| Brain tumors have one of the poorest outcomes of all human cancers. Glioblastoma multiforme (GBM, World Health Organization grade IV glioma) is the most aggressive and, unfortunately, most common type of primary brain tumors in adults with a median survival of approximately 14 months, despite advances in surgery and adjuvant therapies. The failure of current treatments for GBMs primarily attributes to their invasiveness and resistance to chemo- and radio-therapies, resulting in frequent relapse or progression. Currently, the mechanisms underlying GBM tumorigenesis are not fully known. The cancer stem cell (CSC) hypothesis suggests that GBM is driven by a subpopulation of glioma stem cells (GSCs) which also contributes to their resistance to therapy. Therefore, it is important for both cancer research and clinical practice to further explore the intrinsic molecular basis behind the tumorigenicity of GSCs. Great efforts have been made to understand the mechanisms underlying the stem cell properties and tumorigenicity of GSCs. In this study, we will isolate and identify GSCs from glioma tissues, and explore new markers for GSC. Then, the mechanisms underlying the tumorigenicity and the formation of GSCs will be studied.Part I: Brain tumor stem like cells identified by neural stem cell marker CD15. In this part, we cultured 7 neurosphere cell lines derived from glioma tissues, which are enriched for GSCs, from 15 cases of primary brain tumors. These GSC cell lines can be maintained for 5-20 passages. At present, many studies used stem cell marker CD133 to identify GSCs. However, recent studies indicated that CD133 is not a universal marker for GSC and there are CD133 negative GSCs. Genetic mouse models demonstrated that gliomas are originated from transformed neural stem cells (NSCs), and there are many similarities between GSCs and NSCs. Therefore, we investigated the expression of CD15, one surface marker for NSCs, in GSCs derived from astrocytoma and ependymoma. CD15~+ cells isolated from these tumor spheres had properties of GSCs including self-renewal, multi-differentiation, and recapitulating the phenocopy of primary tumors. CD15 exhibited stable expression in long term cultured tumor spheres which sustained GSCs properties. Furthermore, CD15~+CD133- cells isolated from early or late passages of tumor spheres showed similar characteristics of GSCs. Examination of glioma samples by immunohistochemistry showed that CD15 was expressed in a subset of human brain tumors. Therefore, CD15 can be used as one marker of stem like cells derived from brain tumors which might contain CD133- GSCs.Part II. Overexpression of ZNF217 in glioblastoma contributes to the maintenance of glioma stem cells regulated by hypoxia-inducible factors. It has been established that GSCs reside in a specialized hypoxic niche, which can regulate the tumorigenic capacity of GSCs primarily through the hypoxia-inducible factors (HIFs), HIF1αand HIF2α. ZNF217 is an oncogene frequently amplified in many kinds of tumors. It is associated with aggressive tumor behavior and poor clinical prognosis, but its role in gliomas is poorly known. Gene expression and copy number analysis from TCGA data reveal that ZNF217 is amplified in 32% and overexpressed in 71.2% of GBMs. Quantitative RT-PCR and Western blotting of a cohort of glioma samples showed that ZNF217 was highly expressed in gliomas and increased with tumor grade. Analysis of a molecular database demonstrated that ZNF217 expression correlated with poor survival of glioma patients. Investigation of ZNF217 expression in GSCs, non-GSCs and normal neural stem cells (NSCs) indicated that ZNF217 was more highly expressed in GSCs than in non-GSCs and NSCs. Knockdown of ZNF217 in GSCs by siRNA inhibited their growth and promoted their differentiation. Interestingly, ZNF217 was upregulated in GSCs and the GBM cell line U87 when exposed to the hypoxic environment of 1% oxygen. Knockdown of either HIF1αor HIF2α, which play a central role in the hypoxia induced responses of these cells, inhibited ZNF217 expression. In addition, ZNF217 upregulation was compromised under hypoxia in U87 and GSCs when either HIF1αor HIF2αwas targeted by siRNA. HIF2αknockdown inhibited ZNF217 expression more efficiently in both normoxia and hypoxia than HIF1αknockdown. Therefore, ZNF217 is overexpressed in GBMs and contributes to the maintenance of GSCs which is regulated by HIFs released by the hypoxic environment of the tumor.Part III. LIN28 facilitates the transformation of human neural stem cells and promotes glioblastoma tumorgenicity. The pluripotency factor LIN28 is activated in many cancers arising outside the central nervous system, but its role in brain tumors is poorly understood. Using immunohistochemistry, we detected LIN28 protein in a subset of gliomas, with higher expression in GBMs than in lower grade tumors. Quantitative real-time PCR revealed that LIN28 is more highly expressed in glioma-derived stem like cells (GSCs) than in normal human neural stem cells (hNSC) and serum cultured GBM cell lines. Knockdown of LIN28 with lentivirus encoding LIN28 shRNA in GSC cell line HSR-GBM1 inhibited their growth and clonogenicity. Gain of function of LIN28 in GSC cell line JHH-GBM14 with LIN28 lentivirus caused larger and more invasive xenografts when injected into brains of immunocompromised mice. qPCR demonstrated that LIN28 over-expression in JHH-GBM14 resulted in down-regulation of let-7b and let-7g and up-regulation of HMGA2, one important target of let-7. LIN28 over-expression also increased the invasiveness of JHH-GBM14 as revealed by Matrigel invasion assay, which may contribute to the increased tumorigenicity of GBM14 in vivo. To find whether the stem cell factor LIN28 plays a role in the development of high grade gliomas, which are thought to arise from normal hNSC, we examined whether LIN28 would facilitate transformation of hNSC. We infected these cells with lentivirus encoding LIN28 together with dominant negative R248W p53 (DN-p53), constitutively active KRAS (CA-KRAS) and hTERT (hNSC-LIN28/DN-p53/CA-KRAS/hTERT), which are all commonly dysregulated in GBM. As controls, hNSC were also infected with DN-p53/CA-KRAS/hTERT, LIN28/DN-p53/hTERT, DN-p53/hTERT, or with GFP. To test their tumorigenecity in vivo, hNSC-LIN28/DN-p53/CA-KRAS/hTERT were injected into the brains of immunocompromised mice. Eight weeks after injection, 66.7% (12 of 18) of the mice developed invasive brain tumors resembling GBMs. In contrast, control injections including hNSC-DN-p53/hTERT (10 mice), hNSC-LIN28/DN-p53/hTERT (5 mice) and hNSC-GFP (15 mice) did not generate tumors over 6 months. Interestingly, hNSC-DN-p53/CA-KRAS/hTERT cultures proliferated very poorly, suggesting that both LIN28 and activated KRAS may play key roles in transformation. Our data show that LIN28 is expressed in human GBMs and that LIN28 can promote tumorigenicity in orthotopic GBM mouse models and facilitates the transformation of human neural stem cells.In summary, our study identified GSCs from brain tumor tissues, and elucidated that CD15 is one marker for GSCs. Based on these findings, we demonstrated that oncogene ZNF217 is important for the tumorigenicity of GSCs and is regulated by hypoxia condition, one of the critical factors of GSC microenvironment. Then we find that the pluripotency factor LIN28 promotes tumorigenicity of GSCs and facilitates the transformation of normal human NSCs. Our findings provide a deeper understanding of the machanisms of tumorigenicity of glioma and potential targets to eliminate GSCs. |
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