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

项目总结 ERBB家族成员通过受体酪氨酸激酶的异常信号在病因中起关键作用 乳腺癌的风险。大约20%-25%的浸润性乳腺癌表现出ERBB2基因的扩增 或该基因编码的受体酪氨酸激酶的结构性激活。有大量的 实验和临床数据表明，ERBB2过表达的乳腺癌细胞有升高的 转移潜能。尽管曲妥珠单抗和曲妥珠单抗对ERBB2的靶向治疗有初步反应 Pertuzumab，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。作为一个组件 在内吞机制中，由肿瘤易感基因101(TSG101)编码的蛋白质对 泛素化货运蛋白的细胞内运输、分类和溶酶体降解，包括 ERBB2和其他受体酪氨酸激酶。TSG101是人口贩运的中心节点，因为它能够 同时结合Cargo蛋白以及含有PTAP氨基酸基序的调节蛋白，并 泛素连接酶，修饰货物复合体的分类和运输。TSG101本身拥有一个 在其C末端附近有内在的PTAP基序，我们收集了初步证据表明该结构域可以 与TSG101 N端的PTAP结合槽结合。此外，我们可以证明 将固有的PTAP基序突变为AtaA氨基酸序列，从而阻断分子内 结合通道导致TSG101的稳态水平显著降低，同时 其货运蛋白ERBB2、EGFR和IGF-R1的表达下降。这样做的直接目标是 探索性项目是扩展体外研究的初步发现，并开发TSG101-ATA Knockin小鼠模型使用基于CRISPR/Cas9的基因编辑方法来牢固地建立这种干扰 固有的PTAP结构域与其结合林的分子内结合增加了PTAP的周转和 降低TSG101在体内的表达。然后，我们将把这个突变的TSG101-Ataa敲门模型应用于 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发展。 加快TSG101及其货物周转是防治ERBB2的新途径-- 阳性乳腺癌，这一探索性项目的集体结果将在体内提供强大的 针对特定分子内联系的一类新疗法的开发证据 在TSG101内。

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.