Notch1 Signaling Pathway Regulates GSCs Invasion And Migration Through PI3K/Akt/mTOR Signaling Pathway Activity Medicated By SDF-1/CXCR4 Axis

Glioblastoma is the most malignant and invasive glioma. The tumor cells always invade and migrate to the surrounding normal brain tissue, even given surgery and supplement by radiotherapy and chemotherapy combination therapy, only can limitedly improve patients’ life quality and prolong progression-free survival, but cannot be cured. Cancer stem cells(CSCs) is first discovered in leukemia, and later it is also found in breast cancer, lung cancer and other epithelial tumors. Glioma stem cells(GSCs) is also confirmed to exist among malignant glioma. GSCs is a group of tumor cells with self-renewal, unlimited proliferation and multi-differentiation ability, which expresses the neural stem cells phenotype, and often concentrates around tumor necrosis, microvascular edge and tumor edge where invasion occurs. GSCs is very likely to get involved in tumor initiation, development, invasion and spread, drug-resistant and eventually lead to tumor recurrence. The importance of GSCs’ role in tumor cell invasion and migration is also getting more and more attention by researchers from China and abroad.PI3K/Akt/m TOR signaling pathway is highly activated in GSCs and GBM. It is an important signaling pathway in regulating tumor cell invasion and migration. PI3 K pathway can regulate Focal Adhesion Kinase(FAK) and the expression and activation of cytoskeleton-associated protein. FAK attends in and regulates the formation of focal adhesion. It is an important regulatory protein during cell migration movement, and its binding to PI3 K is necessary for FAK to promote cell migration. Besides, PI3 K inhibitors can inhibit the promoting effect of FAK on cell migration in a concentration dependent manner. The activation of PI3 K pathway can also promote the expression of matrix metalloproteinases(MMPs), while MMPs can degrade the majority of all kinds of protein components in extracellular matrix, destroy histological barrier and promote tumor cell to invade. In the maintenance of GSCs phenotype, the effect of Notch signaling pathway activation is the most significant. Notch proteins is first discovered in Drosophila research. Among vertebrates, the receptors of Notch signaling pathway include Notch1-4, and its ligand includes Delta-like-1,-3,-4 and Jagged-1,-2.Notch pathway activation refers to after Notch receptor binding with ligand and twice proteases hydrolysis, the activated form of Notch protein generated- Notch intracellular domain. When NICD is released into the nucleus, it goes through a series of reaction to activate downstream target genes, including HES and HEY, then the corresponding biological effects produce, normally Hes1 is used as the activation signal of Notch signaling pathway. In human brain tumors, Notch signaling pathway is aberrantly activated, it maintains GSCs self-renewal, inhibits its differentiation and promotes its proliferation. It is found that in pancreatic cancer, esophageal cancer, breast cancer and other tumors, after Notch pathway activation, it can start Epithelial-mesenchymal transition on tumor cells, which can enhance their migration and invasion, among which in gliomas expression intensity of Notch1 increases as pathological grade increases, and it is associated with survival of patients. In acute T lymphoblastic leukemia, renal clear cell cancer, bladder cancer and other tumors, it is found that Notch1 signaling pathway can regulate PI3K/Akt/m TOR signaling pathway activation. This means that the Notch1 signaling pathway may regulate invasion and migration of GSCs through PI3K/Akt/m TOR signaling pathway.Chemokine receptor CXCR4 expressed in glioma cells and associated with tumor grade, combined with itsspecificity ligand SDF-1(also known as stromal cell-derived factor, CXCL12),playing an extremely important role in malignant glioma cell chemotaxis guide migration. Studies indicate that CXCR4 are over expressed in transcriptional level and protein level in the glioma with strong invasion ability while glioma cells with weak invasion ability have no over expression of CXCR4.In GSCs, research finds after CXCR4 is activated by its ligand SDF-1, tumor cell invasion and migration is promoted through activation of PI3K/Akt/m TOR signaling pathway.In summary, these studies indicate that GSCs are tumor cells with strong invasion ability, while Notch1 signaling pathway plays an important role in maintaining GSCs phenotype. In many tumor invasion and migration, it also plays a very important role, which means Notch1 signaling pathway plays a significant role in maintaining GSCs invasion ability. We hypothesize, the high expression Notch1 signaling pathway in GSCs can regulate PI3K/Akt/m TOR signaling pathway activity by mediated SDF-1/CXCR4 axis, thereby enhancing GSCs invasion and migration ability.This research mainly discusses the role of activated Notch1 signaling pathway in GSCs invasion and migration, and its potential mechanism, the research includes three parts:1, The expression of Nestin,Notch1, Hes1 in high-grade glioma tissues. Firstly, 35 cases of high grade glioma tissues and 12 cases of temporal lobe epilepsy brain tissue samples from hospital surgical specimens are given immunohistochemical to detect the expression of Nestin, Notch1 and Hes1; then, applying immunofluorescence staining to detect the co-expression of Nestin and Notch1, Nestin and Hes1, Nestin and CD31 in Glioblastoma tissue. Experimental results show that Nestin, Notch1 and Hes1 are negative or weakly positive expression in the control brain tissue, while the positive expression rate are increased in high-grade glioma tissue specimens, and it mainly distributes around the microvascular. Nestin positive cells distribute around blood vessels which express CD31.The majority of cells positively express Nestin also positively presente Notch1 and Hes1.2, Notch1 signaling pathway regulates GSCs invasion migration via PI3K/Akt/m TOR signaling pathway. Culturing and obtaining glioma cell spheres from U87 and U251 cell lines, and using CD133 MACS cell isolation kit to isolate and obtain CD133+ glioma cells. CD133 and Nestin are detected by immunofluorescence to identify whether they are GSCs; Applying immunofluorescence to detect expression of Nestin and Notch1, p Akt and Hes1 in GSCs; Applying the recombinant lentivirus aimed at Notch1 silencing and divides into the corresponding Control group, sh RNA-NC group, sh RNA-Notch1 group and Control group, vehicle group and DAPT group; PCR and Western blot test detect Notch1 m RNA and protein and Hes1, Akt, p Akt, pm TOR protein expression in the respective treatment groups, Transwell invasion and migration experiments respectively detect invasion and migration of cells in each group.The experimental results are as follows: Immunofluorescence experiment results show that cell sphere selected through serum-free culture and MACS by U87 and U251 cell all express stem cell as markers CD133 and Nestin, which can be identified as GSCs(U87), GSCs(U251); Co-expression of Nestin and Notch1 existed in GSCs, and coexpression also exists between Hes1 and p Akt; Compared with control group, after the inhibitory activity of Notch1 signaling pathway, GSCs invasion and migration ability declined obviously, the Akt protein levels do not change significantly, but p Akt,pm TOR protein expression significantly decreases.3, Notch1 signaling pathway regulates GSCs invasion and migration through PI3K/Akt/m TOR signaling pathway activity medicated by SDF-1/CXCR4 axis. Applying Immunofluorescence to detect the expression of CXCR4 and Hes1 in GSCs; Adding CXCR4 specific inhibitor AMD3100 in GSCs and GSCs with low and stable expression of Notch1. The experiment is divided into seven groups, including Sh RNA-Notch 1 group, DAPT group, AMD3100 group, Sh RNA-Notch1+AMD3100 group, sh RNA-NC group, the pharmaceutical vehicle group and Control group. Transwell invasion and migration experiments are used respectively to detect the invasion and migration of cells in each group. Western blot experiment are used to test the expression of Notch1, Hes1, SDF-1, CXCR4, Akt, p Akt, pm TORproteinin corresponding treatment groups. The experimental results are as follows: Co-expression of Hes1 and CXCR4 existed in GSCs; Compared with control group, after Notch1 signaling pathway inhibition, there is no significant change of SDF-1 protein expression, while CXCR4 protein expression significantly reduced; Compared with control group, the invasion and migration ability of sh RNA-Notch1 group, DAPT group, AMD3100 group and sh RNA-Notch1+CXCR4 inhibit group significantly reduce, and there is no significant difference in invasion and migration reduce level of the four groups; Compared with control group, there is no significant change of Akt protein levels in sh RNA-Notch1 group, DAPT group, AMD3100 group, and Sh RNA-Notch1+AMD3100 group, while p Akt and pm TOR protein levels significantly reduce, and there is no significant difference in reducing level of the four groups. conclusion: 1, Compared with control brain tissue, positive rate of Nestin, Notch1 and Hes1 in high-grade glioma tissue specimens increase, and they mainly distribute around the microvascular; Nestin positive cells distribute around the vascular distribution of CD31 expression, and most of Nestin positive cells also show Notch1 and Hes1 positive. 2, Notch1 signaling pathway can regulate invasion and migration of GSCs, and it is realized by regulating the phosphorylation of Akt, and then regulating its downstream signaling implementation. 3, Notch1 signaling pathway can regulate GSCs invasion and migration through SDF-1/CXCR4 axis regulation of PI3K/Akt/m TOR signaling pathway activity.