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）发展机制，因为这可能导致肿瘤细胞中易感分子过程的鉴定，从而发现更有效的抗癌药物和/或化学预防药物。由于有证据表明结肠SC是结直肠癌的起源细胞，发展识别结肠SC的能力（Aim 1）应该使分离SC群体成为可能，从而研究SC在结直肠癌发展中的作用机制。由于APC基因的突变引发了大多数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，那么将基于APC的功能变化与沿正常隐窝轴的结肠细胞特性变化相关联（Aim 2）应该揭示基于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的底部，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）来研究新分离的人类结肠隐窝亚段（SC所在隐窝的底部三分之一）来识别结肠SC的潜在标记物。因为有证据表明，当SC含有突变APC（如FAP）时，隐窝SC群体扩大，并且有更多的SC，我们的第一个假设是(a)蛋白质组学分析将显示，在组织学上正常的FAP隐窝中，某些蛋白质（斑点强度）的表达明显高于对照隐窝；(b)至少一些此类蛋白产生的抗体在正常隐窝底部显示染色，并且在FAP中隐窝底部阳性染色细胞的数量增加。鉴定SC标志物将使研究人员能够培养SC并研究SC在肿瘤发生过程中的变化。在Aim 2中，我们将使用正常的人类隐窝标本来区分沿隐窝轴的APC磷酸化、亚细胞定位和与结合蛋白相互作用的不同模式。我们的第二个假设是，APC浓度和沿隐窝轴的亚细胞定位的已知差异与APC磷酸化和与其他蛋白质结合的特定模式相关。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过度群体和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.