Regulatory Mechanisms Of Glioma Cell Stemness And Biological Behaviors And The Potential Therapeutic Significance

Glioma is the most common primary tumor in the central nervous system. According to the WHO pathological standards of tumor malignancy, glioma could be classified into four grades(WHO I-IV). Glioblastoma(GBM, WHO IV) is the most malignant and lethal glioma, with a high risk of recurrence and dismal therapeutic efficiency. Glioma stem cells(GSCs) are a proportion of glioma cells with stem cell-like features. Previous studies showed that GSCs could initiate tumor, self-renew and differentiate into heterogeneous tumor cells, thus promote tumor growth. Although accounting for a small subset of tumor cells, GSCs are privileged to promote glioma malignant behaviors, such as tumor proliferation, invasion and angiogenesis. Therefore, investigation of the mechanisms underlying the functions of GSCs is crucially important, and may promote the development of GSC-targeting therapeutics to benefit patients with GBM.GSCs exhibit “stem cell-like” phenotypes(i.e., stemness), including expressing specific stem cell markers(such as SOX2, Olig2 and CD133), self-renewal ability, multi-lineage differentiation potency, and in vivo tumor-reconstruction capacity. The stemness features are pivotal for GSC maintenance and proliferation, and are crucial for GSC-driven tumor malignancy. A growing number of evidences have indicated that the maintenance of GSCs mainly relied on: 1) the expressions of stem cell markers; 2) their specific intrinsic regulatory mechanism(genomics, epigenomics, transcriptomics, etc.); 3) their dynamic interplays with microenvironmental cells or factors(tumor-associated macrophages(TAMs), endothelial cells and secreted growth factors, etc.). In this study, we perform microarray screening and identify membrane protein CD9 is preferentially expressed in GSCs acting as a new marker of GSCs. We also demonstrate that CD9, miRNA-663 and miRNA-126 are crucial for the stem-like phenotypes and malignant behaviors(cell proliferation, invasion and angiogenesis) of glioma cells. Finally, we uncovered that TAMs secrete pleiotrophin(PTN) to promote PTPRZ1 signaling in GSCs and support tumor growth. The main findings and conclusions of this study are summarized as follows:1. Identification of CD9 as a GSC marker and its regulation on GSC maintenance and tumorigenesis: 1) CD9 is preferentially expressed in GSCs and is co-localized with the GSC markers SOX2, Olig2 and CD133. 2) CD9 disruption apparently inhibits GSC self-renewal and proliferation. 3) CD9 disruption potently inhibits GSC tumorigenesis.2. The regulation of CD9 on the stability of membrane receptor gp130: 1) CD9 interacts with gp130 in GSCs. 2) Silencing CD9 significantly suppresses gp130 expression. 3) CD9 stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation.3. The effect of CD9 on the activation of gp130-mediated STAT3 pathway: 1) CD9 disruption apparently inhibits STAT3 signaling activation. 2) Constitutively-activated STAT3 partially compromises the suppressive effect of CD9 disruption on GSC maintenance and tumorigenesis.4. The expression of miRNA-663 and its prognostic value: 1) miRNA-663 expression is significantly reduced in GBM tumor tissues relative to the normal brain tissues and its level is negatively correlated with the glioma grades. 2) miRNA-663 is an independent prognostic marker for glioma, especially for GBM.5. The effect of miRNA-663 on glioma malignant behaviors and the underlying mechanism: 1) Enforced expression of miRNA-663 markedly suppresses the proliferation and invasion of glioma cells in vitro and retards GBM tumor growth in vivo. 2) PIK3 CD and CXCR4 are direct targets of miRNA-663. 3) Enforced miRNA-663 impairs the expressions of PIK3 CD and CXCR4. 4) The expression of miRNA-663 is negatively correlated with the level of PIK3 CD or CXCR4 in GBM tissues. 5) miRNA-663 inhibits AKT signaling activity and reduces the expressions of CCND1, MMP2 and MMP7 that related with tumor proliferation and invasion. 6) Ectopic PIK3 CD or CXCR4 rescues the proliferation and invasion of GBM cells disrupted by miRNA-663.6. The combined therapeutic effects of miRNA-663 inducible expression and CXCR4 inhibitor AMD3100. 1) Doxycycline induced miRNA-663 expression combines with AMD3100 treatment to suppress GBM tumor growth in vivo. 2) Enforced expression of miRNA-663 combines with AMD3100 to suppress CXCR4 expression and CXCR4 signaling activation thus prolongs animal survival.7. The expression of miRNA-126 in GSCs and its function on GSC maintenance and GSC-induced tumor angiogenesis: 1) miRNA-126 expression is decreased in GSCs, and is inversely correlated with the level of the GSC marker SOX2 in GBM tissues. 2) Ectopic expression of miRNA-126 potently retards the proliferation and self-renewal of GSCs. 3) Enforced expression of miRNA-126 suppresses GSC-induced migration and tube formation ability of endothelial cells. 4) Overexpression of miRNA-126 significantly suppresses the production of VEGFA from GSCs and inhibits the microvessel density(MVD) in the GSC-derived xenografts.8. The mechanism underlying the function of miRNA-126 on GSCs: 1) HIP1 is a direct target of miRNA-126. 2) miRNA-126 overexpression inhibits HIP1 expression in GSCs. 3) HIP1 is required for GSC maintenance, tumorigensis and pro-angiogensis. 4) HIP1 interacts with EGFR to promote the activations of AKT and STAT3 signalings in GSCs. 5) Introduction of HIP1 largely compromises the suppressive effect of miRNA-126 on GSC maintenance and GSC-induced tumor angiogenesis. 6) Enforced expression of HIP1 restores EGFR expression and rescues the activations of AKT and STAT3 pathways impaired by miRNA-126.9. The combined therapeutic effects of miRNA-126 expression and anti-angiogenic agent Bevacizumab(Beva): 1) Doxycycline-induced miRNA-126 expression compromises the Beva treatment induced GSC enrichment. 2) miRNA-126 expression combines with Beva treatment to suppress GSC-driven tumor growth. 3) miRNA-126 expression combines with Beva treatment to prolong survival of tumor-bearing mice.10. The expression of miRNA-126 in human glioma and its correlation with tumor microvessel parameters: 1) miRNA-126 expression is reduced in glioma tumor tissues relative to the normal brain tissues and its level is negatively correlated with the glioma grades. 2) miRNA-126 expression is inversely associated with tumor microvessel parameters. 3) miRNA-126 is a potential prognostic marker for patients with glioma.11. Identification of PTN as a M2 TAM-secreted factor and its role in mediating the pro-tumorigenic effect of M2 TAMs: 1) PTN is preferentially expressed by tumor-supportive TAMs. 2) PTN is essential for the tumor-promoting effect of M2 TAMs on GSC tumor growth.12. The effect of PTN/PTPRZ1 signaling on GSC maintenance and tumorigenesis, and its therapeutic significance: 1) PTN receptor PTPRZ1 is preferentially expressed by GSCs. 2) PTN-PTPRZ1 signaling promotes GSC maintenance and proliferation. 3) Disrupting PTPRZ1 expression potently inhibits GSC tumor growth and prolongs animal survival. 4) Blocking the PTN-PTPRZ1 paracrine signaling by anti-PTPRZ1 antibody potently inhibits GSC tumor growth.13. Identification of PTN/PTPRZ1 downstream signaling: 1) PTN-PTPRZ1 signaling mainly activates the PI3K-AKT pathway in GSCs. 2) PTPRZ1 interacts with Fyn in GSCs. 3) PTN-PTPRZ1 signaling promotes Fyn kinase phosphorylation, thus activating AKT pathway in GSCs.Taken together, the conclusions of this study are listed as follows:1. As a new marker of GSCs, CD9 binds to gp130 and stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation to promote the BMX-mediated STAT3 activation, thus maintaining GSC self-renewal and tumorigenic potential.2. miRNA-663 targets PIK3 CD and CXCR4 to suppress the activation of PI3K-AKT signaling, thus impairs tumor progression and favors patient survival.3. miRNA-126 targets HIP1 to impair EGFR expression and to suppress the signaling activities of AKT and STAT3 pathway, thus inhibits the self-renewal and pro-angiogenic ability of GSCs.4. M2 TAM-secreted PTN binds to its receptor PTPRZ1 preferentially expressed by GSCs to activate the downstream Fyn-AKT pathway, which promotes the maintenance and tumor propagation of GSCs.These results will enlarge our understandings of the mechanisms underlying GSC maintenance and biological behaviors, facilitate the identification of specific GSC markers and promote the development of GSC-targeting strategies.