Retinoic acid-induced 2 (RAI2) is a novel antagonist of Wnt/β-catenin signaling pathway and increases the chemosensitivity of colorectal cancer cells to oxaliplatin and fluorouracil
Retinoic acid-induced 2 (RAI2) is a novel antagonist of Wnt/β-catenin signaling pathway and increases the chemosensitivity of colorectal cancer cells to oxaliplatin and fluorouracil
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Retinoic Acid-induced 2 (RAI2) 是 Wnt/β-catenin 信号通路的新型拮抗剂，可增加结直肠癌细胞对奥沙利铂和氟尿嘧啶的化疗敏感性
作者：
Weitao Zhang;Lu Kong;Hongbin Zhu;Decong Sun;Quanli Han;Bin Yan;Zhi Cui;Weiwei Zhang;Shurong Zhang;Xindan Kang;Guanghai Dai;Niansong Qian;Wenji Yan
目的：Wnt/β-catenin信号的异常激活参与了结直肠癌干细胞的维持和化疗耐药。维甲酸诱导的2(RAI2)在结直肠癌中被证实是一种肿瘤抑制因子。本研究进一步探讨了RAI2在Wnt/β-catenin信号转导中的作用。方法：据报道，C端结合蛋白2(CtBP2)作为一种转录辅助调节因子，以多种复杂的方式参与Wnt信号转导。用TCGA数据集分析RAI2和CtBP2在结直肠癌中的相关性，并用免疫共沉淀(Co-IP)法探讨RAI2和CtBP2在结直肠癌细胞中的相互作用。采用双荧光素酶报告基因分析和免疫荧光法分别检测RAI2对Wnt信号转导活性和β-连环蛋白定位的影响。Western blotting分析Wnt信号转导相关靶基因的表达。球体形成实验检测RAI2对干细胞样特性的影响。结果：Co-IP证实了RAI2与CtBP2的相互作用。此外，通过对TCGA数据集的分析，发现RAI2与CtBP2在结直肠癌中的表达呈负相关。RAI2在人结肠癌细胞(LoVo和HCT116)中的重新表达抑制了Wnt信号的荧光活性，增加了β-catenin的磷酸化，并抑制了其核转位，下调了靶基因c-Myc、cyClinD1、ASCL2和LGR5。相反，突变的RAI2不能与CtBP2相互作用，没有上述作用。33.89%的结直肠癌患者RAI2低表达，这与β-连环蛋白的磷酸化程度降低(r=0.8866，P<0.0001)、5年无复发生存率低(P=0.0029)和总生存率(OS)低(P=0.0102)有关。在LoVo和HCT116细胞中RAI2的恢复抑制了CRC细胞的干细胞样特性，并增加了这些细胞对奥沙利铂和氟尿嘧啶的化疗敏感性。结论：RAI2低表达可作为独立的预后不良指标。Rai2通过与CtBP2相互作用或下调CtBP2来抑制Wnt信号，从而抑制干细胞样属性并增加结直肠癌细胞的化疗敏感性。
Objective: Aberrant activation of Wnt/β-catenin signaling contributes to the maintenance of cancer stem cells and chemoresistance in colorectal cancer (CRC). Retinoic acid-induced 2 (RAI2) was proved to be a tumor suppressor in CRC in our previous report. In this study, the role of RAI2 in Wnt/β-catenin signaling was further investigated. .Methods: As a transcriptional co-regulator, C-terminal Binding Protein 2 (CtBP2) was reported to be involved in Wnt signaling in multiple and complex ways. The correlation of RAI2 and CtBP2 in CRC was analyzed by TCGA dataset, and the interaction between RAI2 and CtBP2 was explored by co-immunoprecipitation (Co-IP) in CRC cells. The effect of RAI2 on the activity of Wnt signaling and the location of β-catenin was detected by Dual-luciferase reporter assay and Immunofluorescence respectively. Western blotting analysis was performed to detect the expression of target genes involved in Wnt signaling. Sphere formation assay was employed to detect the effect of RAI2 on stem cell like properties. Cell viability assay was used to detect the chemosensitivity of cells before and after transfection of RAI2..Results: The interaction between RAI2 and CtBP2 was confirmed by Co-IP in CRC cells. Besides, the negative correlation of RAI2 and CtBP2 in CRC was found by analyzing the TCGA dataset. Re-expression of RAI2 in human colon cancer cells (LoVo and HCT116) suppressed the fluorescent activity of Wnt signaling, increased the phosphorylation, and inhibited nuclear translocation of β-catenin, with down-regulation of target genes like c-Myc, CyclinD1, ASCL2 and LGR5. In contrast, the mutated RAI2, which can’t interact with CtBP2, had no above effects. We observed low expression of RAI2 in 33.89% (101/298) of CRC patients, which was significantly associated with reduced phosphorylation of β-catenin (r=0.8866, P＜0.0001), poor 5-year relapse-free survival (RFS) (P = 0.0029), and overall survival (OS) (P = 0.0102). Restoration of RAI2 in LoVo and HCT116 cells inhibited stem cell-like properties of CRC cells and increased chemosensitivity of these cells to oxaliplatin and fluorouracil. .Conclusion: low expression of RAI2 can serve as an independent poor prognostic marker. RAI2 inhibits Wnt signaling by interacting with or down-regulating CtBP2, resulting in repression of stem cell-like properties and increased chemosensitivity of CRC cells.