Study Of Key Molecular Pathways In Chordoma And Classification Of Chordoma Using Genomics Technology

Chordomas are rare malignant bone tumors arising from notochordal remnants, thus a major challenge in studying chordomas is the lack of normal tissue to serve as a control. The low prevalence of this disease is another difficulty. T gene is the most well studied gene in chordomas. Both familial and sporadic chordomas have been associated with the T gene, although population heterogeneity was observed. The brachyury protein, which is encoded by the T gene, has been used as a diagnostic marker, but not as a prognostic indicator of chordoma, with inconsistent study results. So, further study on the role of T gene in chordomas and identifying new genes and pathways involved in chordoma are still necessary. By sequencing miRNAs and mRNAs of 2 tumor and 2 normal notochord samples, we found that TGFβ signaling pathway was inactivated by downregulation of TGFβ3. We also found somatic mutations in two regulators of TRAF6, which is a key gene in TGFβ signaling pathway by exome sequencing. Furthermore, downregulation of TGFβ3 was due to dramatic upregulation of its inhibitor miR-29 in chordomas (R2=0.87) and due to somatic copy number loss at TGFβ3 locus in more than half of the tumors tested. Somatic copy number gains in miR-29 genes bl/c and b2/a were observed in most chordomas. These results suggested the important roles of TGFβ3 and miR-29 in the inactivation of TGFβ signaling pathway. Then we test the expression of TGFβ3 and miR-29 in 8 chordomas and 8 notochords using qPCR, and found that TGFβ3 was downregulation in chordomas and miR-29 was upregulated in chordomas. The upregulation of miR-29 and downregulation of TGFβ3 were also found in a chordoma cell line UM-Chorl and proliferation inhibition was observed by TGFβ3 treatment. Moreover, in a zebrafish model in which TGFβ3 was knocked down by morpholino-oligomers, chordoma formation was revealed by H&E (haematoxylin and eosin) staining and immunohistochemistry of brachyury in larvae. These results demonstrated, for the first time, that the dramatic inhibition of TGFβ3 by multiple somatic changes was a major cause of chordoma development. To investigate the role of Tgene isoforms in chordoma,22 skull base chordomas,3 chordoma cell lines and 9 infant notochords, which were used as normal controls, were collected. We first conducted ddPCR (droplet digital PCR) to quantify the absolute expression levels of the long and short isoforms of the T gene (T-long and T-short, respectively) and revealed that T-long was dominantly expressed in all chordomas and chordoma cell lines, but not in the notochords. The distinct expression patterns of these T gene isoforms may contribute to the pathogenesis of chordomas. We investigated the methylation profile of 9 conventional chordomas using methylation chip. Cluster analysis revealed two subclasses of chordoma, one of which was more close to normal tissues. There were differences in MRI (Magnetic Resonance Imaging) and H&E staining results between the two subclasses, indicating the existance of subclasses within conventional chordomas, though experiment using more samples were still needed.