Novel Signaling Pathways Regulating Pancreatic Cancer Pathogenesis

 描述（由申请人提供）：HPK 1是哺乳动物Ste 20相关的丝氨酸/苏氨酸激酶，已显示其调节造血细胞中的NF κ B B和JNK途径。本实验室首次发现HPK 1是一种新的胰腺导管癌（PDC）抑癌基因。我们的数据显示，HPK 1在正常胰腺导管细胞中表达，但在>95%的PDCs中丢失。HPK 1的丢失发生在早期PanIN中，并且与人类和Kras PDC模型中从早期PanIN到PDC的进展密切相关。在PDC细胞中恢复HPK 1表达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（3）。此外，我们还证明了PDC中HPK 1的丢失是由CUL 7/Fbxw 8泛素连接酶通过26 S蛋白酶体介导的，这需要HPK 1激酶活性和自磷酸化。CUL 7/Fbxw 8泛素连接酶对HPK 1的靶向降解构成了抑制HPK 1活性的负反馈环（4）。为了进一步研究HPK 1的调节机制，我们发现COP 9信号体6（CSN 6）通过CUL 7/Fbxw 8泛素连接酶调节HPK 1。此外，我们的初步数据表明，HPK 1通过内吞作用下调Axl，并通过p120 GAP作为Ras的负调节剂发挥作用，并且HPK 1的缺失在PDC的发展中起关键作用。鉴于Kras突变和HPK 1丢失均发生在早期PanIN中，因此研究HPK 1降解及其下游信号传导的机制以确定参与PanIN进展的关键分子靶点将非常重要。本提案的主要目的是研究CSN 6调节CUL 7/Fbxw 8泛素连接酶和HPK 1功能的机制，并使用小鼠模型研究HPK 1在Axl、Ras信号传导和Kras驱动的PanIN向PDC的发育和进展中的机制和肿瘤抑制功能。我们的研究不仅将为CSN 6对CUL 7/Fbxw 8泛素连接酶和HPK 1的调控提供新的见解，以及HPK 1调控p120 GAP、Axl和Ras信号的新机制，还将确立HPK 1作为Ras信号的新负调控因子和PDC中的肿瘤抑制因子。通过对参与HPK 1调控的新途径的剖析，结合我们的新小鼠模型，我们将鉴定用于早期检测的分子标记物，并通过靶向CSN 6-CUL 7/Fbxw 8-HPK 1-Ras途径开发新的基于机制的PDC治疗策略。因此，拟议的研究与改善PDC患者的结局高度相关。我们有三个具体目标：具体目标1：研究HPK 1抑制胰腺癌中Ras信号通路的机制具体目的2：研究CSN 6调节胰腺癌中CUL 7/Fbxw 8泛素连接酶和HPK 1的分子机制具体目的3：利用KrasG 12 D胰腺癌小鼠模型研究HPK 1拮抗Kras信号通路的抑癌作用

 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