Manipulating hTERT splicing in lung cancer cells.

操纵肺癌细胞中的 hTERT 剪接。

基本信息

批准号：
9267141
负责人：
Andrew Todd Ludlow
金额：
$ 14.47万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9267141
关键词：
3&apos Untranslated Regions A549 Alternative Splicing Award Binding Binding Sites Bioinformatics Biological Biological Assay Biology Cancer cell line Candidate Disease Gene Cell Line Cell model Cells Cellular biology Clinical Trials Code Data Development Dominant-Negative Mutation Environment Enzymes Epithelial Cells Excision Faculty Follow-Up Studies Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Transcription Goals Growth H1299 Human In Vitro Introns Kinetics Laboratory Research Lead Length Lesion Link Luciferases Malignant Neoplasms Malignant neoplasm of lung Mediating Medical center Mentors Mentorship Messenger RNA MicroRNAs Minisatellite Repeats Modeling Molecular Molecular Biology Muscle Neoplasm Metastasis Non-Small-Cell Lung Carcinoma Pathway Analysis Pathway interactions Patients Pattern Pharmaceutical Preparations Phase Phenotype Play Premalignant Protein Isoforms Proteins RNA Splicing RNA interference screen RNA-Binding Proteins RNA-Directed DNA Polymerase Recurrent disease Regulation Reporter Reporter Genes Research Resources Role Sampling Scientist Series Signal Transduction Spices Surveys Telomerase Telomerase Inhibitor Telomerase inhibition Telomere Length Maintenance Telomere Maintenance Telomere Shortening Testing Therapeutic Tissues Training Trans-Activators Transcript Transcriptional Activation Variant Xenograft Model Xenograft procedure animal imaging cancer cell cancer initiation cancer type career design experimental study in vivo in vivo Model insight knock-down loss of function mRNA Precursor neoplastic cell new therapeutic target next generation sequencing novel novel strategies prevent programs protein complex public health relevance response screening small hairpin RNA small molecule small molecule inhibitor standard of care success telomere therapeutic target tool transcriptome sequencing tumor tumor progression

项目摘要

DESCRIPTION (provided by applicant): The vast majority (>85%) of human cancers use telomerase as the primary telomere length maintenance mechanism. My long-term goal is to establish an independent research laboratory, where I will elucidate the molecular mechanisms by which alternative splicing plays a role in telomere length maintenance mechanisms. This basic biological information will be important for my long-term goals for designing telomerase specific cancer therapeutics. The K99/R00 career award will help me in achieving my goals as a research scientist by advancing my training in: gene expression regulation, alternative splicing, next generation sequencing, and molecular biology under the co-mentorship of Dr. Woodring Wright, as well as cancer cell biology and models of cancer cell phenotypes under the co-mentorship of Dr. Jerry Shay. This training and guidance will compliment my previous training in gene expression, muscle biology, and cell signaling. The scientific environment at UT Southwestern Medical Center is extremely stimulating and will provide me with the facilities necessary for the completion of the mentored phase of this project and will prepare me to transition smoothly into the independent faculty phase. hTERT expression is developmentally regulated and has several splice variants. Only full-length hTERT mRNA produces catalytically active telomerase. Upon differentiation into mature tissue telomerase activity is down-regulated and only non-catalytically active hTERT splice variants remain. However, upon transformation to cancer, the majority of cancer types express full-length hTERT and have functional telomerase activity that helps to maintain the already very short telomeres in precancerous lesions. The transcriptional activation of hTERT has been widely studied, but the regulation of hTERT alternative splicing is almost entirely unknown. In Aim 1, during the K99 phase, I will define the role of NOVA1 that I identified in two different RNAi screens, in the regulation of hTERT splicing, telomere length, and telomerase activity. I will determine if NOVA1 binds hTERT pre-mRNA directly (CLIP) or in a protein complex (RIP). I will also define the role of NOVA1 in cancer cell alternative splicing by RNA sequencing. In Aim 2, during the K99 phase, I will use two xenograft models of tumor progression to determine if reduced levels of NOVA1 result in less metastases and tumor progression via inhibition of telomerase. During the R00 phase, Aim 3, I will characterize the regulation of NOVA1's 3'UTR in cancer cells and the potential of using a NOVA1 3'UTR reporter for small molecule screening to find a novel telomerase inhibitor. I will also continue to decipher the "hTERT splicing code" I discovered during my mentored phase with the goal of finding novel means to inhibit telomerase in cancer. These studies should lead to a robust research program into how alternative spicing participates and regulates telomere length maintenance mechanisms in cancer cells.

描述（由应用程序提供）：绝大多数（> 85％）的人类癌症将端粒酶作为主要的端粒长度维护机制。我的长期目标是建立一个独立的研究实验室，我将阐明替代剪接在端粒长度维持机制中起作用的分子机制。这种基本的生物学信息对于我设计特定于端粒酶癌症的长期目标将很重要。 K99/R00职业奖将通过推进我的培训来帮助我实现我作为研究科学家的目标：在伍德·赖特（Wooding Wright）博士的同事下，在Wooding Wright博士的同事下，替代剪接，下一代测序和分子生物学，以及癌症细胞生物学和癌症细胞表型模型下的癌症细胞表型。这种训练和指导将补充我以前在基因表达，肌肉生物学和细胞信号传导方面的培训。 UT西南医学中心的科学环境极具刺激性，将为我提供完成该项目修订阶段所需的设施，并将为我准备顺利过渡到独立的教师阶段。开发了HTERT表达，并具有多种剪接变体。仅全长HTERT mRNA会产生催化活性的端粒酶。分化为成熟组织端粒酶活性后，仅仍存在非催化性HTERT剪接变体。但是，在转化为癌症后，大多数癌症类型表达全长HTERT并具有功能性端粒酶活性，有助于维持预科损伤中已经很短的端粒。 HTERT的转录激活已被广泛研究，但是HTERT替代剪接的调节几乎完全未知。 AIM 1，在K99阶段，我将定义我在两个不同RNAi屏幕中确定的Nova1的作用，在调节HTERT剪接中，端粒长度和端粒酶活性。我将确定NOVA1是否直接结合HTERT PRE-MRNA（夹子）或蛋白质复合物（RIP）。我还将通过RNA测序来定义NOVA1在癌细胞替代剪接中的作用。在AIM 2中，在K99阶段，我将使用两个异种移植模型的肿瘤进展模型来确定NOVA1的水平降低是否导致转移和肿瘤进展减少，而肿瘤进展是否会抑制端粒酶。在R00阶段，AIM 3中，我将表征癌细胞中Nova1的3'UTR的调节，以及使用Nova1 3'Utr报告基因进行小分子筛选以找到新型端粒酶抑制剂的潜力。我还将继续破译我在修订阶段发现的“ HTERT剪接代码”，目的是寻找新颖的手段来抑制癌症中的端粒酶。这些研究应导致一项强大的研究计划，以探讨替代性的调味参与和调节癌细胞中端粒长度维持机制的方式。