Epidermal growth factor receptor in glioblastoma

We compiled the current state of knowledge about the epidermal growth factor receptor (EGFR) in glioblastoma. Glioblastoma is one of the most common primary brain tumours and has an unfavourable prognosis despite aggressive treatment. These factors stimulate new research trials and a recent area of interest of neuro-oncologists is EGFR. This molecule is frequently altered in glioblastoma and constitutes the potential target for therapy. We decided to review the literature on biological structure of that molecule, its biological activity and the role in GBL with potential targeting it in the future neuro-oncological practice.     

Epidermal growth factor receptor and p53 protein expression in human glioblastomas

p53 mutations and amplification of epidermal growth factor receptor (EGFR) gene are the most frequently detected genetic alterations in glioblastomas; thus, these changes seem to delineate two subgroups of glioblastomas: those originated de novo and those originated from preexistent low grade astrocytomas. Paraffin-embedded surgical specimens of 30 human glioblastomas were analyzed immunohistochemically for the presence of p53 protein and EGFR. Approximately half of the cases were p53 protein-positive while one-third were EGFR positive. Only three cases were positive for both p53 protein and EGFR. There was no difference between the average ages of patients with only-p53-positive, and double-negative tumors, while three glioblastomas with both p53 protein and EGFR immunopositivity occured in older patients (mean age 67.0 years, p < 0.02). Patients with only-EGFR-positive tumors were younger, but not significantly (44.3 years, p < 0.1). This study supports the notion that there are two main subpopulations of glioblastoma—with EGFR and with p53 protein overexpression.     

Immunocytochemical distribution of transferrin and its receptor in the developing chicken nervous system

Transferrin is the plasma protein responsible for iron transport in all vertebrates. While transferrin is known to have growth-promoting activity on a variety of cells in culture, the role of transferrin and its membrane receptor in neuronal development is unknown. Using antibodies to transferrin and transferrin receptors, we studied the immunocytochemical localization of transferrin and its receptor in developing chicken neural tissues by the peroxidase-antiperoxidase method. In 5-day-old embryonic brain, germinal cells of the ventricular zone showed a positive reaction for transferrin receptors but were negative for transferrin. By 6-7 days, transferrin-positive cells were seen in the inner layer of the ventricular zone and a few 'patches' of transferrin-positive cells were also seen in the adjacent area. By 10 days, large neurons throughout the brain were strongly positive for transferrin. By 11-16 days, all neurons in the brain showed a strong positive reaction for the protein. Thereafter, the transferrin-positive reaction became gradually weaker in neurons whereas the walls of blood capillaries showed a positive reaction for transferrin. In the adult brain, neurons showed very weak or negative staining. A similar staining pattern for transferrin was observed in the developing spinal cord and dorsal root ganglia (DRG). By 10-12 days, both spinal cord neurons and DRG neurons showed strong reactions for transferrin. Thereafter, the transferrin-positive reaction gradually diminished in older spinal cord neurons and completely disappeared from DRG neurons. Cultured cerebral hemisphere, spinal cord, and DRG neurons showed positive staining reactions for both transferrin and its receptor. Our results suggest that: transferrin is initially taken up by developing neurons from cerebrospinal fluid via receptor-mediated endocytosis; the accumulation of transferrin ultimately reaches a maximum level within immunoreactive neurons and then declines just prior to hatching; in contrast to other CNS neurons, DRG neurons accumulate transferrin only briefly and then become negative for transferrin by immunocytochemistry; and after closure of the blood-brain barrier, transferrin may reach neurons by transport across capillaries into the 'paravascular' spaces. In view of these results, transferrin may play some important but unrecognized role in early neuronal development in vivo as well as in vitro.     

D-serine in the developing human central nervous system

To elucidate the role of D-serine in human central nervous system, we analyzed D-serine, L-serine, and glycine concentrations in cerebrospinal fluid of healthy children and children with a defective L-serine biosynthesis (3-phosphoglycerate dehydrogenase deficiency). Healthy children showed high D-serine concentrations immediately after birth, both absolutely and relative to glycine and L-serine, declining to low values at infancy. D-Serine concentrations were almost undetectable in untreated 3-phosphoglycerate dehydrogenase-deficient patients. In one patient treated prenatally, D-serine concentration was nearly normal at birth and the clinical phenotype was normal. These observations suggest a pivotal role for D-serine in normal and aberrant human brain development.     

Epidermal growth factor receptor expression and activation in neuroendocrine tumours

Epidermal growth factor receptor (EGFR) is expressed in many cancers and is associated with poor prognosis. EGFR activation pathways have been well characterised using tumour cell lines and are known to involve EGFR activation through autophosphorylation. Phosphorylation of downstream signalling molecules, such as ERK1/2 (extra-cellular regulated kinase 1 and 2) and PKB/Akt (protein kinase B), leads to enhanced tumour cell survival and proliferation. Although EGFR expression has been determined in neuroendocrine tumour tissue, its activation and subsequent effects on the downstream signalling molecules, ERK1/2 and Akt, have not been studied. We therefore planned to determine the role of EGFR in neuroendocrine tumours (NETs) by determining its pattern of expression and activation, and the subsequent activation of downstream signalling molecules ERK1/2 and Akt. Paraffin-embedded tumour tissue was available from 98 patients with NETs (39 foregut, 42 midgut, four hindgut, five paragangliomas, and four of unknown origin). Immunohistochemical evaluation was performed for the expression of EGFR, p-EGFR, p-Akt, and p-ERK1/2. Ninety-six percent of tumour samples were positive for EGFR expression; 63% were positive for activated EGFR; 76% were positive for activated Akt; and 96% were positive for activated ERK1/2. Importantly, the histological score for the activation of Akt and ERK1/2 correlated with the histological score for activated EGFR. These data provide a rationale for considering EGFR inhibitors in the treatment of NETs. Additionally, direct inhibition of Akt and ERK1/2 may provide further therapeutic options in the treatment of NETs in the future.     

Epidermal growth factor binding and mitogenic activity on purified populations of cells from the central nervous system

Binding of 125I-epidermal growth factor (EGF) to purified populations of rat astrocytes, oligodendrocytes, and neurons was measured. Astrocytes bound 40,000-100,000 EGF molecules per cell, while oligodendrocytes bound only 6,000-10,000 EGF molecules per cell. In contrast, neurons had little or no capacity to bind 125I-EGF. EGF alone was able to stimulate incorporation of tritiated thymidine fivefold in purified astrocyte cultures incubated in serum-free medium. When EGF was added to the previously described chemically defined medium for astrocytes, incorporation of tritiated thymidine in purified astrocytes was equivalent to that observed in medium containing 10% fetal calf serum. Addition of EGF to this chemically defined medium also doubled the proliferative capability of the medium for cultured astrocytes. EGF was maximally effective in stimulating 3H-thymidine incorporation at concentrations between 1-10 ng/ml.     

Epidermal growth factor receptor in adult retinal neurons of rat, mouse, and human

During development, the epidermal growth factor receptor (EGFR) regulates proliferation and differentiation of many types of cells, including precursors of neurons and glia. In the adult, EGFR continues to drive the growth and differentiation of epithelial cells but is absent from glia in the CNS. However, the localization and functions of EGFR in adult neurons are not well defined. By using immunohistochemistry and Western blotting, we have identified EGFR and its ligands in adult retinal ganglion cells in the normal rat, mouse, and human retina. EGFR and its ligands were also present in certain other adult retinal neurons, for example, horizontal cells and amacrine cells, and had different distribution patterns among these species. In addition, we found that EGFR was expressed in the rat retinal ganglion cell line RGC-5. One of the EGFR ligands, EGF, caused a cell shape change and increased neurofilament phosphorylation in RGC-5 cells. The expression of EGFR in postmitotic, terminally differentiated adult retinal neurons suggests that EGFR has pleiotropic functions. In addition to the conventional mitogenic role in adult epithelial cells, EGFR must serve a different, nonmitogenic function in adult neurons. Our work localizes EGFR and its ligands in the adult retinas of several species as a step toward investigating the nonmitogenic functions of EGFR in adult neurons.     

Epidermal growth factor receptor induced Erk phosphorylation in the suprachiasmatic nucleus

A significant functional role for epidermal growth factor receptor (EGFR) in the suprachiasmatic nucleus (SCN) is suggested by the confluence of several recent findings: (1) EGFR is highly expressed in the SCN, (2) the EGFR ligand TGFalpha is expressed and apparently locally released in the SCN, and (3) EGFR activation local to the 3rd ventricle suppresses circadian locomotor behavior and drinking/feeding activities. However, it has not yet been shown that binding of EGFR in the SCN can activate the key signaling pathways associated with its function. Here, we report that EGF treatment induced Erk1/2 phosphorylation in the rat SCN cells. The observation indicates functional EGFR in the SCN and motivates further investigation of the functional role of these receptors.     

Vascular endothelial growth factor and the nervous system

Vascular endothelial growth factor (VEGF) is an angiogenic factor essential for the formation of new blood vessels during embryogenesis and in many pathological conditions. A new role for VEGF as a neurotrophic factor has recently emerged. In the developing nervous system, VEGF plays a pivotal role not only in vascularization, but also in neuronal proliferation, and the growth of coordinated vascular and neuronal networks. After injury to the nervous system, activation of VEGF and its receptors may restore blood supply and promote neuronal survival and repair. There is a growing body of evidence that VEGF is essential for motor neurone survival, and that aberrant regulation of VEGF may play a role in the degeneration of neurones in diseases such as amyotrophic lateral sclerosis.     

Fibroblast growth factor receptor 3 signaling regulates injury-related effects in the peripheral nervous system

Fibroblast growth factor receptor (FGFR) signaling is crucial for neural development and regeneration. Here we investigated the L5 spinal ganglion and the sciatic nerve of intact Fgfr3-deficient mice after nerve injury. Quantification of sensory neurons in the L5 spinal ganglion revealed no significant differences between wild-type and Fgfr3-deficient mice. Seven days after nerve lesion, the normally occurring neuron loss in wild-type mice was not found in Fgfr3-deficient animals, suggesting that FGFR3 signaling is involved in the cell death process. Morphometric analysis of the sciatic nerve showed similar numbers of myelinated axons, but the axonal and myelin diameter was significantly smaller in Fgfr3-deficient mice compared to the wild types. Evaluation of regenerating myelinated axons of the sciatic nerve revealed no differences between both mouse strains 7 days after crush injury. Our results suggest that FGFR3 signaling seems to be involved in processes of damage-induced neuron death and axonal development.     

Epidermal growth factor receptor extracellular domain mutations in primary glioblastoma

Aims: Novel missense mutations of the epidermal growth factor receptor (EGFR) extracellular domain have been recently described in a large series of glioblastomas. Methods: The exons 2, 3, 7, 8 and 15 coding for the EGFR extracellular domain were sequenced in a series of 161 consecutive primary glioblastomas and correlated with clinical features of patients in order to determine whether these alterations are linked to specific clinical characteristics of the disease. Results: Missense mutations were observed in 18 cases (11.2%), and 4 novel mutations were detected, including G178C, A271C, C818A and C1860G. Mutations of the EGFR extracellular domain were not associated with overall survival or with age at onset of the disease. In contrast, the EGFR extracellular domain mutations were significantly associated with patients' gender. Indeed, 15 mutations were observed in men vs. 3 in women ( P  < 0.05). EGFR extracellular domain mutations were also strongly associated with EGFR amplification ( P  < 0.0001). Conclusions: To our knowledge, EGFR extracellular domain mutations are the first genomic abnormalities associated with gender in primary glioblastomas, although a link between mutations of the EGFR tyrosine kinase domain and gender has been previously made in lung cancer.     

Epidermal growth factor receptor expression regulates proliferation in the postnatal rat retina

Epidermal growth factor (EGF) is known to promote proliferation of both retinal progenitors and Muller glia in vitro, but several questions remain concerning an in vivo role for this factor. In this study, we investigated whether the EGF receptor (EGFR) is necessary for the maintenance of normal levels of progenitor and Muller glial proliferation in vivo. Here, we show that (1) mice with homozygous deletion of the Egfr gene have reduced proliferation in late stages of retinal histogenesis, (2) EGF is mitogenic for Müller glia in vivo during the first two postnatal weeks in the rodent retina, (3) the effectiveness of EGF as a Müller glial mitogen declines in parallel with the decline in EGFR expression as the retina matures, and (4) following damage to the retina from continuous light exposure, EGFR expression is up-regulated in Müller glia to levels close to those in the neonatal retina, resulting in a renewed mitotic response to EGF. Together with previous results from other studies, these data indicate that the downregulation of a growth factor receptor is one mechanism by which glial cells maintain mitotic quiescence in the mature nervous system.     

Nerve growth factor receptors in the central nervous system

Nerve growth factor (NGF) is well known to be involved in the development, survival, and maintenance of sympathetic and neural crest-derived sensory neurons in the peripheral nervous system. Over the last 10-15 years, however, the role of NGF as a necessary trophic substrate for magnocellular cholinergic neurons in the central nervous system (CNS) has emerged. Because the trophic effects of NGF are initiated by its interaction with membrane-bound receptors, the characterization, localization, and function of these specific NGF receptors are essential to understanding the many actions of NGF. The first part of this review will summarize briefly the presence and possible role of NGF in the CNS, with the remainder of the review focusing on what is known about the receptor to NGF.     

Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system

NGF acts on responsive neurons by binding to specific NGF receptors on axonal termini, after which a critical biochemical signal is retrogradely transported to the cell body. The identity of the signal(s) is unknown; candidates include NGF itself or some other "second messenger." A possible second messenger is the NGF receptor. As a first step in assessing the possible role of NGF receptor in the generation of the NGF-dependent signal, and in understanding the economy of NGF receptor synthesis and utilization, we determined whether the NGF receptor is retrogradely transported. Using immunohistochemical staining with a monoclonal antibody (192-IgG) against rat NGF receptor, we looked for accumulation of NGF receptor molecules distal (retrograde transport), as well as proximal (anterograde transport), to sites of axonal ligation or transection. By 10-12 hr in both the ligated sciatic nerve and the lesioned fimbria-fornix, accumulated NGF receptor was detected proximal and distal to the ligation/lesion site. The transported receptor presumably was located in sympathetic and sensory neurons in the sciatic nerve and in forebrain cholinergic neurons projecting from the medial septum to the hippocampus. In both anatomical sites, accumulation of NGF receptor on the proximal (anterograde) side occurred in streams of fine axonal processes, whereas staining on the distal (retrograde) side occurred in varicose or granular configurations. These results raise the possibility that the NGF receptor has a role in the mechanism of NGF beyond the initial binding event at the plasma membrane of the axonal terminus.     

Apoptosis in the basal ganglia of the developing human nervous system

Programmed cell death (PCD) plays a crucial role in the development of the central nervous system through controlling neuronal numbers and adequate synaptic connections. PCD has been considered to occur in the form of apoptosis. To examine how apoptosis occurs in the developing human brain, we performed a morphometric TUNEL study, using a commercially available kit (ApopTag Kit, Oncor Inc.). We examined apoptotic cells in the basal ganglia of 47 fetuses and newborns without macroscopical and microscopical evident congenital anomalies. Gestational age ranged from 12 to 40 weeks. The numerical density as well as the labeling index of TUNEL-positively labeled nuclei were evaluated. In the caudate nucleus and putamen, TUNEL-labeled cells were observed around the 12th week of gestation. The numerical density of total cells was significantly decreased, whereas the labeling index of apoptotic cells was significantly increased with advanced gestational age. In the globus pallidus, the numerical density of total cells decreased with advancing gestational age, while the labeling index of apoptotic cells increased between the 20th and 28th week, followed by a decrease until the 40th week. The analysis of TUNEL-positive cells revealed a different reaction pattern for the various basal ganglia with regard to the timing and degree of the apoptotic process in regulating cell numbers.     

Expression of NGF receptor in the developing and adult primate central nervous system

Monoclonal antibodies against human NGF receptor have been used for immunocytochemical localization of NGF receptors in the CNS of macaques and baboons at various stages of development. In the adult, neurons in most brain regions are devoid of detectable NGF receptors. However, abundant NGF receptor immunoreactivity is present on a population of neurons in basal forebrain, which, on the basis of appearance and pattern of distribution, probably correspond, at least in part, to magnocellular cholinergic neurons of this region. NGF receptors were also associated with the vasculature in most brain regions. NGF receptor immunoreactivity is present on Mueller glia of neural retina. In macaque fetuses, approximately 1 month prenatally, retinal Mueller glia possess lower levels of receptor, while higher levels of receptor are present in the retinal nerve fiber layer. In fetal cerebellum, abundant receptor immunoreactivity is present on Purkinje cells, granule cells of the premigratory zone of the external granule layer, and neurons of the deep nuclei. Immunoreactivity decreases with subsequent development and is absent in the adult. In cerebellum, levels of NGF receptor assayed by affinity crosslinking to radioiodinated NGF, and levels of NGF receptor mRNA assayed by Northern blot analysis decrease dramatically during the last month of fetal life.     

Expression of tumor necrosis factor alpha in the developing nervous system.

We present evidence that tumor necrosis factor alpha (TNF-alpha) is transiently expressed at specific times during embryogenesis in precisely defined areas of the nervous system in two different classes of vertebrates. In murine embryos, TNF-alpha was detected in the brain, neural tube and peripheral mixed spinal nerves. In the chick embryo, TNF-alpha was observed in the brain neuroepithelium and in the developing Purkinje neurons of the cerebellum. Western immunoblot analysis revealed that brain tissue from both mouse and chick embryos contained a 50 kDa protein showing immunoreactivity with anti-TNF-alpha antibody. These results suggest that TNF-alpha participates in the normal development of the vertebrate brain and spinal cord.     

Epidermal growth factor receptor immunoreactivity in rat brain. Development and cellular localization

In rat brain, distinct epidermal growth factor-receptor immunoreactivity (EGFR-IR) first appeared in astroglia at about day 16 postnatal, reached maximum intensity at 19 days and then became much weaker as the animals reached adulthood. EGFR-IR was also observed in cerebellar Purkinje cells as early as 11 days postnatal and was maintained into adulthood. In adult and aged animals the most prominent EGF receptor immunostaining occurred in cerebral cortex neurons (layers IV and V) that had the morphology of basket cells. Immunoreactive neurons were abundant in the cingulate, frontal, frontoparietal and striate cortices. In the frontoparietal cortex, EGFR positive neurons were most numerous in the motor area, diminishing laterally towards the somatosensory area. The localization and time of appearance of EGFR-IR did not appear consistent with a direct mitogenic role of the EGF domain in astroglia proliferation during development. However, the EGFR may be involved in neuronal survival and/or neuron-glia signalling.