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依赖于肿瘤内的一小部分细胞。这一概念最初是基于这样一种观察，即当对许多不同类型的癌细胞进行各种体外或体内试验以测定其增殖潜力时，只有少数细胞能够广泛增殖。这一观察结果提出了恶性肿瘤是由具有巨大增殖潜力的一小部分独特的癌症干细胞和增殖潜力非常有限的分化程度更高的癌细胞组成的。我们的研究小组已经在人类胰腺癌中发现了一种高度致瘤性的癌细胞群，其功能相当于癌症干细胞。这项提议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治疗方法的方法。这种癌症干细胞群代表了一小部分细胞，因此需要专门的微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。使用的方法将包括散弹枪方法，其中整个细胞裂解物将被消化，肽将使用毛细管等电聚焦在pH尺寸上分离。然后使用无孔反相高效液相色谱进一步分离蛋白质，并根据蛋白质数据库的LC-MS/MS对肽进行鉴定，我们预计鉴定约2500种蛋白质。然后使用生物信息学方法和使用获得的蛋白质谱的KEGG通路数据库研究活性信号通路。然后将蛋白质组学鉴定的信号通路与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寻找与Sonic Hedgehog（SHH）通路相关的失调通路，以及Wnt、Notch和Bmi-1等其他通路。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）的抵抗力。更深入地了解胰腺癌干细胞生长和调控的信号通路，可能为开发针对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.