Aberrant activation of Wnt/β-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of β-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to β-catenin stabilization, increase in β-catenin/T-cell factor (TCF)–mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target β-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce β-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of β-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced β-catenin reduction, suggesting that BBR affects β-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E–binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap–binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of β-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of β-catenin. Our findings indicate that BBR antagonizes β-catenin pathway by inhibiting β-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated β-catenin variants that are currently undruggable.

中文翻译：

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小檗碱抑制肝细胞癌细胞中涉及 4E-BPs 的 β-连环蛋白翻译

Wnt/ β -catenin 轴的异常激活发生在几种胃肠道恶性肿瘤中，原因是腺瘤性结肠息肉病（结肠直肠癌）的失活突变或β -catenin 本身的激活突变[肝细胞癌 (HCC)]。这些导致β-连环蛋白稳定，增加β-连环蛋白/T 细胞因子 (TCF) 介导的转录激活和靶基因表达，其中许多与肿瘤进展有关。在研究可以靶向癌细胞中β-连环蛋白的药物时，我们观察到植物化合物小檗碱 (BBR) 是一种有效的 AMP 活化蛋白激酶 (AMPK) 激活剂，可以减少β- 以剂量依赖性方式在 HCC 细胞中表达和下游信号传导。对了解机制的更深入分析表明，BBR 诱导的β-连环蛋白减少与 AMPK 激活无关，并且不涉及转录或翻译后机制。用蛋白质合成抑制剂放线菌酮预处理可拮抗 BBR 诱导的β -catenin 减少，表明 BBR 影响β-连环蛋白翻译。BBR 治疗还拮抗了哺乳动物雷帕霉素靶标 (mTOR) 的活性，并与翻译复合物中真核翻译起始因子 4E 结合蛋白 (4E-BP) 1 的募集增加有关，7-甲基帽结合测定表明这一点，表明对帽依赖性翻译的抑制。有趣的是，敲低 4E-BP1 和 4E-BP2 显着减弱了 BBR 诱导的β-连环蛋白水平降低及其下游靶基因的表达。此外，4E-BP 敲低的细胞对 BBR 诱导的细胞死亡具有抗性，并且在β-连环蛋白的药理抑制后对 BBR 重新敏感。我们的研究结果表明，BBR通过抑制β -catenin 通路来拮抗β -catenin 通路。-catenin 翻译和 mTOR 活性，从而降低 HCC 细胞存活率。这些还表明 BBR 可用于靶向表达目前无法成药的突变/激活β-连环蛋白变体的 HCC。