DEFINING THE IN VIVO ROLE OF DAP1 IN AUTOPHAGY AND COLITIS

描述(申请人提供)：结直肠癌(CRC)的病因是复杂和多因素的，然而炎症性肠病(IBD)被证明是CRC的一个强烈的危险因素。自噬是降解和回收细胞部件的过程，与IBD密切相关。自噬在IBD和CRC中都可能是重要的。不幸的是，由于遗传工具的不足，自噬在IBD和CRC中的作用仍未得到充分的研究。DAP1可能提供了CRC、IBD和自噬之间的联系。DAP1内含子SNP与UC相关，DAP1在许多肿瘤中高度过度表达，包括结直肠癌。在转化的细胞系中，DAP1负向调节饥饿条件下的自噬。此外，Dap1被mTOR磷酸化，mTOR是结肠炎和肿瘤发生中的一个重要分子。因此，我们假设，通过调节自噬，Dap1的表达有助于溃疡性结肠炎的易感性，然后与IBD一起，加速肿瘤的发生，导致结直肠癌。我们的长期目标是阐明Dap1在IBD中的作用，然后测试它在IBD启动的CRC中的作用。我们相信，这种方法将使我们能够更好地理解自噬和mTOR在这些疾病中的时空作用，并为调节IBD和CRC中这些途径的治疗提供有针对性的方法。这项提案的重点将是测试以下目标：目标1.确认DAP1负向调节自噬，并受原代细胞中mTOR的调节。我们将通过培养DAP1缺陷的原代细胞来实现这一点，并分析饥饿和/或mTOR抑制剂处理后的自噬。目的2.确定Dap1在原代细胞中对结肠炎的作用。我们将原代培养缺乏DAP1表达的结肠上皮和巨噬细胞，分析细菌和病毒刺激后的炎性细胞因子。目的3.明确DAP1在体内对结肠炎的作用。我们将测试Dap1缺陷是否能保护小鼠免受病毒感染后Atg16L1基因缺陷小鼠的胃肠道病变的影响，确定Dap1-/-小鼠是否对IL10缺乏引起的结肠炎具有抵抗力，并分析Dap1-/-小鼠对结肠损伤的炎症反应。

DESCRIPTION (provided by applicant): The etiology of colorectal cancer (CRC) is complex and multifactorial, however Inflammatory Bowel Disease (IBD) is demonstrated to be a strong risk factor for CRC. Autophagy, the process of degrading and recycling cellular parts, is strongly associated with IBD. Autophagy is likely to be of importance in both IBD and CRC. Unfortunately, the role of autophagy in IBD and CRC remains understudied due to insufficient availability of genetic tools. Dap1 may provide a link between CRC, IBD, and autophagy. An intronic SNP within DAP1 is associated with UC, and Dap1 is highly over-expressed in many tumors, including CRC. Dap1 negatively regulates autophagy in starvation conditions in transformed cell lines. Furthermore, Dap1 is phosphorylated by mTOR, an important molecule in colitis and tumorigenesis. Therefore, we hypothesize that by regulating autophagy, Dap1 expression contributes to ulcerative colitis susceptibility, then in conjunction with IBD, accelerates tumorigenesis resulting in colorectal cancer. Our long-term goals are to delineate the role of Dap1 in IBD, then to test its role in IBD-initiated CRC. We believe this approach will allow us to better understand the spatio-temporal role of autophagy and mTOR in these diseases, and provide a targeted approach to therapies modulating these pathways in IBD and CRC. The focus of this proposal will be to test the following aims: Aim 1. Confirm that Dap1 negatively regulates autophagy, and is regulated by mTOR in primary cells. We will approach this by culturing Dap1 deficient primary cells and analyze autophagy after starvation and/or treatment with mTOR inhibitors. Aim 2. Define Dap1 contributions to colonic inflammation in primary cells. We will culture primary colonic epithelium and macrophages that lack Dap1 expression, analyze inflammatory cytokines after stimulating with bacteria and virus. Aim 3. Define contribution of Dap1 to colitis in vivo. We will test if Dap1 deficiency protects mice from gastrointestinal pathologies occurring in mice hypomorphic for Atg16L1 after virus infection, determine if Dap1-/- mice are resistant to colitis induced by IL10 deficiencies, and analyze inflammatory responses to colonic wounding in Dap1-/- mice.