Identification of a GABP Feedback Loop and its Role in Tumor Cell Immortality

GABP 反馈环路的识别及其在肿瘤细胞永生中的作用

基本信息

批准号：
10403601
负责人：
Nicholas Oliver Stevers
金额：
$ 4.24万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-03 至 2023-07-02
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10403601
关键词：
Ablation Adult Binding Binding Proteins Binding Sites Brain Neoplasms CRISPR/Cas technology Cell Aging Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats Coiled-Coil Domain Complex DNA Binding Data Development Doxycycline Exhibits Exons Feedback Genes Genomic DNA Glioblastoma Growth Homologous Gene Human Impairment Implant In Vitro Investigation Knock-out Knockout Mice Length LoxP-flanked allele Luciferases Maintenance Malignant Neoplasms Malignant neoplasm of urinary bladder Measures Mediating Molecular Monitor Mus Mutate Mutation Population Production Protein Isoforms Proteins RNA RNA-Directed DNA Polymerase Repetitive Sequence Resolution Role Site Small Interfering RNA Somatic Cell Stains TERT gene Telomerase Telomerase inhibition Therapeutic Transactivation U251 Untranslated RNA Up-Regulation Variant Western Blotting Work beta-Galactosidase bioluminescence imaging cell growth design dimer expression vector in vivo knock-down melanoma mutant neoplastic cell new therapeutic target promoter protein complex recruit senescence stem cells targeted treatment telomere therapeutic target transcription factor tumor tumor growth tumorigenesis vector

项目摘要

Project Summary A fundamental hallmark of human cancers is their ability to overcome replicative senescence and achieve cellular immortality. Normally silenced during differentiation in somatic cells, 90% of human tumors reactivate Telomerase Reverse Transcriptase (TERT) expression early during tumorigenesis to achieve cellular immortality. TERT, the catalytic subunit of telomerase, complexes with the RNA template molecule TERC and other proteins and binds to and extends the repetitive sequences at chromosomal ends, known as telomeres. Recently, non-coding mutations in the TERT promoter (TERTp) have been described in many cancers. These TERTp mutations are the most common non-coding mutation across all cancers and are the most frequent mutations in many cancers, such as 83% of IDH wildtype glioblastomas (GBM), the most common and deadly form of adult brain tumors. These mutations result in the formation of canonical E26 Transformation Specific (ETS) binding motifs that work in tandem with TERTp native ETS sites to recruit a specific ETS transcription factor, the GA-binding protein (GABP) complex. Our lab has demonstrated that this recruitment of GABP is necessary for TERT reactivation and maintenance of tumor cell immortality. Furthermore, our lab has shown, through CRISPR-cas9 mediated mutation of the tetramer forming subunit, GABPB1L (B1L), that the tetrameric form of GABP is necessary for this maintenance of tumor cell immortality. Interestingly, while CRISPR-Cas9 mediated ablation of B1L does not show a reduction in TERT expression, it does show a large increase in the GABP dimer specific subunit, GABPB1S (B1S). Importantly, it seems that this increase in B1S expression may be due to a GABP tetramer mediated negative feedback loop. Furthermore, preliminary evidence suggests that this increase in B1S may allow B1S containing dimers to bind to the mutant TERTp and maintain TERT expression. Broadly, this study aims to elucidate the molecular mechanisms underlying the maintenance of TERT expression during states of B1L elimination. In doing so, this study will determine the mechanisms underlying B1S expression upregulation during B1L reduction and will determine if B1S-containing GABP dimers are competent regulators of the mutant TERTp. Telomerase has long been an attractive therapeutic target for the reversal of tumor cell immortality; however, attempts to target telomerase have been mostly unsuccessful due to telomerase expressing stem cells. Prior studies suggest that targeting B1L in TERTp mutant cancers would allow for tumor specific inhibition of telomerase, however, our data have revealed that this approach is more complicated than previously appreciated. Importantly, the findings from these investigations will guide the design and development of effective and durable therapies targeting the GABP-TERT axis for the reversal of tumor cell immortality in TERTp mutant cancers such as glioblastoma, melanoma, bladder cancer, and many others.

项目概要人类癌症的一个基本特征是它们能够克服复制衰老和实现细胞永生。 90% 的人类肿瘤的体细胞分化过程中通常处于沉默状态在肿瘤发生早期重新激活端粒酶逆转录酶 (TERT) 表达，以实现细胞永生。 TERT，端粒酶的催化亚基，与 RNA 模板分子形成复合物 TERC 和其他蛋白质结合并延伸染色体末端的重复序列，称为端粒。最近，TERT 启动子 (TERTp) 中的非编码突变已在许多研究中得到描述。癌症。这些 TERTp 突变是所有癌症中最常见的非编码突变，并且是许多癌症中最常见的突变，例如 83% 的 IDH 野生型胶质母细胞瘤 (GBM) 成人脑肿瘤的常见且致命的形式。这些突变导致规范 E26 的形成转化特异性 (ETS) 结合基序与 TERTp 天然 ETS 位点协同工作，以招募特定的 ETS 转录因子，GA 结合蛋白 (GABP) 复合物。我们的实验室已经证明，这 GABP 的募集对于 TERT 重新激活和维持肿瘤细胞永生是必要的。此外，我们的实验室已经表明，通过 CRISPR-cas9 介导的四聚体形成亚基突变， GABPB1L (B1L)，认为四聚体形式的 GABP 对于维持肿瘤细胞的永生性是必需的。有趣的是，虽然 CRISPR-Cas9 介导的 B1L 消融并未显示 TERT 减少表达，它确实显示 GABP 二聚体特异性亚基 GABPB1S (B1S) 的大量增加。重要的是，它 B1S 表达的增加似乎可能是由于 GABP 四聚体介导的负反馈环所致。此外，初步证据表明，B1S 的增加可能允许含有二聚体的 B1S 结合突变的 TERTp 并维持 TERT 表达。总的来说，这项研究旨在阐明分子 B1L 消除状态期间维持 TERT 表达的机制。这样做，这研究将确定 B1L 减少过程中 B1S 表达上调的机制，并将确定包含 B1S 的 GABP 二聚体是否是突变体 TERTp 的有效调节剂。端粒酶有长期以来一直是逆转肿瘤细胞永生性的一个有吸引力的治疗靶点；然而，试图瞄准由于表达端粒酶的干细胞，端粒酶大多不成功。先前的研究表明在 TERTp 突变癌症中靶向 B1L 将允许端粒酶的肿瘤特异性抑制，然而，我们的数据显示，这种方法比以前想象的更复杂。重要的是，这些研究的结果将指导有效且持久的疗法的设计和开发靶向 GABP-TERT 轴以逆转 TERTp 突变癌症中的肿瘤细胞永生性，例如胶质母细胞瘤、黑色素瘤、膀胱癌等。