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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 ERBB 家族成员受体酪氨酸激酶的异常信号在病因学中起着关键作用乳腺癌。大约 20-25% 的浸润性乳腺癌表现出 ERBB2 基因座扩增或由该基因编码的受体酪氨酸激酶的组成型激活。有一个实质性的机构实验和临床数据表明，ERBB2 过度表达的乳腺癌细胞的转移潜力。尽管使用曲妥珠单抗和帕妥珠单抗后，大多数患者最终会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。作为一个组件肿瘤易感基因 101 (TSG101) 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 ERBB2 和其他受体酪氨酸激酶。 TSG101 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可以改变货物复合物的分类和运输。 TSG101本身具有内在 PTAP 基序靠近其 C 末端，我们收集了初步证据表明该结构域可以与 TSG101 N 末端的 PTAP 结合槽结合。此外，我们可以证明将内在的 PTAP 基序突变为 ATAA 氨基酸序列，从而阻断分子内结合方式导致 TSG101 的稳态水平非常显着降低，同时其货物蛋白 ERBB2、EGFR 和 IGF-R1 的表达下降。本次活动的近期目标探索性项目旨在扩展体外研究的初步结果并开发 TSG101-ATAA 使用基于 CRISPR/Cas9 的基因编辑方法的敲入小鼠模型牢固地建立了这种破坏内在 PTAP 结构域与其结合域的分子内关联增加了周转率降低体内 TSG101 的表达。然后我们将应用这个突变体 TSG101-ATAA 敲入模型评估降低 TSG101 的稳态水平是否足以共同下调致癌作用 ERBB2 并预防转基因小鼠中 ERBB2 诱发的乳腺癌的发生和进展。加速 TSG101 及其货物的周转代表了预防和治疗 ERBB2 的新方法- 阳性乳腺癌，这个探索性项目的集体结果将提供强大的体内开发针对特定分子内关联的新型疗法的证据在 TSG101 内。