Abstract A27: Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma
Authors: Josep Roma, Ana Almazán-Moga, Patricia Zarzosa, Pablo Velasco, Carla Molist, Irina Giralt, Isaac Vidal, Natalia Navarro, Miguel Segura, Aroa Soriano, Josep Sánchez de Toledo, Soledad Gallego
Journal: Clinical Cancer Research (2018)

Abstract The Hedgehog (HH) pathway is known to develop an oncogenic role in some cancers, including rhabdomyosarcoma (RMS). However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. Currently, a consistent activation of the pathway is well established and generally accepted in RMS. However, despite controversy regarding the importance of mutation-driven constitutive activation of the pathway in RMS patients, the activator mutations reported to date can only account---in the best scenario---for the activation of the pathway in small subsets of patients; therefore, the mechanism of activation remains elusive for the majority of cases. Purposes of the Study: The main purpose of this study is to demonstrate the importance of the ligand-dependent activation of Hedgehog pathway in this neoplasia. A secondary purpose is the description and characterization of a deleterious effect of the SMO inhibitor vismodegib, which promotes cell invasiveness throughout activation of Unfolded Protein Response (UPR). Experimental Procedures: The expression of Hedgehog ligands was studied in RMS samples by quantitative PCR, Western blot, and immunohistochemistry. Functional assays (cell cycle, cell proliferation, cell invasion, and apoptosis) and animal model studies were carried out with cells transduced with shRNAs (against the 3 Hedgehog ligands and Gli1) or treated with Hedgehog-specific inhibitors (vismodegib and MEDI-5304). Finally, the molecular characterization of an off-target effect of vismodegib was also made by cDNA microarrays, Transwell assay, Western blot, and shRNA-mediated TRIB3 inhibition. Summary of New Data: Results showed a prominent expression of HH ligands (especially Indian and Desert Hedgehog) in RMS cells and tumors, thereby supporting the existence of autocrine ligand-dependent activation of the pathway in this neoplasia. Moreover, although the shRNA-mediated downregulation of the ligands was not complete and performed separately for each ligand, the effects observed on tumor growth were remarkable. Thus, the downregulation of each single HH ligand clearly impaired tumor growth, suggesting that the participation of all ligands is crucial for RMS pathogenesis. Interestingly, a first description of pernicious off-target effect of vismodegib is also reported. Unexpectedly, vismodegib enhanced the invasive properties of all 3 cell lines by 2-fold. The mechanistic characterization of this biologic response suggested an off-target effect of vismodegib on cell invasiveness and chemoresistance via UPR activation. Given that the cell lines studied covered all the main subtypes of RMS, the results strongly suggest that UPR activation may be a general mechanism of cell invasiveness in RMS under vismodegib treatment and not a negligible peculiarity of a single cell line. Conclusions: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols for RMS and other ligand-dependent cancers. Moreover, the description of a pernicious off-target effect of vismodegib may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers or even the premature halting of some of them owing to survival worsening. Therefore, the findings herein described may have significant translational implication not only for RMS but also for other HH ligand-dependent cancers. Citation Format: Josep Roma, Ana Almazán-Moga, Patricia Zarzosa, Pablo Velasco, Carla Molist, Irina Giralt, Isaac Vidal, Natalia Navarro, Miguel Segura, Aroa Soriano, Josep Sánchez de Toledo, Soledad Gallego. Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A27.