Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; Liverpool University Hospitals, National Health Service Foundation Trust, Liverpool, United Kingdom.
Cell Mol Gastroenterol Hepatol. 2020;10(1):113-132. doi: 10.1016/j.jcmgh.2020.01.010. Epub 2020 Jan 28.
BACKGROUND & AIMS: In patients with autoimmune atrophic gastritis and achlorhydria, hypergastrinemia is associated with the development of type 1 gastric neuroendocrine tumors (gNETs). Twelve months of treatment with netazepide (YF476), an antagonist of the cholecystokinin B receptor (CCKBR or CCK2R), eradicated some type 1 gNETs in patients. We investigated the mechanisms by which netazepide induced gNET regression using gene expression profiling.
We obtained serum samples and gastric corpus biopsy specimens from 8 patients with hypergastrinemia and type 1 gNETs enrolled in a phase 2 trial of netazepide. Control samples were obtained from 10 patients without gastric cancer. We used amplified and biotinylated sense-strand DNA targets from total RNA and Affymetrix (Thermofisher Scientific, UK) Human Gene 2.0 ST microarrays to identify differentially expressed genes in stomach tissues from patients with type 1 gNETs before, during, and after netazepide treatment. Findings were validated in a human AGS gastric adenocarcinoma cell line that stably expresses human CCK2R, primary mouse gastroids, transgenic hypergastrinemic INS-GAS mice, and patient samples.
Levels of pappalysin 2 (PAPPA2) messenger RNA were reduced significantly in gNET tissues from patients receiving netazepide therapy compared with tissues collected before therapy. PAPPA2 is a metalloproteinase that increases the bioavailability of insulin-like growth factor (IGF) by cleaving IGF binding proteins (IGFBPs). PAPPA2 expression was increased in the gastric corpus of patients with type 1 gNETs, and immunohistochemistry showed localization in the same vicinity as CCK2R-expressing enterochromaffin-like cells. Up-regulation of PAPPA2 also was found in the stomachs of INS-GAS mice. Gastrin increased PAPPA2 expression with time and in a dose-dependent manner in gastric AGS cells and mouse gastroids by activating CCK2R. Knockdown of PAPPA2 in AGS cells with small interfering RNAs significantly decreased their migratory response and tissue remodeling in response to gastrin. Gastrin altered the expression and cleavage of IGFBP3 and IGFBP5.
In an analysis of human gNETS and mice, we found that gastrin up-regulates the expression of gastric PAPPA2. Increased PAPPA2 alters IGF bioavailability, cell migration, and tissue remodeling, which are involved in type 1 gNET development. These effects are inhibited by netazepide.
在自身免疫性萎缩性胃炎伴胃酸缺乏的患者中，高胃泌素血症与 1 型胃神经内分泌肿瘤（gNET）的发展相关。12 个月的 netazepide（YF476）治疗（一种胆囊收缩素 B 受体（CCKBR 或 CCK2R）拮抗剂）根除了一些患者的 1 型 gNET。我们通过基因表达谱分析研究了 netazepide 诱导 gNET 消退的机制。
我们从参加 netazepide 2 期试验的 8 例高胃泌素血症和 1 型 gNET 患者中获得了血清样本和胃体活检标本。对照样本来自 10 例无胃癌的患者。我们使用来自总 RNA 的扩增和生物素化 sense 链 DNA 靶标以及 Affymetrix（Thermofisher Scientific，英国）Human Gene 2.0 ST 微阵列，以鉴定在接受 netazepide 治疗前后的 1 型 gNET 患者胃组织中差异表达的基因。在稳定表达人 CCK2R 的人 AGS 胃腺癌细胞系、原代小鼠胃上皮细胞、转基因高胃泌素血症 INS-GAS 小鼠和患者样本中验证了发现。
与治疗前的组织相比，接受 netazepide 治疗的 gNET 患者的 Pappalysin 2（PAPPA2）信使 RNA 水平显著降低。PAPPA2 是一种金属蛋白酶，通过切割 IGF 结合蛋白（IGFBPs）增加胰岛素样生长因子（IGF）的生物利用度。1 型 gNET 患者的胃体中 PAPPA2 表达增加，免疫组织化学显示其定位于与表达 CCK2R 的肠嗜铬样细胞相同的附近。在 INS-GAS 小鼠的胃中也发现了 PAPPA2 的上调。胃泌素通过激活 CCK2R 随时间增加和呈剂量依赖性增加胃 AGS 细胞和小鼠胃上皮细胞中的 PAPPA2 表达。用小干扰 RNA 敲低 AGS 细胞中的 PAPPA2 显著降低了它们对胃泌素的迁移反应和组织重塑。胃泌素改变了 IGFBP3 和 IGFBP5 的表达和切割。
在对人类 gNET 和小鼠的分析中，我们发现胃泌素上调了胃 PAPPA2 的表达。增加的 PAPPA2 改变了 IGF 的生物利用度、细胞迁移和组织重塑，这些都参与了 1 型 gNET 的发展。这些作用被 netazepide 抑制。
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