Regulation of caspase 9 RNA splicing in NSCLC cancer

描述（由申请人提供）：今天，肺癌是工业化国家男性和女性死亡的主要原因，估计占美国所有癌症死亡的28%。 非小细胞肺癌（NSCLC）代表了大多数肺癌，预后不良，中位生存期不到12个月。 大多数患者存在不可切除的疾病，目前的化疗和放疗治疗方案充其量是姑息性的。 因此，需要新的策略来治疗NSCLC以影响这种疾病。 在这项研究中，我们专注于NSCLC模型，以检查调节NSCLC细胞/肿瘤的化疗敏感性、生成和维持的远端信号传导机制。 具体来说，该资助申请的重点是调节hnRNP L功能和caspase 9的选择性剪接的细胞信号传导途径。 半胱天冬酶9的表达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。 将该外显子盒包含到成熟转录物中产生促凋亡半胱天冬酶9（半胱天冬酶9a），而排除产生抗凋亡半胱天冬酶9 b。 我们实验室的研究表明，非小细胞肺癌肿瘤存在类似于抗细胞凋亡/化疗耐药表型的caspase 9/caspase 9 b比例失调。 本实验室的后续研究表明，caspase 9的选择性剪接在NSCLC细胞的锚定非依赖性生长（AIG）、EGFR突变诱导的非转化人支气管上皮细胞的AIG和厄洛替尼敏感性中具有重要功能。 从机制上讲，我们的实验室确定了外显子3中的外显子剪接沉默子（C9/E3-ESS），其调节caspase 9前体mRNA的外显子3，4，5和6盒的包含。 hnRNP L与该RNA顺式元件结合，并抑制外显子盒的包含。 重要的是，丝氨酸52上的hnRNP L的磷酸化（仅在转化细胞中观察到）是抑制外显子3、4、5、6盒所需的。 最后，hnRNP L的ser 52磷酸化被证明是NSCLC细胞通过半胱天冬酶9的选择性剪接的致瘤能力所需的介体。 这些关键机制对转化细胞是特异性的，可转移到>70%的NSCLC，并且在致癌途径的极端远端。 因此，这些远端机制是开发新抗癌疗法的合理且高度期望的靶点。 本研究的总体目标是确定调节外显子3定义的机制和细胞信号传导途径，从而确定caspase 9的选择性剪接。 此外，我们建议进行临床前研究，以确定这些机制以及caspase 9 b的特异性靶向是否是治疗NSCLC的有效靶点，并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.