Nelson Lau

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.

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.

Suppression of glioblastoma tumorigenicity by the Kruppel-like transcription factor KLF6

The Kruppel-like transcription factor KLF6 is a novel tumor-suppressor gene mutated in a significant fraction of human prostate cancer. It is localized to human chromosome 10p14–15, a region that displays frequent loss of heterozygosity in glioblastoma multiforme (GBM). Indeed, mutations of the KLF6 gene have recently been reported in this tumor type. In this study, we report that the expression of KLF6 is attenuated in human GBM when compared with primary astrocytes. Expression of KLF6 in GBM cells reverts their tumorigenicity both in vitro and in vivo, which is correlated with its transactivation of the p21/CIP1/WAF1 promoter. Additionally, KLF6 inhibits cellular transformation induced by several oncogenes (c-sis/PDGF-B, v-src, H-Ras, and EGFR) that are components of signaling cascades implicated in GBM. Our results provide the first evidence of functional tumor suppression by KFL6, and its loss may contribute to glial tumor progression.

Expression and hypoxic regulation of angiopoietins in human astrocytomas

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and edema in human astrocytomas by its interaction with cognate endothelial-specific receptors (VEGFR1/R2). Tie1 and Tie2/Tek are more recently identified endothelial-specific receptors, with angiopoietins being ligands for the latter. These angiogenic factors and receptors are crucial for the maturation of the vascular system, but their role in tumor angiogenesis, particularly in astrocytomas, is unknown. In this study, we demonstrate that the angiopoietin family member Ang1 is expressed by some of the astrocytoma cell lines. In contrast to VEGF, Ang1 is down regulated by hypoxia. Ang2 was not overexpressed. Expression profiles of low-grade astrocytoma specimens were similar to those of normal brain, with low levels of Ang1, Ang2, and VEGF expression. Glioblastoma multiforme expressed higher levels of Angl, but not to the same degree as pseudopalisading astrocytoma cells around necrotic and hypoxic zones expressed VEGF, as shown in previous studies. Ang2 expression in the highly proliferative tumor vascular endothelium was also increased, as was phosphorylated Tie2/Tek. The expression profile of these angiogenic factors and their endothelial cell receptors in human glioblastomas multiforme was similar to that in a transgenic mouse model of glioblastoma multiforme. These data suggest that both VEGF and angiopoietins are involved in regulating tumor angiogenesis in human astrocytomas.

Pathological and Molecular Progression of Astrocytomas in a GFAP:12V-Ha-Ras Mouse Astrocytoma Model

We previously characterized a genetically engineered mouse astrocytoma model with embryonic astrocyte-specific, activated (12)V-Ha-RAS (GFAP-RAS) transgenesis. The GFAP-RAS line Ras-B8 appears normal at birth, but 50% of mice die by 4 months from low- and high-grade astrocytomas. We examined the development and progression of astrocytomas in the Ras-B8 genetically engineered mouse. At embryonic day 16.5 (E16.5), there were no pathological differences compared to control littermates, aside from transgene expression. Diffuse astroglial hyperplasia was the first distinguishing feature in the 1-week-old Ras-B8 mice; however, these astrocytes were not transformed in vitro or in vivo. From 3 to 8 weeks the incidence of low-grade astrocytomas progressively increased with 85% of 12-week-old mice harboring low- or high-grade astrocytomas, the latter characterized by increased proliferation, nuclear atypia, and angiogenesis. Tp 53 mutations were detected in both astrocytoma grades, with high-grade astrocytomas expressing elevated levels of epidermal growth factor receptor and vascular endothelial growth factor, plus decreased levels of PTEN and p16, similar to human astrocytomas. We postulate that expression of (12)V-Ha-RAS in astroglial precursors induces astroglial hyperplasia, but transformation and subsequent progression requires additional molecular alterations resulting from aberrant activated p21-RAS. Of interest, many of these acquired alterations occur in human astrocytomas, further validating GFAP-RAS as a useful model for studying astrocytoma development and progression.

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-