Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells

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.

A Novel Notch–YAP Circuit Drives Stemness and Tumorigenesis in Embryonal Rhabdomyosarcoma

“…Intriguingly, the transcriptional co-activator YAP1 (but not its homolog TAZ, encoded by WWTR1 ) (Fig.1B) and the Notch pathway readout genes HEY1 and HES1 (Fig.1C) were also increased in both cell lines, suggesting YAP and Notch expression may support stemness. To gain insight into the mechanism of increased Notch pathway activation, we investigated the expression of Notch ligands and receptors previously implicated in eRMS tumorigenesis (7,10,13,59), and found increased expression of the Notch ligands JAG1 and DLL1 (Fig.1D) and the Notch receptors NOTCH1 and NOTCH3 (Fig.1E). …”

“…Notch signaling is upregulated in several human cancer types (reviewed in (9)). In eRMS, Notch receptors 1–4, ligands JAG1 and DLL1, the transcription factor RBPJ, and the Notch target genes HEY1 and HES1 have been found upregulated (7,10–14). Functionally, Notch signaling promotes eRMS proliferation and self-renewal (7,15), inhibits myogenic differentiation (7), and increases eRMS cell mobility and invasiveness (11,12).…”

PAX7 is a required target for microRNA-206-induced differentiation of fusion-negative rhabdomyosarcoma

“…Knockdown of these targets reduced proliferation as previously shown. 11, 20, 24, 34, 35 This finding suggests that single targets and even a subset of targets are insufficient to induce differentiation to the extent induced by miR-206 overexpression. Thus, target identification collectively is a key to understanding the mechanism of miR-206-induced differentiation in RMS further suggesting the ‘1 miRNA-1 target' model to be insufficient to explain complex phenotypes induced by miRNAs.…”

“…20 NOTCH signaling is also increased in RMS and NOTCH inhibition promotes RMS differentiation. 34, 35, 36 The paired-box transcription factors, PAX3 and PAX7, are expressed in embryonic muscle progenitors and function upstream of Myf5 and MyoD1 to promote myoblast and satellite cell proliferation and self-renewal. 7 PAX3/PAX7 are normally downregulated in myogenic differentiation and their exogenous overexpression inhibits differentiation.…”

The Role of Notch3 in Cancer

“…Notch3 is overexpressed in rhabdomyosarcoma cell lines compared with normal myoblasts and has been shown to induce proliferation via a Hes1‐dependent mechanism . Downregulation of Notch3 in vitro resulted in increased differentiation of alveolar rhabdomyosarcoma cells and a reduction in cellular proliferation . It has been suggested that osteosarcoma maintains an undifferentiated state by depressing of Notch3 and its downstream target Hes5 [147].…”