Targeting the PTAP domain of TSG101 in ERBB2-associated mammary cancer

靶向 ERBB2 相关乳腺癌中 TSG101 的 PTAP 结构域

• 批准号: 9377349
• 负责人: Kay-Uwe Wagner
• 金额: $ 7.58万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9377349
• 关键词: Address; Amino Acid Motifs; Amino Acid Sequence; Autophagocytosis; Binding; Biochemical; Biological; Breast Cancer Cell; Breast Cancer Prevention; C-terminal; CRISPR/Cas technology; Cell Cycle Arrest; Cell Death; Cell Line; Cell Proliferation; Cells; Clinical Data; Complex; Degradation Pathway; Development; Disease; Drug Targeting; EGFR gene; ERBB2 gene; Epidermal Growth Factor Receptor; Etiology; Exhibits; Family member; Generations; Genes; Genetic Models; Genetically Engineered Mouse; Goals; Growth; Human; In Vitro; Investigation; Knock-in; Knock-in Mouse; Knock-out; Malignant - descriptor; Malignant Neoplasms; Mammary Neoplasms; Mammary Tumorigenesis; Mammary gland; Metastatic to; Modality; Modeling; Mouse Mammary Tumor Virus; Movement; Mus; Mutate; N-terminal; Neoplastic Epithelial Cell; Oncogenic; Outcome; Pathway interactions; Patients; Pertuzumab; Phenotype; Phosphotransferases; Pilot Projects; Play; Property; Proteins; Receptor Activation; Receptor Protein-Tyrosine Kinases; Relapse; Role; Signal Transduction; Sorting - Cell Movement; TSG101 gene; Tertiary Protein Structure; Transgenic Mice; Trastuzumab; Variant; Work; base; cancer initiation; cancer subtypes; cancer therapy; design; drug development; evidence base; genetic regulatory protein; in vivo; in vivo Model; malignant breast neoplasm; mammary epithelium; member; mouse model; mutant; neoplastic cell; novel strategies; novel therapeutics; overexpression; prevent; promoter; protein function; receptor; receptor downregulation; response; targeted treatment; therapeutic target; trafficking; ubiquitin ligase

PROJECT SUMMARY Aberrant signaling through receptor tyrosine kinases of ERBB family members plays a key role in the etiology of breast cancer. Approximately 20-25% of invasive breast cancers exhibit an amplification of the ERBB2 locus or a constitutive activation of the receptor tyrosine kinase encoded by this gene. There is a substantial body of experimental and clinical data that shows that ERBB2-overexpressing breast cancer cells have an elevated metastatic potential. Despite initial response to targeted therapies against ERBB2 using trastuzumab and pertuzumab, the majority of patients eventually relapse and succumb to metastatic disease. Therefore, novel therapeutic strategies are needed to prevent and treat this aggressive breast cancer subtype. As a component of the endocytic machinery, the protein encoded by the Tumor Susceptibility Gene 101 (TSG101) is crucial for the intra-cellular trafficking, sorting, and lysosomal degradation of ubiquitinated cargo proteins including ERBB2 and other receptor tyrosine kinases. TSG101 is a central node in trafficking as it is able to simultaneously bind cargo proteins as well as PTAP amino acid motif-containing regulatory proteins and ubiquitin ligases that modify the sorting and trafficking of cargo complexes. TSG101 itself possesses an intrinsic PTAP motif near its C-terminal end, and we gathered preliminary evidence that this domain can associate with the PTAP binding groove at the N-terminus of TSG101. Moreover, we can demonstrate that mutating the intrinsic PTAP motif into an ATAA amino acid sequence and thereby blocking the intramolecular binding modality leads to a very significant decrease in the steady-state level of TSG101 and a simultaneous decline in the expression of its cargo proteins ERBB2, EGFR, and IGF-R1. The immediate objectives of this exploratory project are to extend the preliminary findings from in vitro studies and to develop a TSG101-ATAA knockin mouse model using the CRISPR/Cas9-based gene editing approach to firmly establish that disrupting the intramolecular association of the intrinsic PTAP domain with its binding grove increases the turnover and reduces the expression of TSG101 in vivo. We will then apply this mutant TSG101-ATAA knockin model to assess whether lowering the steady-state level of TSG101 will be sufficient to co-downregulate oncogenic ERBB2 and prevent the onset and progression of ERBB2-indcued mammary cancer in transgenic mice. Accelerating the turnover of TSG101 and its cargos represents a novel approach to prevent and treat ERBB2- positive breast cancer, and the collective results from this exploratory project will provide strong in vivo evidence for the development of a new class of therapeutics that target specific intramolecular associations within TSG101.

项目摘要 通过ERBB家族成员的受体酪氨酸激酶的异常信号传导在病因学中起关键作用 乳腺癌大约20-25%的浸润性乳腺癌表现出ERBB 2基因座扩增 或由该基因编码的受体酪氨酸激酶的组成性激活。有大量的 实验和临床数据表明，ERBB 2过表达乳腺癌细胞具有升高的 转移潜能尽管对使用曲妥珠单抗的ERBB 2靶向治疗有初步应答， 在帕妥珠单抗治疗后，大多数患者最终复发并死于转移性疾病。因此，小说 需要治疗策略来预防和治疗这种侵袭性乳腺癌亚型。含 肿瘤易感基因101（TSG 101）编码的蛋白质对于内吞机制至关重要。 泛素化货物蛋白的细胞内运输、分选和溶酶体降解，包括 ERBB 2和其他受体酪氨酸激酶。TSG 101是贩运的中心节点，因为它能够 同时结合货物蛋白以及含有PTAP氨基酸基序的调节蛋白， 泛素连接酶修饰货物复合物的分选和运输。TSG 101本身具有 在其C-末端附近的固有PTAP基序，我们收集了初步证据，表明该结构域可以 与TSG 101的N-末端的PTAP结合沟结合。此外，我们可以证明， 将所述固有PTAP基序突变成ATAA氨基酸序列，从而阻断分子内的 结合方式导致TSG 101的稳态水平非常显著降低，同时 其货物蛋白ERBB 2、EGFR和IGF-R1的表达下降。这一行动的直接目标是 探索性项目是扩展体外研究的初步结果，并开发TSG 101-ATAA 使用基于CRISPR/Cas9的基因编辑方法的敲入小鼠模型，以牢固地建立破坏 内在PTAP结构域与其结合格罗夫的分子内结合增加了周转， 降低TSG 101在体内的表达。然后，我们将该突变TSG 101-ATAA敲入模型应用于 评估降低TSG 101的稳态水平是否足以共同下调致癌基因， ERBB 2并预防转基因小鼠中ERBB 2诱导的乳腺癌的发生和进展。 加速TSG 101及其货物的周转代表了预防和治疗ERBB 2的新方法。 阳性乳腺癌，从这个探索性项目的集体结果将提供强有力的体内 开发靶向特定分子内关联的新型疗法的证据 在TSG 101中。