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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：
9764287
负责人：
Andrew Todd Ludlow
金额：
$ 24.3万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9764287
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 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 anti-cancer cancer cell cancer initiation cancer type career design experimental study in vivo in vivo Model in vivo evaluation 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(申请人提供)：绝大多数(&gt；85%)人类癌症使用端粒酶作为维持端粒长度的主要机制。我的长期目标是建立一个独立的研究实验室，在那里我将阐明选择性剪接在端粒长度维持机制中发挥作用的分子机制。这些基本的生物学信息对于我设计端粒酶特异性癌症疗法的长期目标将是重要的。K99/R00职业生涯奖将帮助我实现作为一名研究科学家的目标，促进我在以下方面的培训：在Woodring Wright博士的共同指导下，基因表达调控、选择性剪接、下一代测序和分子生物学，以及在曾傑瑞·Shay博士的共同指导下，癌细胞生物学和癌细胞表型模型。这次培训和指导将补充我之前在基因表达、肌肉生物学和细胞信号方面的培训。德克萨斯大学西南医学中心的科学环境非常令人兴奋，将为我完成这个项目的指导阶段提供必要的设施，并为我顺利过渡到独立教员阶段做好准备。HTERT的表达受发育调节，有几种剪接变体。只有全长hTERT mRNA才能产生催化活性的端粒酶。在分化为成熟组织后，端粒酶活性下调，仅保留非催化活性的hTERT剪接变体。然而，在转化为癌症后，大多数类型的癌症表达全长hTERT，并具有功能性端粒酶活性，有助于在癌前病变中维持已经非常短的端粒。HTERT的转录激活已被广泛研究，但hTERT选择性剪接的调控几乎完全未知。在目标1中，在K99阶段，我将定义我在两个不同的RNAi屏幕中发现的NOVA1在调节hTERT剪接中的作用，端粒长度和端粒酶活性。我将确定NOVA1是直接结合hTERT前-mRNA(CLIP)还是在蛋白质复合体中结合(RIP)。我还将通过RNA测序确定NOVA1在癌细胞选择性剪接中的作用。在目标2中，在K99阶段，我将使用两种肿瘤进展的异种移植模型来确定NOVA1水平的降低是否通过抑制端粒酶来减少转移和肿瘤进展。在R00阶段，目标3，我将表征NOVA1‘S 3’UTR在癌细胞中的调节，以及使用NOVA1‘3’UTR报告进行小分子筛选以寻找新的端粒酶抑制物的可能性。我还将继续破译我在指导阶段发现的“hTERT剪接密码”，目的是找到抑制癌症端粒酶的新方法。这些研究应该导致一个强有力的研究计划，研究替代香料如何参与和调节癌细胞中的端粒长度维持机制。