Department of Oncology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China.
Division of Digestive and Liver Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
Cell Commun Signal. 2018 Mar 12;16(1):9. doi: 10.1186/s12964-018-0222-5.
The WNT-beta-catenin pathway is known to regulate cellular homeostasis during development and tissue regeneration. Activation of WNT signaling increases the stability of cytoplasmic beta-catenin and enhances its nuclear translocation. Nuclear beta-catenin function is regulated by transcriptional co-factors such as CREB binding protein (CBP) and p300. Hyper-activated WNT-beta-catenin signaling is associated with many cancers. However, its role in inducing stemness to liver cancer cells, its autoregulation and how it regulates tumor suppressor pathways are not well understood. Here we have investigated the role of CBP-beta-catenin signaling on the expression of CD133, a known stem cell antigen and PP2A-PTEN pathway in tumor initiating liver cancer cells.
Human hepatoblastoma cell line HepG2 and clonally expanded CD133 expressing tumor initiating liver cells (TICs) from premalignant murine liver were used in this study. CBP-beta-catenin inhibitor ICG001 was used to target CBP-beta catenin signaling in liver cancer cells in vitro. Western blotting and real time PCR (qPCR) were used to quantify protein expression/phosphorylation and mRNA levels, respectively. CBP and CD133 gene silencing was performed by siRNA transfection. Fluorescence Activated Cell Sorting (FACS) was performed to quantify CD133 positive cells. Protein Phosphatase (PP2A) activity was measured after PP2AC immunoprecipitation.
CBP inhibitor ICG001 and CBP silencing significantly reduced CD133 expression and anchorage independent growth in HepG2 and murine TICs. CD133 silencing in TICs decreased cell proliferation and expression levels of cell cycle regulatory genes, CyclinD1 and CyclinA2. ICG001 treatment and CBP silencing reduced the levels of phosphoPTEN, phospho-AKT, Phospho-beta-catenin in TICs. ICG001 mediated de-phosphorylation of PTEN in TICs was PP2A dependent and partly prevented by co-treatment with PP2A inhibitor okadaic acid.
CBP-beta-catenin signaling promotes stemness via CD133 induction and cell proliferation in TICs. We found a novel functional link between CBP-beta-catenin and PP2A-PTEN-AKT pathway in liver TICs. Therefore, CBP-beta-catenin-PP2A-PTEN-AKT signaling axis could be a novel therapeutic target to prevent liver tumor initiation and cancer recurrence.
WNT-β-连环蛋白途径已知在发育和组织再生过程中调节细胞内稳态。WNT 信号的激活增加了细胞质β-连环蛋白的稳定性，并增强了其核易位。核β-连环蛋白的功能受转录共因子的调节，如 CREB 结合蛋白（CBP）和 p300。高度激活的 WNT-β-连环蛋白信号与许多癌症有关。然而，它在诱导肝癌细胞的干性、自身调节以及如何调节肿瘤抑制途径方面的作用还不是很清楚。在这里，我们研究了 CBP-β-连环蛋白信号对 CD133 表达的作用，CD133 是一种已知的干细胞抗原和 PP2A-PTEN 途径在肿瘤起始性肝癌细胞中的作用。
本研究使用人肝癌细胞系 HepG2 和从癌前鼠肝中克隆扩增的表达 CD133 的肿瘤起始性肝癌细胞（TICs）。CBP-β-连环蛋白抑制剂 ICG001 用于体外靶向肝癌细胞中的 CBP-β 连环蛋白信号。Western 印迹和实时 PCR（qPCR）分别用于定量蛋白表达/磷酸化和 mRNA 水平。通过 siRNA 转染进行 CBP 和 CD133 基因沉默。通过荧光激活细胞分选（FACS）定量 CD133 阳性细胞。PP2A 免疫沉淀后测量蛋白磷酸酶（PP2A）活性。
CBP 抑制剂 ICG001 和 CBP 沉默显著降低了 HepG2 和鼠 TICs 中的 CD133 表达和锚定非依赖性生长。TICs 中的 CD133 沉默降低了细胞增殖和细胞周期调节基因 CyclinD1 和 CyclinA2 的表达水平。ICG001 处理和 CBP 沉默降低了 TICs 中磷酸化 PTEN、磷酸化 AKT 和磷酸化β-连环蛋白的水平。TICs 中 ICG001 介导的 PTEN 去磷酸化依赖于 PP2A，并用 PP2A 抑制剂 okadaic acid 共同处理部分阻止。
CBP-β-连环蛋白信号通过诱导 TICs 中的 CD133 表达和细胞增殖来促进干性。我们在肝 TICs 中发现了 CBP-β-连环蛋白和 PP2A-PTEN-AKT 途径之间的新的功能联系。因此，CBP-β-连环蛋白-PP2A-PTEN-AKT 信号轴可能是预防肝肿瘤起始和癌症复发的新的治疗靶点。
