u-0126 and Astrocytoma

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Astrocytes; Astrocytoma; Brain Neoplasms; Butadienes; Child; Child, Preschool; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Profiling; Guanine Nucleotide Exchange Factors; High-Throughput Nucleotide Sequencing; Humans; Infant; Intracellular Signaling Peptides and Proteins; Male; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; MicroRNAs; Nitriles; RNA-Seq; RNA, Messenger; Sequence Analysis, RNA; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Cells, Cultured; Young Adult

Matrix metalloproteinase-9 (MMP-9) is an important angiogenic and prognostic factor in malignant tumors. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as the death ligand, which induces preferential apoptosis of transformed tumor cells. In this study, we investigated the biological functions of TRAIL, other than its role in induction of apoptosis. We demonstrated that TRAIL induces MMP-9 expression in human astrocytoma cells, which is preceded by activation of extracellular signal-regulated protein kinase (ERK). In addition, TRAIL induces the DNA-binding activity of NF-kappaB, an important transcription factor for MMP-9 induction. The specific MEK inhibitor, U0126, significantly blocks TRAIL-mediated NF-kappaB activation and subsequent MMP-9 induction. These findings indicate that TRAIL treatment in human astrocytoma cells leads to the activation of NF-kappaB and subsequent expression of MMP-9, which are dependent on ERK activation. Collectively, these results suggest that TRAIL has alternative biological functions in addition to its role in inducing apoptosis in human malignant astrocytoma cells.

Topics: Astrocytoma; Butadienes; Cell Line, Tumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Matrix Metalloproteinase 9; NF-kappa B; Nitriles; Protein Kinase Inhibitors; TNF-Related Apoptosis-Inducing Ligand

Tuberous sclerosis (TS) is an autosomal dominant disease associated with the formation of usually benign tumors or hamartomas. The disease is connected with upregulation of mammalian target of rapamycin, central regulator of protein translation, which is usually regarded to be activated by Akt kinase. Here, we show for the first time that in all four brain lesions and one angiomyolipoma from TS patients both extracellular signal-regulated kinase (Erk) and p90 ribosomal S6 kinase 1 activation as well as Erk-dependent phosphorylation of p70 ribosomal S6 kinase 1 are markedly elevated whereas Akt, participating in the classical pathway of mammalian target of rapamycin activation is not always activated. Erk activation is also present in TS-derived cell lines. Importantly, Erk inhibition leads to the decrease of proliferation potential of such lines. These results show that Erk is specifically implicated in the pathogenesis of hamartomas.

Topics: Angiomyolipoma; Animals; Astrocytoma; Brain Neoplasms; Butadienes; Cell Line; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Nitriles; Protein Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Tuberous Sclerosis

Malignant astrocytomas are characterized by extensive vascularization attributed to increased expression of the angiogenic cytokine vascular endothelial growth factor (VEGF). VEGF is elevated in astrocytomas under normal oxygen conditions and undergoes induction in hypoxic stress. Prior studies have shown that both the phosphatidylinositol 3'-kinase (PI3-kinase) and MEK1/2 (MAPK/ERK kinase 1/2) pathways promote proliferation of astrocytoma cells and growth of astrocytic tumors. Whether these pathways regulate growth by modulating angiogenesis as well as proliferation is not clear. In this study, pharmacologic inhibitors were used to specifically inhibit PI3-kinase and MEK1/2 activity in human malignant astrocytoma cell lines, and their effects on VEGF expression were determined. Northern blot analysis of VEGF messenger RNA (mRNA) from cells treated with inhibitors demonstrated cell line-specific responses. The PI3-kinase pathway regulated both the normoxic expression and hypoxic induction of VEGF in 2 cell lines, whereas MEK1/2 regulated only the normoxic expression in the same 2 lines. The third cell line showed no change in VEGF mRNA with inhibition of either of these 2 pathways. This study suggests that modulation of signaling pathways implicated in proliferation of astrocytoma cell lines may have varying effects in vivo depending on the role these pathways play in regulating tumor angiogenesis.

Topics: Astrocytoma; Blotting, Northern; Blotting, Western; Butadienes; Cell Hypoxia; Cell Line; Central Nervous System Neoplasms; Chromones; Endothelial Growth Factors; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase Kinases; Morpholines; Neovascularization, Pathologic; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.

Topics: Adjuvants, Immunologic; Astrocytoma; Butadienes; Calcium; Chemokine CCL2; Chemokine CXCL10; Chemokine CXCL12; Chemokines, CXC; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Interleukin-8; Intracellular Fluid; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Receptors, CXCR4; RNA, Messenger; Stromal Cells; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation