活性氧/NFkB信号通路在脑胶质瘤干细胞分化中的作用及其调控机制

Cancer stem cells (CSCs), which are known for their high resistance to all kinds of treatments and causing cancer recurrence and metastasis, have been isolated in glioblastoma multiforme (GBM). GBM is the most common and aggressive primary brain tumor with poor prognosis which may be ascribed to the presence of glioblastoma stem cells (GSCs). This subgroup of cells seem to reside in special stem cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Our group reported that GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited DNA repair, achieving strikingly killing effects in drug resistant GSCs (Stem Cells. 2013;31:23-34.). Recently, we used several stem-like glioblastoma cell lines derived from patients' tissues by typical neurosphere culture system, and showed that addition of fetal bovine serum to the medium induced an increase of reactive oxygen species (ROS), leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induces their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS leads to a down-regulation of molecules including SOX2, Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine (NAC) in the serum-treated GSCs suppresses the increase of superoxide and largely rescues the expression of SOX2, Olig2, and Notch1, and prevents the serum-induced differentiation phenotype. We also found that serum could cause the activation of NFκB which could be blocked by the antioxidant NAC, suggesting that the activation of NF?B in the serum-induced cells is probably caused by the ROS increase. A selective IKK1 and IKK2 inhibitor BMS-345541 could protect the decrease of CD133 and prevent the increase of a differentiation marker ANXA1. The main objectives of current research are to investigate the role of ROS in GSC aberrant differentiation and its effects in cancer development, to elucidate the underlying mechanisms by which NF?B become activated by ROS, to evaluate the effect and mechanisms of NF?B on GSC aberrant differentiation. It is anticipated that the proposed studies will provide new insights into the aberrant differentiation of GSCs, uncover the role of ROS and NF?B in GSCs aberrant differentiation and identify possible therapeutic strategies to effectively kill cancer stem cells through interfering their aberrant differentiation.

肿瘤干细胞是恶性肿瘤复发转移的根源，诱导肿瘤干细胞分化是癌症治疗的一大难题。我们前期研究发现脑胶质瘤干细胞高度依赖有氧糖酵解进行能量代谢(Stem Cells. 2013)，进一步检测到脑胶质瘤干细胞分化中线粒体内超氧化物显著升高，活性氧相关信号通路明显上调，采用抗氧化物NAC抑制线粒体内超氧化物阻止了肿瘤干细胞分化。然而，活性氧调节肿瘤干细胞分化机制仍未明确。我们通过转录组基因芯片发现NF?B信号通路在分化过程中显著上调，NF?B抑制剂可抑制肿瘤干细胞标记物下调，而NAC可抑制NF?B的上调。因此，拟以脑胶质瘤原代干细胞培养体系及原位接种动物模型，探索活性氧对脑胶质瘤干细胞分化及肿瘤进展的影响；活性氧对NF?B通路的调节机制；NF?B对脑胶质瘤干细胞分化的影响及机制。本研究将丰富对脑胶质瘤干细胞分化的认识，揭示活性氧及NF?B通路在肿瘤干细胞分化中的意义，为脑胶质瘤防治提供新的理论依据。

我们课题组曾报道了脑胶质瘤干细胞线粒体呼吸功能较低，主要依赖有氧糖酵解产生能量，在分化过程中细胞线粒体内的电子传递链呼吸功能显著上调，有氧糖酵解活性下降。在本研究中我们发现氧化应激反应信号通路是脑胶质瘤干细胞分化早期改变最明显的信号通路；并建立了来源于患者的原代脑胶质瘤干细胞系，证实了活性氧在肿瘤干细胞分化中的重要作用。此外，我们还发现经血清诱导分化后肿瘤干细胞有更高的成瘤能力；通过抑制线粒体活性氧可以抑制肿瘤干细胞的分化。本研究进一步阐释了血清诱导肿瘤干细胞分化是通过线粒体呼吸功能上调产生的活性氧激活NFĸB信号通路导致的。本研究首次发现血清诱导肿瘤干细胞分化是通过激活线粒体呼吸链而产生的活性氧诱导，揭示了活性氧在肿瘤干细胞分化中的重要分子机制，具有重大的科学意义；并提示线粒体激活产生的活性氧可能诱导了肿瘤干细胞的分化，促进了肿瘤的发展，同时也提示抗氧化物NAC能够抑制血清诱导的肿瘤干细胞分化；还发现肿瘤干细胞内活性氧很可能通过激活NFκB信号通路促进了脑胶质瘤干细胞的分化，具有重大的临床转化前景，可进一步结合临床实践，验证本研究发现。