Secreted frizzled related protein 3 (SFRP3) is required for tumorigenesis of PAX3-FOXO1-positive alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (aRMS) is a soft tissue sarcoma associated with the skeletal muscle lineage. The majority of aRMS tumors express the fusion protein PAX3-FOXO1 (PF), which has proven chemically intractable. As such, we identified proteins downstream from or cooperate with PF to support tumorigenesis, including SFRP3 (FRZB). Suppression of SFRP3 using lentivirally transduced shRNAs inhibits cell growth in vitro and tumor growth in vivo. This study aims to identify the genetic changes that underlie the SFRP3 suppression-mediated decreased cell growth. We analyzed changes using Gene Ontology (GO) enrichment and found the induced genes were enriched in striated muscle development/differentiation. In contrast, the repressed genes were enriched in response to stimulus and cell cycle/mitosis genes. We also observed as expected downregulation of SFRP3 (FRZB) but also downregulation of Wnt pathway-repressing genes such as CTBP2 (a transcriptional repressor of TCF, similar to CTBP1 ) and NAV2 (which is downstream from APC). Conversely, we noted upregulation of genes including CCND1 (cyclin D1) and SNAI2 (SLUG), both Wnt signaling target genes and WNT6, which is known to inhibit myoblast proliferation but induce myoblast elongation. A human aRMS cell line (Rh28) was lentivirally transduced with an empty vector (V) or doxycycline-inducible SFRP3 shRNAs (sh3, sh5) and selected for on puromycin. Population doubling 18 (PD18) cells were plated at the same confluence and treated for 5 days with either growth media (No) or growth media supplemented with 4 ug/ul doxycycline (Dox) prior to harvesting. Only empty vector cells treated with growth media (VNo) were included in the array as a control. The gene expression affected by the introduction of doxycyline-inducible SFRP3 shRNAs.