Abstract 4259: Active hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS).

Abstract RMS is the most common sarcoma of childhood. The majority of embryonal RMS (ERMS) and a minority of alveolar RMS (ARMS) express HH pathway components. However, a role for the HH signal transduction pathway is incompletely defined in RMS. Co-regulation of GLI1 and Patched1 (PTCH1) in ERMS and fusion-negative ARMS have suggested that the HH pathway is functionally intact in these subtypes. However, HH pathway activity has not been directly tested in RMS. Our aim was to demonstrate that some RMS cells have intact HH signaling pathways and are responsive to HH pathway activation or inhibition. We demonstrated expression of Indian HH, Desert HH, PTCH1, SMO and GLI1 by qRT PCR in Rh18 ERMS cells and Rh41 ARMS cells; Desert HH, PTCH1, SMO, and GLI1 in Rh 30 ARMS cells; and Indian HH, Desert HH, PTCH1, and SMO in RD ERMS cells. We confirmed high GLI1 protein expression by Western blot in Rh41 and Rh18 cells. Since HH signaling is believed to occur in primary cilia, we used immunofluorescence to demonstrate primary cilia in RMS-13 cells. Based on their differing levels of GLI1 expression (high in Rh41 and Rh18 cells and low/absent in Rh28 and RD cells), these RMS cell lines provided an opportunity to up- and down-regulate the HH pathway and measure effects on gene expression and cell function. Exposure of Rh41 cells to Sonic HH-containing conditioned media caused a 12-fold increase in PTCH1 expression and exposure of Rh28 cells to Sonic HH peptide increased GLI1 expression 1.5-fold, suggesting intact HH signal transduction in some RMS cell lines. We down-regulated HH pathway activity with the GLI1 inhibitor GANT61 and assessed Rh41 and RD cell viability by MTT assays. RMS cell viability decreased following exposure to increasing concentrations of GANT61. The reduced cell viability appeared to result at least in part from down-regulation of the anti-apoptotic GLI1 target gene, BCL2. These results suggest a role for HH signaling in RMS cell survival. Understanding the role of HH signaling in RMS biology may contribute to successful introduction of HH pathway inhibitors into RMS treatment. Citation Format: Joon Won Yoon, Marilyn Lamm, King-Fu Leong, Stephen Iannaccone, Philip Iannaccone, David Walterhouse. Active hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS). [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 4259. doi:10.1158/1538-7445.AM2013-4259