Abstract A57: The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis

Abstract

Abstract Introduction: This study aims to understand the contribution of the Wnt pathway inhibitor secreted frizzled related protein 3 (SFRP3) to alveolar rhabdomyosarcoma tumorigenesis. 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 <30%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, our goal is 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. Experimental procedures: Microarray analysis using the Affymetrix U133A platform was performed to compare the transcriptomes of human skeletal muscle (HSMM) cells stably expressing either an empty vector or PF. Only genes that were differentially regulated at least three-fold were considered for further study. The microarray was validated and SFRP3 mRNA levels were assessed using RT-PCR. Lentiviral technology was used to stably express shRNAs in human PF-positive aRMS cell lines Rh28 and Rh30. Cell growth, cell proliferation, apoptosis, and cell cycle analysis were measured by MTT assay, BrdU assay, immunoblot for cleaved caspase 3, and by PI staining followed by analysis by flow cytometry, respectively. For the xenograft tumors in mice, doxycycline-inducible SFRP3 shRNAs were stably expressed in aRMS cells. These cells were injected into the flanks of SCID/beige mice to generate the tumors. Induction of the shRNAs by doxycycline administration began once the tumors were palpable. All animal work was performed under institution approved IACUC protocols. Results: In a microarray analysis of the transcriptomes of human skeletal muscle myoblasts (HSMM) with an empty vector or PF, it was noted that the Wnt pathway was preferentially represented in HSMMs with PF. Of these, SFRP3 was selected for further investigation as its mRNA levels were elevated in PF-positive aRMS cells, but not eRMS or HSMM cells. shRNAs against SFRP3 were generated and stably expressed in Rh28 and Rh30 cells, validated to suppress SFRP3, and shown to profoundly decrease cell growth (p<0.001). Associated with this decreased cell growth was a decrease in cell proliferation (p<0.001), an increase in apoptosis, and an increase in the percentage of cells at G1, suggesting a cell cycle arrest. Consistent with a G1 cell cycle arrest, shRNAs against SFRP3 also increased the levels of p21, an inhibitor of cell cycle progression. To assess the effects of SFRP3 shRNAs on tumor growth in a xenograft model of aRMS, a doxycycline-inducible system was used, allowing the shRNAs to be expressed only after the tumors formed. In this model, suppression of SFRP3 reduced tumor growth and tumor weight by more than three-fold. Further, in a pilot study, the combination of SFRP3 suppression with vincristine, a standard chemotherapeutic agent used in the treatment of aRMS, was more effective at reducing cell growth in vitro than with either treatment alone and ablated tumor growth in vivo. Conclusions: In conclusion, SFRP3 is necessary for the growth of aRMS cells both in vitro and in vivo. Inhibition of SFRP3 combined with standard of care chemotherapy should be explored further as SFRP3 is a promising new target in the treatment of PF-positive aRMS. Citation Format: Julie Kephart, Rosanne Jones, Lisa Crose, Jen-Tsan Ashley Chi, Corinne Linardic. The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A57.