CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPβ) was upregulated by metformin and that two isoforms of CEBPβ reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPβ signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.

CD133是一种细胞表面蛋白，据报道它是癌症干细胞标志物，因此被认为是癌症治疗的潜在靶点。二甲双胍是用于治疗糖尿病的双胍之一，还可以降低癌症发展和癌症干样细胞 (CSC) 的风险，包括 CD133 的表达。然而，二甲双胍降低 CD133 表达的机制尚不清楚。这项研究表明，二甲双胍主要通过腺苷单磷酸 (AMP) 激活的蛋白激酶 (AMPK) 信号传导影响 CD133 P1 启动子，而不是雷帕霉素 (mTOR) 的哺乳动物靶标，从而抑制 CD133 表达。AMPK 抑制可挽救二甲双胍对 CD133 的减少。进一步的实验表明，二甲双胍上调 CCAAT/增强子结合蛋白 β (CEBPβ)，并且 CEBPβ 的两种亚型相互调节 CD133 的表达。具体来说，肝脏富集的激活蛋白（LAP）亚型通过直接结合到P1启动子区域来增加CD133的表达，而肝脏富集的抑制蛋白（LIP）亚型则抑制CD133的表达。与这些发现一致，三维 (3D) 培养测定和药物敏感性测定表明，LAP 过表达细胞形成大球体，并且对 5-氟尿嘧啶 (5-FU) 治疗更耐药，而 LIP 过表达细胞对 5-氟尿嘧啶 (5-FU) 治疗更敏感。 5-FU 并显示出与二甲双胍的联合作用。我们的结果表明二甲双胍-AMPK-CEBPβ信号在调节CD133基因表达中起着至关重要的作用。此外，调节 LAP/LIP 的比例可能是针对 CSC 治疗癌症的一种新策略。