Exploring the Therapeutic Potential of Stem Cell Biology in Gliomas

TSCs中非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动态平衡至关重要。尽管TSCs与正常干细胞在表型和功能上有广泛的相似性，但TSCs的分化潜能并不完全正常。阐明在正常干细胞和TSCs中操作的分化途径对于全面理解肿瘤的发生至关重要，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胶质母细胞瘤的TSCs的非调控分化途径。与正常神经干细胞(NSCs)相比，骨形态发生蛋白(BMP)和睫状神经营养因子(CNTF)介导的Janus激酶/信号转导和转录激活子(JAK/STAT)通路在成人脑源性TSCs中均有不同的生物学效应。与非常早期的胚胎神经干细胞一样，0308 TSCs在BMP的刺激下增殖，并在CNTF刺激后诱导STAT3的边缘激活。然而，与发育后期的正常神经干细胞不同的是，0308细胞对CNTF触发的STAT3激活获得了反应性，并伴随着BMP受体1B(BMPR1B)的表达增加，0308细胞不表达BMPR1B，而BMPR1B继发于Zust同源基因增强子(EZH2)依赖的BMPR1B启动子超甲基化。BMPR1B在0308 TICS中的强制表达，无论是通过转基因表达还是通过启动子去甲基化，都可以完全恢复其分化能力，并不仅通过BMP介导的途径，而且通过CNTF介导的JAK/STAT激活来诱导其致瘤性丧失。一项对54个原发人类胶质母细胞瘤的调查显示，大约20%的人抑制了与启动子高甲基化相关的BMPR1B的表达。综上所述，这些数据暗示，在胶质母细胞瘤TSCs亚群中，BMP发育分化途径的解除调控不仅通过使TIC对正常分化信号脱敏，而且通过将其他细胞抑制信号转化为促增殖信号，从而促进了它们的致瘤表型。用维甲酸等分化诱导剂对GBM TSCs进行了大量的体外和体内鉴定，结果表明这些TSCs具有高效的分化和抑制增殖的作用。我们已经证明，维甲酸治疗通过减少肿瘤中CD133阳性细胞的比例和通过诱导分化为星形胶质细胞来实现细胞抑制作用。CD133阳性细胞是脑TSC的潜在标志。有趣的是，体内用放化疗药物预处理的GBM TICS的一部分，并没有显示出显著的维甲酸介导的分化。阐明其潜在的分子机制将为预测辨证施治的敏感性提供重要线索。TSCs在分化诱导剂方面的特性进一步揭示了传统的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暴露可以有效地诱导大多数GBM TIC分化，但不能诱导传统细胞系分化。这促使我们质疑之前在细胞系中研究的许多潜在的肿瘤抑制基因和/或细胞抑制基因是否没有被识别。鉴于生物信息学方法和实验室积累的干细胞培养技术专业知识在胶质母细胞瘤干细胞中鉴定出的潜在TSG和癌基因的数量不断增加，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.