Exploring the Therapeutic Potential of Stem Cell Biology in Gliomas

对TSCs中去调控分化途径的分子理解：干细胞自我更新和分化之间的微妙平衡受到各种细胞内在和外在因素的控制，这些因素对正常组织的稳态至关重要。尽管TSCs与正常干细胞之间存在广泛的表型和功能相似性，但TSCs的分化潜能并不完全正常。阐明正常干细胞和TSCs的分化途径对于充分理解肿瘤发生至关重要，并可能导致新的治疗靶点。我们已经确定了一组来自人类原发性胶质母细胞瘤的TSCs的不受调节的分化途径。我们证明，与正常神经干细胞（NSCs）相比，骨形态发生蛋白（BMP）介导和睫状体神经营养因子（CNTF）介导的Janus激酶/信号换能器和转录激活因子（Jak/STAT）途径在成人脑源性TSCs中引起不同的生物学后果。与非常早期的胚胎NSCs一样，0308 TSCs在CNTF刺激后会对BMP产生增殖反应，并引发STAT3的边缘激活。然而，与发育后期的正常NSCs不同，在BMP受体1B （BMPR1B）表达增加的过程中获得对cntf触发的STAT3激活的反应性，0308细胞不表达继发于Zeste同源增强子2 （EZH2）依赖性BMPR1B启动子超甲基化的BMPR1B。通过转基因表达或启动子的去甲基化，在0308 tic中强制表达BMPR1B，不仅可以通过bmp介导的途径，还可以通过cntf介导的Jak/STAT激活，完全恢复它们的分化能力，并诱导它们的致瘤性丧失。一项对54例原发性人胶质母细胞瘤的调查显示，大约20%的人抑制与启动子超甲基化相关的BMPR1B的表达。综上所述，这些数据表明，在胶质母细胞瘤TSCs的一个子集中，BMP发育分化途径的解除不仅使TIC对正常分化信号脱敏，而且还将细胞抑制信号转化为促增殖信号，从而有助于其致瘤表型。通过使用维甲酸等分化诱导剂对GBM TSCs进行的大量体外和体内表征表明，这些TSCs能够有效地分化并停止增殖。我们已经证明，维甲酸治疗通过降低肿瘤中CD133阳性细胞（一种假定的脑TSCs标记物）的比例，并通过诱导分化为星形胶质细胞谱系，达到细胞抑制作用。有趣的是，在体内用放疗和化疗药物预处理的GBM tic的一个子集没有显示显着的视黄酸介导的分化。阐明其分子机制将为预测分化治疗方法的敏感性提供重要线索。从诱导分化剂的角度对TSCs进行表征，进一步揭示了传统胶质瘤细胞系在血清中生长的局限性。例如，维甲酸处理和CNTF暴露可在大多数GBM tic中诱导分化，但在传统细胞系中却没有。这促使我们质疑以前在细胞系中研究的许多潜在的肿瘤抑制因子和/或细胞抑制基因是否未被识别。鉴于生物信息学方法和实验室积累的干细胞培养技术经验在胶质母细胞瘤TSCs中发现的潜在tsg和致癌基因数量不断增加，我们建立了筛选系统来研究这些基因在干细胞培养中的功能。

Molecular understanding of deregulated differentiation pathways in TSCs: The delicate balance between stem cell self-renewal and differentiation is controlled by various cell intrinsic and extrinsic factors that are critical for normal tissue homeostasis. Despite extensive phenotypic and functional similarities between TSCs and normal stem cells, the differentiation potentials of TSCs are not entirely normal. Elucidation of the differentiation pathways operative in both normal stem cells and TSCs will be critical for fully understanding tumorigenesis and will likely lead to novel therapeutic targets. We have identified a set of deregulated differentiation pathways in TSCs derived from human primary glioblastoma. We demonstrated that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathways elicit distinct biological consequences in adult brain derived TSCs compared to normal neural stem cells (NSCs). Like very early embryonic NSCs, 0308 TSCs proliferate in response to BMP and elicit marginal STAT3 activation after CNTF challenge. However, unlike normal NSCs in later developmental stages that acquire responsiveness to CNTF-triggered STAT3 activation in association with increased expression of BMP receptor 1B (BMPR1B), 0308 cells do not express BMPR1B secondary to Enhancer of Zeste homolog 2 (EZH2)-dependent BMPR1B promoter hypermethylation. Forced expression of BMPR1B in 0308 TICs either by transgene expression or demethylation of the promoter fully restores their differentiation capabilities and induces loss of their tumorigenicity not only via a BMP-mediated pathway but also by CNTF-mediated Jak/STAT activation. A survey of 54 primary human glioblastomas reveals that approximately 20% have suppressed expression of BMPR1B associated with promoter hypermethylation. Taken together, these data implicate that deregulation of the BMP developmental differentiation pathway in a subset of glioblastoma TSCs contributes to their tumorigenic phenotype by not only desensitizing TIC to normal differentiation cues, but also by converting otherwise cytostatic signals to pro-proliferative signals. Extensive in vitro and in vivo characterization of GBM TSCs by using differentiation-inducing agents such as retinoic acid demonstrated that these TSCs differentiate efficiently and stop proliferation. We have demonstrated that retinoic acid treatment achieve cyctostatic effect by decreasing the proportion of CD133 positive cells, a putative marker for brain TSCs, from tumors and by inducing differentiation into astroglial lineage. Interestingly, a subset of GBM TICs pretreated with radiation and chemotherapeutic agents in vivo, do not reveal significant retinoic acid-mediated differentiation. Elucidation of underlying molecular mechanism will provide important clue for predicting sensitivity of differentiation therapeutic approach. Characterization of TSCs in aspect of differentiation-inducing agents further revealed the limitation of traditional glioma cell lines grown in serum. For example, retinoic acid treatment and CNTF exposure potently induce differentiation in most GBM TICs but not of traditional cell lines. This prompted us to question whether many of potential tumor suppressors and/or cytostatic genes previously studied in cell lines, were not recognized. Given the ever increasing number of potential TSGs and oncogenes in glioblastoma TSCs identified from bioinformatics approach and technical expertise of stem cell culture accumulated in the laboratories, we have set up screening systems to study the function of these genes in stem cell cultures.