Department of Neuro-Oncology, Hugo W. Moser Research Institute at Kennedy Krieger, Baltimore, MD 21205, USA.
Oncogene. 2011 Aug 4;30(31):3454-67. doi: 10.1038/onc.2011.58. Epub 2011 Mar 7.
It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell-matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.
有必要了解分化剂影响肿瘤起始癌症干细胞的机制。为此，我们研究了神经胶质瘤干细胞样细胞（GBM-SCs）对全反式视黄酸（RA）的细胞和分子反应。GBM-SCs 在生长因子补充的无血清培养基中作为非贴壁神经球生长。RA 处理迅速诱导形态变化，诱导 G1/G0 到 S 期过渡的生长停滞，降低 cyclin D1 表达并增加 p27 表达。免疫荧光和 Western blot 分析表明，RA 诱导了谱系特异性分化标记物 Tuj1 和 GFAP 的表达，并降低了神经干细胞标记物如 CD133、Msi-1、巢蛋白和 Sox-2 的表达。RA 处理显著降低了神经球形成能力，抑制了神经球在软琼脂中形成集落的能力，并抑制了它们在皮下和颅内异种移植中增殖的能力。表达微阵列分析确定了 RA 处理后 48 小时内改变的约 350 个基因。受影响的途径包括视黄酸信号转导和代谢、细胞周期调控、谱系决定、细胞黏附、细胞-基质相互作用和细胞骨架重塑。Notch 信号通路是这些对 RA 有反应的通路中最突出的。Notch 信号通路的下调是基于 HES 和 HEY 家族成员的下调得到证实的。Notch 胞内结构域的组成性激活挽救了 GBM 神经球免受 RA 诱导的分化和干细胞耗竭。我们的发现确定了 RA 靶向 GBM 来源的干细胞样肿瘤起始细胞的机制，以及适用于神经胶质瘤分化治疗的新靶点。
