its relationship with other genes, and the demonstration of the efficacy of cyclopamine and Gli 1 siRNA Shh inhibition in vitro. Citation Format: R Garcia-Lopez, B Vera-Cano, A Vacas-Oleas, J de la Rosa, G Gallo-Oller, M H. Shahi, X Fan, M M. Alonso, J A. Rey, Javier S. Castresana. Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5053. doi:10.1158/1538-7445.AM2013-5053