Sonic Hedgehog Signaling Regulates Proliferation And Differentiation Of Human Brain Glioma Stem Cells

As the most frequently malignant brain tumor in central nervous system (CNS), recurrence is the most important problem in treating glioma due to its unlimited proliferation and invasive growth. Brain glioma stem cells take up a small portion of tumor population,and are capable of self-renew, proliferation and differention to the phenotype of parent tumor. When being transplanted continuously, BGSCs can form the same tumor as the parent one. Thus BGSCs are the "seeds" of glioma cells with any differented degree, play a key role in initiation, progression and recurrence of glioma. That is why we choose BGSCs as a cell model.Hedgehog (HH) signaling pathway was found to be correlated with human tumor formation in recent studies. Ectopic activation of sonic hedgehog signaling pathway was detected in many tumors, and blocking the pathway with specific inhibitor leads to inhibition of tumor growth. Overexpression of HH may give rise to brain tumor in CNS. Moreover, cyclopamine, a specific inhibitor of SHH signaling pathway, blocks the growth of several primary gliomas, medulloblastomas and glioma cell lines. HH signaling may play a key role in the genesis of brain tumor, especially glioma. SHH signaling is essential to maintenance and survival of many gliomas, medulloblastomas and the model system; SHH signal may participate in ininitiation of glioma, and usually affect neural precursor cells or neural stem cells. HH signaling take effect through GLI-1, and the expression of GLI-1 is a reliable marker of cell reaction to the HH signal. The expression level of GLI-1 may correlated with tumor grade. HH signal may also control proliferation and survival of tumor through Cyclins, anti- apoptosis factor BCL2 and cooperation with other signals, such as Notch signaling. Since SHH signaling pathway plays a key role in self-renew, proliferation and differentiation of NSCs or neural precursor cells, and ectopic activation of SHH signal was detected in many gliomas (including medulloblastomas), as well as some other tumors and cell lines, we hypothesized that there may be ectopic activation of SHH signal in BGSCs, seeds of glioma, which may play a key role in initiation and progression of glioma. But how did it happen? How did these changes decide the fate of BGSCs and control its proliferation and differentiation?In Part 1 of our research, BGSCs were sorted through immunomagnetic beads marking by CD133 and cultured in vitro, and character as a stem cell was identified by stem cell markers (CD133 and Nestin) and differentiated cell markers [ microtubule-associated protein 2(MAP2), glial acidic fibrillary protein(GFAP) and myelin basic protein (MBP)] , ultrastructure observing with electron microscope(EM)and engrafting to severe combined immunodeficiency mice(SCID mice) for tumorigenesis test.The results were as following:Only a small subset of CD133+ glioma cells in glioma cell lines and fresh specimens from various pathologic grade could express stem cell markers CD133 and Nestin, view ultrastructure of a stem cell and be capacity of serial passage in culture. These CD133+ cells possese a marked capacity for multipotent differentiation and could differentiate into tumor cells expressing MAP2,β-TubulinⅢ, GFAP and MBP; When engrafted into SCID mice, they can generate and form tumors that phenotypically resembl the tumor from the patient. Therefore, the small fraction of CD133+ cells identified stem cells is BGSCs , so called as tumor stem cells in glioma.In Part 2 of the research, BGSCs in vitro were adopted as model. Genes and proteins expression of SHH signaling pathway were detected during BGSCs proliferation and differentiation. Proteins expression of SHH signaling pathway and cell type were also detected in glioma specimens and cell lines. The relationship between proliferation and differentiation of BGSCs has been analysed. On this basis, a retroviral-mediated expression system containing double strands DNA for RNA interference on human SHH gene was constructed. Expression and function of SHH signaling pathway in BGSCs were blocked by RNA interference. Immunity fluorescence technique, RT-PCR, Western Blotting and flow cytometry were used to evaluate effects on proliferation, differentiation, survival and tumorigenesis of BGSCs.The results were as following:1,Expression of Shh,Gli-1 and Bmi-1 protein were detected in glioma specimens and cell lines but not in normal adult brain tissue. More intensity of expression were detected in high grade glioma compared with low grade glioma.2,Shh and Gli-1 expressed in relativly differentiated and immature cells of MAP2, GFAP and MBP; No expression or weak expression in mature cells; no expression in CD133 positive cells . It implies that expression of Shh and Gli-1 may be correlated to iniatiation and maintenance of glioma differentiation whatever the cell type is.3,Expression of Bmi-1 was detected in all CD133, MAP2, GFAP and MBP positive cells, but more intensity in CD133 and MAP2 positive cells as compared with GFAP and MBP positive cells;also more intensity of Bmi-1 expression in immature cells than in mature cells,which leads to the implication that strong expression of Bmi-1 may be correlated to maintenance of undifferentiation or low-differentiation of glioma cells.4,In proliferating BGSCs, no expression of SHH mRNA and protein, weak expression of GLI-1 mRNA and protein and strong expression of BMI-1 mRNA and protein were detected. which implies that the maintenance of weak Gli-1 signal may be correlated to stem cell state and survival.5,Expression of Shh and Gli-1 in differentiating BGSCs increased gradually, then tapered to disappearance; Expression of CD133 tapered too, but the expression of MAP2, GFAP and MBP appeared and increased gradually. Which suggests that proteins expression of SHH signaling pathway may participate in initiation and phase control of BGSCs differentiation.6,A retroviral-mediated expression system containing double strands DNA for RNA interference on human sonic hedgehog (SHH) gene was successfully constructed.7,When SHH signaling was blocked by RNAi, proliferation of BGSCs were inhibited and viable cells decreased; Cells in G0/G1 phase decreased,in S phase was exhausted or held up, and apoptotic or necrotic cells increased. Which hints that SHH signaling could promote proliferation , cell division and survival, and may implement by controlling cell cycle, promoting cell division and decreasing cell apoptosis or necrosis.8,When SHH signaling was blocked by RNAi, differentiation of BGSCs were inhibited and dead cells incresed. At the 3rd day after RNAi, CD133,β-TubulinⅢ, GFAP and MBP positive cells ratio increased in RNAi group as compared with No-RNAi group and Negative-RNAi group. A inference could be drawn that SHH signaling promoted differentiation and survival of BGSCs.9,No tumor was observed in SCID mice engrafted with BGSCs after RNAi during 8 weeks, while autologus BGSCs without RNAi could form tumor after transplantation even at 2 order of magnitude lower. Which implies that BGSCs with SHH signaling blocked by RNAi decreased in capacity for tumorigenesis after transplantation. Still more orthotope transplantation with numbers of animals should be taken .It was general believed that the expression of genes and proteins of SHH signaling pathway plays a key role in proliferating and differentiating BGSCs. Which promoted not only proliferation and survival but also differentation of BGSCs. It may implement by controlling cell cycle, promoting cell division and decreasing cell apoptosis or necrosis. However, SHH signaling is not the only key signal pathways in proliferation, differentiation and survival of BGSCs, some other signals such as Notch, WNT, EGF and bFGF may also be at work. It is supposed that numerous signals are involved in controlling the proliferation,differentiation and survival of BGSCs. Further extensive researches on these key factors could shed light on mechanisms that control initiation, progression and recurrence of glioma at a more advanced level.