Characterization of Wnt/β-catenin signaling in rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. b-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/b-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed b-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of b-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including b-catenin, glycogen synthase kinase-3b, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of b-catenin, stabilization of the active cytosolic form and nuclear translocation of b-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/b-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

Secreted Frizzled-Related Protein 3 (SFRP3) Is Required for Tumorigenesis of PAX3–FOXO1-Positive Alveolar Rhabdomyosarcoma

“…No consistent or robust differences in the number of differentiated (as assessed by ability to form myotubes and express the myogenic marker MF20) or senescent cells were observed in response to the stable expression of SFRP3 shRNAs in Rh28 cells (data not shown). Similarly, we saw no evidence of Wnt pathway activation (which is required for terminal myogenic differentiation) in aRMS cells stably expressing SFRP3 shRNAs, although in response to Wnt3a-conditioned media and LiCl (a GSK3β inhibitor) they retain the ability to activate β-catenin signaling, similarly to seen previously (15). However, in Rh28 cells both shRNAs caused a 16-22% increase in the percentage of cells accumulated in the G 1 phase of the cell cycle (Fig.…”

“…4K) were upregulated in response to both SFRP3 sh3 and sh5. Given the requirement for Wnt signaling during myogenic differentiation, we next examined the same tumors for evidence of Wnt pathway activation using nuclear β-catenin immunohistochemical staining and Axin2 expression (15, 33-35). While we could not detect an increase in nuclear β-catenin staining in those tumors bearing the SFRP3 shRNAs under conditions of dox treatment (data not shown), we did find Axin2 modestly upregulated (Fig.…”

“…The role of Wnt in RMS, like other developmental pathways Hedgehog, Hippo and Notch, is only beginning to be understood (15, 16, 26, 52). Unlike in most adult epithelial cancers, where Wnt/β-catenin activity is upregulated and oncogenic, in a p53 -/- /c-fos -/- mouse model of eRMS and in human eRMS cell lines, β-catenin activity is downregulated.…”

“…In a murine model of eRMS, Wnt signaling was found downregulated, and activation of Wnt signaling promoted differentiation (13). Similarly, activation of Wnt signaling in human eRMS and aRMS cell lines in vitro promoted differentiation (13-15). After identifying changes in the Wnt pathway in a microarray comparing the transcriptomes of human skeletal muscle myoblast (HSMM) cells with and without PAX3-FOXO1 expression, SFRP3 was noted to be upregulated in response to PAX3-FOXO1 expression and investigated further.…”

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

“…This finding is further supported by work in the mouse p53 −/− Fos −/− ERMS model, in which the canonical WNT/β-catenin pathway was actively suppressed by DKK1 and sFRP4 (30). Despite the active suppression of WNT/ β-catenin pathway in RMS, Annavarapu et al (31) recently showed that human ERMS and ARMS cell lines are responsive to exogenous WNT3A, inducing stabilization of β-catenin, altering proliferation, and enhancing differentiation in a subset of RMS cell lines. However, these studies failed to identify a role for the WNT/β-catenin pathway in modulating ERMS selfrenewal or in suppression of tumor growth in vivo.…”