Abstract
Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.
Highlights
Pediatric rhabdomyosarcoma (RMS) is a skeletal musclederived soft-tissue sarcoma affecting children and adolescents
All the Notch3IC-over-expressing RMS cell lines were insensitive to DAPT (Figure 4D and E, and Figure S2D). These findings suggest that forcing Notch3 activation increases RMS cell proliferation and renders cells GSI-resistant, irrespective of their fusion oncoprotein expression or the inhibition of other Notch paralogs
We previously demonstrated that Notch3 activation, in part through the expression of its target gene HES1, prevents the in vitro differentiation of both embryonal and PAX3-FOXO1-positive alveolar RMS cell lines and sustains the in vivo growth of PAX3
Summary
Pediatric rhabdomyosarcoma (RMS) is a skeletal musclederived soft-tissue sarcoma affecting children and adolescents. It accounts for approximately 50% of all pediatric soft-tissue sarcomas and for 7–8% of all childhood malignancies [1]. Pediatric RMS includes two major histological subtypes, embryonal and alveolar [2]. Embryonal RMS has a favorable prognosis with survival rates of about 90% when nonmetastatic. 70% of alveolar RMSs harbor t(2;13) or t(1;13) chromosomal translocations that result in PAX3-FOXO1 or PAX7FOXO1 oncoprotein expression. PAX3-FOXO1 may be a key biomarker patients’ risk-stratification being correlated to the poorest outcome [3]. Despite improvement in multimodality treatments for high risk RMS, the management of those patients remains challenging, with a 5-year overall survival less than 30%. Understanding the molecular pathways that contribute to the pathogenesis and self-propagation of the most aggressive tumor forms is urgently needed
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