<p>Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition.</p><p>First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression. </p><p>Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. In conclusion, SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/10503 |
| Date | January 2015 |
| Creators | Kephart, Julie Grondin |
| Contributors | Linardic, Corinne M |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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