Targeting insulin receptor splicing for treatment of rhabdomyosarcoma

 描述（由申请人提供）：替代性前信使RNA（前mRNA）剪接已成为蛋白质组多样性和基因表达的主要机制。这种多样性已经被最近的工作所强调，这些工作阐明了作为肿瘤发生过程的一部分的细胞刺激（例如DNA损伤和上皮细胞向间质细胞转化，EMT）的应答的选择性剪接基因网络。虽然自发现以来，选择性剪接在许多细胞过程中的重要性已被认识到，但对其调控机制的了解仍然很少。实体瘤的特征在于短暂和慢性缺氧，并且肿瘤细胞适应缺氧的能力对于肿瘤的生长至关重要。 肿瘤进展。我们最近已经表明，刺激血管内皮细胞增殖和血管生成诱导的主要血管生成因子，VEGF，是依赖于胰岛素样生长因子（IGF）信号。此外，人血管内皮细胞和源自儿科肉瘤的细胞主要表达胰岛素受体基因（IN-R）的可变剪接形式，其对IGF-2具有高亲和力。重要的是，IN-R在缺氧时发生剪接，小儿肉瘤细胞分泌IGF-2。因此，IN-R剪接在儿科肉瘤的进展中似乎是至关重要的，既作为肿瘤细胞自分泌生长的受体，又作为血管细胞旁分泌生长的受体，因此是血管生成的受体。该提案的主要目标是了解缺氧时IN-R前体mRNA剪接的机制和后果。这项工作将测试的假设，调节元件和剪接因子的调制响应缺氧，并参与选择性剪接的IN-R，以促进肿瘤的发生。为了确定IN-R缺氧诱导的剪接的效应物，我们将使用细胞测定来筛选缺氧诱导的剪接，所述细胞测定稳健地再现了在癌细胞中观察到的IN-R剪接的变化。我们将利用缺氧诱导的剪接试验来鉴定RNA序列及其各自的结合伴侣，这是调节IN-R前mRNA剪接所必需的。此外，我们将使用已建立的小鼠异种移植试验和新型反义寡核苷酸（ASO）来调节IN-R剪接，并确定剪接亚型在肿瘤进展中的作用，以及作为治疗干预的新靶点。

 DESCRIPTION (provided by applicant): Alternative pre-messenger RNA (pre-mRNA) splicing has emerged as a predominant mechanism for proteomic diversity and gene expression. This diversity has been emphasized by recent work that elucidates a network of alternatively spliced genes in response to cell stimuli that are part of the tumorigenic process (DNA damage and Epithelial to Mesenchymal Transition, EMT, for example). Though the importance of alternative splicing in a number of cell processes has been recognized since its discovery, the mechanisms underlying its regulation remain poorly understood. Solid tumors are characterized by both transient and chronic hypoxia, and the ability for tumor cells to adapt to hypoxia is essential for tumor progression. We have shown recently that stimulation of vascular endothelial cell proliferation and angiogenesis induced by the major angiogenic factor, VEGF, is dependent upon insulin-like growth factor (IGF) signaling. Additionally, both human vascular endothelial cells and cells derived from pediatric sarcomas express predominantly the alternatively spliced form of the Insulin Receptor gene (IN-R), that has high affinity for IGF-2. Importantly, IN-R is spliced in response to hypoxia, and pediatric sarcoma cells secrete IGF-2. Consequently, IN-R splicing appears critical in progression of pediatric sarcomas both from acting as a receptor for autocrine growth of tumor cells, and for paracrine growth of vascular cells, and hence angiogenesis. The primary goal of this proposal is to understand the mechanisms and consequences of IN-R pre-mRNA splicing in response to hypoxia. This work will test the hypothesis that regulatory elements and splicing factors are modulated in response to hypoxia and are involved in the alternative splicing of IN-R to contribute to tumorigenesis. In order to determine the effectors of IN-R hypoxia- induced splicing, we will be screening hypoxia induced splicing using a cellular assay which robustly recapitulates changes in IN-R splicing that are seen in cancer cells. We will utilize the hypoxia-induced splicing assay to identify RNA sequences and their respective binding partners that are necessary for regulation of IN-R pre-mRNA splicing. Furthermore, we will use established mouse xenograft assays and novel antisense oligonucleotides (ASOs) to modulate IN-R splicing and determine the role of the spliced isoforms in tumor progression and as novel targets for therapeutic intervention.