Monoallelic Loss of the Imprinted Gene Grb10 Promotes Tumor Formation in Irradiated Nf1+/- Mice

Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.印记基因（Imprinted gene）仅从亲本的单个等位基因进行表达，若杂合性缺失累及该表达等位基因，即可导致蛋白质表达完全丧失。遗传改变在肿瘤发生过程中极为常见，但印记基因在此过程中的作用尚未得到充分阐释。早期研究中，我们对携带神经纤维瘤病I型（Neurofibromatosis I）肿瘤抑制基因（NF1）杂合突变的小鼠进行诱变，以构建辐射后NF1患者所发生的放疗相关第二恶性肿瘤模型。通过对本团队小鼠模型构建的肿瘤细胞系开展表达分析，我们发现Grb10的表达普遍缺失。Grb10属于印记基因，对细胞系与原发性肿瘤的多态性分析结果显示，在本团队小鼠模型中发生的各类Nf1突变肿瘤内，其表达等位基因通常会发生缺失。我们开展功能实验，以探究Grb10的恢复表达或缺失是否会改变肿瘤生长的核心特征。在Nf1突变肿瘤中恢复Grb10的表达，可降低细胞增殖能力、减少软琼脂集落形成，并下调Ras信号通路（Ras）活性。与之相反，在未转化的小鼠胚胎成纤维细胞中沉默Grb10，可显著提升细胞增殖能力，并升高Ras-GTP水平。组成型激活的MEK（MEK）的表达可挽救肿瘤细胞，使其免受Grb10介导的集落形成减少效应。本研究证实，Grb10的缺失可在体内肿瘤发生过程中出现，并对未转化的原代细胞产生功能性影响。在肿瘤中，Grb10的缺失可独立促进Ras信号通路的过度激活，进而推动细胞过度增殖——这正是肿瘤发生发展的早期特征之一。借助成熟的Nf1突变癌症小鼠模型，本研究揭示了印记基因在肿瘤发生过程中的全新功能角色。