Examination of ceramide signaling in the crosstalk between pancreatic cancer cells and the tumor microenvironment

项目总结/摘要 背景：鞘脂代谢失调改变胰腺癌的进展， 作为开发新疗法的潜在靶点受到了相当大的关注。细胞内 神经酰胺水平对于调节细胞衰老、凋亡和细胞周期停滞是必需的， 对压力刺激的反应，如化疗。我们实验室的初步数据显示 对于调节神经酰胺合成和外泌体生物发生的基因， 进展小鼠胰腺导管腺癌细胞系的原位稳定移植 在免疫缺陷小鼠中表达靶向该基因的shRNA显著降低了胰腺肿瘤负荷 与对照组相比。此外，我们的初步数据还表明，该基因调节外泌体 从胰腺癌细胞系分泌。基于我们令人兴奋的初步数据，我设计了一项研究， 破译该基因调节胰腺肿瘤发生的分子机制。 目的/假设：考虑到该基因在调节癌细胞外泌体分泌和抑制肿瘤细胞增殖中的复杂功能， 和细胞内神经酰胺水平，我提出以下中心假设：该基因的功能， 胰腺癌是多种多样的，在其发生和发展过程中可能发挥不同的作用。整体 本研究的目的是确定该基因控制胰腺癌发生的机制。 具体目的：本研究的具体目的是：目的1）评价该基因在正常小鼠中的功能 胰腺和胰腺肿瘤形成和进展目的2）确定 该基因调节胰腺肿瘤生长目的2A）评估该基因改变细胞活性的能力 目的2B）评估外泌体通过该基因途径分泌的能力 来调节胰腺肿瘤的扩张。研究设计：本文提出的方法严格确定 在PDA形成和发展过程中，使用已建立的功能缺失研究来研究该基因的功能。 PDA的小鼠和细胞培养模型。我将在一个广泛使用的小鼠模型中切除我们感兴趣的基因， 胰腺癌，以确定该基因调节胰腺癌形成和进展的能力。 此外，我建议使用我们的实验室产生的小鼠胰腺癌（MPDA）细胞系进行机制分析。 一组稳定表达的构建体，允许敲除和过表达研究，这将阐明 外泌体分泌和细胞内神经酰胺水平调节在胰腺癌发生中作用 由这个基因介导。影响：胰腺癌预计将成为第二大癌症原因- 到2020年，美国的相关死亡人数。鉴于这一显著的预计发病率增加，胰腺癌仍然是 这是人类整体健康的主要问题。与NIH国家癌症中心的总体使命一致 研究所，拟议的研究可能会确定新的治疗目标的发展新的战略至关重要 预防、治疗和治愈病人。

Project Summary/Abstract Background: Deregulation of sphingolipid metabolism alters pancreatic cancer progression and has thus received appreciable attention as a potential target for development of novel therapeutics. Intracellular ceramide levels are imperative for regulation of cellular senescence, apoptosis, and cell cycle arrest in response to stress stimuli such as chemotherapeutics. Preliminary data from our lab indicate a prominent role for a gene that regulates ceramide synthesis and exosome biogenesis, in the maintenance of pancreatic tumor progression. Orthotopic implantation of murine pancreatic ductal adenocarcinoma (PDA) cell lines stably expressing shRNA targeting this gene into immunodeficient mice significantly reduced pancreatic tumor burden when compared to controls. In addition, our preliminary data also indicate that this gene regulates exosome secretion from pancreatic cancer cell lines. Based on our exciting preliminary data, I have designed a study to decipher the molecular mechanisms by which this gene modulates pancreatic tumorigenesis. Objective/Hypothesis: Given this gene’s complex function in regulation of both cancer cell exosome secretion and intracellular ceramide levels, I propose the following central hypothesis: The function of this gene in pancreatic cancer is multiplex, potentially playing distinct roles during initiation and progression. The overall objective of this study is to determine the mechanisms by which this gene controls pancreatic carcinogenesis. Specific Aims: The specific aims of this study are: Aim 1) Evaluate the function of this gene in normal murine pancreas and pancreatic neoplasia formation and progression Aim 2) Determine the mechanism(s) by which this gene regulates pancreatic tumor growth Aim 2A) Assess the ability of this gene to alter cellular activities downstream of ceramide signaling Aim 2B) Evaluate the ability of exosomes secreted via this gene’s pathway to modulate pancreatic tumor expansion. Study Design: The approach proposed herein rigorously determines the function of this gene during PDA formation and progression using loss of function studies in established mouse and cell culture models of PDA. I will ablate our gene of interest in a widely-used mouse model of pancreatic cancer to determine the ability of this gene to regulate pancreatic cancer formation and progression. In addition, I propose mechanistic assays using murine pancreatic cancer (MPDA) cell lines generated by our group which stably express constructs allowing knockdown and overexpression studies that will illuminate the effect of exosome secretion and regulation of intracellular ceramide levels on pancreatic carcinogenesis mediated by this gene. Impact: Pancreatic cancer is projected to become the 2nd leading cause of cancer- related death in the US by 2020. Given this notable projected increase in incidence, pancreatic cancer remains a major problem for overall human health. Consistent with the overall mission of the NIH National Cancer Institute, the proposed studies may identify new therapeutic targets critical for development of new strategies to prevent, treat, and cure patients.