Abstract：
The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation. Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.
摘要：
RAS-MAPK途径在癌症和称为RASopathies的发育性疾病中被异常调节。虽然通常评估Ras对各种癌细胞系增殖的影响，尚不清楚Ras如何影响细胞分化。在这里，我们实施C2C12成肌细胞细胞系来系统研究Ras突变体和Ras途径药物对分化的影响。我们首先提供的证据表明，Pax7+祖细胞的少量池补充了准备分化的转运扩增细胞的主要池。我们的数据表明，Ras亚型在分化培养中具有不同的作用，其中K-Ras耗竭增加，H-Ras耗竭降低终末分化。因此，该测定可以为Ras生物学和Ras驱动的疾病提供重要的新见解。与此相符,我们发现所有致癌Ras突变体都能阻断转运扩增细胞的终末分化。相比之下,RASopathy相关的K-Ras变异体较少能够阻断分化。对七个致癌Ras突变体的八种靶向Ras途径药物的分析显示，与触发细胞死亡相比，它们的等位基因特异性活性和恢复正常分化的独特能力。特别是,MEK抑制剂曲美替尼可以广泛恢复分化，而mTOR抑制剂雷帕霉素广泛抑制分化。我们期望Ras途径突变体和药物对细胞分化的影响的这种定量评估具有补充癌细胞增殖数据的巨大潜力。
