Proteomic Pathways for Pancreatic Cancer Stem Cells

描述（由申请人提供）：新出现的证据表明，肿瘤生长和繁殖的能力取决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，即当在各种体外或体内测定中测定许多不同类型的癌细胞的增殖潜力时，只有少数细胞能够广泛增殖。这一观察结果产生了这样的想法，即恶性肿瘤由具有巨大增殖潜力的不同癌症干细胞的一小部分以及具有非常有限增殖潜力的更分化的癌细胞组成。我们的研究小组已经在人类胰腺癌中鉴定出一种高度致瘤性的癌细胞群，其功能是癌症干细胞。该提案的目标是检查胰腺癌干细胞亚群中的蛋白质表达，作为一种可能帮助我们开发更有效疗法的方法。这种癌症干细胞群体代表了少量细胞，因此需要专门的微蛋白质组学方法来分析与细胞信号传导途径相关的蛋白质表达和鉴定的变化。所使用的方法将涉及鸟枪法，其中全细胞裂解物将被消化，并且肽在pH维度上使用毛细管等电聚焦分离。然后使用无孔RP-HPLC进一步分级分离蛋白质，并基于肽的LC-MS/MS对蛋白质数据库进行鉴定，我们预计可鉴定约2500种蛋白质。然后将使用生物信息学方法和KEGG通路数据库使用获得的蛋白质谱研究活性信号通路。然后将通过蛋白质组学鉴定的信号通路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。我们将专门使用这些方法来寻找与Sonic Hedgehog（SHH）通路相关的失调通路，以及其他通路，如Wnt，Notch和Bmi-1。公共卫生相关性拟议的研究项目可能提供一种研究少量胰腺癌干细胞蛋白质组学的方法，并可能进一步促进识别相对于非干细胞群体可能失调的关键信号通路。这些蛋白质通路可能是干细胞独特特性的原因，例如它们的增殖潜力，以及对治疗干预（包括药物和放射治疗）的抗性。对胰腺癌干细胞生长和调控中所涉及的信号通路的更深入了解可能为开发相对于非癌症干细胞群体特异性靶向癌症干细胞群体的治疗方式提供关键信息。

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.