Investigation of Chordoma-like Tumors in Zebrafish Embryos Induced by SB-505124 Treatment Open Access
Bellah, Jeffrey (2017)
Abstract
Chordomas are rare, malignant bone cancers that affect the axial skeleton and are believed to develop from the remnants of the notochord. Signaling by Nodal, via the TGF-beta signaling pathway, is important in the control of proliferation and differentiation in notochord development. One of the genes Nodal influences, Brachyury, is known to be a marker for chordomas. We have found that zebrafish embryos treated with the drug SB-505124, which inhibits the TGF-beta signaling pathway, develop tumors that resemble chordomas in their expression of ta, a homolog of Brachyury. We initially hoped to use microarray analyses of SB-505124-induced tumors and control notochord tissue to compare expression patterns of SB-505124-induced tumors with those of chordomas. In attempting to validate the microarray through in situ hybridizations, there were numerous inconsistencies, which suggest that the microarray results are in error, though we do not currently understand the reason why. Though the protocol for our RNAseq analysis of the tumors is still being refined before comparison with human chordoma datasets, the initial dataset has proven to be more verifiable than the microarray analyses. As an additional comparison, we have conducted in situ hybridizations using probes for zebrafish orthologs of published diagnostic markers for chordomas. The results suggest significant similarity to chordomas, since 4 of the 11 diagnostic markers tested so far have exhibited expression of a zebrafish ortholog in the tumors. We have identified that the signaling of the ligands tgfb2 and tgfb3 from the notochord to the receptor tgfbr1b on nearby tissues is the most likely signaling event that SB-505124 is inhibiting during tumorigenesis due to their mRNA expression patterns, which may lead to tumorigenesis in a cell non-autonomous fashion. Our inability to recreate the tumor phenotype using alternate methods of inhibiting TGF-beta signaling suggests that SB-505124 may possess a novel activity in addition to inhibiting TGF-beta signaling that plays an important role in tumorigenesis. This does not exclude a role for the inhibition of TGF-beta signaling in tumorigenesis though, because preliminary results suggest that the inhibition of TGF-beta signaling via alternative inhibitors increases tumorigenesis in low dosage SB-505124 treatments.
Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Future Directions for a Zebrafish Chordoma Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Figures and Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Figure 1: SB-505124 induces tumor formation
Figure 2: Tumor phenotype penetrance is correlated to SB-505124 concentration
Figure 3: Initial characterization of 3-4dpf SB-505124 treated embryos
Figure 4: Histology of control notochord and tumors
Figure 5: Notochord dissection
Figure 6: SB-505124-induced tumors express ngs, ehd2b, and c7a
Figure 7: Microarray entries of known SB-505124-induced tumor markers
Figure 8: SB-505124-induced tumors express ptrfb and sncga
Figure 9: SB-505124-induced tumors express 4 chordoma diagnostic marker genes
Table 1: Expression of ALK4, ALK5, and ALK7 orthologs in zebrafish
Figure 10: Expression patterns of TGF-beta and Activin ligands
Figure 11: Morpholino-induced knockdown of tgfb2/tgfb3 does not cause notochord tumors
Figure 12: Alternative inhibitors of ALK4, ALK5, and ALK7 do not cause notochord tumors
Figure 13: Cotreatment with SD-208 increases the penetrance of the SB-505124-induced tumor phenotype
Figure 14: Transgenic lines expressing fluorescent protein in notochord and tumor cells
Figure 15: Decreased tumor formation in rapamycin cotreatment
Figure 16: Rapamycin cotreatment significantly decreases the tumor prevalence in SB-505124 treated embryos
Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Figure A1: Expression of tgfbr1a in wild-type embryos
Figure A2: Expression of tgfbr1b in wild-type embryos
Figure A3: Expression of acvr1ba in wild-type embryos
Figure A4: Expression of acvr1bb in wild-type embryos
Figure A5: Expression of acvr1c in wild-type embryos
Figure A6: Expression of acvr1 in wild-type embryos
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