Targeting insulin receptor splicing for treatment of rhabdomyosarcoma

 描述(由申请人提供)：替代前信使RNA(前信使RNA)剪接已成为蛋白质组多样性和基因表达的主要机制。最近的工作强调了这种多样性，这些工作阐明了作为肿瘤形成过程一部分的细胞刺激(例如DNA损伤和上皮向间充质转化，EMT)反应的选择性剪接基因网络。尽管自发现以来，选择性剪接在许多细胞过程中的重要性已经被认识到，但其调控机制仍然知之甚少。实体瘤具有暂时性和慢性缺氧的特点，而肿瘤细胞对缺氧的适应能力对于 肿瘤进展。我们最近发现，主要的血管生成因子血管内皮生长因子对血管内皮细胞增殖和血管生成的刺激作用依赖于胰岛素样生长因子(IGF)信号。此外，人类血管内皮细胞和来自儿童肉瘤的细胞主要表达胰岛素受体基因(IN-R)的选择性剪接形式，该基因与IGF-2具有高亲和力。重要的是，IN-R在低氧条件下被剪接，儿童肉瘤细胞分泌IGF-2。因此，IN-R剪接似乎在儿童肉瘤的进展中起关键作用，既是作为肿瘤细胞自分泌生长的受体，也是血管细胞旁分泌生长的受体，从而促进血管生成。这一建议的主要目的是了解IN-R前-mRNA剪接对低氧的反应的机制和后果。这项工作将检验这一假设，即调节元件和剪接因子在低氧反应中被调节，并参与IN-R的选择性剪接，从而促进肿瘤的发生。为了确定IN-R缺氧诱导剪接的效应器，我们将使用一种细胞实验来筛选缺氧诱导的剪接，该实验有力地概括了在癌细胞中看到的IN-R剪接的变化。我们将利用低氧诱导的剪接实验来鉴定调控IN-R前体mRNA剪接所必需的RNA序列及其各自的结合伙伴。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，并确定剪接的异构体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.