Role of sonic hedgehog inhibition in nb and characterization of stemness gene expression in neuroblastoma and ewing sarcoma/pnet

Resumen

Neuroblastoma (NB) and Ewing Sarcoma (ES) are two aggressive forms of childhood cancer, affecting the autonomic nervous system and soft tissues and bones, respectably. This thesis work is focused on the analysis of stem cell-like populations in these tumors, through molecular characterization of stemness gene expression. Moreover, the study of expression and inhibition of the Sonic Hedgehog signaling pathway (Hh) in these two tumor entities was performed. We found that Hh signaling (was active in NB and ES, as most pathway components are expressed, being GLI1 over-expressed in cell lines and a subset of tumors. Furthermore, SHH ligand was found to be present in cell lines and tumors and GLI1 up-regulation was achieved in response to ligand treatment, suggesting an autocrine mechanism of aberrant activation. Hh signaling was modulated by means of pharmacological antagonist (cyclopamine) binging to Shh co-receptor SMO. The biological effects produced upon Hh inhibition were: decrease in proliferation and tumorigenic potential, together with increased apoptosis. Also a dramatic decrease in the percentage of CD15+ population was found. Our data suggest a central role for aberrant Hh signaling in NB and ES tumorigenesis, implying that this kind of tumors might be dependent on Hh signaling. Furthermore growth impairment was observed also in cells growing as neurospheres, a culture mode selecting for neural stem cells, previously shown to model important aspects of the original neural tumor. Our data suggest that tumor stem-like cells are possibly contained in NB studied, as they display an immature stem-like phenotype, expressing stem cell markers as well as markers of chemioresistence. The cells also were able to grow in stem cell media, forming neurospheres and displaying a distinct stem cell marker expression. Epigenetic regulation of stem cell marker CD133 was also analyzed. We found that several NB and ES cell lines featured a half-methylated CD133 promoter, all of which contained a low percentage of CD133+ cells. In addition, we were able to restore CD133 expression at RNA and protein level after demethylation treatment, suggesting that methylation plays an important role on CD133 expression in NB and ES.