Proteomic Pathways for Pancreatic Cancer Stem Cells

描述(由申请人提供)：新出现的证据表明，肿瘤的生长和繁殖能力依赖于肿瘤内的一小部分细胞。这一概念最初是基于这样的观察，即当在不同的体外或体内试验中检测许多不同类型的癌细胞的增殖能力时，只有少数细胞能够广泛增殖。这一观察结果引发了人们的想法，即恶性肿瘤是由一小部分不同的癌症干细胞组成的，这些干细胞具有很大的增殖潜力，以及更分化的癌细胞，它们的增殖潜力非常有限。我们的研究小组已经在人类胰腺癌中发现了一种高度致癌的癌细胞群体，它具有癌症干细胞的功能。这项建议的目标是检测胰腺癌干细胞亚群中的蛋白质表达，以此作为一种可能帮助我们开发更有效治疗方法的方法。这一癌症干细胞群体代表了一小部分细胞，因此需要专门的微型蛋白质组学方法来描述与细胞信号通路相关的蛋白质表达和鉴定的变化。所使用的方法将涉及一种鸟枪法，即整个细胞裂解产物将被消化，并使用毛细管等电聚焦在pH维度上分离多肽。然后，这些蛋白质将使用无孔反相高效液相色谱进一步分离，并根据蛋白质数据库中多肽的LC-MS/MS进行鉴定，我们预计将在那里鉴定约2500种蛋白质。然后，将使用生物信息学方法和KEGG途径数据库，使用所获得的蛋白质图谱来研究活跃的信号通路。蛋白质组学确定的信号通路随后将与来自寡核苷酸阵列的数据进行比较，以获得相应的基因组数据。我们将使用这些方法专门搜索与Sonic Hedgehog(SHH)途径相关的调控异常的途径，以及其他途径，如Wnt、Notch和Bmi-1。公共卫生相关性拟议的研究项目可能为研究少量胰腺癌干细胞的蛋白质组学提供一种方法，并可能进一步促进识别可能相对于非干细胞群体而言调控失调的关键信号通路。这些蛋白质途径可能与干细胞的独特性质有关，例如它们的增殖能力，以及对治疗干预(包括药物和放射治疗)的抵抗力。进一步了解胰腺癌干细胞生长和调控所涉及的信号通路可能会为发展针对癌症干细胞群体和非癌症干细胞群体的治疗方法提供关键信息。

DESCRIPTION (provided by applicant): Emerging evidence has shown that the capacity of a tumor to grow and propagate is dependent on a small subset of cells within a tumor. This concept was originally based on the observation that when cancer cells of many different types were assayed for their proliferative potential in various in vitro or in vivo assays, only a minority of cells were able to proliferate extensively. This observation gave rise to the idea that malignant tumors are comprised of a small subset of distinct cancer stem cells which have great proliferative potential, as well as more differentiated cancer cells, which have very limited proliferative potential. Our research group has identified a highly tumorigenic cancer cell population within human pancreatic cancers that functions as a cancer stem cell. The goal of this proposal is to examine protein expression in this subpopulation of pancreatic cancer stem cells as an approach that may help us develop more effective therapies. This cancer stem cell population represents a small number of cells so that specialized micro-proteomic approaches will be needed to profile changes in protein expression and identification related to cell signaling pathways. The methods used will involve a shotgun approach where the whole cell lysates will be digested and the peptides separated using capillary isolectric focusing in the pH dimension. The proteins will then be further fractionated using nonporous RP-HPLC and identified based upon LC-MS/MS of the peptides against protein databases where we expect to identify around 2500 proteins. The active signal pathways will then be studied using bioinformatic methods and the KEGG pathway database using the protein profiles obtained. The signal pathways identified by proteomics will then be compared to data from oligonucleotide arrays to obtain corresponding genomic data. We will use these methods specifically to search for dysregulated pathways related to the Sonic Hedgehog(SHH) pathway and also for other pathways such as Wnt, Notch and Bmi-1. PUBLIC HEALTH RELEVANCE The proposed research project may provide a methodology for studying the proteomics of small numbers of pancreatic cancer stem cells and may further facilitate identification of key signaling pathways that may be dysregulated relative to the non-stem cell population. These protein pathways may be responsible for the unique properties of stem cells such as their proliferative potential, as well as resistance to therapeutic interventions (including both drug and radiation treatment). Greater understanding of the signaling pathways involved in pancreatic cancer stem cell growth and regulation may provide critical information for development of therapeutic modalities specifically targeting the cancer stem cell population relative to the non-cancer stem cell population.