Figure 6. YAP is upregulated and pro-proliferative in aRMS.

(A) Phosphorylated MST1/2 (MST1 T183, MST2 T180), MST1, phosphorylated (S127) YAP, and YAP levels in HSMMs (proliferating and differentiated) and RMS cell lines as measured by immunoblot analysis. ERK1 was used as a loading control. (B) Left: Representative images from RMS TMAs immunostained for YAP protein. Scale bars: 100 μm. Right: Quantitation of YAP-immunostained RMS TMAs. Muscle, n = 11; eRMS, n = 58; aRMS, n = 72. Error bars represent SEM. *P < 0.0001, Mann-Whitney test. (C) YAP1 shRNA knockdown validation in HSMM^PF+H+M and Rh28 cell lines as measured by immunoblot analysis. YAP1 knockdown aRMS cells were defective in proliferation, as measured by BrdU incorporation (D). YAP1 loss induced senescence in aRMS cells as measured by β-gal staining (E). (F) Model of RASSF4-mediated suppression of the Hippo pathway in aRMS. PAX3-FOXO1 transcriptionally upregulates RASSF4 through a 5′ enhancer region. Subsequent RASSF4 protein associates with MST1 and inhibits signaling to MOB1. While YAP expression is elevated in aRMS cells and tumors, a connection between RASSF4 and YAP remains to be elucidated. Both MST1 inhibition and YAP expression support cell proliferation and senescence evasion to promote tumorigenesis in aRMS cells.