Interaction between RAS and Hedgehog Signaling in Rhabdomyosarcoma

横纹肌肉瘤是儿童最常见的软组织肉瘤。在分子水平上，胚胎亚型（ERMS）的特征在于例如Hedgehog（HH）靶基因如GLI 1的过表达和H-、K-或NRAS（oncRAS）中的致癌突变。我们证明，散发性ERMS不显示典型的HH信号传导活性，因此不与SMO抑制剂反应。然而，所有oncRAS亚型通过MEK/ERK轴抑制源自散发性ERMS的细胞系中GLI 1/GLI 1 mRNA/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GANT 61敏感，因此oncRAS显然是一种非常强的促肿瘤发生刺激，其允许同时抑制其他促肿瘤发生刺激。由于GANT 61增加ERK的磷酸化，因此GLI与MEK抑制剂的组合可能在ERMS中产生非常强的抗肿瘤作用，反之亦然。我们还在杂合Ptch+/-小鼠的ERMS中诱导了oncRas突变的表达。与散发性ERMS相反，该模型中的ERMS由Ptch种系突变启动，基底细胞痣综合征患者也是如此。Ptch相关ERMS显示典型的Hh信号传导活性，并且对SMO抑制剂治疗敏感。OncKRas和oncHRas增强Ptch相关ERMS的发生和生长，而oncNRas不增强。有趣的是，这些效应仅发生在出生后ERMS前体病变中诱导oncRas突变时，而不是在成熟ERMS阶段诱导时。结合oncRas-种系突变不会导致小鼠ERMS的事实，可能oncRas突变仅在已经启动的ERMS前体病变和/或ERMS干细胞中诱导时才有利于ERMS生长。相反，它们确实影响肿瘤体积，至少在小鼠中没有。由于oncRAS突变增加了源自散发性ERMS的细胞系的增殖速率，因此细胞系可能具有在Ptch相关ERMS中根本不存在的特异性oncRAS易感细胞亚群。此外，当肿瘤细胞对突变敏感时，oncRas突变可能仅在ERMS发展期间的非常特定的时间窗内诱导时才影响ERMS生长。该窗口对于各个oncRas亚型可能不同，这可以解释Ptch小鼠模型中ERMS前体病变中诱导oncNRas突变后缺乏生长变化。这些问题将在本赠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.