Tissue-Regulated Lineage-Specific Splicing in Glioblastoma Pathogenesis

项目总结 谱系特定的选择性剪接，其中剪接以组织调控的方式发生，涉及进化 保守的替代外显子在大脑发育中起着决定性的作用。组织特异性蛋白的作用 另一外显子在恶性转化和肿瘤发展中的作用尚未确定。世系的影响- 胶质母细胞瘤(GBM)发病机制的特异性剪接仍不清楚，但在概念上是有吸引力的 鉴于这一过程在决定潜在的基底膜起源的祖先细胞的命运方面的盛行。 我们发现，大脑富集型剪接因子PTBP1介导了ANXA7的谱系特异性选择性剪接 肿瘤抑制基因。PTBP1在神经和神经胶质前体细胞中表达，并与 ANXA7前mRNA跳过外显子6；这会产生剪接的ANXA7亚型2(I2)。在世系规范期间， PTBP1被下调，这允许包括外显子6的未剪接ANXA7 I1(I1)的表达。 确定I2剪接变体在大脑中的图案化表达仅限于 代表潜在的GBM细胞来源，但这些细胞中几乎不存在I1。我们的初步数据 说明谱系特异性剪接可以增强基因机制以解除对致癌基因的调控 小路。具体地说，我们展示了针对致癌受体酪氨酸激酶(RTK)的ANXA7 I1，而不是I2 如EGFR、MET和PDGFRA对内体降解的作用。在成人大脑中，PTBP1几乎不存在；在 GBM、PTBP1异常持续，RTK水平升高。然而，我们证明了在PTBP1上 下调和/或ANXA7 I1重新表达，RTK信号转导减少，GBM致瘤性降低。 因此，我们假设GBM受益于持续的PTBP1表达，因为这抑制了 ANXA7 I1，阻止RTK内体降解，并维持GBM中的RTK信号。我们的具体目标 1.确定ANXA7 I1调节内体和细胞转运的机制 EGFR。在这里，我们将确定ANXA7 I1与其相互作用和调节的详细机制 以EGFR的胞内和胞外囊泡转运为原型的RTK。2.演示ANXA7 I1是 用于多种RTK和RTK靶向治疗的信号调制器 退化。在这里，我们将证明ANXA7 I1有能力通过多个 促肿瘤RTK调节TKI反应，因此对体内GBM有广泛的影响。这些 需要机械性的和有针对性的目标来更好地定义谱系特定的剪接过程在 肿瘤发生，这方面的研究还不够深入。我们的结果将确定ANXA7 I1足以 同时抑制多个RTK的信号转导，因此有可能揭示新的治疗方向。 这项工作将确定异常的谱系特异性选择性剪接在GBM发病机制中的作用， 提供了对恶性转化过程的更广泛的理解，并提供了潜在的 为未来针对发育调节剪接因子的治疗性研究奠定基础。

PROJECT SUMMARY Lineage-specific alternative splicing, where splicing occurs in a tissue-regulated manner involving evolutionary conserved alternative exons, has a determinative role in brain development. The role of tissue-specific alternative exons in malignant transformation and tumor development is undefined. The impact of lineage- specific splicing on glioblastoma (GBM) pathogenesis remains unclear but is conceptually attractive given the prevalence of this process in determining the fate of ancestral cells of potential GBM origin. We found that brain-enriched splice factor PTBP1 mediates lineage-specific alternative splicing of the ANXA7 tumor suppressor gene. PTBP1 is expressed in neural and glial precursor cells (NPCs/GPCs) and binds ANXA7 pre-mRNA to skip exon 6; this produces spliced ANXA7 isoform 2 (I2). During lineage specification, PTBP1 is downregulated and this allows the expression of unspliced ANXA7 I1 (I1), which includes exon 6. We determined that the patterned expression of the I2 splice variant in the brain is restricted to lineages that represent potential GBM cells of origin but that I1 is virtually absent in these cells. Our preliminary data illustrate that lineage-specific splicing can augment genetic mechanisms to deregulate oncogenic pathways. Specifically, we showed ANXA7 I1, but not I2, targets oncogenic receptor tyrosine kinases (RTKs) such as EGFR, MET and PDGFRA for endosomal degradation. In the adult brain, PTBP1 is nearly absent; in GBM, PTBP1 aberrantly persists and RTK levels are elevated. However, we demonstrate that upon PTBP1 knockdown and/or, ANXA7 I1 re-expression, RTK signaling is reduced, and GBM tumorigenicity is diminished. Therefore, we hypothesize that GBMs benefit from persistent PTBP1 expression as this suppresses ANXA7 I1, prevents RTK endosomal degradation, and sustains RTK signaling in GBM. Our Specific Aims are: 1. Define the Mechanism by which ANXA7 I1 Regulates Endosomal and Cellular Trafficking of EGFR. Herein, we will determine the detailed mechanism by which ANXA7 I1 interacts with and regulates endosomal and extracellular vesicle transport of EGFR as a prototype RTK. 2. Demonstrate ANXA7 I1 is a Signaling Modulator for Multiple RTKs and RTK-Targeted Therapies by Perpetuating Endosomal Degradation. Herein, we will demonstrate that ANXA7 I1 is competent to attenuate signaling through multiple pro-tumorigenic RTKs, modulate TKI response, and thus has a broad impact on GBM in vivo. These mechanistic and focused aims are needed to better define the role of lineage-specific splicing processes in tumorigenesis, which remains understudied. Our results will determine that ANXA7 I1 is sufficient to simultaneously inhibit signaling by multiple RTKs and thus holds potential to reveal a new therapeutic direction. This work will define the role of aberrant lineage-specific alternative splicing in GBM pathogenesis, offer a broader understanding of this process in malignant transformation, and provide a potential foundation for future studies to therapeutically target developmentally regulated splicing factors.