Hypoxia and hypoxia-inducible factor-1 (HIF-1) play a critical role in glioblastoma multiforme (GBMs). CXCR4 is involved in angiogenesis and is upregulated by HIF-1alpha. CXCR4 is a chemokine receptor for stromal cell-derived factor-1 (SDF-1)alpha, also known as CXCL12. We hypothesized that CXCR4 would be upregulated by hypoxia in GBMs. First, we investigated the expression of HIF-1alpha and CXCR4 in GBMs. CXCR4 was consistently found colocalized with HIF-1alpha expression in pseudopalisading glioma cells around areas of necrosis. In addition, angiogenic tumor vessels were strongly positive for CXCR4. Next, we tested the in vitro effect of hypoxia and vascular endothelial growth factor (VEGF) on the expression of CXCR4 in glioma cell lines and in human brain microvascular endothelial cells (HBMECs). Exposure to hypoxia induced significant expression of CXCR4 and HIF-1alpha in glioma cells, whereas treatment with exogenous VEGF increased CXCR4 expression in HBMECs. We also transfected U87MG glioma cells with an HIF-1alpha construct and observed that CXCR4 was upregulated in these cells even in normoxic conditions. We then used a lentivirus-mediated shRNA expression vector directed against HIF-1alpha. When exposed to hypoxia, infected cells failed to show HIF-1alpha and CXCR4 upregulation. We performed migration assays under normoxic and hypoxic conditions in the presence or absence of AMD3100, a CXCR4 inhibitor. There was a significant increase in the migration of U87MG and LN308 glioma cells in hypoxic conditions, which was inhibited in the presence of AMD3100. These studies demonstrate the critical role played by hypoxia and CXCR4 in glioma cell migration. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at two levels. First, through HIF-1alpha in the pseudopalisading tumor cells themselves and, secondly, by the VEGF-stimulated angiogenic response in HBMECs. We believe this knowledge may lead to a potentially important two-pronged therapy against GBM progression using chemotherapy targeting CXCR4.
T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents1. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse2,3. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment3. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance4. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine–receptor interaction as a CNS ‘entry’ signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications.
BackgroundGlioblastoma (GBM) is the most common and malignant primary intracranial human neoplasm. GBMs are characterized by the presence of extensive areas of necrosis and hypoxia. Hypoxia and its master regulator, hypoxia inducible factor 1 (HIF-1) play a key role in glioma invasion.ResultsTo further elucidate the functional role of HIF-1α in glioma cell migration in vitro and in invasion in vivo, we used a shRNA approach to knock down HIF-1α expression complemented with genome-wide expression profiling, performed in both normoxic and hypoxic conditions. Our data show that knock down of HIF-1α in glioma cells significantly impairs their migration in vitro as well as their ability to invade into the brain parenchyma in vivo. Next, we assessed the role that HIF-1α plays in maintaining the characteristics of cancer stem cells (CSCs). By using the tumor sphere forming assay, we demonstrate that HIF-1α plays a role in the survival and self-renewal potential of CSCs. Finally, expression profiling experiments in glioma cells provided detailed insight into a broad range of specific biological pathways and processes downstream of HIF-1α. We discuss the role of these processes in the migratory and invasive properties, as well as the stem cell biology of glioblastomasConclusionsOur data show that knock down of HIF-1α in human and murine glioma cells impairs their migration in vitro and their invasion in vivo. In addition, our data suggest that HIF-1α plays a role in the survival and self-renewal potential of CSCs and identify genes that might further elucidate the role of HIF-1α in tumor migration, invasion and stem cell biology.
Purpose: High-grade glioma treatment includes ionizing radiation therapy. The high invasiveness of glioma cells precludes their eradication and is responsible for the dismal prognosis. Recently, we reported the down-regulation of MHC class I (MHC-I) products in invading tumor cells in human and mouse GL261gliomas. Here, we tested the hypothesis that whole-brain radiotherapy (WBRT) up-regulates MHC-I expression on GL261 tumors and enhances the effectiveness of immunotherapy. Experimental Design: MHC-I molecule expression on GL261 cells was analyzed in vitro and in vivo by flow cytometry and immunohistochemistry, respectively. To test the response of established GL261gliomas to treatment, mice with measurable (at CT imaging) brain tumors were randomly assigned to four groups receiving (a) no treatment, (b) WBRT in two fractions of 4 Gy, (c) vaccination with irradiated GL261cells secreting granulocyte-macrophage colony-stimulating factor, or (d) WBRTand vaccination. Endpoints were tumor response and survival. Results: An ionizing radiation dose of 4 Gy maximally up-regulated MHC-I molecules on GL261 cells in vitro. In vivo,WBRT induced the expression of the h2-microglobulin light chain subunit of the MHC class I complex on glioma cells invading normal brain and increased CD4+ and CD8+ Tcell infiltration. However, the survival advantage obtained with WBRTor vaccination alone was minimal. In contrast,WBRT in combination with vaccination increased long-term survival to 40% to 80%, compared with 0% to 10% in the other groups (P < 0.002). Surviving animals showed antitumor immunity by rejecting challenge tumors. Conclusion: Ionizing radiation can be successfully combined with peripheral vaccination for the treatment of established high-grade gliomas.
Angiogenesis is a critical step required for sustained tumor growth and tumor progression. The stimulation of endothelial cells by cytokines secreted by tumor cells such as vascular endothelial growth factor (VEGF) induces their proliferation and migration. This is a prominent feature of high-grade gliomas. The secretion of VEGF is greatly upregulated under conditions of hypoxia because of the transcription factor hypoxiainducible factor (HIF)-1alpha, which controls the expression of many genes, allowing rapid adaptation of cells to their hypoxic microenvironment. Flavopiridol, a novel cyclin-dependent kinase inhibitor, has been attributed with antiangiogenic properties in some cancer cell lines by its ability to inhibit VEGF production. Here, we show that flavopiridol treatment of human U87MG and T98G glioma cell lines decreases hypoxia-mediated HIF-1alpha expression, VEGF secretion, and tumor cell migration. These in vitro results correlate with reduced vascularity of intracranial syngeneic GL261 gliomas from animals treated with flavopiridol. In addition, we show that flavopiridol downregulates HIF-1alpha expression in the presence of a proteasome inhibitor, an agent that normally results in the accumulation and overexpression of HIF-1alpha. The potential to downregulate HIF-1alpha expression with flavopiridol treatment in combination with a proteasome inhibitor makes this an extremely attractive anticancer treatment strategy for tumors with high angiogenic activity, such as gliomas.
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