Abstract
Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser430. Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS.
Highlights
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children
Our study focused on fascaplysin, because cyclin-dependent kinase 4 (Cdk4) may play an important role in regulating the function of PAX3-FOXO1, suggested by previous studies showing that a loss of Ink4a/ARF, which leads to activation of Cdk4, promoted PAX3-FOXO1’s oncogenic function [4,5,6]
We hypothesized that the activities of PAX3-FOXO1 would be regulated by its functional interactions with Cdk4 and sought to determine whether Cdk4 could phosphorylate the PAX3-FOXO1 protein
Summary
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Histopathologically, two subtypes of RMS have been identified, embryonal (ERMS) and alveolar (ARMS), each with distinct clinical and genetic characteristics. In a recent study using primary human skeletal muscle cells, a cell type relevant to RMS, Linardic et al [4] showed that expression of PAX3-FOXO1, accompanied by the a loss of expression of tumor suppressor p16INK4A, could promote these cells to bypass the senescence growth arrest checkpoint and proliferate inappropriately. High frequencies of ARMS tumor formation occurred only in mice with Pax3-foxo knock-in accompanied by a conditional Trp or INK4a/ARF loss of function, suggesting that expression of PAX3-FOXO1 is necessary but not sufficient to induce ARMS at high frequencies. These observations implied that the activity of PAX3-FOXO1 requires activation of another signaling pathway, which is possibly mediated by the loss of INK4a/ARF function
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