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: 10343740
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10343740
• 关键词: 19p13; Accounting; Address; Affinity Chromatography; Anchorage-Independent Growth; Antisense RNA; Applications Grants; Automobile Driving; Back; Biological; CDK4 gene; CDKN2A gene; CRISPR/Cas technology; Cancer Etiology; Cancer cell line; Cause of Death; Cell Survival; Cessation of life; Chemotherapy and/or radiation; Chromosomes; Clinical Trials; Consensus Sequence; Coupled; Cyclin-Dependent Kinase Inhibitor 2A; Cytoplasm; Data; Developed Countries; Development; Disease; Down-Regulation; Elements; Exclusion; Exons; Foundations; Fractionation; Gene Silencing; Genes; Germ Cells; Goals; Human; Investigation; Laboratories; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Messenger RNA; Modification; Molecular; Mutagenesis; Mutate; Mutation; Non-Small-Cell Lung Carcinoma; Nuclear; Oligonucleotides; PD-1/PD-L1; Pathway interactions; Patients; Phenotype; Production; Prognosis; Proteomics; Proto-Oncogene Proteins c-akt; RNA; RNA Splicing; Regulatory Pathway; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Small Interfering RNA; Survival Rate; Techniques; Technology; Testing; Transcript; Tumor Suppressor Proteins; Tumor-Suppressor Gene Inactivation; Tumorigenicity; United States; Unresectable; Untranslated RNA; Validation; Variant; Veterans; Woman; anti-cancer therapeutic; base; combat; experimental study; gene product; lung cancer cell; men; mortality; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; palliative; precision medicine; small hairpin RNA; small molecule inhibitor; targeted treatment; transcriptomics; tumor; tumorigenesis; tumorigenic

Lung cancer is the leading cause of cancer death for veterans and in the world, for both men and women in industrialized countries, accounting for an estimated 28% of all cancer deaths in the United States. Non-small cell lung cancer (NSCLC) represents the majority of these cancers,, and NSCLC has a 5-year survival rate of ~23% with current treatment options palliative. Hence, new therapeutic strategies are needed to impact this disease, and elucidation of new mechanisms controlling the tumorigenicity of NSCLC cells will provide the foundation upon which to build. In this regard, the gene, cyclin dependent kinase inhibitor 2A (CDKN2A), that encodes the tumor suppressors, p16(INK4A) and p14(ARF), is found inactivated or deleted in NSCLC at a high percentage (e.g., ~53% deletion rate). Importantly, our laboratory linked CDKN2A inactivation to dysregulation of the RNA splicing of an uncharacterized, long non-coding RNA (lncRNA) located on Chromosome 19p13.12 (ENSG00000267053). Specifically, exon 3 inclusion/exclusion into the mature RNA transcript was dysregulated in NSCLC cell lines with CDKN2A mutated/deleted, and thus, we now term this lncRNA, Cyclin Dependent Kinase Inhibitor 2A-regulated lncRNA 1 (CyKILR-1). Initial cell signaling studies using small molecule inhibitors also identified the AKT/CDK4 axis as a regulatory pathway upstream of CyKILR-1 RNA splicing linked to CDKN2A inactivation. In human NSCLC, CyKILR-1 splice variant expression correlated with lower patient survival. These findings are congruent with studies showing the linkage of the aberrant expression of non-coding RNAs located at Chromosome 19p13.12 to cancer development. In preliminary studies, specific downregulation of CyKILR-1 splice variants in NSCLC cells induced dramatic decreases in cell survival and anchorage-independent growth. Initial mechanistic studies examining CyKILR-1 Exon 3 revealed a consensus sequence for nuclear localization (TCTAGCTCAGCCC). Fractionation experiments then demonstrated differential localization between the two splice variants with CyKILR-1B (exon 3 excluded) localized to the cytoplasm in contrast to the nuclear localization of CyKILR-1A (exon 3 included). The culmination of these data forms the hypothesis that inactivation of the tumor suppressor encoding gene, CDKN2A, in NSCLC regulates the inclusion of exon 3 producing the CyKILR-1 splice variants, which localize to the cytoplasm and imparts pro-survival phenotypes to NSCLC cells and tumors. To examine this hypothesis in depth, we propose in this application the following independent, but synergistic specific aims (SAs): SA1: To determine the biological relevance of CyKILR-1 RNA splicing in NSCLC; SA2: To determine the regulating mechanism of CyKILR-1 RNA splicing; and SA3: To determine the cell signaling pathways regulated by CyKILR-1 splice variants. These specific aims are focused on scientific lines of investigation to address the need for new and precise therapies for treating NSCLC, which when combined with newer therapies (e.g., PD- L1/PD-1 interaction blockers; IMFINZI/durvalumab) may produce more durable results and a longer 5 yr survival. For example, the validation of our hypothesis in Aim 1 (i.e., CyKILR-1 splice variants regulate NSCLC cell survival) will show that targeting the this novel lncRNA mechanism is a promising approach to compromise lung cancer development and tumor maintenance for those tumors presenting with a CDKN2A inactivation mutation or deletion. With RNA-based therapies showing efficacy in clinical trials, these mechanisms become enticing targets for new therapeutics with the added benefit of being tailored to specific oncogenotypes. Aims 2 and 3 synergize with Aim 1 by determining the cellular mechanisms regulating both CyKILR-1 RNA splicing and the downstream signaling pathways modulated by CyKILR-1 splice variants. These findings will then be related back to Aim 1 in regard to specific tumor biological phenotypes, which would allow clinicians to use specific targeted therapies.

肺癌是退伍军人和全世界癌症死亡的主要原因， 在发达国家，估计占美国所有癌症死亡的28%。非小 细胞肺癌（NSCLC）占这些癌症的大多数，NSCLC的5年生存率为 ~23%，目前的治疗选择是姑息性的。因此，需要新的治疗策略来影响这一点。 疾病，并阐明控制NSCLC细胞致瘤性的新机制将提供 建立在什么基础上。在这方面，基因，细胞周期蛋白依赖性激酶抑制剂2A（CDKN 2A）， 编码肿瘤抑制因子p16（INK 4A）和p14（ARF），在NSCLC中被发现失活或缺失， 百分比（例如，~53%缺失率）。重要的是，我们的实验室将CDKN 2A失活与失调联系起来， 位于染色体19p13.12上的未表征的长非编码RNA（lncRNA）的RNA剪接 （ENSG00000267053）。具体而言，成熟RNA转录物中外显子3的包含/排除是 在CDKN 2A突变/缺失的NSCLC细胞系中，这种lncRNA表达失调，因此，我们现在将其称为细胞周期蛋白 依赖性激酶抑制剂2A调节的lncRNA 1（CyKILR-1）。使用小分子的初始细胞信号传导研究 分子抑制剂也将AKT/CDK 4轴鉴定为CyKILR-1 RNA上游的调节途径 与CDKN 2A失活相关的剪接。在人NSCLC中，CyKILR-1剪接变体表达与 降低患者生存率。这些发现与研究结果一致，表明异常的 位于染色体19p13.12的非编码RNA的表达与癌症的发生有关。初步 研究表明，NSCLC细胞中CyKILR-1剪接变体的特异性下调诱导细胞凋亡显著减少。 生存和锚定独立的增长。检查CyKILR-1外显子3的初步机制研究显示， 核定位共有序列（TCTAGCTCAGCCC）。分馏实验， 证明了两种剪接变体与CyKILR-1B（外显子3除外）之间的差异定位 与CyKILR-1A（包括外显子3）的核定位相反，CyKILR-1A定位于细胞质。的 这些数据的最终结果形成了肿瘤抑制基因编码基因的失活， CDKN 2A在NSCLC中调节外显子3的包含，产生CyKILR-1剪接变体，其定位于 细胞质并赋予NSCLC细胞和肿瘤促存活表型。为了检验这一假设， 为了更深入地了解本申请，我们在本申请中提出了以下独立但协同的具体目标（SA）： 确定CyKILR-1 RNA剪接在NSCLC中的生物学相关性; SA 2：确定CyKILR-1 RNA剪接在NSCLC中的调节作用。 CyKILR-1 RNA剪接的机制;和SA 3：为了确定由CyKILR-1 RNA剪接调节的细胞信号传导途径， CyKILR-1剪接变体。这些具体目标的重点是科学的调查路线，以解决 需要新的和精确的治疗NSCLC的疗法，当与较新的疗法（例如，PD- L1/PD-1相互作用阻滞剂; IMFINZI/durvalumab）可能产生更持久的结果， 生存例如，我们在目标1中的假设的验证（即，CyKILR-1剪接变体调节NSCLC 细胞存活）将表明，靶向这种新的lncRNA机制是一种有前途的方法， 对于那些呈现CDKN 2A失活的肿瘤， 突变或缺失。随着基于RNA的疗法在临床试验中显示出疗效，这些机制成为 新疗法的诱人靶点具有针对特定癌基因型定制的额外益处。目标2 和3通过确定调控CyKILR-1 RNA剪接的细胞机制与Aim 1协同作用， 以及由CyKILR-1剪接变体调节的下游信号传导途径。这些发现将被 与目标1有关的特定肿瘤生物学表型，这将允许临床医生使用 具体的靶向治疗。