Analyzing Adhesion and Signaling Functions for PTPN12 in Invasive Glioma Cells

胶质母细胞瘤（GBM）是一种恶性脑癌，对所有治疗方式都有抵抗力。这 耐药性在很大程度上是由于高侵袭性和低增殖性的癌细胞群 这些肿瘤无法手术切除，化疗和放疗也难以治愈。虽然伟大的 关于癌基因、肿瘤抑制因子和其他促进GBM细胞增殖的途径， 增殖，我们了解相对较少的机制，驱动GBM细胞侵袭， 大脑微环境因此，PI的小组进行了遗传和生化 筛选以鉴定调节GBM中侵袭性细胞生长的粘附和信号传导因子。 这些努力将非受体蛋白酪氨酸磷酸酶PTP-PEST/PTPN 12鉴定为 在侵袭性GBM细胞中的关键信号效应子。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 关键的粘着斑信号蛋白，特别是Crk相关底物（p130 Cas）。在 特别是，我们已经发现，PTP-PEST在局灶性粘连介导的相互作用 p130 Cas和含valosin蛋白（Vcp）之间的关系，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。这些发现促使我们 假设PTP-PEST通过调节磷酸化对GBM细胞侵袭是必需的- 粘着斑蛋白底物的依赖性泛素化。为了验证这个假设，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， p130 Cas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GBM细胞中PTP-PEST产生的磷酸降解决定子序列。 p130 Cas，并确定它们如何通过募集Vcp和 促进蛋白酶体对p130 Cas的降解;（3）在特定位点对Vcp进行基因突变 p130 Cas的磷酸化依赖性泛素化所需，并分析细胞侵袭 使用GBM的三维培养系统和临床前小鼠模型;和（4） 通过p130 Cas和Vcp定量人PTP-PEST信号传导的水平和空间模式 GBM样品和原代癌细胞培养系统。我们将这些数据与患者 存活率以及对诸如替莫唑胺和贝伐单抗的治疗的应答。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， 但可能会导致新的策略来靶向侵袭性细胞和阻断肿瘤进展。

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.