Matthias M. Feldkamp

Proliferation of human malignant astrocytomas is dependent on Ras activation

Overexpression and activation of receptor tyrosine kinases, such as platelet derived growth factor receptors (PDGFRs) and epidermal growth factor receptor (EGFR), leads to proliferation of human malignant astrocytoma cells. Although oncogenic mutations affecting Ras are not prevalent in human malignant astrocytomas, we have investigated whether levels of activated Ras.GTP might be elevated in these tumors secondary to the mitogenic signals originating from activated receptor tyrosine kinases. In support of this hypothesis high levels of Ras.GTP, similar to those found in oncogenic Ras transformed fibroblasts, were present in four established human malignant astrocytoma cell lines which express PDGFRs and EGFR, and 20 operative malignant astrocytoma specimens. Stimulation of PDGFRs and EGFRs induced tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2, suggesting a mechanism by which Ras may be activated in human malignant astrocytoma cells. Furthermore, blocking Ras activation by expression of the Ha-Ras-Asn17 dominant-negative mutant, or by farnesyl transferase inhibitors, decreased in vitro proliferation of the human astrocytoma cell lines. These results support the hypothesis that proliferative signals from receptor tyrosine kinases expressed by human malignant astrocytoma cells utilize the Ras mitogenic pathway. Pharmacological inhibitors of the Ras pathway may therefore be of therapeutic value in these presently terminal tumors.

Meningiomas: role of vascular endothelial growth factor/vascular permeability factor in angiogenesis and peritumoral edema.

OBJECTIVE Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) is a potent angiogenic growth factor implicated in the tumor angiogenesis/metastasis of a number of human cancers. Activation of receptors for VEGF/VPF is specifically mitogenic to endothelial cells and increases their permeability. Although extensive literature exists regarding VEGF/VPF in human astrocytomas, little is known about its potential biological role(s) in meningiomas. Our interest in meningiomas was initiated by the observation that some meningiomas are extremely vascular and are occasionally associated with a considerable degree of peritumoral brain edema, both potentially related to the biological attributes of VEGF/VPF. METHODS As a first test of this hypothesis, we examined a cohort of 18 meningiomas for expression of VEGF/VPF at the messenger ribonucleic acid and protein levels and correlated expression with pathological characteristics, vascularity, and degree of peritumoral edema. RESULTS The majority of meningiomas expressed VEGF/VPF at both the messenger ribonucleic acid and protein levels. Corresponding serial sections were stained with an endothelial cell marker to obtain a microvascular density count, which positively correlated (P = 0.0005) with expression of VEGF/VPF. Furthermore, meningiomas with a large amount of peritumoral edema, as determined from the preoperative computed tomographic scans or magnetic resonance imaging scans, had elevated expression of VEGF/VPF (P = 0.05). CONCLUSION These data suggest that VEGF/VPF may play a role in both meningioma vascularity and peritumoral edema.

Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by Ras.

Vascular endothelial growth Factor (VEGF) has been identified as a key angiogenic factor involved in the growth and malignant progression of tumours. Glioblastoma multiforme (GBM) are the most common primary human brain tumours, histo‐pathologically characterized by intense tumour angiogenesis. GBMs do not harbour oncogenic Ras mutations, but there is a functional up‐regulation of Ras signaling through activation of receptor tyrosine kinases overexpressed by these tumours. We demonstrate that Ras pathway activation regulates VEGF secretion in astrocytoma cell lines. Ras pathway inhibition was carried out using genetic and pharmacologic techniques. Astrocytoma cells that were transfected to express the dominant inhibitory mutant H‐RasN17 demonstrated a reduction in VEGF secretion under both normoxic and hypoxic conditions. Cells treated with the farnesyl transferase inhibitor L‐744,832 demonstrated similar reductions in VEGF secretion. Furthermore, astrocytoma cells expressing a constitutively phosphorylated and truncated EGF‐R common in GBMs (EGFRvIII or p140EGF‐R) demonstrate further elevations in Ras activation, resulting in a further increase in VEGF secretion. We have previously demonstrated that activation of Ras plays a vital role in transducing mitogenic signals in human malignant astrocytoma cells. Our present results further extend the role of Ras activation in modulating tumour angiogenesis in these tumours. We propose that Ras may contribute to the angiogenic switch in astrocytomas. Int. J. Cancer, 81:118–124, 1999.

Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects

While 25% of human cancers harbor oncogenic Ras mutations, such mutations are not found in astrocytomas. We have previously demonstrated that the activation of receptor tyrosine kinases expressed by malignant human astrocytoma cells and specimens results in functional upregulation of the Ras signalling pathway and increased levels of activated Ras•GTP. Farnesyl transferase inhibitors (FTIs) are promising anti-cancer agents in early clinical trials, which may exert their effect through pharmacological inhibition of the Ras signalling pathway. In this study we establish the anti-tumorigenic properties of the FTI L-744,832 against a panel of malignant human astrocytoma cell lines. Furthermore, we demonstrate the multiple mechanisms by which L-744,832 exerts its effect. L-744,832 demonstrates both cytostatic and cytotoxic effects on astrocytoma cells, and cells expressing a truncated constitutively phosphorylated Epidermal Growth Factor Receptor common in high-grade astrocytomas (EGFRvIII/p140EGF-R) demonstrate increased sensitivity to the agent. L-744,832 is capable of inducing apoptosis in astrocytoma cells under anchorage-dependent conditions; this process occurs in a p53-independent manner and is associated with increased expression of Bax and Bak. L-744,832 also induces cell cycle arrest at both the G1/M and G2/S checkpoints; this process is also independent of p53 mutational status. Cell cycle arrest in drug-treated cells can be accompanied by induction of p21WAF1/CIP1, but this induction is not necessary for the cell cycle inhibitory effects, nor is it dependent on functional p53. Finally, angiogenesis in astrocytomas has been shown to be dependent on secretion of Vascular Endothelial Growth Factor (VEGF) by tumour cells, particularly under hypoxic conditions. L-744,832 potently inhibits the secretion of VEGF under hypoxic conditions. These combinations of mechanisms suggest that these tumours, despite the absence of oncogenic Ras mutations, will be amenable to growth inhibition by FTIs, through a combination of anti-proliferative, pro-apoptotic, and anti-angiogenic effects.

Signal transduction pathways and their relevance in human astrocytomas

Aberrations in a number of signal transduction pathwayshave been identified as playing a key rolein the molecular pathogenesis of astrocytomas and theirprogression to high grade glioblastoma multiforme (GBM). GBMsare characterized by overexpression of the Platelet DerivedGrowth Factor Receptors (PDGFR) and their ligands (PDGF),as well as the Epidermal Growth Factor Receptor(EGF-R). These receptors activate the Ras pathway, akey cellular signal transduction pathway, culminating in theactivation of a wide range of Ras-dependent cellularevents. GBMs have also been found to eitheroverexpression or lose expression of various Protein KinaseC (PKC) isoforms. Major strides are being madein developing pharmacological agents which specifically inhibit thesegrowth factor receptors and intracellular signal transduction pathways.Elucidating the role of these pathways in GBMsis thus of major clinical importance, as thesenovel molecularly-targeted agents may prove of use inthe clinical management of GBMs in the future.

Neurofibromatosis type 1 peripheral nerve tumors: aberrant activation of the ras pathway

BACKGROUND Neurofibromatosis Type 1 (NF1) is an autosomal dominant transmitted cancer predisposing syndrome, with peripheral nerve tumors being a prominent feature. The NF1 gene encodes a large cytoplasmic protein called neurofibromin, which is a major negative regulator of Ras, a key protein in a major signal transduction pathway. It is hypothesized, based on data from neurogenic sarcoma cell lines, that loss of neurofibromin leads to increased levels of activated Ras-GTP, and subsequent uncontrolled mitogenic signals to the nucleus. However, it is not known whether aberrant activity of the Ras pathway is also a prevalent molecular pathogenetic mechanism in actual peripheral nerve tumors. METHODS To investigate whether aberrant Ras activity was present, and varied with increased tumorigenic potential in peripheral nerve tumors, we have recently developed and published an enzymatic luciferase-based assay that allows measurement of Ras activity in tissues for the first time. RESULTS AND CONCLUSIONS Neurofibromin, the gene product of the NF1 gene, was not expressed in the NF1 tumors. Levels of activated Ras-GTP in NF1 neurogenic sarcomas and NF1 plexiform neurofibromas were approximately 15 and 5 times higher, respectively, compared with non-NF1 schwannomas, supporting the hypothesis that aberrant activity of this key signaling pathway is important in the pathogenesis of these tumors. In this article we review this data, the molecular genetics of NF1, and the current knowledge of the role of neurofibromin in cellular control. Our understanding of the molecular pathogenic mechanisms of NF1 tumors should be transferable to sporadic peripheral nerve tumors, and allow development of biological therapies directed against relevant targets such as Ras.

Neurofibromatosis type 1: piecing the puzzle together.

Neurofibromatosis type 1 (NF1) was first described in 1882 and is characterized by a diverse spectrum of clinical manifestations, including neurofibromas, café au lait spots, and Lisch nodules. NF1 is also noted for the higher risk of associated malignancies, making it the most common tumour-predisposing disease in humans. Transmitted in an autosomal dominant manner, the NF1 gene was cloned in 1990, and belongs to the family of tumour suppressor genes. Since then, there has been an explosion in our understanding of how the gene product, neurofibromin, functions in normal cellular physiology, and how its loss in NF1 relates to the wide spectrum of clinical findings, including NF1-associated tumours. Neurofibromin is a major negative regulator of a key signal transduction pathway in cells, the Ras pathway, which transmits mitogenic signals to the nucleus. Loss of neurofibromin leads to increased levels of activated Ras (bound to GTP), and thus increased downstream mitogenic signaling. Our understanding of neurofibromins role within cells has allowed for the development of pharmacological therapies which target the specific molecular abnormalities in NF1 tumours. These include the farnesyl transferase inhibitors, which inhibit the post-translational modification of Ras, and other agents which modulate Ras-mediated signaling pathways.

The Farnesyltransferase Inhibitor L‐744,832 Inhibits the Growth of Astrocytomas through a Combination of Antiproliferative, Antiangiogenic, and Proapoptotic Activities

The 21-kD cellular protein Ras is known to play a critical role in a wide range of cellular events, including proliferation, cytoskeletal rearrangement, development, and apoptosis. Activated Ras is bound to GTP, whereas Ras•GDP represents the inactive protein. Up to 30% of human malignancies express a mutant form of this protein in which Ras is constitutively present in its activated GTP-bound form. Such oncogenic mutations have not been identified in glioblastoma multiforme (GBM). We have however demonstrated that Ras activation is critical in the proliferation of these tumors1 as well as important in the secretion of the angiogenic factor vascular endothelial growth factor (VEGF2), with Ras activation occurring secondary to overexpression of the receptor tyrosine kinases platelet derived growth factor receptor (PDGF-R) and epidermal growth factor receptor (EGF-R).1 As farnesyltransferase inhibitors (FTIs) inhibit the posttranslational modification of Ras, preventing the association of Ras with its upstream activators at the cell surface, we hypothesized that astrocytoma cells would be growth inhibited by such agents. The FTI L-744,832 was provided by Allen Oliff (Merck Research Laboratories, West Point, Pennsylvania). Six astrocytoma cell lines were evaluated, including five established lines (U87, U118, U138, U343, and U373). In addition, U118:EGFRvIII cells were evaluated, which express the constitutively activated truncated receptor EGFRvIII, expressed in a large subset of GBMs. We previously showed that U118:EGFRvIII cells constitutively express higher levels of activated Ras•GTP. Cells were treated with L-744,832 at doses ranging from 10 nM to 100 μM, and the concentration of vehicle was kept constant in all wells (0.1% methanol). Cells were grown in 96-well plates, and proliferation was measured using the tetrazolium compound MTS, which is bioreduced by viable cells into a colored formazan product. Dose-response curves (Hill plots) were constructed by modeling a log normal doseresponse relationship. Logistic regression (probit analysis) was used to estimate the LD50 for each cell line as the dose at which proliferation was reduced to 50% of control wells. All astrocytoma cell lines were growth inhibited by L-744,832, with IC50 ranging from 5.3 to 17.4 μM. Cells expressing EGFRvIII were more sensitive to L-

A persistent median artery causing carpal tunnel syndrome in a patient with chronic renal failure: case report.

A 63-year-old woman presented with carpal tunnel syndrome 6 years after being diagnosed as having chronic renal failure and 1 year after the start of hemodialysis treatment. Symptoms were in the hand contralateral to the side of the arteriovenous fistula used for hemodialysis. During surgery, a large, patent, noncalcified median artery was found pulsating in the carpal tunnel; this pulsation was causing the compression of the median nerve. The flexor retinaculum was decompressed, and the median artery was transposed. This is the first reported case of a persistent median artery in a hemodialysis patient who has symptoms contralateral to the vascular access. The pathophysiology and epidemiology of carpal tunnel syndrome in chronic renal failure patients is reviewed. In addition, the role of the median artery in the vascularization of the hand and the management of a persistent median artery causing carpal tunnel syndrome are reviewed.