DEFINING THE IN VIVO ROLE OF DAP1 IN AUTOPHAGY AND COLITIS

描述（由申请人提供）：结直肠癌（CRC）的病因复杂且多因素，但炎症性肠病（IBD）被证明是结直肠癌的一个重要危险因素。自噬，降解和循环细胞部分的过程，与IBD密切相关。自噬可能在IBD和CRC中都很重要。不幸的是，由于遗传工具的可用性不足，自噬在IBD和CRC中的作用仍未得到充分研究。Dap1可能在结直肠癌、IBD和自噬之间提供了联系。DAP1中的内含子SNP与UC相关，DAP1在包括CRC在内的许多肿瘤中高度过表达。Dap1负调控转化细胞系在饥饿条件下的自噬。此外，Dap1被mTOR磷酸化，mTOR是结肠炎和肿瘤发生的重要分子。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一起加速肿瘤发生，导致结直肠癌。我们的长期目标是描述Dap1在IBD中的作用，然后测试其在IBD引发的CRC中的作用。我们相信这种方法将使我们更好地了解自噬和mTOR在这些疾病中的时空作用，并为IBD和CRC中调节这些途径的治疗提供有针对性的方法。本建议的重点将是测试以下目标：证实Dap1负向调控自噬，并在原代细胞中受mTOR调控。我们将通过培养Dap1缺陷原代细胞和分析饥饿和/或mTOR抑制剂治疗后的自噬来解决这个问题。目标2。确定Dap1在原代细胞结肠炎症中的作用。我们将培养缺乏Dap1表达的原代结肠上皮细胞和巨噬细胞，分析细菌和病毒刺激后的炎症因子。目标3。确定Dap1对体内结肠炎的作用。我们将测试Dap1缺乏是否能保护小鼠免受病毒感染后Atg16L1低形态小鼠的胃肠道病变，确定Dap1-/-小鼠是否对IL10缺乏诱导的结肠炎具有抗性，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.