A MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting [RNA-seq]
Rhabdomyosarcoma (RMS) is a pediatric mesenchymal-derived malignancy encompassing Fusion Positive (FP)-RMS expressing PAX3/7-FOXO1 and Fusion Negative (FN)-RMS often mutated in the RAS pathway. RMS expresses the master myogenic transcription factor MYOD that, paradoxically, is unable to support differentiation while essential for tumor cell survival. We identify here SKP2, an oncogenic E3-ubiquitin ligase, as a critical driver of tumorigenesis in FN-RMS. SKP2 is overexpressed in RMS at the highest levels among several adult and pediatric cancers and its expression is maintained by MYOD through an intronic enhancer within the gene. In FN-RMS SKP2 promotes cell cycle progression and prevents differentiation directly targeting p27Kip1 and p57Kip2, respectively, unlocking a transcriptional myogenic program, partly MYOD-dependent, resulting in de novo expression of terminal muscle differentiation markers. SKP2 depletion strongly affects stemness and tumorigenic features in vitro and prevents in vivo tumor growth. The in vitro effects are mirrored by the SKP2 inhibitor SMIP004. Moreover, the investigational NEDDylation inhibitor MLN4924 hampers SKP2 functions restraining FN-RMS cell survival and tumor growth. Our results uncover a MYOD-SKP2 axis crucial for the crosstalk between transcriptional and post-translational mechanisms that contribute to FN-RMS tumorigenesis and broaden the understanding of MYOD function. Furthermore, they suggest inhibition of NEDDylation as a potential therapeutic approach in this tumor. Overall design: To investigate the function of SKP2 in the regulation of myogenic differentiation, senescence and tumor growth in RMS, we infected JR1 and RD cell lines with lentiviral vectors expressing an shRNA targeting human SKP2 (shSKP2.2) or a non-targeting control shRNA (shSCR). We then perfomed gene expression profiling analysis using data from RNA-seq of JR1 and RD cell lines expressing shSKP2 and compared the results with their correspondant shSCR controls.