Abstract 2440: FEM1A regulation of the NF-κB/YY1/miR-29 circuitry in rhabdomyosarcoma

Introduction: Rhabdomyosarcoma (RMS), a malignant tumor related to defective skeletal muscle differentiation, is the most common soft tissue neoplasm of children, and metastatic RMS continues to have a poor prognosis. The canonical form of transcription factor NF-κB, a p50/p65 heterodimer, regulates normal myogenic differentiation, and is dysregulated in RMS tumorigenesis. NF-κB is active in myoblasts and blocks myogenic differentiation; for myogenic differentiation to occur, NF-κB must be down-regulated, which leads to down-regulation of YY1 and de-repression of miR-29, a microRNA that promotes myogenic differentiation. Conversely, NF-κB is up-regulated in RMS, leading to activation of YY1, down-regulation of miR-29, and suppression of myogenic differentiation. The mechanisms of NF-κB regulation in these contexts are incompletely understood. FEM1A is a gene most highly expressed in cardiac and skeletal muscle. We have shown that FEM1A is up-regulated early during skeletal muscle differentiation, and is consistently down-regulated in RMS, both in human RMS and in mouse RMS genetic models. However, whether down-regulation of FEM1A plays a role in RMS tumorigenesis, or is just a marker of defective myogenic differentiation, is unknown. In macrophages, FEM1A has been shown to inhibit canonical NF-κB, both by directly binding p50, and by binding and preventing the degradation of p105, the precursor of p50 that also acts as a cytoplasmic inhibitor of NF-κB activation. Therefore, we tested the hypothesis that over-expression of FEM1A in RMS cells decreases NF-κB activation, which in turn results in decreased expression of YY1, leading to increased expression of miR-29, promoting RMS myogenic differentiation. Methods: Cultured RMS13 rhabdomyosarcoma cells were used. To determine if FEM1A regulates NF-κB/YY1/miR-29 circuitry, resulting in RMS13 differentiation, we transfected RMS13 cells with HA-FEM1A using X-treme Gene HP (Roche). Results: Using immunoblotting, we observed that FEM1A overexpression decreases p50 and p65 translocation to the nucleus, decreases YY1 protein levels, and increases myosin heavy chain expression, a marker of skeletal muscle differentiation, in RMS13 cells. Using qPCR, we observed that FEM1A overexpression increases miR-29 gene expression in RMS13 cells. Conclusion: These data demonstrate that FEM1A negatively modulates NF-κB and YY1, and promotes miR-29 expression and myogenic differentiation, in RMS cells. This indicates that FEM1A down-regulation may play a role in RMS tumorigenesis, rather than just being a marker of defective differentiation. Results from our study have the potential to improve understanding of RMS tumor biology, and could allow the FEM1A/NF-κB/YY1/miR-29 circuitry to become a target for molecular-based therapy of RMS. Citation Format: Samira C. Grifoni, Joseph F. Maher. FEM1A regulation of the NF-κB/YY1/miR-29 circuitry in rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2440.