Department of Surgical Oncology, the First Affiliated Hospital of Xi'an Jiaotong University, 277 Yanta W. Road, Xi'an, 710061, Shaanxi, China.
Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, 33136-1015, USA.
J Exp Clin Cancer Res. 2019 Jan 9;38(1):13. doi: 10.1186/s13046-018-1021-y.
The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.
Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.
Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.
IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.
食管腺癌（EAC）在美国和西方国家的发病率正在迅速上升。巴雷特食管（BE）的发展及其向 EAC 的进展与慢性胃食管反流病（GERD）有关。在 GERD 条件下，BE 和 EAC 细胞暴露于酸性胆盐（ABS）会引起高水平的氧化应激和 DNA 损伤。在这项研究中，我们研究了胰岛素样生长因子结合蛋白 2（IGFBP2）在调节 ABS 诱导的 DNA 双链断裂中的作用。
本研究采用实时 RT-PCR、western blot、免疫组织化学、免疫荧光、共免疫沉淀、流式细胞术和环己酰亚胺（CHX）追踪实验。为了模拟 GERD 条件，在 2D 和 3D 器官型培养模型中使用酸性胆盐混合物（pH4）。通过在 EAC 细胞中过表达和敲低 IGFBP2，研究了 IGFBP2 在 ABS 诱导的 DNA 损伤中的功能和机制作用。
与癌前 Barrett 细胞系相比，EAC 细胞系中 IGFBP2 的 mRNA 和蛋白水平均较高，并且 IGFBP2 在 EAC 中频繁过表达（31/57）。用 ABS 处理 EAC 细胞，模拟 GERD 条件，可诱导 IGFBP2 表达水平升高。在 FLO1 细胞（内源性 IGFBP2 水平较高）中敲低内源性 IGFBP2 后，短暂暴露于 ABS 会导致 DNA 双链断裂和凋亡显著增加。另一方面，在 OE33 细胞（内源性 IGFBP2 水平较低）中过表达外源性 IGFBP2 可对 ABS 诱导的双链断裂和凋亡产生保护作用。我们发现，IGFBP2 是 ABS 诱导的 EGFR 和 DNA-PKcs 核内积累和磷酸化所必需的，这对于 DNA 损伤修复活性是必要的。通过共免疫沉淀实验，我们在酸性胆盐处理后检测到 IGFBP2 与 EGFR 和 DNA-PKcs 的共定位。我们进一步通过环己酰亚胺追踪实验证明，IGFBP2 可促进 ABS 暴露后 EGFR 蛋白的稳定性。
IGFBP2 通过稳定和激活 EGFR-DNA-PKcs 信号轴，保护 EAC 细胞免受 ABS 诱导的 DNA 损伤和凋亡。
