Dysregulation of apoptotic pathway/s in chordoma: searching for prognostic markers and study of fas and fasl involvement in zebrafish model

Chordoma is a rare malignant bone tumor arising from notochord remnants, characterized by local invasiveness and variable tendency for recurrency. Given the implication of apoptosis in notochord regression, we studied in 32 tumours the expression by RT-PCR of 8 proapoptotic genes mapping in 1p36 region, showing loss of heterozigosity in most chordomas analyzed (83%). TNFRSF8 and TNFRSF14 genes are differently expressed compared to control (nucleus pulposus, NP) in 50% tumors, while DFFA, DFFB, CASP9, TNFRSF1B, TNFRSF14 and TP73 showed occasional different pattern of expression from control. The comparison of the expression profile of each tumour with that of the control didn’t reveal any overlapping. As the apoptotic pathway mediated by FAS-FASL was found to be involved in notochord regression, we studied their expression in 34 chordoma and in 3 derived cell lines to verify their possible dysregulation. Since most analyzed samples express the receptor, but not the ligand, a possible implication of FAS/FASL pathway dysfunction during tumorigenesis has been hypothesized. At this purpose we started an in vivo study on zebrafish (Danio rerio) animal model. We investigated zfas/zfasl expression in embryos' total RNA during different developemental stages and observed that zfas has maternal and ubiquitous zygotic expression, while zfasl has maternal and specific developmental stages expression (2-4, 64-100 cells, 48, 72, 120 hpf, 8, 10, 14 dpf, 6, 9 mm). Notochordal cells were then sorted by FACS, following the injection of a luciferase gene construct activated by a specific physalipherous cell factor, to study zfas/zfasl expression in notochord. A preliminary analysis at 24hpf and 48hpf revealed that zfas is expressed at both stages, while zfasl is expressed in none of them; further stages will be studied to better define zfas/zfasl developmental expression profile. We also observed zfasl expression in the notochord by in situ hybridization at the stage of 9mm larva, when the chondrification process of this structure begins. Further analyses will be performed to determine the expression timing of zfas and zfasl to guide functional studies aimed at silencing these two genes. Possible appearance of aberrant phenotypes during notochord regression will be studied and it will be verified whether the reactivation of zfas/zfasl pathway might rescue normal phenotype.