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

• 批准号: 10212283
• 负责人: Nicholas Oliver Stevers
• 金额: $ 4.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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/10212283
• 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; Image; 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; 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

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.

项目摘要 人类癌症的基本标志是他们克服复制性衰老和 实现细胞永生。通常在分化的体细胞中沉默的人类肿瘤中有90％ 在肿瘤发生期间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模板分子的复合物 TERC和其他蛋白质与在染色体末端的重复序列结合并扩展 端粒。最近，在许多人中已经描述了TERT启动子（TERTP）中的非编码突变 癌症。这些TERTP突变是所有癌症中最常见的非编码突变，是 许多癌症中最常见的突变，例如83％的IDH WildType胶质母细胞瘤（GBM），最常见 成人脑肿瘤的常见和致命形式。这些突变导致典型E26的形成 转换特定（ET）结合基序，与TERTP天然ETS位点同时起作用以招募A 特定的ETS转录因子，GA结合蛋白（GABP）复合物。我们的实验室证明了这一点 GABP的募集对于TERT重新激活和维持肿瘤细胞的不朽所必需。 此外，我们的实验室通过CRISPR-CAS9介导的四聚体形成亚基的突变表明， GABPB1L（B1L），GABP的四聚体形式对于维持这种肿瘤细胞的不朽而言是必需的。 有趣的是，尽管CRISPR-CAS9介导B1L的消融并未显示出TERT的减少 表达，它确实显示出GABP二聚体特异性亚基GABPB1S（B1S）的大幅度增加。重要的是，它 似乎B1S表达的增加可能是由于GABP四聚体介导的负反馈回路。 此外，初步证据表明，B1s的增加可能允许包含二聚体的B1结合 到突变体TERTP并保持TERT表达。广泛地，这项研究旨在阐明分子 在B1L消除状态下维持TERT表达的基础机制。这样，这个 研究将确定B1L降低期间B1S表达上调的基础机制，将 确定含B1S的GABP二聚体是否是突变体TERTP的合格调节剂。端粒酶具有 长期以来一直是肿瘤细胞永生逆转的有吸引力的治疗靶标。但是，试图定位 由于端粒酶表达干细胞，端粒酶主要不成功。先前的研究表明 在TERTP突变体癌中靶向B1L将允许肿瘤特异性抑制端粒酶，但是，我们的 数据表明，这种方法比以前所欣赏的要复杂。重要的是， 这些调查的发现将指导有效耐用疗法的设计和开发 靶向GABP-TERT轴以逆转TERTP突变体癌症（例如 胶质母细胞瘤，黑色素瘤，膀胱癌等。