Manipulating hTERT splicing in lung cancer cells.

 描述（由申请人提供）：绝大多数（>85%）人类癌症使用端粒酶作为主要的端粒长度维持机制。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我设计端粒酶特异性癌症治疗的长期目标很重要。K99/R 00职业奖将帮助我实现我作为研究科学家的目标，通过推进我在以下方面的培训：基因表达调控，选择性剪接，下一代测序和分子生物学在Woodring Wright博士的共同指导下，以及癌细胞生物学和癌细胞表型模型在Jerry Shay博士的共同指导下。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训练。UT西南医学中心的科学环境非常刺激，将为我提供完成该项目指导阶段所需的设施，并为我顺利过渡到独立教师阶段做好准备。hTERT表达受发育调节，并具有几种剪接变体。只有全长hTERTmRNA产生催化活性端粒酶。在分化成成熟组织后，端粒酶活性下调，仅保留非催化活性的hTERT剪接变体。然而，在转化为癌症时，大多数癌症类型表达全长hTERT，并具有功能性端粒酶活性，有助于维持癌前病变中已经非常短的端粒。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筛选中鉴定的NOVA 1在hTERT剪接调节中的作用， 端粒长度和端粒酶活性。我将确定NOVA 1是否直接（CLIP）或以蛋白复合物（RIP）结合hTERT前mRNA。我还将通过RNA测序确定NOVA 1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NOVA 1水平降低是否通过抑制端粒酶导致更少的转移和肿瘤进展。在R 00阶段，目标3，我将描述NOVA 1的3 'UTR在癌细胞中的调节，以及使用NOVA 1 3' UTR报告基因进行小分子筛选以发现新的端粒酶抑制剂的潜力。我也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大的研究计划，以了解替代香料如何参与和调节癌细胞中的端粒长度维持机制。

 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.