Novel Signaling Pathways Regulating Pancreatic Cancer Pathogenesis

 描述(申请人提供)：HPK1是一种与Ste20相关的哺乳动物丝氨酸/苏氨酸激酶，已被证明调节造血细胞中的NFB和JNK途径。本实验室首次发现HPK1是胰腺导管癌(PDC)中一种新的肿瘤抑制因子。我们的数据显示，HPK1在正常胰腺导管细胞中表达，但在&gt；95%的PDCs中缺失。在人类和Kras PDC模型中，HPK1的丢失发生在早期PAINS，并与从早期PAINS向PDC的进展密切相关。恢复PDC细胞中HPK1的表达会导致细胞周期停滞和生长抑制，这部分是由于 P21和p27的稳定化(3)。此外，我们还证明了PDC中HPK1的丢失是由CUL7/Fbxw8泛素连接酶通过26S蛋白酶体介导的，这需要HPK1的活性和自磷酸化。CUL7/Fbxw8泛素连接酶对HPK1的靶向降解构成了抑制HPK1活性的负反馈回路(4)。为了进一步研究HPK1的调节机制，我们发现COP9信号体6(CSN6)通过CUL7/Fbxw8泛素连接酶来调节HPK1。此外，我们的初步数据表明，HPK1通过内吞作用下调Axl，并通过p120GAP作为RAS的负调控因子，HPK1的缺失在PDC的发展中起着关键作用。鉴于Kras基因突变和HPK1缺失均发生在早期PAINS中，因此研究HPK1降解及其下游信号转导机制以确定参与PAINS进展的关键分子靶点将是极其重要的。本研究的主要目的是研究CSN6调节CUL7/Fbxw8泛素连接酶和HPK1功能的机制，并利用小鼠模型研究HPK1在Ax1、RAS信号和Kras驱动的Panins向PDC的发展和进展中的机制和肿瘤抑制功能。我们的研究不仅将为CSN6对CUL7/Fbxw8泛素连接酶和HPK1的调控以及HPK1调控p120GAP、Ax1和RAS信号的新机制提供新的见解，还将建立HPK1作为一种新的RAS信号负调控因子和PDC中的肿瘤抑制因子。通过对参与HPK1调控的新途径的剖析，结合我们的新小鼠模型，我们将寻找早期检测的分子标记，并通过靶向CSN6-CUL7/Fbxw8-HPK1-RAS通路来开发新的基于机制的PDC治疗策略。因此，建议的研究对改善PDC患者的预后具有高度的相关性。我们有三个特异性目的：特异性目的1：研究HPK1抑制胰腺癌Ras信号转导的机制；特异性目的2：研究CSN6调节胰腺癌CUL7/Fbxw8泛素连接酶和HPK1的分子机制；特异性目的3：利用KrasG12D胰腺癌小鼠模型，研究HPK1在拮抗Kras信号转导中的抑瘤作用。

 DESCRIPTION (provided by applicant): HPK1 is a mammalian Ste20-related serine/threonine kinase, which has been shown to regulate NFB and JNK pathways in hematopoietic cells. Our lab is the first to discover that HPK1 is a novel tumor suppressor in pancreatic ductal carcinoma (PDC). Our data show that HPK1 is expressed in normal pancreatic ductal cells, but is lost in >95% PDCs. Loss of HPK1 occurs in early PanINs and is strongly associated with the progression from early PanINs to PDC both in human and Kras PDC models. Restoring HPK1 expression in PDC cells causes cell cycle arrest and growth inhibition, which is due in part to the stabilization of p21 and p27 (3). In addition, we have also demonstrated that loss of HPK1 in PDC is mediated by CUL7/Fbxw8 ubiquitin ligase through 26S proteasome, which requires HPK1 kinase activity and autophosphorylation. Targeted degradation of HPK1 by CUL7/Fbxw8 ubiquitin ligase constitutes a negative-feedback loop to restrain HPK1 activity (4). To further examine the mechanisms of HPK1 regulation, we showed that COP9 signalosome 6 (CSN6) regulates HPK1 through CUL7/Fbxw8 ubiquitin ligase. Furthermore, our preliminary data showed that HPK1 down- regulates Axl via endocytosis and functions as a negative regulator of Ras through p120GAP and that loss of HPK1 plays a critical role in the development of PDC. Given the fact that both Kras mutations and loss of HPK1 occurs in early PanINs, it would be extremely important to examine the mechanisms of HPK1 degradation and its downstream signaling to identify key molecular targets involved in the progression of PanINs. The primary objectives of this proposal are to examine the mechanisms by which CSN6 regulates the functions of CUL7/Fbxw8 ubiquitin ligase and HPK1 and to examine the mechanisms and tumor suppressor functions of HPK1 in Axl, Ras signaling and Kras-driven development and progression of PanINs to PDC using mouse models. Our proposed studies will not only provide new insights into the regulation of CUL7/Fbxw8 ubiquitin ligase and HPK1 by CSN6 and the novel mechanisms by which HPK1 regulates p120GAP, Axl, and Ras signaling, but also establish HPK1 as a novel negative regulator of Ras signaling and a tumor suppressor in PDC. Through the dissection of novel pathways involved in HPK1 regulation combined with our new mouse models, we will identify molecular markers for early detection and develop novel mechanism-based strategy for PDC treatment by targeting CSN6-CUL7/Fbxw8-HPK1-Ras pathways. Therefore the proposed research is highly relevant to improving the outcome of PDC patients. We have three specific Aims: Specific Aim 1: To study the mechanisms by which HPK1 inhibits Ras signaling in pancreatic cancer Specific Aim 2: To examine the molecular mechanisms by which CSN6 regulates CUL7/Fbxw8 ubiquitin ligase and HPK1 in pancreatic cancer Specific Aim 3: To study the tumor suppressor function of HPK1 in antagonizing Kras signaling using KrasG12D pancreatic cancer mouse models