Background TWIST (TW), a bHLH transcription factor with an uninvestigated role in medulloblastoma (MB), has important roles in human cancer and development. Normal cerebellar granule cell precursor development requires sonic hedgehog (Shh) signaling, but mutations causing aberrant Shh signaling can give rise to MB. In vitro, TW directly regulates expression of GLI1, a Shh signaling component. Also GLI1 and TW are co-expressed in rhabdomyosarcoma, a tumor associated with aberrant Shh signaling. Thus we hypothesize that TW plays a role in MB oncogenesis by interacting with Shh signaling. To investigate we analyzed expression of TW and Shh signaling genes in MB cell lines and tumor samples and characterized in vitro Shh signaling inhibition of two MB cell lines using the Smoothened (SMO) inhibitor cyclopamine (cyclo).
Methods Expression of TW and the Shh signaling pathway genes (PATCH, SMO, GLI1, GLI2 and GLI3) were quantified in 16 MB cell lines and primary tumor samples by qRT-PCR and standardized to GAPDH. TWIST expression was analyzed by immunohistochemistry (IHC) using a TW specific polyclonal antibody in selected tumor tissue samples. In DAOY and RES 256 cell lines we analyzed the concentration dependent effects of cyclo (2.5μM, 5μM, 10μM) on total cell number at 24, 48, 72, and 96 hour time points compared to media, vehicle and a related alkaloid compound, tomatadine. Further analysis of cyclo treated RES 256 was performed by flow cytometry (FC) to quantify necrotic cells, apoptotic cells, and the cell cycle phases.
Results TW, PATCH, SMO, GLI2, and GLI3 were highly expressed in the majority of tumors and cell lines with GLI1 having an overall lower expression. IHC staining in tumor sections revealed high TW levels. In RES256 and DAOY, treatment with 10μM cyclo significantly reduced cell number and altered cell morphology. Based on FC data, the reduction of cell number at 10 μM cyclo was not due to increased cell necrosis or apoptosis. However, increased %G2 cells at 48 and 96 hours suggest cyclo may promote cell-cycle arrest at the G2/M checkpoint.
Conclusion High levels of TW suggest a potential in the oncogenesis of MB. Inhibition of Shh signaling by cyclo, in MB, reduces cell proliferation and can provide a useful tool for future investigations of TW interactions with Shh signaling.