High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor.

BACKGROUND: Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma. METHODS: Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown. RESULTS: EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α. CONCLUSION: EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the growth factor through ErbB1.

Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma.

Inositol-requiring enzyme 1 (IRE1) is a proximal endoplasmic reticulum (ER) stress sensor and a central mediator of the unfolded protein response. In a human glioma model, inhibition of IRE1alpha correlated with down-regulation of prevalent proangiogenic factors such as VEGF-A, IL-1beta, IL-6, and IL-8. Significant up-regulation of antiangiogenic gene transcripts was also apparent. These transcripts encode SPARC, decorin, thrombospondin-1, and other matrix proteins functionally linked to mesenchymal differentiation and glioma invasiveness. In vivo, using both the chick chorio-allantoic membrane assay and a mouse orthotopic brain model, we observed in tumors underexpressing IRE1: (i) reduction of angiogenesis and blood perfusion, (ii) a decreased growth rate, and (iii) extensive invasiveness and blood vessel cooption. This phenotypic change was consistently associated with increased overall survival in glioma-implanted recipient mice. Ectopic expression of IL-6 in IRE1-deficient tumors restored angiogenesis and neutralized vessel cooption but did not reverse the mesenchymal/infiltrative cell phenotype. The ischemia-responsive IRE1 protein is thus identified as a key regulator of tumor neovascularization and invasiveness.

CpG-oligonucleotides for cancer immunotherapy : review of the literature and potential applications in malignant glioma.

Bacterial DNA and synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are strong activators of both innate and specific immunity, driving the immune response towards the Th1 phenotype. CpG-ODNs have been successfully used in several experimental models of allergies or infections and are now entering clinical trials for these diseases. In this review, we will focus on their potential applications in cancers. CpG-ODN can be used alone to activate locally the innate immunity and trigger a tumor-specific immune response, overcoming the need for identification of a relevant tumoral antigen. Other promising approaches combined CpG-ODN with tumor antigens, monoclonal antibodies or dendritic cells. Preclinical models have shown impressive results and several clinical trials are on-going worldwide. So far, the toxicity observed in humans appeared limited, and objective responses have been observed in a few patients. In malignant gliomas, intra-tumoral injections of CpG-ODN represent a practical approach. Indeed, human gliomas display a locally invasive pattern of growth and rarely metastasize, making local treatment clinically relevant.

CpG-oligodeoxynucleotide rejection of a neuroblastoma in A/J mice does not induce a paraneoplastic disease.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are powerful immunostimulating agents that are currently entering clinical trials in various human diseases. Concerns exist about potential auto-immune diseases triggered by such treatment. We thus investigated whether tumor rejection induced by CpG-ODN treatment could lead to a harmful auto-immune reaction against the nervous system (neurological paraneoplastic disease) at the time of acute tumor rejection, or in long-term surviving animals. Mice bearing established neuroblastomas were treated with intra-tumoral injections of CpG-ODN, resulting in tumor inhibition and tumor rejection in one-third of the animals. Immunocytochemistry and Western blot studies revealed no specific anti-neuronal antibodies. None of the animals developed neurological disabilities and histological studies of the nervous system were normal. CpG-ODN can therefore trigger neuroblastoma rejection without inducing neurological paraneoplastic disease.

D-dimer elevation and paresis predict thromboembolic events during bevacizumab therapy for recurrent malignant glioma.

BACKGROUND: The major side-effects of bevacizumab in glioma treatment are venous thromboembolic events (VTE). We retrospectively evaluated factors potentially predictive of thromboembolic events. PATIENTS AND METHODS: Bevacizumab, alone or in combination with chemotherapy was used as salvage therapy for recurrence in malignant glioma every two weeks. None but one patient received anti-coagulants. Before each bevacizumab cycle differential blood cell count, kidney and liver parameters, D-dimers, neurological status, body-mass index, vital signs and signs of venous thrombosis were assessed. RESULTS: Thirty-eight patients received 428 cycles of bevacizumab. In five patients (13%), six VTE were observed. These complications were preceded four weeks before the onset of symptoms by D-dimer elevation above 0.865 mg/l [p<0.0001; sensitivity=89% (95% confidence interval=83-93%); specificity=89% (95% CI=52-100%)]. An existing hemiparesis constituted a 27-fold risk elevation for thrombotic complication (p<0.0001, χ(2)-test). CONCLUSION: D-Dimer elevation or hemiparesis predict VTE under bevacizumab and chemotherapy, four weeks before the event becomes clinically apparent. Future investigations should determine if prophylactic anti-coagulants for patients at risk may reduce the risk of VTE.