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.

横纹肌肉瘤（Rhabdomyosarcoma, RMS）是一种儿科起源的间叶源性恶性肿瘤，包含表达PAX3/7-FOXO1融合基因的融合阳性型（Fusion Positive, FP）-RMS，以及常发生RAS通路突变的融合阴性型（Fusion Negative, FN）-RMS。RMS表达关键肌源性转录因子MYOD，矛盾的是，该因子既无法支持细胞分化，却又是肿瘤细胞存活所必需的。本研究鉴定出SKP2——一种致癌性E3泛素连接酶，是融合阴性型横纹肌肉瘤（FN-RMS）发生的关键驱动因素。SKP2在多种成人及儿科癌症中高表达，且在RMS中的表达水平位居所有癌症之首；其表达由MYOD通过该基因内含子增强子维持。在FN-RMS中，SKP2分别直接靶向p27Kip1与p57Kip2，促进细胞周期进展并抑制细胞分化，解锁部分依赖MYOD的转录性肌源性程序，最终导致终末肌肉分化标志物的从头表达。SKP2沉默会显著影响体外实验中的干细胞干性与肿瘤发生特征，并可抑制体内肿瘤生长。SKP2抑制剂SMIP004可重现上述体外效应。此外，研究性NEDD化抑制剂MLN4924可抑制SKP2功能，从而限制FN-RMS细胞存活与肿瘤生长。本研究结果揭示了MYOD-SKP2调控轴，该轴是转录与翻译后机制交互调控的关键环节，参与FN-RMS的肿瘤发生过程，并加深了对MYOD功能的理解。此外，研究提示NEDD化抑制可作为该肿瘤的潜在治疗手段。整体实验设计：为探究SKP2在RMS中对肌源性分化、细胞衰老及肿瘤生长的调控作用，我们用携带靶向人SKP2的短发夹RNA（shRNA）载体shSKP2.2，或非靶向对照shRNA（shSCR）的慢病毒载体感染JR1与RD细胞系。随后通过RNA测序对表达shSKP2的JR1及RD细胞系进行基因表达谱分析，并将结果与对应的shSCR对照组进行比较。