ADAM12 is selectively overexpressed in human glioblastomas and is associated with glioblastoma cell proliferation and shedding of heparin-binding epidermal growth factor.
ABSTRACT: ADAMs (a disintegrin and metalloproteinases) are multifunctional molecules involved in cell-cell fusion, cell adhesion, membrane protein shedding, and proteolysis. In the present study, we examined the mRNA expression of 13 different ADAM species with putative metalloproteinase activity in human astrocytic tumors, nonneoplastic brain tissues, and other intracranial tumors by reverse transcriptase-polymerase chain reaction, and found that prototype membrane-anchored ADAM12 (ADAM12m) is predominantly expressed in glioblastomas. Real-time quantitative polymerase chain reaction indicated that the expression level of ADAM12m is remarkably at least 5.7-fold higher in glioblastomas (n = 16) than in nonneoplastic brain tissues (n = 6), low grade (n = 7) and anaplastic astrocytic tumors (n = 9) (P < 0.05 for each group), and intracranial neurinomas (n = 5) (P < 0.01). In situ hybridization showed that glioblastoma cells are responsible for the gene expression. By immunohistochemistry, ADAM12m was predominantly immunolocalized on the cell membranes of glioblastoma cells. Immunoblotting analysis demonstrated that ADAM12m is expressed as an activated N-glycosylated form of approximately 90 kd in glioblastoma tissues. There was a direct correlation between the mRNA expression levels of ADAM12m and proliferative activity (MIB1-positive cell index) of gliomas (r = 0.791, P < 0.0001; n = 32). Protein bands consistent with the soluble form of heparin-binding epidermal growth factor, a substrate of ADAM12m, were observed by immunoblotting in glioblastoma samples with the ADAM12m expression, and inhibited by treatment with ADAM inhibitor of the glioblastomas. These data demonstrate for the first time that among the 13 different ADAM species, ADAM12m is highly expressed in human glioblastomas, and suggest the possibility that ADAM12m plays a role in the prominent proliferation of the glioblastomas through shedding of heparin-binding epidermal growth factor.
Project description:We studied the expression and distribution of the microtubule-severing enzyme spastin in 3 human glioblastoma cell lines (U87MG, U138MG, and T98G) and in clinical tissue samples representative of all grades of diffuse astrocytic gliomas (n = 45). In adult human brains, spastin was distributed predominantly in neuronsand neuropil puncta and, to a lesser extent, in glia. Compared with normal mature brain tissues, spastin expression and cellular distribution were increased in neoplastic glial phenotypes, especiallyin glioblastoma (p < 0.05 vs low-grade diffuse astrocytomas). Overlapping punctate and diffuse patterns of localization wereidentified in tumor cells in tissues and in interphase and mitotic cells ofglioblastoma cell lines. There was enrichment of spastin in the leading edges of cells in T98G glioblastoma cell cultures and in neoplastic cell populations in tumor specimens. Real-time polymerase chain reaction and immunoblotting experiments revealed greater levels of spastin messenger RNA and protein expression in theglioblastoma cell lines versus normal human astrocytes. Functional experiments indicated that spastin depletion resulted in reduced cell motility and higher cell proliferation of T98G cells. Toour knowledge, this is the first report of spastin involvement incellmotility. Collectively, our results indicate that spastinexpression in glioblastomas might be linked to tumor cell motility, migration, and invasion.
Project description:Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo-glycoprotein encoded by PDPN gene is over-expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR-29b and miR-125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 3' untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR-29b and miR-125a are downregulated in glioblastomas and also in CD133-positive cells. Taken together, these results suggest that miR-29b and miR-125a represent potential therapeutic targets in glioblastoma.
Project description:Glioblastomas (GBMs) diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixture of neoplastic and nonneoplastic cells that remain after surgery form the biological context for adjuvant therapeutic intervention and recurrence. We performed RNA-sequencing (RNA-seq) and histological analysis on radiographically guided biopsies taken from different regions of GBM and showed that the tissue contained within the contrast-enhancing (CE) core of tumors have different cellular and molecular compositions compared with tissue from the nonenhancing (NE) margins of tumors. Comparisons with the The Cancer Genome Atlas dataset showed that the samples from CE regions resembled the proneural, classical, or mesenchymal subtypes of GBM, whereas the samples from the NE regions predominantly resembled the neural subtype. Computational deconvolution of the RNA-seq data revealed that contributions from nonneoplastic brain cells significantly influence the expression pattern in the NE samples. Gene ontology analysis showed that the cell type-specific expression patterns were functionally distinct and highly enriched in genes associated with the corresponding cell phenotypes. Comparing the RNA-seq data from the GBM samples to that of nonneoplastic brain revealed that the differentially expressed genes are distributed across multiple cell types. Notably, the patterns of cell type-specific alterations varied between the different GBM subtypes: the NE regions of proneural tumors were enriched in oligodendrocyte progenitor genes, whereas the NE regions of mesenchymal GBM were enriched in astrocytic and microglial genes. These subtype-specific patterns provide new insights into molecular and cellular composition of the infiltrative margins of GBM.
Project description:There are emerging reports that the family of a disintegrin and metalloproteinases (ADAM) are involved in the maintenance of the malignant phenotype of glioblastomas. Notably, ADAM proteases 10 and 17 might impair the immune recognition of glioma cells via the activating immunoreceptor NKG2D by cleavage of its ligands from the cell surface. Glioblastoma-initiating cells (GIC) with stem cell properties have been identified as an attractive target for immunotherapy. However, GIC immunogenicity seems to be low.Here,we show that ADAM10 and ADAM17 are expressed on the cell surface of GIC and contribute to an immunosuppressive phenotype by cleavage of ULBP2. The cell surface expression of ULBP2 is enhanced upon blocking ADAM10 and ADAM17, and treatment with ADAM10 and ADAM17specific inhibitors leads to enhanced immunerecognition of GIC by natural killer cells.Therefore, ADAM10 and ADAM17 constitute suitable targets to boost an immune response against GIC.
Project description:TEM1/endosialin is an emerging microvascular marker of tumor angiogenesis. We characterized the expression pattern of TEM1/endosialin in astrocytic and metastatic brain tumors and investigated its role as a therapeutic target in human endothelial cells and mouse xenograft models.In situ hybridization (ISH), immunohistochemistry (IH) and immunofluorescence (IF) were used to localize TEM1/endosialin expression in grade II-IV astrocytomas and metastatic brain tumors on tissue microarrays. Changes in TEM1/endosialin expression in response to pro-angiogenic conditions were assessed in human endothelial cells grown in vitro. Intracranial U87MG glioblastoma (GBM) xenografts were analyzed in nude TEM1/endosialin knockout (KO) and wildtype (WT) mice.TEM1/endosialin was upregulated in primary and metastatic human brain tumors, where it localized primarily to the tumor vasculature and a subset of tumor stromal cells. Analysis of 275 arrayed grade II-IV astrocytomas demonstrated TEM1/endosialin expression in 79% of tumors. Robust TEM1/endosialin expression occurred in 31% of glioblastomas (grade IV astroctyomas). TEM1/endosialin expression was inversely correlated with patient age. TEM1/endosialin showed limited co-localization with CD31, alphaSMA and fibronectin in clinical specimens. In vitro, TEM1/endosialin was upregulated in human endothelial cells cultured in matrigel. Vascular Tem1/endosialin was induced in intracranial U87MG GBM xenografts grown in mice. Tem1/endosialin KO vs WT mice demonstrated equivalent survival and tumor growth when implanted with intracranial GBM xenografts, although Tem1/endosialin KO tumors were significantly more vascular than the WT counterparts.TEM1/endosialin was induced in the vasculature of high-grade brain tumors where its expression was inversely correlated with patient age. Although lack of TEM1/endosialin did not suppress growth of intracranial GBM xenografts, it did increase tumor vascularity. The cellular localization of TEM1/endosialin and its expression profile in primary and metastatic brain tumors support efforts to therapeutically target this protein, potentially via antibody mediated drug delivery strategies.
Project description:Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor-related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-alpha (PKC-alpha) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-alpha small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate-induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-alpha/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-alpha and PI3K could provide a new treatment paradigm for glioblastomas.
Project description:PURPOSE:Glioblastoma is the most common adult primary malignant intracranial cancer. It is associated with poor outcomes because of its invasiveness and resistance to multimodal therapies. Human adipose-derived mesenchymal stem cells (hAMSC) are a potential treatment because of their tumor tropism, ease of isolation, and ability to be engineered. In addition, bone morphogenetic protein 4 (BMP4) has tumor-suppressive effects on glioblastoma and glioblastoma brain tumor-initiating cells (BTIC), but is difficult to deliver to brain tumors. We sought to engineer BMP4-secreting hAMSCs (hAMSCs-BMP4) and evaluate their therapeutic potential on glioblastoma. EXPERIMENTAL DESIGN:The reciprocal effects of hAMSCs on primary human BTIC proliferation, differentiation, and migration were evaluated in vitro. The safety of hAMSC use was evaluated in vivo by intracranial coinjections of hAMSCs and BTICs in nude mice. The therapeutic effects of hAMSCs and hAMSCs-BMP4 on the proliferation and migration of glioblastoma cells as well as the differentiation of BTICs, and survival of glioblastoma-bearing mice were evaluated by intracardiac injection of these cells into an in vivo intracranial glioblastoma murine model. RESULTS:hAMSCs-BMP4 targeted both the glioblastoma tumor bulk and migratory glioblastoma cells, as well as induced differentiation of BTICs, decreased proliferation, and reduced the migratory capacity of glioblastomas in vitro and in vivo. In addition, hAMSCs-BMP4 significantly prolonged survival in a murine model of glioblastoma. We also demonstrate that the use of hAMSCs in vivo is safe. CONCLUSIONS:Both unmodified and engineered hAMSCs are nononcogenic and effective against glioblastoma, and hAMSCs-BMP4 are a promising cell-based treatment option for glioblastoma.
Project description:BACKGROUND: Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas. METHODS: MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models. RESULTS: MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed. CONCLUSIONS: This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.
Project description:Glioblastomas are the most common and malignant intracranial tumors with a low survival rate. Dysregulation of long non-coding RNAs and RNA-binding protein causes various diseases, including cancers. However, the function of LINC00680 and TTN-AS1 in the progression of glioblastomas is still elusive. In this study, we detected that LINC00680 and TTN-AS1 were upregulated in glioblastoma cells. RNA-binding protein EIF4A3 could prolong the half-life of LINC00680 and TTN-AS1. Knockdown of EIF4A3, LINC00680, and TTN-AS1 impaired proliferation, migration, and invasion and inhibited the growth of tumor in vivo and promoted apoptosis of glioblastoma cells. miR-320b was proven to be a target of LINC00680 and TTN-AS1. They interacted with miR-320b as competing endogenous RNAs, which resulted in the reduction of binding between transcriptional factor EGR3 (early growth response 3) mRNA and miR-320b. The accumulation of EGR3 promoted expression of plakophilin (PKP)2, which could activate the epidermal growth factor receptor (EFGR) pathway, leading to the malignant biological behaviors of glioblastoma cells. In summary, LINC00680 and TTN-AS1 promoted glioblastoma cell malignant biological behaviors via the miR-320b/EGR3/PKP2 axis by being stabilized by EIF4A3, which may provide a novel strategy for glioblastoma therapy.
Project description:MMPs (matrix metalloproteinases) and the related "a disintegrin and metalloproteinases" (ADAMs) promote tumorigenesis by cleaving extracellular matrix and protein substrates, including N-cadherin. Although N-cadherin is thought to regulate cell adhesion, migration, and invasion, its role has not been characterized in glioblastomas (GBMs). In this study, we investigated the expression and function of posttranslational N-cadherin cleavage in GBM cells as well as its regulation by protein kinase C (PKC). N-Cadherin cleavage occurred at a higher level in glioblastoma cells than in non-neoplastic astrocytes. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA) increased N-cadherin cleavage, which was reduced by pharmacological inhibitors and short interfering RNA (siRNA) specific for ADAM-10 or PKC-alpha. Furthermore, treatment of GBM cells with PMA induced the translocation of ADAM-10 to the cell membrane, the site at which N-cadherin was cleaved, and this translocation was significantly reduced by the PKC-alpha inhibitor Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole] or PKC-alpha short hairpin RNA. In functional studies, N-cadherin cleavage was required for GBM cell migration, as depletion of N-cadherin cleavage by N-cadherin siRNA, ADAM-10 siRNA, or a cleavage-site mutant N-cadherin, decreased GBM cell migration. Together, these results suggest that N-cadherin cleavage is regulated by a PKC-alpha-ADAM-10 cascade in GBM cells and may be involved in mediating GBM cell migration.