Characterising a role for EDI3 in metastasis in vivo

最近，我们确定了甘油磷酸二酯酶，子宫内膜癌鉴别3 （EDI3; GPCPD1）作为一项更大的研究的一部分，目的是表征子宫内膜癌转移的标志物。我们观察到转移的原发性肿瘤表达的EDI3比非转移性肿瘤高约6倍。此外，EDI3与较差的预后相关。这些观察结果在一组卵巢癌患者中也得到了证实，表明EDI3是一种有希望的转移标志物。我们最初的工作还将EDI3描述为甘油磷酸二酯酶，它将其底物甘油磷酸胆碱（GPC）水解为胆碱和甘油-3-磷酸。由于一些报告显示胆碱代谢物，特别是磷胆碱，在特别侵袭性的癌症类型中升高，人们对胆碱代谢的兴趣最近有所增加。此外，胆碱是产生磷脂酰胆碱所必需的，磷脂酰胆碱是细胞膜中含量最多的磷脂，也是几种信号脂质的前体。甘油-3-磷酸，EDI3的第二个产物，进一步代谢为信号脂质磷脂酸（PA）和溶血磷脂酸（LPA）。它也可以转化为甘油醛-3-磷酸，这是糖酵解的早期代谢物，在癌症中也会发生改变。在一些癌细胞中，用siRNA敲除EDI3使磷脂胆碱水平逆转到非转化细胞中的水平，并改变了许多关键的脂质代谢物，包括PA和LPA。EDI3的进一步表征确定了细胞迁移的关键作用，这是转移的必要步骤。有趣的是，在转移传播过程中发生的另外两个过程——细胞粘附和扩散——依赖于与迁移相关的信号机制，也受到EDI3的影响。然而，迄今为止关于EDI3的所有结果都是通过体外细胞培养模型进行的，这使我们能够研究EDI3的基本功能，但缺乏体内微环境的复杂性和存在的多种细胞类型。因此，本课题拟利用已建立的乳腺癌模型，在体内研究EDI3对乳腺癌转移的影响。将在具有高或低水平EDI3表达的肿瘤细胞的小鼠中建立乳腺癌原位模型。将分析不同组织中的原发肿瘤和转移瘤的EDI3表达、增殖和迁移标志物、肿瘤细胞在肿瘤和微环境中的行为以及胆碱代谢。我们的总体目标是更好地了解EDI3在转移过程中的作用，并最终确定其作为转移标志物和治疗靶点的适用性。

Recently, we identified the glycerophosphodiesterase, Endometrial cancer Differential 3 (EDI3; GPCPD1) as part of a larger study with the goal to characterise markers of endometrial cancer metastasis. We observed that primary tumours that went on to metastasize expressed approximately six times higher EDI3 than the non-metastasizing tumours. In addition, EDI3 was associated with worse prognosis. These observations were also confirmed in a cohort of ovarian cancer patients, suggesting that EDI3 is a promising marker of metastasis. Our initial work also characterised EDI3 as a glycerophosphodiesterase, which hydrolyses its substrate glycerophosphocholine (GPC) to choline and glycerol-3-phosphate. Interest in choline metabolism has recently increased due to several reports showing that choline metabolites, in particular phosphocholine, are elevated in particularly aggressive cancer types. Furthermore, choline is needed for the production of phosphatidylcholine, the most abundant phospholipid in the membrane of cells, and precursor to several signalling lipids. Glycerol-3-phosphate, the second EDI3 product, is further metabolised to signalling lipids phosphatidic acid (PA), and lysophosphatidic acid (LPA). It can also be converted to glyceraldehyde-3-phosphate, an early metabolite in glycolysis, which too is altered in cancer. Knocking down EDI3 with siRNA in several cancer cells reversed phosphocholine levels to that found in non-transformed cells, and altered many key lipid metabolites, including PA and LPA. Further characterisation of EDI3 identified a critical role in cellular migration, a step necessary for metastasis. Interestingly, two other processes - cell adhesion and spreading - which occur during metastatic dissemination and are dependent on signalling mechanisms similar to those involved in migration, are also influenced by EDI3. However, all of the results described on EDI3 so far were performed using in vitro cell culture models, which allowed us to study the basic functions of EDI3, but lacked the complexity of the in vivo microenvironment and the multitude of cell types present. Therefore, the current proposal will use an established model of breast cancer to study the influence of EDI3 on metastasis in vivo. An orthotopic model of breast cancer will be developed in mice with tumour cells expressing either high or low levels of EDI3. Both the primary tumour and resulting metastases in different tissues will be analysed for EDI3 expression, markers of proliferation and migration, behaviour of the tumour cells within the tumour and the microenvironment, and choline metabolism. The overall goal is that we gain a better understanding of the role of EDI3 in the metastasis process, and ultimately its suitability as a metastatic marker and therapeutic target.