Izmir Biomedicine and Genome Center (IBG), Dokuz Eylul University Health Campus, 35340 Izmir, Turkey; Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylul University, Izmir 35340, Turkey.
Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylul University, Izmir 35340, Turkey.
Cell Signal. 2021 Jun;82:109972. doi: 10.1016/j.cellsig.2021.109972. Epub 2021 Mar 6.
Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with "hepatoblast-like" appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.
富含亮氨酸重复的 G 蛋白偶联受体 5（LGR5）是一种新定义的内胚层衍生器官中的干细胞标志物，如小肠、结肠和胰腺。最近，LGR5 被证明是肝脏再生和干细胞维持的重要因素。此外，LGR5 的表达在包括肝细胞癌在内的各种癌症中高度上调。在此，我们证明 LGR5 表达特异性地存在于具有“肝前体细胞样”外观的某些 HCC 细胞系中，其特征在于表达肝胎儿/祖细胞标志物。值得注意的是，经典 Wnt 途径的激活显著增加了这组细胞系中 LGR5 的表达，而在间充质样细胞系 SNU-475 和 SNU-449 中，它不会引起任何 LGR5 表达的诱导。此外，我们表明，LGR5 配体 R-Spo1 处理肝前体细胞样 HCC 细胞系 HuH-7 和 Hep3B 可显著扩增 LGR5 表达的诱导，LRP6 和 β-连环蛋白的磷酸化导致 TCF/LEF 活性增强，无论是单独还是与 Wnt3a 联合使用。一致地，LGR5 基因的沉默减弱了 R-Spo1/Wnt3a 对 TCF/LEF 活性的协同刺激作用，而 LGR5 的过表达增强了它。相反，LGR5 的过表达不会改变 R-Spo1/Wnt3a 在间充质样 HCC 系 SNU-449 中诱导的 TCF/LEF 活性。重要的是，LGR5 过表达细胞在 R-Spo1/Wnt3a 处理后表达几种 Wnt 靶基因和与干细胞相关的基因，包括 EpCAM 和 CK19。LGR5 过表达细胞还具有增加的球体形成、迁移和侵袭能力，并且 R-Spo1/Wnt3a 刺激增强了 LGR5 过表达细胞的这些能力。此外，LGR5 的异位过表达独立于 R-Spo1/Wnt3a 刺激显著增加细胞增殖率。此外，体外管形成实验表明，仅在用 R-Spo1/Wnt3a 处理的 LGR5 过表达细胞中增强毛细血管管的发芽。最后，R-Spo1/Wnt3a 在斑马鱼异种移植模型中显著促进 LGR5 过表达细胞的体内扩散。我们的研究揭示了 LGR5 通过在肝前体细胞样 HCC 中激活经典 Wnt/β-连环蛋白信号传导发挥促肿瘤作用。总之，我们的结果表明，LGR5/R-Spo1/Wnt3a 产生了一个轴，介导了肝前体细胞样亚群 HCC 中侵袭表型的获得，并且可能代表了治疗 Wnt/β-连环蛋白途径异常激活的 HCC 肿瘤的有价值的靶标。
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