The role of RNA splicing in non-small cell lung cancer

RNA剪接在非小细胞肺癌中的作用

• 批准号: 9235511
• 负责人: CHARLES E. CHALFANT
• 金额: --
• 依托单位: VA VETERANS ADMINISTRATION HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9235511
• 关键词: Accounting; Address; Alternative Splicing; Anchorage-Independent Growth; Apoptotic; Applications Grants; Binding; Biological; Biophysics; CASP9 gene; Cancer Etiology; Cancer Model; Caspase; Cause of Death; Cell Survival; Cessation of life; Cisplatin; Clinical; Coupled; Data; Developed Countries; Development; Disease; Distal; Down-Regulation; Elements; Epithelial Cells; Epithelium; Event; Exclusion; Exons; Foundations; Funding; Generations; Genetic; Goals; Heterogeneous-Nuclear Ribonucleoprotein L; Human; In Vitro; Investigation; KRAS2 gene; Laboratories; Link; Lung; Lung Neoplasms; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Mediator of activation protein; Molecular Conformation; Mutation; Non-Small-Cell Lung Carcinoma; Oncogenes; Oncogenic; Paclitaxel; Pathway interactions; Patients; Phenotype; Phosphorylation; Play; Protein Isoforms; Purines; RNA; RNA Recognition Motif; RNA Sequences; RNA Splicing; RNA immunoprecipitation sequencing; Radiation; Recombinants; Regulation; Reporting; Repression; Resistance; Role; Signal Transduction; Small Interfering RNA; Specificity; Surface Plasmon Resonance; Survival Rate; Therapeutic; Transcript; Tumorigenicity; United States; Unresectable; Validation; Veterans; Woman; anti-cancer therapeutic; base; cancer cell; cell transformation; chemotherapy; combat; in vivo; mRNA Precursor; men; metaplastic cell transformation; mortality; mouse model; mutant; neoplastic cell; next generation; novel; novel therapeutic intervention; novel therapeutics; outcome forecast; palliative; preference; small hairpin RNA; transcriptome sequencing; tumor; tumorigenic

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 the 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 regard, we are focusing on NSCLC models for examining distal signaling mechanisms that modulate the generation and maintenance of NSCLC cells/tumors. Specifically, this grant application begins with a focus on the consequence of expressing caspase 9b (C9b) in lung epithelium. The expression of C9b is regulated by alternative RNA splicing via the inclusion or exclusion of a four exon cassette (exons 3,4,5,6). Inclusion of this exon cassette into the mature transcript produces the pro-apoptotic caspase 9 (caspase 9a) while the exclusion produces the anti-apoptotic and survival/oncogenic signaling factor, caspase 9b. Studies from our laboratory have demonstrated that NSCLC tumors present with a dysregulated (e.g. low) ratio of caspase 9/caspase 9b analogous to an anti-apoptotic/chemotherapy resistance phenotype. Subsequent studies by our laboratory demonstrated that the expression of C9b had important functions in the anchorage-independent growth (AIG) of NSCLC cells, AIG induced by oncogenic mutation in non-transformed human bronchial epithelial cells, and chemotherapy sensitivity (e.g. cisplatinum and paclitaxel). Mechanistically, our laboratory identified an exonic splicing silencer (C9/E3-ESS) in exon 3 that regulates the inclusion of the exon 3,4,5,6 cassette of caspase 9 pre-mRNA. The RNA trans-factor, hnRNP L, was shown to associate with this RNA cis-element, repress the inclusion of the exon cassette, and induce caspase 9b expression. 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. Our proposed studies will dramatically extend these previous findings by first determining whether C9b is a key oncogenic signaling factor in the transformation of lung epithelial cells. The next set of proposed studies will determine how hnRNP L becomes activated to drive the expression of C9b. Our last set of proposed studies extend the role of hnRNP L in regard to NSCLC. In stark contrast with our findings on the phosphorylation of Ser52 in hnRNP L in transformed cells, downregulation of hnRNP L in non-transformed cells had no effect on RNA splicing events important in maintaining oncogenic phenotypes (e.g. AIG). Further investigations by our laboratory determined that the lack of effect on RNA splicing events in non-transformed cells was due to a lack of phosphorylation of Ser52 in hnRNP L. Thus, these findings suggest that the phosphorylation of hnRNP L (i.e. activation in transformed cells) mediates specific RNA splicing events important in cell survival, proliferation, AIG, and tumor formation versus constitutive functions of non- phosphorylated hnRNP L. Therefore, we hypothesize that the phosphorylation of hnRNP L on Ser52 is required for modulating a specific subset of splicing events, which are important for NSCLC cells to develop and maintain transformed phenotypes. Our proposed studies will serve to determine this specific “cluster” of RNA splicing events and further investigate the biological relevance of these events in maintaining the oncogenic phenotypes of NSCLC cells.

如今，肺癌是工业化国家男性和女性死亡的主要原因 据估计，这些国家的癌症死亡人数占美国所有癌症死亡人数的 28%。 癌症（NSCLC）占肺癌的大多数，预后较差，中位生存期为 大多数患者出现无法切除的疾病，目前的治疗选择是 化疗和放疗充其量只是姑息治疗，因此需要新的治疗策略。 NSCLC 为了影响这种疾病，在这方面，我们重点关注用于检查远端的 NSCLC 模型。 具体而言，调节 NSCLC 细胞/肿瘤的生成和维持的信号传导机制。 拨款申请首先关注肺上皮中表达 caspase 9b (C9b) 的结果。 C9b 的表达通过包含或排除四个外显子的选择性 RNA 剪接进行调节 盒（外显子 3、4、5、6）包含在成熟转录物中会产生促凋亡。 caspase 9 (caspase 9a)，而排除会产生抗凋亡和存活/致癌信号因子， 我们实验室的研究表明 NSCLC 肿瘤存在失调。 （例如低）Caspase 9/Caspase 9b 的比率类似于抗凋亡/化疗耐药表型。 本实验室后续研究表明C9b的表达在 NSCLC 细胞的贴壁依赖性生长 (AIG)，非转化细胞中致癌突变诱导的 AIG 人支气管上皮细胞和化疗敏感性（例如顺铂和紫杉醇）。 我们的实验室在外显子 3 中发现了一个外显子剪接沉默子 (C9/E3-ESS)，它调节包含 Caspase 9 前体 mRNA 的外显子 3,4,5,6 盒显示 RNA 反式因子 hnRNP L 与相关。 该 RNA 顺式元件抑制外显子盒的包含，并诱导 caspase 9b 表达。 重要的是，hnRNP L 在 ser52 上的磷酸化（仅在转化细胞中观察到）是 外显子 3,4,5,6 盒的抑制最后，hnRNP L 的 ser52 磷酸化被证明是必需的。 通过 Caspase 9 的选择性剪接调节 NSCLC 细胞的致瘤能力。这些关键 机制是转化细胞特有的，可转化为 > 70% 的 NSCLC，并且位于最远端 因此，这些远端机制是合理且高度期望的目标。 我们提出的新抗癌疗法的开发将极大地扩展以前的这些研究。 首先确定 C9b 是否是肺转化中关键的致癌信号因子 下一组拟议的研究将确定 hnRNP L 如何被激活以驱动 C9b的表达。 我们最后一组提出的研究扩展了 hnRNP L 在 NSCLC 中的作用，与此形成鲜明对比。 我们对转化细胞中 hnRNP L 中 Ser52 磷酸化的发现，以及 hnRNP L 中 hnRNP L 下调的发现 非转化细胞对维持致癌表型很重要的RNA剪接事件没有影响 （例如 AIG）我们实验室的进一步研究确定，对 RNA 剪接事件缺乏影响。 未转化的细胞是由于 hnRNP L 中 Ser52 缺乏磷酸化所致。因此，这些发现表明 hnRNP L 的磷酸化（即转化细胞中的激活）介导特定的 RNA 剪接事件 与非细胞的组成功能相比，对于细胞存活、增殖、AIG 和肿瘤形成很重要 hnRNP L 被磷酸化。因此，我们认为 hnRNP L 在 Ser52 上的磷酸化是必需的 用于调节剪接事件的特定子集，这对于 NSCLC 细胞的发育和维持非常重要 我们提出的研究将有助于确定这个特定的 RNA 剪接“簇”。 事件并进一步研究这些事件在维持致癌表型方面的生物学相关性 NSCLC 细胞。