epidermal-growth-factor and Neurofibromatosis-1

The overall aim of this systematic review was to identify risk factors for onset and natural progression, which were shown to increase, decrease, or have a null association with risk of primary brain tumour. For onset, the project was separated into two phases. The first phase consisted of a systematic search of existing systematic reviews and meta-analyses. Moderate to high methodological quality reviews were incorporated and summarized with relevant observational studies published since 2010, identified from a systematic search performed in phase 2. For natural progression, only the first phase was conducted. Standard systematic review methodology was utilized. Based on this review, various genetic variants, pesticide exposures, occupational farming/hairdressing, cured meat consumption and personal hair dye use appear to be associated with increased risk of onset amongst adults. The specific EGF polymorphsm 61-A allele within Caucasian populations and having a history of allergy was associated with a decreased risk. For progression, M1B-1 antigen was shown to increase the risk. High birth weight, pesticide exposure (childhood exposure, and parental occupational exposure) and maternal consumption of cured meat during pregnancy may also increase the risk of onset of childhood brain tumours. Conversely, maternal intake of pre-natal supplements (folic acid) appeared to decrease risk. Children with neurofibromatosis 2 were considered to have worse overall and relapse free survival compared to neurofibromatosis 1, as were those children who had grade III tumours compared to lesser grades.

Topics: Age of Onset; Brain Neoplasms; Disease Progression; Epidermal Growth Factor; Humans; Ki-67 Antigen; Mutation; Neurofibromatosis 1; Neurofibromatosis 2; Pesticides; Risk Factors; Von Hippel-Lindau Tumor Suppressor Protein

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Topics: Animals; Disease Models, Animal; Epidermal Growth Factor; Humans; Mice; Neurofibromatosis 1; Neurofibrosarcoma; Sarcoma; Vascular Endothelial Growth Factor A

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Cafe-au-Lait Spots; Catalytic Domain; DNA Mutational Analysis; Epidermal Growth Factor; ErbB Receptors; Guanosine Triphosphate; HEK293 Cells; Humans; K562 Cells; Neurofibromatosis 1; Neurofibromin 1; Oncogenes; Phosphorylation; Point Mutation; Protein Binding; Protein Domains; Protein Interaction Maps; Proto-Oncogene Proteins p21(ras); Signal Transduction

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Transport System A; Cafe-au-Lait Spots; Epidermal Growth Factor; Female; Genes, Reporter; Genetic Association Studies; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Male; MAP Kinase Signaling System; Membrane Proteins; Models, Molecular; Mutation, Missense; Neurofibromatosis 1; Neurofibromin 1; Peptide Fragments; Point Mutation; Protein Conformation; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins p21(ras); Recombinant Fusion Proteins

Neurofibromatosis type 1 (NF1) is characterized by a wide variation in clinical presentation and in some cases progression to malignant tumor. Epidermal growth factor (EGF) is an important mitogen for Schwann cells and is involved in the development of malignant tumors in NF1 patients. We hypothesized that EGF +61 G/A functional polymorphism, which represents constitutional all-life exposure to higher EGF expression and circulating levels, may predispose for precocious and more aggressive manifestations of disease. We found that clinical findings of intestinal polyps are significantly more frequent in patients with G homozygous genotype (P = 0.023). Those carriers of GG genotype have earlier onset of café-au-lait spots and Lisch nodules appearance (P = 0.030 and P = 0.017, respectively). Nevertheless, the EGF overexpressing genotype-GG, is not associated with higher risk for malignant progression or severity of disease. EGF polymorphism may play a role in the earlier onset of NF1 pigment cell-related manifestations and in intestinal polyps' development. Further studies in larger samples should confirm the absence of risk for having higher severity grade or malignant phenotype in NF1 patients.

Topics: Adult; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Humans; Male; Neurofibromatosis 1; Phenotype; Polymerase Chain Reaction; Polymorphism, Genetic

Neurofibromatosis type 1 (NF1) is characterized by systemic development of neurofibromas. Early clinical diagnosis can be ambiguous, and genetic diagnosis can be prohibitively difficult. Dysregulation of a number of growth factors has been suggested to be a mechanism of pathogenesis. This study was performed to assess the contribution of circulating growth factors for diffuse tumorigenesis and the diagnostic value of circulating growth factor identification in serum.. The growth stimulation of neurofibroma-derived cells by serum from NF1 patients was tested, and serum growth factor levels in a cohort of NF1 patients (n = 39) between the ages of 7 and 70 years were analyzed.. Concentrations of midkine (MK) and stem cell factor, but not epidermal growth factor, were substantially increased in serum of NF1 patients when compared with healthy controls. Within the NF1 group, MK levels increased dramatically at puberty from an average of 0.79 ng/mL in patients <18 years to 1.18 ng/mL in patients >18 years old. Stem cell factor and MK concentrations above a defined threshold in serum of NF1 patients are of diagnostic benefit for 96% of patients in the cohort tested. Furthermore, serum from NF1 patients enhanced proliferation of human neurofibroma-derived primary Schwann cells and endothelial cells substantially better than normal serum.. Enhanced circulating growth factor levels contribute to diffuse tumorigenesis in NF1 and may provide the basis for molecular diagnosis.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Cell Proliferation; Cell Transformation, Neoplastic; Child; Child, Preschool; Cohort Studies; Cytokines; Endothelial Cells; Epidermal Growth Factor; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Midkine; Neurofibromatosis 1; Schwann Cells; Stem Cell Factor

We have found that EGF-R expression is associated with the development of the Schwann cell-derived tumors characteristic of neurofibromatosis type 1 (NF1) and in animal models of this disease. This is surprising, because Schwann cells normally lack EGF-R and respond to ligands other than EGF. Nevertheless, immunoblotting, Northern analysis, and immunohistochemistry revealed that each of 3 malignant peripheral nerve sheath tumor (MPNST) cell lines from NF1 patients expressed the EGF-R, as did 7 of 7 other primary MPNSTs, a non-NF1 MPNST cell line, and the S100(+) cells from each of 9 benign neurofibromas. Furthermore, transformed derivatives of Schwann cells from NF1(-/-) mouse embryos also expressed the EGF-R. All of the cells or cell lines expressing EGF-R responded to EGF by activation of downstream signaling pathways. Thus, EGF-R expression may play an important role in NF1 tumorigenesis and Schwann cell transformation. Consistent with this hypothesis, growth of NF1 MPNST lines and the transformed NF1(-/-) mouse embryo Schwann cells was greatly stimulated by EGF in vitro and could be blocked by agents that antagonize EGF-R function.

Topics: Animals; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Mice, Mutant Strains; Neurilemmoma; Neurofibromatosis 1; Neurofibromin 1; Proteins; Rats; Tumor Cells, Cultured

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by abnormalities affecting multiple tissues derived from the neural crest. The peripheral neurofibromas are numerous and sometimes reach several hundred in number. In this study, the possible involvement of several growth factors in neurofibroma growth was investigated in vitro. When explants of neurofibroma tissue were cultured, macrophage-like cells with pseudopodia migrated out first, and later took on a slender fusiform shape. These cells contained S-100 protein and were identified as Schwann cells. They did not proliferate under standard culture conditions. Nerve growth factor (NGF) was helpful in maintaining the differentiated phenotype of Schwann cells, but did not stimulate their proliferation. Immunohistochemical staining for type IV collagen revealed that some large flattened polygonal cells had a mesh of type IV collagen on the surface. These cells were perineurial cells. The proliferation of cells derived from neurofibroma was stimulated by basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-alpha). In comparison with skin fibroblasts, the cells derived from neurofibroma responded to these growth factors at considerably lower concentrations. Stimulation by EGF at physiological concentrations indicated the possible involvement of EGF in the development of neurofibromas.

Topics: Cell Division; Collagen; Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Substances; Humans; Nerve Growth Factors; Neurofibromatosis 1; S100 Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

We hypothesized that skin fibroblasts from patients with neurofibromatosis (NF) may have abnormalities of growth in tissue culture to correlate with the clinical abnormalities of overgrowth and malignancy seen in this disease. Using five lines of NF cells, age- and passage-matched to normal controls, we found that NF fibroblasts grew more slowly and stopped growing at a lower population density than normal cells (P less than 0.0005). The same cells also incorporated [3H]thymidine at a lower rate than normal skin fibroblasts (9,330 +/- 3,240 versus 42,100 +/- 6,840; P less than 0.01). The addition of epidermal growth factor to the medium stimulated the growth of both the normal and the NF fibroblasts; however, the stimulation of the NF fibroblasts was inadequate to fully correct the slow growth rate (P less than 0.025). NF cells (N = 5) were found to be morphologically different from normal skin fibroblasts (N = 5) in culture by light microscopy. NF cells were larger (approximately 9 X 10(4) X 2 X 10(4) versus 2 X 10(4) X 2 X 10(4) A), pleomorphic, and failed to form confluent monolayers when growth ceased. Speculation These data indicate that there may be an underlying abnormality of growth regulation in neurofibromatosis. The slow growth of neurofibromatosis fibroblasts, and their diminished response to epidermal growth factor, provides a means for studying the growth abnormality of neurofibromatosis in tissue culture. In addition, the expression of this abnormality may serve as a marker for the disease.

Topics: Culture Techniques; Epidermal Growth Factor; Fibroblasts; Humans; Neurofibromatosis 1; Skin

Topics: Cell Division; Cells, Cultured; Epidermal Growth Factor; Fibroblasts; Humans; Neurofibromatosis 1

Neurofibromatosis (NF) is an autosomal dominant disease characterized by growth abnormalities of epithelial, mesothelial, and endothelial elements. We recently reported abnormal growth and morphology of NF fibroblasts in tissue culture. Because epidermal growth factor (EGF) is known to stimulate the growth of fibroblasts in tissue culture, we studied the binding of commercial iodine 125-labeled EGF to age- and passage-matched confluent NF (N = 6) and normal (N = 4) fibroblasts. Fibroblasts were maintained at 37 degrees C for 2, 30, 60, 120, and 240 minutes in a medium in which the cells grow slowly (Dulbecco's Eagle medium) and one in which they grow normally (Ham's F-12 medium). Binding assays were done in both serum-free media according to accepted procedures. The EGF binding did not differ in the two media, and pooled data are presented. These data demonstrate no significant differences in the early binding of EGF to normal and NF fibroblasts (4,682 +/- 1,092 versus 3,441 +/- 826 cpm/10(6) cells; 20,000 cpm/ng; p > 0.15 at 30 minutes). At one hour, however, differences suggestive of abnormal EGF binding become apparent (12,495 +/- 1,989 versus 3,172 +/- 853 cpm/10(6) cells; 20,000 cpm/ng; p < 0.0025). We conclude that there may be a membrane defect in NF which is reflected by diminished EGF binding.

Topics: Cells, Cultured; Epidermal Growth Factor; Fibroblasts; Humans; Neurofibromatosis 1; Peptides