Dual or multi-targets therapy targeting epidermal growth factor receptor variant III (EGFRvIII) and other molecular may relax the constraint for glioblastoma (GBM), putting forward the urgent requirement of finding candidate molecules. Here, the insulin-like growth factor binding protein-3 (IGFBP3) was considered a candidate, whereas the mechanisms of IGFBP3 production remain unclear. We treated GBM cells with exogenous transforming growth factor β (TGF-β) to simulate the microenvironment. We found that TGF-β and EGFRvIII transactivation induced the activation of transcription factor c-Jun, which specifically bound to the promoter region of IGFBP3 through Smad2/3 and ERK1/2 pathways and promoted the production and secretion of IGFBP3. IGFBP3 knockdown inhibited the activation of TGF-β and EGFRvIII signals and the malignant behaviors triggered by them in vitro and in vivo. Collectively, our results indicated a positive feedback loop of p-EGFRvIII/IGFBP3 under administration of TGF-β, blocking IGFBP3 may be an additional target in EGFRvIII-expressing GBM-selective therapeutic strategy.
Abstract Background Glioblastoma (GBM) characterized by immune escape is the most malignant primary brain tumors, which has strong immunosuppressive effect. Programmed death ligand-1 (PD-L1) is a recognized immunosuppressive member on the surface of tumor cells, and plays a crucial role in immune evasion of tumors. Actually, little is known about the regulation of PD-L1 expression in GBM. Insulin-like growth factor binding protein 3 (IGFBP3) is upregulated in GBM and is related to poor patient prognosis. However, it remains unclear whether IGFBP3 plays a role in the regulation of PD-L1 expression in GBM. Methods The role of IGFBP3 in the glioma immune microenvironment was investigated using the CIBERSORT algorithm. The correlation between IGFBP3 and PD-L1 expression was analyzed using TCGA and CGGA databases. QRT-PCR, immunoblotting and RNA-seq were used to examine the regulatory effect of IGFBP3 on PD-L1 expression. Co-culture assay, cell counting kit (CCK-8), qRT-PCR, ELISA and flow cytometry were performed to explore the function of IGFBP3 in inducing immunosuppression. The biological role of IGFBP3 was verified using immunohistochemical, immunofluorescence and mice orthotopic tumor model. Results In this study, we analyzed immune cells infiltration in gliomas and found that IGFBP3 may be associated with an immunosuppressive microenvironment. Then, by analyzing TCGA and CGGA databases, our results showed that IGFBP3 and PD-L1 expression were positively correlated in GBM patients, but not in LGG patients. In vitro experiments conducted on different GBM cell lines revealed that the overexpression of IGFBP3 led to an increase in PD-L1 expression, which was reversible upon knockdown IGFBP3. Mechanistically, IGFBP3 activated the JAK2/STAT3 signaling pathway, leading to an increase in PD-L1 expression. Additionally, co-culture experiments results showed IGFBP3 overexpression induced upregulation of PD-L1 expression promoted apoptosis in Jurkat cells, and this effect was blocked by IGFBP3 antibody and PDL-1 inhibitors. Importantly, in vivo experiments targeting IGFBP3 suppressed tumor growth and significantly prolonged the survival of mice. Conclusions This research demonstrated IGFBP3 is a novel regulator for PD-L1 expression in GBM, and identified a new mechanism by which IGFBP3 regulates immune evasion through PD-L1, suggesting that IGFBP3 may be a potential novel target for GBM therapy.
Glioblastoma multiforme (GBM) is the most malignant intracranial tumor in adults, characterized by extensive infiltrative growth, high vascularization, and resistance to multiple therapeutic approaches. Among the many factors affecting the therapeutic effect, the immunosuppressive GBM microenvironment that is created by cells and associated molecules via complex mechanisms plays a particularly important role in facilitating evasion of the tumor from the immune response. Accumulating evidence is also revealing a close association of the gut microbiota with the challenges in the treatment of GBM. The gut microbiota establishes a connection with the central nervous system through bidirectional signals of the gut–brain axis, thus affecting the occurrence and development of GBM. In this review, we discuss the key immunosuppressive components in the tumor microenvironment, along with the regulatory mechanism of the gut microbiota involved in immunity and metabolism in the GBM microenvironment. Lastly, we concentrate on the immunotherapeutic strategies currently under investigation, which hold promise to overcome the hurdles of the immunosuppressive tumor microenvironment and improve the therapeutic outcome for patients with GBM.
5-Demethylnobiletin is the active ingredient in citrus polymethoxyflavones that could inhibit the proliferation of several tumor cells. However, the anti-tumor effect of 5-Demethylnobiletin on glioblastoma and the underlying molecular mechanisms are remains unknown. In our study, 5-Demethylnobiletin markedly inhibited the viability, migration and invasion of glioblastoma U87-MG, A172 and U251 cells. Further research revealed that 5-Demethylnobiletin induces cell cycle arrest at the G0/G1 phase in glioblastoma cells by downregulating Cyclin D1 and CDK6 expression levels. Furthermore, 5-Demethylnobiletin significantly induced glioblastoma cells apoptosis by upregulating the protein levels of Bax and downregulating the protein level of Bcl-2, subsequently increasing the expression of cleaved caspase-3 and cleaved caspase-9. Mechanically, 5-Demethylnobiletin trigged G0/G1 phase arrest and apoptosis by inhibiting the ERK1/2, AKT and STAT3 signaling pathway. Furthermore, 5-Demethylnobiletin inhibition of U87-MG cell growth was reproducible in vivo model. Therefore, 5-Demethylnobiletin is a promising bioactive agent that might be used as glioblastoma treatment drug.
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