Browsing by Subject "Rhabdomyosarcoma"

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children and adolescents, is characterized by skeletal muscle features. The Ras-driven subset, which includes the embryonal (eRMS) and pleomorphic (pRMS) histologic subtypes, is an aggressive high risk subgroup with a 5-year survival rate of <30%. Recently the YAP oncoprotein, which is ordinarily silenced by the Hippo tumor suppressor pathway, was found to be highly upregulated in RMS tumors. However, the role of YAP in the Ras-driven subset was unknown.

In patient-derived Ras-driven eRMS cell lines, we suppressed YAP genetically via shRNAs. YAP suppression decreased cell proliferation, increased myogenic differentiation, and promoted apoptosis in vitro and in vivo in subcutaneous xenografts. Pharmacologic inhibition by the YAP-TEAD inhibitor verteporfin also decreased cell proliferation and tumor growth. In a genetically defined model of Ras-driven RMS, constitutively active YAPS127A can serve as the initial oncogenic alteration whereby YAPS127A is sufficient for senescence bypass in primary skeletal muscle myoblasts, but requires expression of hTERT and oncogenic Ras for tumorigenesis in vivo. Importantly these tumors are histologically consistent with human Ras-driven RMS.

To understand the impact of YAP signaling on cell stemness, we cultured eRMS cells as 3D spheres. These spheres are enriched in stem cell genes, as well as in YAP and Notch signaling. The Notch pathway is another developmental pathway that is also highly upregulated in eRMS and contributes to tumorigenesis. Using the spheres as a model, we uncovered a bidirectional signaling circuit between YAP and Notch that regulates stemness. Active Notch signaling upregulates YAP, and YAP in turn upregulates the Notch ligands JAG1 and DLL1 and the transcription factor RBPJ. This circuit controls expression of several stem cell genes including SOX2, which is functionally required for eRMS cell stemness. Silencing this circuit for therapeutic purposes may be challenging, since the inhibition of one node (for example pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. Supporting the existence of this circuit beyond a model system, nuclear Notch and YAP protein expression are correlated in human eRMS tumors, and YAP suppression in vivo decreases both Notch signaling and SOX2 expression. In preliminary studies, we also analyzed the differential effects of the three Ras isoforms on eRMS tumorigenesis, Ras-Notch, and Ras-YAP signaling, and developed a method to culture the alveolar RMS subtype as spheres. In conclusion, the YAP oncoprotein drives Ras-driven tumorigenesis by promoting cell growth, survival, and stemness, and through signaling interactions with the Notch pathway. This study also provides rationale for combination therapies targeting YAP and Notch for the treatment of Ras-driven RMS.

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 <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed 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.

First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression.

Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. In conclusion, SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.