Proteomic Analyses for GI Stem Cell Markers & Mechanisms

描述（由申请人提供）：我们广泛的长期目标是表征基于GI干细胞（SC）的结直肠癌（CRC）发生机制，因为这可能导致识别肿瘤细胞中的脆弱分子过程，从而发现更有效的抗癌药物和/或化学预防剂。由于有证据表明结肠SC是CRC的起源细胞，因此开发鉴定结肠SC的能力（目标1）应使SC群体的分离成为可能，并因此研究SC机制在CRC发展中的作用。由于APC基因突变引发了大多数CRC病例，并且由于最近的证据表明基于APC的机制调节SC群体大小，因此将基于APC的功能变化与沿正常隐窝轴（Aim 2）的结肠细胞特性变化（沿着）相关联，应阐明基于APC的机制如何参与SC的调节以及SC机制如何参与CRC。鉴于目前缺乏结肠SC的标记物，SC鉴定的必要条件是知道它们位于隐窝底部，因此在我们提出的研究中需要使用新鲜组织而不是培养的细胞。在目标1中，我们将使用一种创新的策略，通过应用蛋白质组学分析（2D凝胶/质谱法）来研究新鲜分离的人结肠隐窝子部分（SC所在的隐窝底部三分之一），以确定结肠SC的潜在标志物。因为有证据表明，当SC含有突变APC时，（如在FAP中）隐窝SC群体扩大并且存在实质上更多的SC，我们的第一个假设是（a）蛋白质组学分析将显示某些蛋白质的表达在组织学上表现正常的FAP隐窝中的斑点强度显著高于对照隐窝，和（B）产生的针对至少一些这样的蛋白质的抗体将在正常的隐窝底部显示染色，并且在FAP中阳性染色的隐窝底部细胞的数量增加。鉴定SC标记物将使研究者能够生长SC并研究肿瘤发生期间的SC变化。在目标2中，我们将使用正常的人类隐窝标本区分不同的模式，沿着隐窝轴，APC磷酸化，亚细胞定位和与结合蛋白的相互作用。我们的第二个假设是，已知的APC浓度和亚细胞定位沿着隐窝轴的差异与APC磷酸化和结合其他蛋白质的特定模式相关。这些相关性将提示APC调节SC群体大小的机制，以及APC突变导致SC过度增殖和CRC的机制。

DESCRIPTION (provided by applicant): Our broad long-term objective is to characterize GI stem cell (SC) based mechanisms underlying colorectal cancer (CRC) development because this could lead to identification of vulnerable molecular processes in tumor cells and thus to discovery of more effective anti-cancer drugs and/or chemopreventive agents. Since evidence indicates that colonic SC are the cells of origin of CRC, developing the ability to identify colonic SC (Aim 1) should make possible isolation of SC populations and, consequently, studies into the role of SC mechanisms in CRC development. Because a mutation in the APC gene initiates most CRC cases, and because recent evidence indicates that APC-based mechanisms regulate SC population size, then correlating changes in APC-based functions with changes in colonocyte properties along the normal crypt axis (Aim 2) should shed light on how APC-based mechanisms are involved in the regulation of SC and how SC mechanisms are involved in CRC. Given that markers for colonic SC are currently lacking, a necessary condition for SC identification is knowing that they reside at the bottom of the crypt, and thus use of fresh tissues rather than cultured cells is required in our proposed study. In Aim 1 we will use an innovative strategy to identify potential markers for colonic SC by applying proteomic analysis (2D gels/mass spectrometry) to the investigation of freshly isolated human colonic crypt subsections (bottom third of crypts where SC reside). Because evidence indicates that when SC contain mutant APC (as in FAP) the crypt SC population expands and there are substantially more SC, our first hypothesis is that (a) proteomic analysis will show that expression of some proteins (spot intensity) in histologically normal-appearing FAP crypts is significantly higher than in control crypts, and (b) antibodies generated to at least some such proteins will show staining at the normal crypt bottom and increases in the number of positively-stained crypt bottom cells in FAP. Identifying SC markers will enable investigators to grow SC and study SC changes during tumorigenesis. In Aim 2 we will use normal human crypt specimens to distinguish varying patterns, along the crypt axis, of APC phosphorylation, subcellular localization and interactions with binding proteins. Our second hypothesis is that known differences in APC concentration and subcellular localization along the crypt axis correlate with specific patterns in APC phosphorylation & binding to other proteins. These correlations will suggest mechanisms by which APC regulates SC population size and by which APC mutation leads to SC overpopulation and CRC.