Truncated GLI1 In Glioblastoma

描述（由申请人提供）：在约40-60%的人类基因中观察到选择性剪接;然而，其对人类癌症的全面影响仍不清楚。在这个建议中，我们将功能性地描述一个新的，可变剪接的转录因子，发现于 PI实验室，即tGLI 1（截短的神经胶质瘤相关癌基因同源物1）。tGLI 1属于锌指转录因子GLI 1家族，GLI 1基因的整个外显子3和部分外显子4存在框内缺失。与GLI 1一样，tGLI 1对音刺猬配体有反应，进行核转运，并调节已知的GLI 1靶基因。然而，我们的初步结果表明，tGLI 1可能不同于GLI 1在调节肿瘤表型，转录靶点和表达模式。经工程改造表达tGLI 1的多形性胶质母细胞瘤（GBM）异种移植物在生长、侵袭性和血管生成方面似乎比GLI 1更具有攻击性。tGLI 1已经获得了增强几种侵袭和血管生成促进基因表达的能力。tGLI 1在我们检测的近50%的GBM标本中高度表达，但在正常脑或其他正常组织中检测不到。这些观察结果使我们假设新的tGLI 1转录因子作为功能获得性GLI 1发挥作用，其调节一组独特的非GLI 1靶向基因的表达，并且由于其在GBM中的过表达及其增强对肿瘤生长、侵袭和血管生成重要的基因的能力，tGLI 1支持GBM的一些主要特征，即高度增殖，浸润和血管分布。提出了三个具体目标来检验这一假设。(1)确定tGLI 1是否介导GBM的恶性表型。目前还没有系统的研究探讨tGLI 1在不同GBM恶性表型中的作用。在这里，我们将通过敲入和敲低方法创建具有不同水平tGLI 1的颅内GBM异种移植物，检查异种移植物的生长，侵袭性和血管生成，并监测携带异种移植物的小鼠的存活情况。(2)阐明tGLI 1调节GBM中基因表达的机制。tGLI 1已经获得了增强几种基因表达的能力;然而，这种独特能力的潜在机制目前尚不清楚。在这里，我们将研究两种潜在的机制：a）tGLI 1结合到其靶基因启动子内的独特DNA元件，而这些元件不被GLI 1识别，从而调节GLI 1不靶向的基因;和B）tGLI 1已经获得了与不与GLI 1相互作用的转录调节因子相互作用的能力，因此B调节GLI 1不靶向的基因。(3)研究导致GBM中tGLI 1 mRNA合成的选择性剪接过程。tGLI 1 mRNA合成的选择性剪接过程目前尚不清楚。我们将确定前mRNA和RNA结合剪接因子内的顺式作用元件，它们共同介导tGLI 1 mRNA合成的选择性剪接。我们的研究将是将tGLI 1定义为GBM恶性肿瘤的新介质，并了解tGLI 1功能的全谱和tGLI 1 mRNA合成的分子基础的第一步。

DESCRIPTION (provided by applicant): Alternative splicing is observed in ~40-60% of human genes; however, its full impact on human cancers is still not well understood. In this proposal, we will functionally characterize a novel, alternatively spliced transcription factor discovered in the PI's laboratory, namely, tGLI1 (truncated glioma-associated oncogene homolog 1). Belonging to the GLI1 family of zinc finger transcription factors, tGLI1 has an in-frame deletion of entire exon 3 and part of exon 4 of the GLI1 gene. Like GLI1, tGLI1 responds to sonic hedgehog ligand, undergoes nuclear transport, and regulates known GLI1 target genes. However, our preliminary results showed that tGLI1 may differ from GLI1 in regulating tumor phenotypes, transcriptional targets and expression pattern. Glioblastoma multiforme (GBM) xenografts engineered to express tGLI1 appeared to be more aggressive in growth, invasiveness, and angiogenesis than those with GLI1. tGLI1 has gained the ability to enhance expression of several invasion- and angiogenesis-promoting genes. tGLI1 was highly expressed in nearly 50% of GBM specimens we had examined, but undetectable in normal brain or other normal tissues. These observations have led us to hypothesize that the novel tGLI1 transcription factor behaves as a gain-of-function GLI1 that regulates expression of a unique set of genes not targeted by GLI1, and that because of its overexpression in GBM and its ability to enhance genes important for tumor growth, invasion and angiogenesis, tGLI1 supports some of the predominant features of GBM, i.e. high degrees of proliferation, infiltration and vascularity. Three Specific Aims are proposed to test this hypothesis. (1) Determine whether tGLI1 mediates malignant phenotypes of GBM. There has been no systematic study that investigated the role of tGLI1 in distinctive GBM malignant phenotypes. Here, we will create intracranial GBM xenografts with differential levels of tGLI1 via knock-in and knock-down approaches, examine the xenografts for growth, invasiveness and angiogenesis, and monitor the xenograft-carrying mice for survival. (2) Elucidate the mechanisms by which tGLI1 regulates gene expression in GBM. tGLI1 has gained the ability to enhance expression of several genes; however, the underlying mechanisms for this unique ability is currently unknown. Here, we will investigate two potential mechanisms: a) tGLI1 binds to unique DNA elements, not recognized by GLI1, within its target gene promoters and thereby regulates genes not targeted by GLI1; and b) tGLI1 has gained the ability to interact with transcription regulators that do not interact with GLI1, thereb regulating genes not targeted by GLI1. (3) Investigate alternative splicing process that leads to tGLI1 mRNA synthesis in GBM. Alternative splicing process for tGLI1 mRNA synthesis is presently unknown. We will identify the cis-acting elements within the pre-mRNA and the RNA-binding splicing factors that together mediate alternative splicing for tGLI1 mRNA synthesis. Our study will be the first step towards defining tGLI1 as a novel mediator of GBM malignancy, and understanding the full spectrum of tGLI1 functionality and the molecular basis for tGLI1 mRNA synthesis.