Role of IPMK in generation of small intestinal carcinoid

5-羟色胺分泌性小肠神经内分泌肿瘤(SI-Net)是小肠的主要恶性肿瘤。当临床确诊时，患者通常表现为晚期疾病和较差的5年预后(约36%)。手术是SI-Net最常见的治疗方法。家族性SI-Net的遗传基础不同于散发性SI-Net。唯一报道的与家族性SI-Net相关的突变是IPMK(肌醇多聚磷酸多激酶)基因的四个碱基对缺失突变。IPMK蛋白全长416个氨基酸，突变后截短为333个氨基酸，消除了C-末端的ATP结合位点和部分核定位信号。IPMK杂合突变表现出其激酶活性和核定位的降低。IPMK是高阶肌醇合成途径中的限速酶，产生肌醇五磷酸。在哺乳动物细胞中，IPMK也扮演着PI3K的角色。IPMK主要集中在细胞核中，IPMK功能的丧失会损害P53的转录。由于P53功能缺失与多种肠癌密切相关，我们希望研究IPMK突变(在SI-Net患者中发现)在P53介导的细胞凋亡或细胞周期停滞中在恶性肿瘤发生和肿瘤形成中的重要性。IPMK纯合性功能丧失和杂合性突变型IPMK如何损害P53介导的细胞凋亡和细胞周期停滞是完全未知的，值得研究以了解SI-Net起源的分子机制。利用Cre-Lox技术，我们建立了肠上皮细胞特异性IPMK条件纯合子和杂合子KO小鼠，以了解IPMK介导SI-Net发育的机制。我们提出了三个具体目标--目标1.确定IPMK基因剂量如何影响小鼠SI-Net的范围。将进行组织病理学、免疫组织化学和生化分析，以了解IPMK的杂合性和纯合性丢失如何以年龄依赖的方式影响肠道内稳态和SI-Net的生成程度。目的2.确定IPMK对于促进细胞增殖和细胞周期调节是否必要和充分。将进行细胞生物学和流式细胞术分析，以了解IPMK在调节细胞增殖和凋亡中的关键功能。目的3.确定P53是否是IPMK信号诱导细胞凋亡和细胞周期停滞所必需的。通过对P53靶基因(PTG)的RNA序列分析，我们将确定IPMK在控制P53介导的细胞周期停滞和细胞凋亡中的关键作用，因为IPMK是家族性SI网络中唯一已报道的突变。当临床确诊时，患者通常表现为晚期疾病和较差的5年预后(约36%)。手术是SI-Net最常见的治疗方法。这项研究将有助于了解疾病进展的机制，并开发新的治疗方法。

Serotonin secreting small intestinal neuroendocrine tumors (SI-NET) are primary malignancies of the small bowel. When clinically recognized, afflicted patients often present with advanced disease and a poor 5-year prognosis (~36%). Surgery is the most common treatment of SI-NET. The genetic basis of Familial SI-NET is distinct from sporadic SI-NET. The only reported mutation linked to familial SI-NET is four base pair deletion mutations in the IPMK (Inositol polyphosphate multikinase) gene. IPMK protein is 416 amino acids in length,and the mutation truncates it to 333, eliminating the C-terminal ATP binding site and partial nuclear localization signal. IPMK heterozygous mutation exhibits a reduction of its kinase activity and nuclear localization. IPMK is the rate-limiting enzyme in the higher-order inositol synthesis pathway and generates inositol penta-phosphate. In mammalian cells, IPMK also acts as a PI3kinase. IPMK is primarily enriched in the nucleus, and loss of function of IPMK impairs p53 mediated transcription. As loss of p53 function is highly linked to several intestinal cancers, we like to investigate the importance of IPMK mutant (found in SI-NET patients) in p53 mediated apoptosis or cell cycle arrest in the origination of malignancies and tumor formation. How, homozygous loss of function of IPMK and a heterozygous mutant form of IPMK impair p53 mediated apoptosis and cell cycle arrest is entirely unknown and worth investigating to understand the molecular mechanism of SI-NET origination. Using Cre-Lox technology, we developed an intestinal epithelial cell-specific IPMK conditional homo and heterozygous KO mouse to understand the mechanism of IPMK mediated SI-NET development. We propose three specific aims—Aim 1. Determine how IPMK gene dosage affects the extent of SI-NET in mice. The histo-pathological, immuno-histochemical, and biochemical analysis will be performed to understand how hetero and homozygous loss of IPMK affects intestinal homeostasis and the extent of SI-NET generation in an age dependent manner. Aim 2. To determine if IPMK is necessary and sufficient to enhance cell proliferation and cell cycle regulation. Cell biological and flow-cytometric analysis will be performed to understand the critical function of IPMK in regulating cell proliferation and apoptosis. Aim 3. Determine if p53 is necessary for IPMK signaling-induced apoptosis and cell cycle arrest. By performing RNA seq analysis of p53 target genes (PTG), we will determine the critical role of IPMK in controlling p53 mediated cell cycle arrest and apoptosis As IPMK is the only reported mutation in familial SI NET the Serotonin secreting small intestinal neuroendocrine tumors (SI-NET) are primary malignancies of the small bowel. When clinically recognized, afflicted patients often present with advanced disease and a poor 5-year prognosis (~36%). Surgery is the most common treatment of SI-NET. This study will help understand the mechanism of disease progression and developing new therapeutics.