Regulation of caspase 9 RNA splicing in NSCLC cancer

描述(申请人提供)：今天，肺癌是工业化国家男性和女性的主要死亡原因，估计占美国所有癌症死亡人数的28%。非小细胞肺癌(NSCLC)占肺癌的大多数，预后较差，中位生存期不到12个月。大多数患者目前患有不能切除的疾病，目前的化疗和放射治疗方案充其量是姑息的。因此，需要在治疗非小细胞肺癌方面采取新的策略，以影响这种疾病。在这项研究中，我们将重点放在NSCLC模型上，以检测调节NSCLC细胞/肿瘤的化疗敏感性、生成和维持的远端信号机制。具体地说，GRAN的应用集中在调节hnRNP L功能和Caspase 9选择性剪接的细胞信号通路上。Caspase 9的表达是通过包含或排除四个外显子盒(外显子3、4、5和6)的选择性剪接来调节的。将这个外显子盒包含到成熟转录本中会产生促凋亡的caspase 9(Caspase 9a)，而排除这个外显子盒会产生抗凋亡的caspase 9b。我们实验室的研究表明，NSCLC肿瘤存在类似于抗凋亡/化疗耐药表型的caspase 9/caspase 9b比例失调。本实验室随后的研究表明，Caspase 9的选择性剪接在非小细胞肺癌细胞的锚定非依赖性生长(AIG)、未转化的人支气管上皮细胞中EGFR突变诱导的AIG以及厄洛替尼的敏感性中具有重要作用。从机制上讲，我们的实验室在外显子3中发现了一个外显子剪接沉默(C9/E3-ESS)，它调节caspase 9前-mRNA的外显子3、4、5和6盒的包含。HnRNP L被证明与这个RNA顺式元件结合，并抑制外显子盒的夹杂。重要的是，hnRNP L在Ser52上的磷酸化(仅在转化细胞中观察到)是抑制外显子3、4、5、6盒所必需的。最后，通过Caspase9的选择性剪接，hnRNP L的Ser52磷酸化被证明是非小细胞肺癌细胞致瘤能力的必需中介。这些关键机制是转化细胞所特有的，可翻译到70%的非小细胞肺癌细胞，并且位于致癌途径的最远端。因此，这些远端机制是开发新的抗癌治疗药物的看似合理和非常理想的靶点。这项研究的总体目标是确定调控外显子3定义的机制和细胞信号通路，从而调节caspase 9的选择性剪接。此外，我们正在提议进行临床前研究，以确定这些机制以及caspase 9b的特异性靶向是否是治疗NSCLC的有效靶点，以及提高目前临床使用的化疗药物的有效性。

DESCRIPTION (provided by applicant): Today, lung cancer is the leading cause of death in both men and women in industrialized countries, accounting for an estimated 28% of all cancer deaths in the United States. Non-small cell lung cancers (NSCLC) represent the majority of lung cancers and carry a poor prognosis with a median survival of less than 12 months. Most patients present with unresectable disease and current treatment options of chemotherapy and radiation are palliative at best. Therefore, new strategies are needed in the treatment of NSCLC in order to impact this disease. In this study, we are focusing on NSCLC models for examining distal signaling mechanisms that modulate the chemotherapy sensitivity, generation, and maintenance of NSCLC cells/tumors. Specifically, the grant application focuses on the cell signaling pathways regulating both hnRNP L function and the alternative splicing of caspase 9. The expression of caspase 9 is regulated by alternative splicing via the inclusion or exclusion of a four exon cassette (exons 3, 4, 5, and 6). Inclusion of this exon cassette into the mature transcript produces the pro-apoptotic caspase 9 (caspase 9a) while the exclusion produces the anti-apoptotic caspase 9b. Studies from our laboratory have demonstrated that NSCLC tumors present with a dysregulated ratio of caspase 9/caspase 9b analogous to an anti-apoptotic/chemotherapy resistance phenotype. Subsequent studies by our laboratory demonstrated that the alternative splicing of caspase 9 had important functions in anchorage-independent growth (AIG) in NSCLC cells, AIG induced by EGFR mutation in non-transformed human bronchial epithelial cells, and erlotinib sensitivity. Mechanistically, our laboratory identified an exonic splicing silencer (C9/E3-ESS) in exon 3 that regulates the inclusion of the exon 3, 4, 5, and 6 cassette of caspase 9 pre-mRNA. hnRNP L was shown to associate with this RNA cis-element, and repress the inclusion of the exon cassette. Importantly, phosphorylation of hnRNP L on ser52 (observed only in transformed cells) was required for repression of the exon 3,4,5,6 cassette. Lastly, ser52 phosphorylation of hnRNP L was shown as a required mediator of the tumorigenic capacity of NSCLC cells via the alternative splicing of caspase 9. These key mechanisms are specific to transformed cells, translatable to >70% of NSCLCs, and at an extreme distal point in oncogenic pathways. Therefore, these distal mechanisms are plausible and highly desired targets for the development of new anti-cancer therapeutics. The overall goal of this study is to determine the mechanisms and cell signaling pathways regulating the definition of exon 3, and thus the alternative splicing of caspase 9. Furthermore, we are proposing pre-clinical studies to determine whether these mechanisms as well as specific targeting of caspase 9b are effective targets for treating NSCLC as well as enhancing the effectiveness of current chemotherapeutic agents used in the clinic.