Mechanistic Studies of MUC4 and NGAL in Pancreatic Adenocarcinoma

描述(申请人提供)：胰腺癌仍然是人类已知的最致命的癌症，主要是因为它缺乏早期症状，导致诊断延迟，治疗效果缺乏一致性。MUC4是一种大小的膜锚定粘蛋白，在大多数人类肿瘤中异常过表达。我们先前已报道MUC4通过多种机制促进胰腺肿瘤的生长和转移。在这些研究的同时，利用免疫组织化学技术在包含有良好特征的肿瘤组织的组织微阵列中，我们观察到在胰腺癌进展过程中中性粒细胞明胶酶相关脂钙蛋白(NGAL)的表达增加，也被称为脂钙蛋白2或子宫钙蛋白。有趣的是，NGAL的表达与MUC4在这些组织中的表达相关。免疫细胞化学显示NGAL和MUC4在胰腺癌组织切片中共表达。我们课题组最近的研究表明，NGAL是一个潜在的诊断生物标志物，其表达早在癌前阶段Panin-2就上调了。此外，初步研究表明，在胰腺癌细胞系Capan-1中，MUC4基因敲除会导致NGAL表达减少。在本研究中，我将使用细胞系Colo-357、Capan-1、MiaPaCa和Panc-1以及来自人类和小鼠模型的健康和肿瘤胰腺组织进一步研究胰腺癌中NGAL和MUC4之间的联系，以追求以下假设：MUC4通过HER2调节胰腺癌中NGAL的表达，这种联系将被证明与NGAL作为胰腺癌替代生物标记物的预后能力高度相关。为了进行这项调查，我制定了三个具体目标。在第一个具体目标中，我将使用基因敲除和过表达实验以及Western blotting、定量-实时聚合酶链式反应和免疫组织化学来确定NGAL和MUC4在胰腺癌中的表达是否可靠地关联，以及这种关联是否存在于翻译和/或转录水平。在第二个目标中，我将以目前已知的上下游蛋白和通路为起点，阐明NGAL和MUC4表达的联系途径。具体地说，根据文献和我的初步结果，我将首先评估MUC4是否通过HER2调节NGAL的表达。在第三个目标中，我将使用我们实验室开发的自发产生胰腺癌的具有免疫功能的小鼠模型，以评估组织和血浆NGAL与组织MUC4水平在胰腺癌发生发展过程中的相关性。通过这些目标，我期望最终阐明MUC4和NGAL之间的生化联系，同时改进现有证据，增强NGAL作为胰腺癌早期诊断和预后替代生物标志物的效用。 公共卫生相关性：胰腺癌仍然是最致命的人类恶性肿瘤，主要是由于在晚期疾病中缺乏早期诊断和糟糕的治疗效果。初步证据表明，MUC4基因位于NGAL的上游，它是胰腺癌中最早和分化最高的上调基因之一，与胰腺癌的转移和致癌潜能呈正相关。NGAL是一种分泌型蛋白，在胰腺癌组织和胰腺癌患者的外周血中也有过表达。本研究旨在明确MUC4和NGAL表达相关的机制，从而支持外周血NGAL作为胰腺癌早期诊断和预后标志物的应用。

DESCRIPTION (provided by applicant): Pancreatic cancer remains the most lethal cancer known to man, owing primarily to its lack of early symptom resulting in late diagnosis and lack of consistent treatment efficacy. MUC4 is a large-sized membrane- anchored mucin which is aberrantly overexpressed by most human carcinomas. We have previously reported that MUC4 promotes pancreatic tumor growth and metastasis by multiple mechanisms. In parallel to these studies, using immunohistochemistry in tissue microarrays containing well characterized tumors tissues, we observed elevated expression of neutrophil gelatinase associated lipocalin (NGAL), also known as Lipocalin 2 or uterocalin, during pancreatic cancer progression. Interestingly, the expression of NGAL correlated with MUC4 expression in these tissues. Immunocytochemistry showed co-expression of NGAL and MUC4 in pancreatic cancer tissue sections. Recent studies by our group have shown that NGAL is a potential diagnostic biomarker and its expression is upregulated as early as the preneoplastic PanIN-2 stage. Additionally, preliminary studies have shown that, in the pancreatic cancer cell line CaPan-1, MUC4 knockdown leads to decreased expression of NGAL. In the present study, I will further investigate the link between NGAL and MUC4 in pancreatic cancer using the cell lines CoLo-357, CaPan-1, MiaPaCa, and PANC-1 as well as healthy and neoplastic pancreatic tissue from both humans and murine models in pursuit of the hypothesis that MUC4, acting through HER2, regulates NGAL expression in pancreatic cancer and that this connection will prove highly relevant to the prognostic abilities of NGAL as a pancreatic cancer surrogate biomarker. In pursuit of this investigation I have formulated three specific aims. In the first specific aim, I will use knockdown and overexpression experiments as well as Western blotting, Quantitative-Real Time PCR, and immunohistochemistry to determine if expression of NGAL and MUC4 are reliably linked in pancreatic cancer and if this link is present at the translational and/or transcriptional level. In the second aim, I will elucidate the pathway by which NGAL and MUC4 expression are linked using currently-known upstream and downstream proteins and pathways as a starting-point. Specifically, based on literature and my preliminary results, I will begin by assessing if MUC4 regulates NGAL expression through HER2. In the third aim, I will use an immunocompetent murine model genetically manipulated to spontaneously produce pancreatic cancer that has been developed in our lab to assess the correlation between tissue and plasma NGAL and tissue MUC4 levels throughout the development of and advancement of pancreatic cancer. Through these aims, I expect to conclusively elucidate the biochemical connection between MUC4 and NGAL while improving on current evidence potentiating NGAL's utility as both an early diagnostic and prognostic surrogate biomarker for pancreatic cancer. PUBLIC HEALTH RELEVANCE: Pancreatic cancer remains the most lethal human malignancy, owing mostly to a lack of early diagnosis and abysmal treatment efficacy in late-stage disease. Preliminary evidence suggests that MUC4, one of the earliest and most differentially upregulated genes in pancreatic cancer and which positively correlates with metastatic and tumorigenic potential, is upstream of NGAL, a secreted protein also found to be overexpressed in pancreatic tumor tissues as well as in the peripheral blood of pancreatic cancer patients. This study aims to define the mechanism by which MUC4 and NGAL expressions are linked, thereby bolstering the evidence for peripheral blood NGAL's use as a potential early diagnostic and prognostic marker in pancreatic cancer.