The involvement of the circFOXM1–miR–432–Gα12 axis in glioma cell proliferation and aggressiveness

Accumulating evidence indicates that circFOXM1 (Hsa_circ_0025033) is highly expressed in several cancers; however, the function of circFOXM1 in glioma and the molecular mechanism have not been well explored. In the present study, we found that expression of circFOXM1 was upregulated in both glioma tissues and cell lines. In addition, circFOXM1 knockdown suppressed glioma-cell proliferation, activated apoptosis in vitro, and repressed tumour growth in vivo. Moreover, we clarified that circFOXM1 binds with miR-432, which was downregulated in glioma cells. Furthermore, we indicated that Gα12, a direct target of miR-432, was highly expressed in glioma cells, and Gα12 silencing might limit the progression of glioma. Rescue assays indicated that Gα12 reversed the inhibitory effect of circFOXM1 silencing on glioma-cell tumorigenesis. In conclusion, circFOXM1 acts as a sponge of miR-432 to promote the proliferation and aggressiveness of glioma cells through the Gα12 signalling pathway.

Downregulation of TET1 Promotes Glioma Cell Proliferation and Invasion by Targeting Wnt/β-Catenin Pathway

Glioma is the most common malignant tumor in adult brain characteristic with poor prognosis and low survival rate. Despite the application of advanced surgery, chemotherapy, and radiotherapy, the patients with glioma suffer poor treatment effects due to the complex molecular mechanisms of pathological process. In this paper, we conducted the experiments to prove the critical roles TET1 played in glioma and explored the downstream targets of TET1 in order to provide a novel theoretical basis for clinical glioma therapy. RT-qPCR was adopted to detect the RNA level of TET1 and β-catenin; Western blot was taken to determine the expression of proteins. CCK8 assay was used to detect the proliferation of glioma cells. Flow cytometry was used to test cell apoptosis and distribution of cell cycle. To detect the migration and invasion of glioma cells, wound healing assay and Transwell were performed. It was found that downregulation of TET1 could promote the proliferation migration and invasion of glioma cells and the concomitant upregulation of β-catenin, and its downstream targets like cyclinD1 and c-myc were observed. The further rescue experiments were performed, wherein downregulation of β-catenin markedly decreases glioma cell proliferation in vitro and in vivo. This study confirmed the tumor suppressive function of TET1 and illustrated the underlying molecular mechanisms regulated by TET1 in glioma.

Microglial Cytokines Induce Invasiveness and Proliferation of Human Glioblastoma through Pyk2 and FAK Activation

Glioblastoma is the most aggressive brain tumor in adults. Multiple lines of evidence suggest that microglia create a microenvironment favoring glioma invasion and proliferation. Our previous studies and literature reports indicated the involvement of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) in glioma cell proliferation and invasion, stimulated by tumor-infiltrating microglia. However, the specific microglia-released factors that modulate Pyk2 and FAK signaling in glioma cells are unknown. In this study, 20 human glioblastoma specimens were evaluated with the use of RT-PCR and western blotting. A Pierson correlation test demonstrated a correlation (0.6–1.0) between the gene expression levels for platelet-derived growth factor β(PDGFβ), stromal-derived factor 1α (SDF-1α), IL-6, IL-8, and epidermal growth factor (EGF) in tumor-purified microglia and levels of p-Pyk2 (Y579/Y580) and p-FAK(Y925) in glioma cells. siRNA knockdown against Pyk2 or FAK in three primary glioblastoma cell lines, developed from the investigated specimens, in combination with the cytokine receptor inhibitors gefitinib (1 μM), DMPQ (200 nM), and burixafor (1 μM) identified EGF, PDGFβ, and SDF-1α as key extracellular factors in the Pyk2- and FAK-dependent activation of invadopodia formation and the migration of glioma cells. EGF and IL-6 were identified as regulators of the Pyk2- and FAK-dependent activation of cell viability and mitosis.

Recombinantly Expressed MeICT, a new Toxin from Mesobuthus Eupeus Scorpion, Inhibits Glioma Cell Proliferation and Downregulates Annexin A2 and FOXM1 Genes

Gliomas are highly invasive and lethal malignancy that do not respond to current therapeutic approaches. Novel therapeutic agents are required to target molecular mechanisms involved in glioma progression. MeICT is a new short-chain toxin isolated from Mesobuthus eupeus scorpion venom. This toxin contained 34 amino acid residues and belongs to chloride channels toxins. In this study, the coding sequence of MeICT was cloned into the pET32Rh vector and a high yield of soluble recombinant MeICT was expressed and purified. Recombinant MeICT-His significantly inhibited the proliferation and migration of glioma cells at low concentration. In vivo studies showed that MeICT was not toxic when administrated to mice at high doses. We also determined the effect of MeICT on the mRNA expression of MMP-2, Annexin A2 and FOXM-2 that are key molecules in the progression and invasion of glioma. Expression of Annexin A2 and FOXM1 mRNA was significantly down-regulated following treatment with MeICT. However, no significant decrease in the expression of MMP-2 gene was identified. In this study a short toxin with four disulfide bonds was successfully produced and its anti-cancer effects was detected. Our findings suggest that recombinant MeICT can be considered as a new potent agent for glioma targeting.

TAMI-45. MICROGLIAL CYTOKINES INDUCE INVASIVENESS AND PROLIFERATION THROUGH PYK2 AND FAK ACTIVATION IN HUMAN GLIOBLASTOMA CELL

Glioblastoma (GBM) is the most aggressive and highly invasive primary brain tumor in adults. Evidence suggests that microglia create a microenvironment favoring glioma invasion and proliferation. Indeed, previous reports indicate the involvement of focal adhesion kinase (FAK) signaling cascades in glioma cell proliferation. Besides, studies from our laboratory support a critical role of Pyk2, a relative of FAK, in glioma invasion by tumor-infiltrating microglia. However, the microglial-released factors modulating Pyk2 and FAK signaling pathways are unknown. In this study, 20 human GBM specimens were evaluated to identify the cytokine expression patterns in purified microglia and FAK and Pyk2 phosphorylation in glioma cell fraction by RT-PCR and western blot. A Pierson correlation test demonstrated a high correlation (0.8-1.0) of gene expression for PDGFα, PDGFβ, SDF-1α, IL-6, IL-8, and EGF in percoll-purified microglia, and pPyk2(Y579/580) and pFAK(Y925) levels in glioma cell fraction. The role of cytokines in cell invasion and proliferation by Pyk2/FAK activation was further investigated in primary cell lines from three patients. Thirty percent up-regulation of pPyk2 and pFAK was detected in glioma cells treated (2 hrs.) with microglia conditioned media (MCM) compared to control cells. siPyk2 or siFAK knockdown identified IL-6 (100 μM) and EGF (1 μM) as key factors of Pyk2- and FAK-dependent activation in all glioma cell lines. Similar results with siPyk2 or siFAK were observed for matrix degradation, invadopodia formation, cell viability, and mitosis. Indeed, Tocilizumab (IL-6R blocker, 100 ng/mL) and Gefitinib (EGFR blocker, 1 μM) reversed the effect of MCM on glioma cell proliferation and invasion in all cell lines evaluated. These findings support a pivotal role of Pyk2 and FAK in enhancing proliferation and invasion of glioma tumors through IL-6 and EGF-dependent pathways. The latter could be of clinical relevance for new therapeutic developments in GBM patients.

RIT1 Promotes Glioma Proliferation and Invasion via the NF-ĸB/p65 pathway

RIT1, a member of the Ras family, has been identified as an oncogene in several malignancies. However, the expression and function of RIT1 in glioma remains to be addressed. In this study, we found RIT1 was upregulated in glioma and was associated with poor prognosis of glioma patients. Manipulating RIT1 levels in glioma cells via RNA interference significantly inhibited glioma cell proliferation and invasion in vitro whereas RIT1 overexpression exhibited the opposite effects. Mechanistically, we demonstrate that RIT1 engaged in the activation of the NF-ĸB pathway in vitro and in vivo. Furthermore, treating RIT1-overexpressing glioma cells with the p65 siRNA partially restrained their proliferation and invasion. Together these results indicate RIT1 contributes to the development and metastasis of glioma via the NF-ĸB pathway and suggest that targeting RIT1 may be a treatment strategy for this disease.