Abstract 2630: Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration.

Abstract

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and is thought to arise from primitive mesenchymal cells committed to skeletal muscle differentiation. Less than 30% of patients with metastatic disease survive even with the most intensive treatment regimens. Approximately 50% of patients with RMS have intermediate risk and the event-free survival of the intermediate risk patients is approximately 70%. However, even within this group, the survival of patients with metastatic embryonal tumors is much poorer (∼50%). Thus understanding the molecular mechanism of RMS metastasis and the development of therapeutic strategies to inhibit RMS metastasis is critical for these patients. We have reported that zinc finger transcription factor CASZ1 functions as a tumor suppressor gene in neuroblastoma. In these studies we found that CASZ1 regulates genes involved in cell movement and many pertaining to skeletal/muscle system development. To understand whether the induction of muscle developmental genes by CASZ1 was an anomaly associated with pathology, we evaluated the expression and regulation of CASZ1 in normal myoblasts. CASZ1 expression significantly increased when the C2C12 murine myoblast cell line differentiated upon serum withdrawal. Knockdown of CASZ1 in myoblasts causes a 30% increase in cell migration (p<0.02) as determined using an Essen IncuCyte scratch-wound healing assay. Furthermore knock-down of CASZ1 in C2C12 cells leads to a 50% decrease in expression of the skeletal muscle development gene myogenin while over-expression of CASZ1 leads to 2-fold increase in myogenin mRNA. Since CASZ1 regulates aspects of normal muscle development, we hypothesized that its dysregulation in RMS may impact tumor progression. To test this hypothesis, we first evaluated the expression of CASZ1 in primary RMS tumors. We found that CASZ1 mRNA level in embryonal RMS patients’ samples is ∼1.5 lower than in normal muscle or alveolar RMS (p<0.001). To investigate the role of CASZ1 in embryonal RMS cell growth and migration, we developed a Tet-On CASZ1 expression system using the RD RMS cell line. Overexpression of CASZ1 does not affect RD cell proliferation based on MTS and IncuCyte cell confluence assays. Over-expression of CASZ reduces cell motility by 20% (p<0.01) using an Essen IncuCyte scratch-wound healing assay. Knockdown of CASZ1 in RD cells increases cell migration by 40% (p<0.01) but does not affect cell proliferation. However when CASZ1 is expressed in the presence of a muscle inducing differentiation cue, 12-O-tetradecanoylphorbol-13-acetate, TPA, the proliferation of RD cells is suppressed an additional 40% (p<0.01). Taken together, our results suggest that regulation of myogenin expression and cell migration by CASZ1 in C2C12 maybe integral to proper myogenic differentiation, and dysregulation of CASZ1 during early myogenesis may contribute to embryonal RMS tumorigenesis and metastasis. Citation Format: Zhihui Liu, Wilson Chan, Carol J. Thiele. Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2013-2630 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.