Interaction between RAS and Hedgehog Signaling in Rhabdomyosarcoma

横纹肌肉瘤(RMS)是儿童最常见的软组织肉瘤。在分子水平上，胚胎亚型(ERMS)的特征是Hedgehog(HH)靶基因的过度表达，如GLI1，以及H-、K-或NRAS(OncRAS)的致癌突变。我们证明，零星的ERM不显示典型的HH信号活性，因此对SMO抑制剂不起作用。然而，所有的oncRAS亚型都通过MEK/ERK轴抑制散发性ERMS来源的细胞系中GLI1/GLI1mRNA/蛋白的表达。然而，oncRAS增加了细胞系的致瘤性。根据文献，由于ERMS细胞系对GLI抑制剂GANT61敏感，oncRAS显然是一种非常强的促肿瘤刺激，允许同时抑制其他促肿瘤刺激。由于反之亦然，GANT61增加了ERK的磷酸化，GLI-和MEK-抑制剂的结合可能会在ERMS中产生非常强的抗肿瘤作用。我们还诱导了杂合Ptch+/-小鼠ERMS中oncRas突变的表达。与散发的ERMS不同，该模型中的ERMS是由Ptch胚系突变启动的，这在基底细胞痣综合征患者中也是如此。PTCH相关的ERM表现出典型的HH信号活性，对SMO抑制剂的治疗敏感。OncKRas和oncHRas促进PTCH相关ERMS的发生和生长，而oncNRas则不能。有趣的是，这些影响只发生在出生后在ERMS前体病变中诱导oncRas突变时，而不是在ERMS成熟阶段诱导时。再加上oncRas胚系突变不会导致小鼠ERMS的事实，可能只有当oncRas突变在已经启动的ERMS前体病变和/或ERMS干细胞中诱导时，才有利于ERMS的生长。相反，它们确实会影响肿瘤体积，至少在小鼠身上不会。由于oncRAS突变增加了来自散发性ERM的细胞系的增殖率，因此这些细胞系可能具有特定的、对oncRAS敏感的细胞亚群，而这些亚群在PTCH相关的ERM中是完全不存在的。此外，可能只有oncRas突变在ERMS发育过程中非常特定的时间窗诱导时才会影响ERMS的生长，而此时肿瘤细胞对突变很敏感。对于各自的oncRas亚型，这个窗口可能是不同的，这可以解释为什么在Ptch小鼠模型中，在ERMS前体病变中诱导oncNRas突变时缺乏生长变化。这些问题将在目前的拨款提案的帮助下得到解决。

Rhabdomyosarcoma (RMS) are the most common soft tissue sarcoma in children. On molecular level the embryonal subtype (ERMS) is characterized by e.g. overexpression of Hedgehog (HH) target genes such as GLI1 and by oncogenic mutations in H-, K- or NRAS (oncRAS). We demonstrate that sporadic ERMS do not show canonical HH signaling activity and thus do not react to SMO inhibitors. However, all oncRAS isoforms suppress the expression of GLI1/GLI1 mRNA/protein in cell lines derived from sporadic ERMS via the MEK/ERK axis. Nevertheless, oncRAS increases the tumorigenicity of the cell lines. Since - according to the literature - ERMS cell lines are sensitive against the GLI inhibitor GANT61, oncRAS apparently is a very strong protumorigenic stimulus that allows for a simultaneous suppression of other protumorigenic stimuli. Since – vice versa - GANT61 increases the phosphorylation of ERK, it is possible that a combination of a GLI- with a MEK-inhibitor may result in very strong antitumoral effects in ERMS. We also have induced the expression of oncRas mutations in ERMS of heterozygous Ptch+/- mice. In contrast to sporadic ERMS the ERMS in this model are initiated by Ptch germline mutations, which is also the case in patients with basal cell nevus syndrome. Ptch-associated ERMS show canonical Hh signaling activity and are sensitive towards a treatment with SMO inhibitors. OncKRas and oncHRas enhance the occurrence and growth of Ptch-associated ERMS, whereas oncNRas does not. Interestingly, these effects only occur when the oncRas mutations are induced in ERMS precursor lesions after birth, but not when they are induced at the full-blown ERMS stage. Together with the fact that oncRas-germline mutations do not result in ERMS in mice, it is possible that oncRas mutations are only advantageous for ERMS growth when they are induced in already initiated ERMS precursor lesions and/or ERMS stem cells. In contrast, they do affect the tumor bulk, at least not in the mouse. Since oncRAS mutations increase the proliferation rate of cell lines derived from sporadic ERMS, it is possible that the cell lines harbor specific, oncRAS-susceptible cellular subpopulations that are simply absent in Ptch-associated ERMS. In addition it is possible that oncRas mutations only affect ERMS growth when induced in a very specific time window during ERMS development, when the tumor cells are susceptible for the mutations. This window may be different for the respective oncRas isoforms, which could explain the lack of growth changes upon an induction of an oncNRas mutation in ERMS precursor lesions in the Ptch mouse model. These questions will be solved with help of the present grant proposal.