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，肺癌已成为工业化男性和女性死亡的主要原因 国家估计占美国所有癌症死亡的28％。非小细胞肺 癌症（NSCLC）代表大多数肺癌，并具有不良预后，中位生存期的中位数 少于12个月。大多数患者出现了无法切除的疾病，以及目前的治疗选择 化学疗法和放射线充其量充其量是姑息性的。因此，需要新的策略来治疗 为了影响这种疾病的NSCLC。在这方面，我们专注于检查识别的NSCLC模型 信号传导机制调节NSCLC细胞/肿瘤的生成和维护。具体来说，这是 授予申请的重点是在肺上皮表达caspase 9b（C9b）的后果。 C9b的表达通过替代RNA剪接通过包含或排除四个外显子调节 录音带（外显子3,4,5,6）。将这个外显盒纳入成熟的转录本会产生促凋亡 caspase 9（caspase 9a）虽然排除产生抗凋亡和生存/致癌信号因子，但 caspase 9b。我们实验室的研究表明，NSCLC肿瘤患有失调 （例如，caspase 9/caspase 9b的比率低）类似于抗凋亡/化学疗法抗性表型。 随后我们的实验室研究表明，C9b的表达在 NSCLC细胞的锚固非依赖性生长（AIG），AIG是由非转化的致癌突变引起的 人支气管上皮细胞以及化学疗法敏感性（例如顺铂和紫杉醇）。机械上， 我们的实验室确定了外显子3中的外部剪接消音器（C9/E3-ESS）调节包含 外显子3,4,5,6 caspase 9前MRNA的盒子。 RNA反系数HNRNP L，显示与 该RNA顺式元素反映了外显子盒的包含，并诱导caspase 9b表达。 重要的是，Ser52上HNRNP L的磷酸化（仅在转化细胞中观察到）才需要 抑制外显子3,4,5,6盒式磁带。最后，显示了HNRNP L的Ser52磷酸化作为所需 NSCLC细胞通过caspase 9的替代剪接的介体。 机制特定于转化的细胞，可翻译成> 70％的NSCLC，在极端的远端 在致癌途径中。因此，这些远端机制是合理的，并且是高度期望的目标 开发新的抗癌疗法。我们提出的研究将极大地扩展这些先前的 首先确定C9B是否是肺转化中的关键致癌信号传导因子 上皮细胞。下一组提出的研究将确定HNRNP L如何激活以驱动 C9b的表达。 我们最后一组提出的研究扩展了HNRNP L在NSCLC方面的作用。与之形成鲜明对比 我们关于在转化细胞中HNRNP L中Ser52磷酸化的发现，HNRNP L在中的下调 未转化的细胞对RNA剪接事件没有影响，对维持致癌表型很重要 （例如AIG）。我们实验室的进一步研究确定，缺乏对RNA剪接事件的影响 未转化的细胞是由于HNRNP L中Ser52缺乏磷酸化所致。这些发现表明 HNRNP L（即转化细胞中的激活）的磷酸化介导了特定的RNA剪接事件 在细胞存活，增殖，AIG和肿瘤形成中重要重要 因此，我们假设需要HNRNP L在Ser52上的磷酸化。 用于调节剪接事件的特定子集，这对于NSCLC细胞开发和维护很重要 转化的表型。我们提出的研究将有助于确定RNA剪接的特定“群集” 事件并进一步研究这些事件在维持致癌表型中的生物学相关性 NSCLC细胞。