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

项目摘要/摘要 背景：鞘磷脂代谢的放松调节改变了胰腺癌的进展，并因此 作为开发新疗法的潜在靶点，受到了相当大的关注。细胞内 神经酰胺水平对调节细胞衰老、细胞凋亡和细胞周期停滞是必不可少的。 对应激刺激的反应，如化疗药物。我们实验室的初步数据表明， 一个调节神经酰胺合成和外切体生物发生的基因，在胰腺肿瘤的维持中 进步。稳定的小鼠胰腺导管腺癌细胞系原位移植 在免疫缺陷小鼠中表达针对该基因的shRNA可显著降低胰腺肿瘤的负担 与对照组相比。此外，我们的初步数据还表明，该基因调节外切体。 胰腺癌细胞株的分泌物。基于我们令人兴奋的初步数据，我设计了一项研究 破译该基因调控胰腺肿瘤发生的分子机制。 目的/假设：鉴于该基因在调节癌细胞外切体分泌方面的复杂功能 和细胞内神经酰胺水平，我提出了以下中心假设：该基因在 胰腺癌是多发性的，在发生和发展过程中可能扮演着不同的角色。整体而言 本研究的目的是确定该基因控制胰腺癌发生的机制。 目的：本研究的目的是：1)评价该基因在正常小鼠中的功能 胰腺和胰腺肿瘤的形成和发展目的2)确定胰腺和胰腺肿瘤形成和发展的机制(S) 该基因调控胰腺肿瘤生长目标评估该基因改变细胞活动的能力 神经酰胺信号的下游目的2B)评估通过该基因的途径分泌的外切体的能力 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 应用功能损失研究探讨该基因在动脉导管未闭形成和发展中的作用 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去除我们感兴趣的基因 以确定该基因调控胰腺癌形成和发展的能力。 此外，我建议使用我们的研究小组产生的小鼠胰腺癌(MPDA)细胞系进行机械分析。 稳定表达构建体的组，允许击倒和过度表达研究，这将阐明 胞外体分泌和细胞内神经酰胺水平调节在胰腺癌发生中的作用 由该基因介导。影响：胰腺癌预计将成为第二大癌症原因-- 到2020年，美国将出现相关死亡病例。考虑到预计发病率的显著增加，胰腺癌仍然 这是影响人类整体健康的一个主要问题。与美国国立卫生研究院国家癌症中心的总体使命一致 研究所提出的研究可能会确定新的治疗靶点，这对开发新的策略至关重要 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.