RESUMO
Microvascular proliferation is a key feature of glioblastoma and neovascularization has been implicated in tumor progression. Glioblastomas use pro-angiogenic factors such as vascular endothelial growth factor (VEGF) for new blood vessel formation. Yet, anti-VEGF therapy does not prolong overall survival so that alternative angiogenic pathways may need to be explored as drug targets. Both glioma cells and glioma-associated endothelial cells produce TGF-ß superfamily ligands which bind TGF-ß receptors (TGF-ßR). The TGF-ßR type III endoglin (CD105), is a marker of proliferating endothelium that has already been studied as a potential therapeutic target. We studied endoglin expression in glioblastoma tissue and in glioma-associated endothelial cells in a cohort of 52 newly diagnosed and 10 recurrent glioblastoma patients by immunohistochemistry and by ex vivo single-cell gene expression profiling of 6 tumors. Endoglin protein levels were similar in tumor stroma and endothelium and correlated within tumors. Similarly, endoglin mRNA determined by ex vivo single-cell gene expression profiling was expressed in both compartments. There was positive correlation between endoglin and proteins of TGF-ß superfamily signaling. No prognostic role of endoglin expression in either compartment was identified. Endoglin gene silencing in T98G glioma cells and in human cerebral microvascular endothelial cells (hCMEC) did not affect constitutive or exogenous TGF-ß superfamily ligand-dependent signaling, except for a minor facilitation of pSmad1/5 signaling in hCMEC. These observations challenge the notion that endoglin might become a promising therapeutic target in glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Endoglina/fisiologia , Glioblastoma/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Linhagem Celular Tumoral , Humanos , Neovascularização PatológicaRESUMO
PURPOSE: Members of the transforming growth factor (TGF)-ß superfamily play a key role in the regulation of the malignant phenotype of glioblastoma by promoting invasiveness, angiogenesis, immunosuppression, and maintaining stem cell-like properties. Betaglycan, a TGF-ß coreceptor also known as TGF-ß receptor III (TßRIII), interacts with members of the TGF-ß superfamily and acts as membrane-associated or shed molecule. Shed, soluble TßRIII (sTßRIII) is produced upon ectodomain cleavage of the membrane-bound form. Elucidating the role of TßRIII may improve our understanding of TGF-ß pathway activity in glioblastoma METHODS: Protein levels of TßRIII were determined by immunohistochemical analyses and ex vivo single-cell gene expression profiling of glioblastoma tissue respectively. In vitro, TßRIII levels were assessed investigating long-term glioma cell lines (LTCs), cultured human brain-derived microvascular endothelial cells (hCMECs), glioblastoma-derived microvascular endothelial cells, and glioma-initiating cell lines (GICs). The impact of TßRIII on TGF-ß signaling was investigated, and results were validated in a xenograft mouse glioma model RESULTS: Immunohistochemistry and ex vivo single-cell gene expression profiling of glioblastoma tissue showed that TßRIII was expressed in the tumor tissue, predominantly in the vascular compartment. We confirmed this pattern of TßRIII expression in vitro. Specifically, we detected sTßRIII in glioblastoma-derived microvascular endothelial cells. STßRIII facilitated TGF-ß-induced Smad2 phosphorylation in vitro and overexpression of sTßRIII in a xenograft mouse glioma model led to increased levels of Smad2 phosphorylation, increased tumor volume, and decreased survival CONCLUSIONS: These data shed light on the potential tumor-promoting role of extracellular shed TßRIII which may be released by glioblastoma endothelium with high sTßRIII levels.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinógenos/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Pessoa de Meia-Idade , Prognóstico , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Proteína Smad2/genética , Taxa de Sobrevida , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transforming growth factor (TGF)-ß is a central molecule maintaining the malignant phenotype of glioblastoma. Anti-TGF-ß strategies are currently being explored in early clinical trials. Yet, there is little contemporary data on the differential expression of TGF-ß isoforms at the mRNA and protein level or TGF-ß/Smad pathway activity in glioblastomas in vivo.Here we studied 64 newly diagnosed and 16 recurrent glioblastomas for the expression of TGF-ß1-3, platelet-derived growth factor (PDGF)-B, and plasminogen activator inhibitor (PAI)-1 mRNA by RT-PCR and for the levels of TGF-ß1-3 protein, phosphorylated Smad2 (pSmad2), pSmad1/5/8 and PAI-1 by immunohistochemistry.Among the TGF-ß isoforms, TGF-ß1 mRNA was the most, whereas TGF-ß3 mRNA was the least abundant. TGF-ß1-3 mRNA expression was strongly correlated, as was the expression of TGF-ß1-3 mRNA, and of the TGF-ß1-3 target genes, PDGF-B and PAI-1. TGF-ß2 and TGF-ß3 protein levels correlated well, whereas the comparison of the other TGF-ßisoforms did not. Positive correlation was also observed between TGF-ß1 and pSmad1/5/8 and between pSmad2 and pSmad1/5/8. Survival analyses indicated that a group of patients with high expression levels of TGF-ß2 mRNA or pSmad1/5/8 protein have inferior outcome.We thus provide potential biomarkers for patient stratification in clinical trials of anti-TGF-ß therapies in glioblastoma.