Truncated GLI1 In Glioblastoma

描述(申请人提供)：在大约40-60%的人类基因中观察到选择性剪接；然而，它对人类癌症的全面影响仍然不是很清楚。在这项提案中，我们将对一种新的、可选择性剪接的转录因子进行功能鉴定 PI的实验室，即tGLI1(截断的胶质瘤相关癌基因同源1)。TGLI1属于锌指转录因子GLI1家族，具有GLI1基因的全部外显子3和部分外显子4的框内缺失。和GLI1一样，tGLI1对音速刺猬配体作出反应，经历核转运，并调节已知的GLI1靶基因。然而，我们的初步结果表明，tGLI1在调节肿瘤表型、转录靶点和表达模式方面可能与GLI1不同。表达tGLI1的多形性胶质母细胞瘤(GBM)异种移植瘤在生长、侵袭和血管生成方面似乎比GLI1更具侵袭性。TGLI1已经获得了增强几个侵袭和血管生成促进基因表达的能力。TGLI1在我们所检测的近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tGLI1转录因子作为一种功能获得的GLI1，调节一组独特的非GLI1靶向基因的表达，并且由于它在GBM中的过度表达和增强对肿瘤生长、侵袭和血管生成重要的基因的能力，tGLI1支持GBM的一些主要特征，即高度增殖、渗透和血管生成。为了检验这一假说，本文提出了三个具体目标。(1)确定tGLI1是否介导了GBM的恶性表型。目前还没有系统的研究来研究tGLI1在不同的GBM恶性表型中的作用。在这里，我们将通过敲入和敲除方法创建不同水平的tGLI1的脑内GBM异种移植，检查异种移植的生长、侵袭性和血管生成，并监测携带异种移植的小鼠的存活情况。(2)阐明tGLI1调控GBM基因表达的机制。TGLI1已经获得了增强几个基因表达的能力；然而，这种独特能力的潜在机制目前尚不清楚。在这里，我们将研究两种可能的机制：a)tGLI1在其靶基因启动子内与GLI1不识别的独特DNA元件结合，从而调节GLI1没有靶向的基因；b)tGLI1获得了与不与GLI1相互作用的转录调节因子相互作用的能力，从而调节GLI1没有靶向的基因。(3)探讨GBM中tGLI1mRNA合成的选择性剪接过程。TGLI1mRNA合成的选择性剪接过程目前尚不清楚。我们将确定前mRNA中的顺式作用元件和RNA结合剪接因子，它们共同介导tGLI1mRNA合成的选择性剪接。我们的研究将是将tGLI1定义为一种新的GBM恶性介质的第一步，并了解tGLI1的全谱功能和tGLI1mRNA合成的分子基础。

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.