Analyzing Adhesion and Signaling Functions for PTPN12 in Invasive Glioma Cells

分析侵袭性胶质瘤细胞中 PTPN12 的粘附和信号传导功能

• 批准号: 10543815
• 负责人: Joseph H McCarty
• 金额: $ 40.1万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10543815
• 关键词: 3-Dimensional; Address; Adhesions; Amino Acid Motifs; Angiogenesis Inhibitors; BCAR1 gene; Biochemical; Brain; Brain Neoplasms; Cell Culture System; Cell Proliferation; Cells; Chemotherapy and/or radiation; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cullin Proteins; Data; Data Correlations; Equilibrium; Event; Excision; Focal Adhesions; Genes; Genetic; Genetically Engineered Mouse; Glioblastoma; Glioma; Gliomagenesis; Growth; Human; Invaded; Lesion; Ligase; Link; Malignant - descriptor; Malignant neoplasm of brain; Mediating; Modality; Modeling; Molecular; Mutate; Oncogenes; Operative Surgical Procedures; Outcome; PTPN12 gene; Pathway interactions; Patients; Pattern; Pest Control; Phosphoric Monoester Hydrolases; Phosphorylation; Population; Protein Dephosphorylation; Protein Tyrosine Phosphatase; Proteins; Proteomics; Recurrence; Recurrent tumor; Refractory; Reporting; Resected; Resistance; Resolution; Role; Sampling; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Site; System; Tertiary Protein Structure; Testing; Tumor Cell Invasion; Tumor Promotion; Tumor Suppressor Proteins; Tyrosine; Ubiquitin; Ubiquitination; bevacizumab; cancer cell; cell growth; chemoradiation; chemotherapy; effective therapy; experimental study; fluorescence imaging; genetic approach; in vivo Model; inducible gene expression; inhibitor; live cell imaging; mouse model; multicatalytic endopeptidase complex; neoplastic cell; patient response; pharmacologic; pre-clinical; protein complex; protein expression; recruit; temozolomide; treatment response; tumor; tumor growth; tumor microenvironment; tumor progression; valosin-containing protein

Glioblastoma (GBM) is a malignant brain cancer that is resistant to all treatment modalities. This resistance is due, in large part, to a population of high invasive and low proliferative cancer cells that elude surgical resection and are refractory to chemotherapy and radiation. While a great deal is known about oncogenes, tumor suppressors, and other pathways that promote GBM cell proliferation, we understand relatively little about mechanisms that drive GBM cell invasion in the brain microenvironment. Therefore, the PI's group performed genetic and biochemical screens to identify adhesion and signaling factors that regulate invasive cell growth in GBM. These efforts identified the non-receptor protein tyrosine phosphatase PTP-PEST/PTPN12 as a critical signaling effector in invasive GBM cells. Here, we present a significant amount of supporting data showing that PTP-PEST promotes GBM cell invasion by regulating the stability of key focal adhesion signaling proteins, particularly Crk-associated substrate (p130Cas). In particular, we have discovered that PTP-PEST in focal adhesions mediates interactions between p130Cas and valosin containing protein (Vcp), a ubiquitin-dependent segregase and key component of the ubiquitin proteasome system. These findings have led to our working hypothesis that PTP-PEST is essential for GBM cell invasion by regulating the phosphorylation- dependent ubiquitination of focal adhesion protein substrates. To test this hypothesis, we will (1) characterize protein domains and motifs that mediate interactions between PTP-PEST, p130Cas, and Vcp, as well as determine how these interactions modulate focal adhesion protein stability in GBM cells; (2) identify PTP-PEST-generated phosphodegron sequences in p130Cas and determine how they regulate focal adhesion dynamics by recruiting Vcp and facilitating p130Cas degradation by the proteasome; (3) genetically mutate Vcp at specific sites required for the phosphorylation-dependent ubiquitination of p130Cas and analyze cell invasion using three-dimensional culture systems and pre-clinical mouse models of GBM; and (4) quantify levels and spatial patterns of PTP-PEST signaling via p130Cas and Vcp in human GBM samples and primary cancer cell culture systems. We will correlate these data with patient survival as well as response to therapies such as temozolomide and bevacizumab. Collectively, these experiments will not only elucidate signaling pathways that control the GBM cell invasive state, but may lead to new strategies to target invasive cells and block tumor progression.

胶质母细胞瘤（GBM）是一种对所有治疗方式都有抵抗力的恶性脑癌。这 耐药性在很大程度上是由于高侵袭性和低增殖性癌细胞群 难以手术切除且对化疗和放疗无效。虽然一个伟大的 已知癌基因、肿瘤抑制因子和其他促进 GBM 细胞生长的途径 增殖，我们对驱动 GBM 细胞侵袭的机制了解相对较少 大脑微环境。因此，PI团队进行了遗传和生化检查 筛选以确定调节 GBM 侵袭性细胞生长的粘附和信号传导因子。 这些工作将非受体蛋白酪氨酸磷酸酶 PTP-PEST/PTPN12 确定为 侵袭性 GBM 细胞中的关键信号传导效应器。在这里，我们提出了大量的 支持数据显示 PTP-PEST 通过调节稳定性促进 GBM 细胞侵袭 关键粘附斑信号蛋白，特别是 Crk 相关底物 (p130Cas)。在 特别是，我们发现粘着斑中的 PTP-PEST 介导相互作用 p130Cas 和包含蛋白 (Vcp) 的 valosin 之间，Vcp 是一种泛素依赖性分离酶， 泛素蛋白酶体系统的关键组成部分。这些发现导致我们的工作 假设 PTP-PEST 通过调节磷酸化对 GBM 细胞侵袭至关重要 粘着斑蛋白底物的依赖性泛素化。为了检验这个假设，我们将 (1) 表征介导 PTP-PEST 之间相互作用的蛋白质结构域和基序， p130Cas 和 Vcp，以及确定这些相互作用如何调节粘着斑 GBM 细胞中的蛋白质稳定性； (2) 鉴定 PTP-PEST 生成的磷酸降解子序列 p130Cas 并确定它们如何通过招募 Vcp 和 促进 p130Cas 被蛋白酶体降解； (3)在特定位点对Vcp进行基因突变 p130Cas 磷酸化依赖性泛素化所需并分析细胞侵袭 使用三维培养系统和 GBM 临床前小鼠模型；和（4） 通过 p130Cas 和 Vcp 量化人类 PTP-PEST 信号传导的水平和空间模式 GBM 样本和原代癌细胞培养系统。我们会将这些数据与患者相关联 生存以及对替莫唑胺和贝伐单抗等疗法的反应。总的来说， 这些实验不仅将阐明控制 GBM 细胞侵袭的信号通路 状态，但可能会导致针对侵袭性细胞和阻止肿瘤进展的新策略。