Role of SFRP3 in PAX3-FOXO1-positive alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (aRMS) is an aggressive cancer of skeletal muscle histogenesis that affects children and adults. A signature aRMS mutation is PAX3-FOXO1, which encodes a “super-transcription factor” thought to illegitimately reactivate embryonic myogenic pathways. PAX3-FOXO1-positive patients with metastasis have a 4-year survival of <10%. Understanding PAX3-FOXO1 pathogenicity is essential for designing novel aRMS therapies. Using primary human skeletal-muscle myoblasts (HSMMs) as precursors, we engineered a genetic model of aRMS and found that PAX3-FOXO1 enables proliferation past the senescence checkpoint. To identify genes downstream of PAX3-FOXO1 supporting this senescence bypass, we performed transcriptome profiling and observed changes in several Wnt pathway members. Most intriguing was SFRP3, which encodes a secreted protein that traps Wnt ligands and inhibits Wnt signaling. Preliminary studies show that SFRP3 is upregulated in human aRMS cells, and that its genetic suppression by shRNA blocks aRMS cell proliferation and (in pilot xenograft studies) aRMS tumorigenesis. Wnt is a conserved developmental signaling network with canonical and non-canonical branches that is found frequently dysregulated in epithelial cancers, but only recently beginning to be therapeutically targeted. Wnt has been minimally studied in RMS. During normal skeletal myogenesis, SFRP3 suppresses canonical Wnt signaling to prevent premature myogenic differentiation. We hypothesize that in aRMS, SFRP3 similarly silences Wnt signaling to promote tumor cell proliferation. Our Specific Aims are to (i) understand how PAX3-FOXO1 and SFRP3 are related, including whether SFRP3 is a transcriptional target of or cooperates with PAX3-FOXO1, (ii) establish the Wnt pathway impacted by SFRP3 by interrogating canonical Wnt pathways and using proteomics to identify the Wnt ligand trapped by SFRP3, and (iii) determine whether SFRP3 is a useful pre-clinical target using conditional expression of shRNAs against SFRP3 in a validated aRMS xenograft system.