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Isotype-specific Ras·GTP-Levels Predict the Efficacy of Farnesyl Transferase Inhibitors against Human Astrocytomas Regardless of Ras Mutational Status¹

Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, M5G-1X8 [M. M. F., N. L., L. R., A. G.]; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, M5G-1X5 [M. M. F., N. L., L. R., A. G.]; Division of Neurosurgery, Toronto Western Hospital, University Health Network, and University of Toronto, M5T-2S8 [M. M. F., A. G.]; and Department of Surgical Oncology, Ontario Cancer Institute/Princess Margaret Hospital, M5T-2S8 [A. G.], Toronto, Ontario, Canada

	ABSTRACT

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Previous studies have demonstrated that astrocytomas express elevated levels of activated Ras·GTP despite the absence of activating Ras mutations. Farnesyl transferase inhibitors (FTIs) exert their antitumor effect in part through inhibition of Ras-mediated signaling. SCH66336 is a potent FTI presently undergoing clinical trials in patients with solid tumors. We evaluated the efficacy of SCH66336 against a panel of eight human astrocytoma cell lines and three human astrocytoma explant xenograft models in NOD-SCID mice. SCH66336 demonstrated variable antiproliferative effects against the cell lines, with IC₅₀ ranging from 0.6 µM to 32.3 µM. Two of the three human glioblastoma multiforme (GBM) xenografts demonstrated substantial growth inhibition in response to SCH66336, with up to 69% growth inhibition after 21 days of treatment. Drug efficacy could be accurately predicted using a combination of the H-, K-, and N-isotype-specific Ras·GTP levels. These data indicate that the absence of Ras mutations does not preclude chemotherapeutic efficacy by FTIs, that Ras is likely a major target of FTIs regardless of Ras mutational status, and that isotype-specific Ras·GTP levels are a promising marker of drug efficacy.

	INTRODUCTION

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Activated GTP-bound Ras is a potent activator of intracellular signaling pathways, and its vital role is exemplified by the presence of oncogenic (activating) Ras mutations in approximately 25% of human malignancies (1) . Inactive Ras·GDP is activated to the GTP-bound form by numerous upstream activators, and Ras·GTP is rapidly inactivated to Ras·GDP through an intrinsic GTPase activity catalyzed by p120-GAP and neurofibromin (2 , 3) . Oncogenic/activating Ras mutations lock Ras in its GTP-bound form (4) , resulting in malignant transformation (5) . Such activating mutations have not been identified in human astrocytomas, including the most malignant form, termed GBM³ (1 , 6) . However, these tumors are characterized by the overexpression of ligand-dependent and -independent growth factor receptors (7, 8, 9, 10, 11, 12, 13) . We have demonstrated in previous studies (14, 15, 16) that receptor-induced Ras activation is a common feature of GBMs and their derived cell lines, regulating both proliferative and angiogenic signals.

FTIs represent a promising novel class of molecularly targeted chemotherapeutic agents. These drugs inhibit the critical initial step in the post-translational modification of Ras (17 , 18) and other farnesylated proteins (19) . This step, catalyzed by farnesyl protein transferase, involves the transfer of a 15-carbon trans,trans-farnesyl moiety from farnesyl PP_i to the cysteine residue on the CAAX (C = cysteine; A = aliphatic amino acid; X = any other amino acid) motif at the COOH-terminal of Ras. Although initially intended to inhibit the proliferation of tumors in which the presence of oncogenic/activating Ras mutations resulted in persistently elevated levels of Ras·GTP, there is growing evidence that such agents also inhibit the proliferation of human cancer cell lines lacking such mutations (20, 21, 22) ; e.g., we have demonstrated recently (22) that the FTI L-744,832 can inhibit the growth of established GBM cell lines by reducing cell cycle progression through both G₁-S and G₂-M, as well as by inducing apoptosis, even under anchorage-dependent conditions. Furthermore, L-744,832 potently inhibits the secretion of VEGF by these cells and, hence, may also demonstrate an antiangiogenic effect in vivo (22) .

The FTI SCH66336 has recently completed Phase I clinical trials (23) with prior studies (24 , 25) demonstrating promising effects against tumor cell lines in culture and in animal studies, although its efficacy against astrocytomas has not been reported previously. In the present paper, we demonstrate that SCH66336 decreases the viability of astrocytoma cells and inhibits the growth of human GBM explant xenografts in NOD-SCID mice. More importantly, we demonstrate that isotype-specific H-, K-, and N-Ras·GTP levels can accurately predict drug efficacy. These findings support the hypothesis that Ras is a major therapeutic target of FTIs even in tumors lacking oncogenic Ras mutations and that tumors with high levels of H-Ras·GTP are most sensitive to growth inhibition by these agents.

	MATERIALS AND METHODS

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Determination of Drug Efficacy against Human Astrocytoma Cell Lines.
SCH66336 was obtained from Schering-Plough Research Institute (a gift of Dr. W. Robert Bishop, Kenilworth, NJ). SCH66336 is a novel trihalobenzocycloheptapyridine FTI (M_r, 638.8) modified from agents identified through a random drug screening program (26) . U373 and U-343Cl2:6 cells were a gift of B. Westermark (Uppsala, Sweden); U118 cells were a gift of C. David James (Mayo Clinic, Rochester, MN); U138 and U87 cells were obtained from the American Type Culture Collection (Rockville, MD); and U251, SF763, and SF767 cells were a gift of Dolores Dougherty (University of California-San Francisco Brain Tumor Research Center). All of the cells were grown at 37°C in a 5% CO₂ incubator in DMEM supplemented with 10% calf serum. To measure cell viability, 1000 cells were plated in 96-well plates and treated with SCH66336 (1 nM-100 µM) for 10 days, with the number of viable cells determined using the Cell Titer 96 AQueous One Solution kit (Promega, Madison, WI; Ref. 27 ). Control cells were grown in medium alone or in medium supplemented with the vehicle (0.1% v/v DMSO). Dose-response curves were determined by modeling a log-normal dose-response relationship, with the IC₅₀ defined as the dose at which the number of cells in the treatment well was 50% of that in control wells (Table 1) .

SCH66336 was dissolved in 20% w/v HPßCD and dosed at 50 mg/kg p.o. bid by oral gavage (volume, 100 µl), with the administered dose adjusted based on twice-weekly mouse weights. Vehicle-treated mice were provided with 20% HPßCD by twice-daily oral gavage. After the tumor volume (V = a²b/2, where a < b) had reached approximately 200–250 mm³, mice were randomized to receive either SCH66336 or HPßCD. The study was continued for 21 days, with twice-weekly mouse weight and tumor volume determination. Mice were sacrificed at the conclusion of the study, with portions of the tumor either flash-frozen in liquid nitrogen (to measure levels of Ras·GTP) or fixed in 10% buffered formalin (IHC analyses). Percentage growth inhibition {100 - [100 x (volume in drug group at day 21 - volume in drug group at day 0)/(volume in vehicle group at day 21 - volume in vehicle group at day 0)]} was calculated to determine the effect of SCH66336 on tumor growth (Fig. 1) . The percentage growth inhibition was calculated twice weekly throughout the study, and the mean of these individual calculations was used to estimate the overall mean percentage growth inhibition. The growth fraction [1 - (percentage growth inhibition/100)] was calculated for use in the regression analyses.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Effect of SCH66336 on the growth of xenograft models was evaluated in NOD/SCID mice bearing s.c. flank XEN01, XEN05, or XEN08 GBM xenografts. Mice were dosed twice daily by oral gavage with either 100 µl of 20% w/v HPßCD or 50 mg/kg SCH66336 in a total volume of 100 µl of 20% w/v HPßCD. SCH66336 demonstrates relatively little antitumor effect against XEN01 xenografts, with a mean tumor growth inhibition of 19.4 ± 11.2% (a). In contrast, SCH66336 demonstrates significant antitumor effect against both XEN05 and XEN08 xenografts, with a mean growth inhibition of 68.7 ± 9.3% for XEN05 (b) and of 63.8 ± 5.0% for XEN08 (c).

View larger version (79K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. a, photomicrographs of sections of xenograft GBM tumors (XEN08 model) from the s.c. space of NOD/SCID mice. Top panels are from vehicle-treated mice (A and B), whereas the bottom panels are from mice treated with 50 mg/kg p.o. bid SCH66336 (C and D). Vehicle-treated tumors demonstrate large regions of central necrosis (H&E staining; B, star; original magnification, x100), with a virtual absence of TUNEL-positive cells within the solid portions of the tumor (A, arrow; original magnification, x400). SCH66336-treated tumors were much smaller and demonstrate smaller regions of central necrosis but frequent regions of geographic necrosis throughout the tumor. Clusters of TUNEL-positive cells were seen throughout the drug-treated tumors (C; original magnification, x200). A number of blood vessels in drug-treated tumors reveal TUNEL-positive cells within the vessel wall, suggesting an antiangiogenic effect of SCH66336 (D; original magnification, x400), a feature that was not observed in the vehicle-treated tumors. b, photomicrographs of sections of xenograft GBM tumors (model XEN08) from the s.c. space of NOD/SCID mice. Left panels are from vehicle-treated mice, whereas the right panels are from mice treated with 50 mg/kg p.o. bid SCH66336 for 20 days. Sections were stained with H&E and probed for Ki-67, VEGF, and Factor VIII. Drug-treated tumors demonstrate an abnormal cytoarchitecture compared with vehicle-treated tumors (H&E). Both drug-treated and vehicle-treated tumors demonstrate the presence of Factor VIII-positive structures. These structures failed to form mature vascularized blood vessels in SCH66336-treated tumors (H&E). Vehicle-treated tumors demonstrate robust VEGF staining, even in solid tumor remote from the central necrosis, whereas drug-treated tumors demonstrate weak VEGF staining, even surrounding areas of necrosis. The Ki-67 labeling index appears uniformly high in vehicle-treated tumors, whereas in SCH66336-treated tumors, most of the tumor demonstrates very low or absent Ki-67 labeling. Original magnification, x100 for H&E and Factor VIII; 200 x for Ki-67 and VEGF.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Immunoprecipitation and Western blot analysis to determine the specificity of isotype-specific antibodies. Membranes were immunoprobed for H-Ras (a) and N-Ras (b). Fibroblasts overexpressing H-Ras demonstrate a strong band at Mr 21,000 when immunoprobed with anti-H-Ras but not anti-N-Ras (Lane 1), whereas the opposite pattern is seen for fibroblasts overexpressing N-Ras (Lane 2). Lanes 3–5, U343 astrocytoma cells immunoprecipitated only with anti-H-, anti-K-, or anti-N-Ras in single immunoprecipitation experiments. These studies demonstrate the specificity of these antibodies for immunoprecipitation and Western blotting. Lanes 6–7, the effect of double immunoprecipitations using U343 cells. Lysates immunoprecipitated with both anti-H- and anti-K-Ras demonstrate strong H-Ras expression in the pellet (Lane 6) and relatively little remaining H-Ras in the supernatant (Lane 7), whereas N-Ras is not detected in the pellet but is present in the supernatant. Similar results were obtained from xenograft lysates (data not shown). These studies thus demonstrate the ability of these double immunoprecipitation experiments to enrich the supernatant for the third Ras isotype.

	RESULTS

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Efficacy of SCH66336 against Astrocytoma Cells and Human GBM Explant Xenografts.
The IC₅₀ of the eight GBM and high-grade astrocytoma cell lines tested are provided in Table 1 . These IC₅₀ were calculated after 10 days of treatment with SCH66336 and ranged from 0.6 µM for SF767 cells to 32.3 µM for U373 cells. The effect of SCH66336 on the xenografts was also variable (Fig. 1) , with a nonstatistically significant growth inhibition (19.4 ± 11.2%; P = 0.41) for the XEN01 tumors (n = 24; 10 vehicle, 14 SCH66336; Fig. 1a ). In contrast, SCH66336 had a statistically significant effect on XEN05 tumors (n = 22; 12 vehicle, 10 SCH66336), with a mean growth inhibition of 68.7 ± 9.3% (P = 0.0002; Fig. 1b ). Consistent with this effect, the mean tumor weight of SCH66336-treated XEN05 tumors was 30.8% less than in vehicle-treated tumors after 21 days of treatment (1.88 ± 0.28 g for vehicle versus 1.30 ± 0.31 g for SCH66336; P = 0.23). Finally, SCH66336 demonstrated significant antitumor effects against XEN08 tumors (n = 17; 9 vehicle, 8 SCH66336), with a mean growth inhibition of 63.8 ± 5.0% (P < 0.0001; Fig. 1c ). Drug-treated XEN08 tumors weighed 1.04 ± 0.19 g at harvest, compared with 1.88 ± 0.23 g for vehicle-treated tumors (a 44.9% reduction; P = 0.0136). HPßCD alone did not affect the growth of these tumors, in comparison with mice given 100 µl of drinking water twice-daily by oral gavage (data not shown; n = 19; P = 0.88).

IHC Analysis of SCH66336-treated Tumors.
Formalin-fixed, paraffin-embedded sections from each GBM xenograft were subjected to IHC analysis. Vehicle-treated tumors grew very large, with extensive central necrosis (Fig. 2a , part B), a feature not found in the much smaller SCH66336-treated tumors. In contrast, the presence of TUNEL-positive tumor cells was unusual in vehicle-treated tumors (Fig. 2a , Part A) but was a common feature of SCH66336-treated GBMs (Fig. 2a , Part C). Individual blood vessels in drug-treated tumors revealed the presence of TUNEL-positive endothelial cells, suggesting an antiangiogenic effect that contributed to the overall decreased tumor growth in SCH66336-treated animals (Fig. 2a , part D). Apoptosis was not a feature of the tumor-associated blood vessels in the vehicle-treated GBMs (data not shown).

Viable regions of vehicle-treated tumors demonstrated extensive Ki-67 and bromodeoxyuridine labeling, many vascularized and Factor VIII-positive blood vessels, and robust VEGF expression by the tumor cells. In contrast, few components of drug-treated tumors were viable, with generally absent Ki-67 and bromodeoxyuridine labeling, a disorganized histological architecture, and weak VEGF expression (Fig. 2b) . Both vehicle- and drug-treated tumors contained numerous Factor VIII-positive vascular structures. However, vehicle-treated tumors were well vascularized, with numerous red blood cells seen in H&E sections from these tumors. In contrast, drug-treated tumors lacked such evidence of vascularization (Fig. 2b) .

Prediction of Drug Efficacy from Isotype-specific Ras·GTP Levels.
Total and isotype-specific Ras·GTP levels were measured in all of the eight human malignant astrocytoma cell lines (Table 1) . Total Ras·GTP levels did not help predict drug efficacy in the cell lines. Furthermore, H-Ras, K-Ras, and N-Ras isotype-specific Ras·GTP levels were relatively poor individual predictors of drug efficacy. However, forward stepwise multiple regression analysis demonstrated that the overall model comprised of the combination of H-, K-, and N-Ras·GTP levels was highly predictive of the IC₅₀ (R² = 0.9255; F = 16.5587; P = 0.0102). As depicted in Table 1 , the "predicted IC₅₀ " from this regression analysis and derived from the overall model closely approximates the "actual IC₅₀ " of the astrocytoma cell lines. As shown in Fig. 4, a–d (top), increasing K- and N-Ras·GTP levels predict for greater drug resistance, whereas increasing H-Ras·GTP levels predict for greater drug efficacy.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Multiple linear regression was used to predict the IC50 for SCH66336 in a panel of eight astrocytoma cells (a–d), as well as the efficacy of SCH66336 against three GBM xenografts in NOD/SCID mice (e–h). To approximate the concept of an IC50, in which higher levels imply greater drug resistance, the model for the xenografts predicts the growth fraction. The growth fraction is defined as the growth of drug-treated tumors in comparison with vehicle-treated tumors, with vehicle-treated tumors given a value of 1.0. The strongest regression model was constructed by excluding total Ras·GTP from the equation but by including each of the isotype-specific Ras·GTP levels. Predicted and actual IC50 levels were closely correlated in the cell lines (a; and Table 1 ), as were predicted, and actual growth fractions in the xenograft tumors (e; and Table 2 ). The horizontal dashed line in each graph represents the mean of all of the values. Leverage plots (b–d and f–h) depict the effect on residuals if that variable was removed from the model (49) . These leverage plots demonstrate that increasing H-Ras·GTP levels correlate with increased drug sensitivity and reduced IC50/growth fraction (b and f), whereas increasing K- or N-Ras·GTP levels correlate with increased drug resistance and increased IC50/growth fraction (c, d, g, and h). Dashed curves around the regression line indicate the 95% confidence interval for each regression line.

	DISCUSSION

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FTIs represent one of the first classes of antineoplastic agents designed through rational drug design. The high prevalence of activating Ras mutations in human cancers (1) initially suggested that approximately 25% of human malignancies may demonstrate sensitivity to such agents (32) . However, two critical observations have made this prediction more complex. First, the presence or absence of Ras mutations does not appear to be the sole determinant of FTI efficacy, at least in cell lines (21) . Second, H-Ras-transformed cells appear to be more sensitive to these agents than those transformed by either K-Ras or N-Ras (20 , 31 , 33 , 34) .

The first observation has cast doubts on whether Ras is a major therapeutic target of FTIs in vivo. This is because in addition to Ras, approximately 0.5% of all of the cellular proteins undergo farnesylation (19) , including nuclear laminin A and B, skeletal muscle phosphorylase kinase, and retinal proteins, as well as other members of the Ras superfamily such as Rap2 proteins and RhoB (17 , 35, 36, 37) . Of these, RhoB in particular has been identified as an important putative antineoplastic target of FTIs (38, 39, 40, 41) . The second observation was initially disappointing in terms of human cancers, because K-Ras mutations occur more frequently in human malignancies than H-Ras mutations (1) . Two factors appear to explain this observation (42) . First, K-Ras4B has a 10–20-fold greater affinity for farnesyl protein transferase, the target of FTIs, necessitating much higher levels to inhibit the post-translational processing of this Ras isotype (43) . Second, N-Ras, K-Ras4A, and K-Ras4B can also act as substrates for geranylgeranyl protein transferase-1 (43) , which catalyzes the addition of a 20-carbon geranylgeranyl isoprenyl group to the cysteine residue of the CAAX motif at the COOH terminal of proteins such as Ras. Under physiological conditions, these Ras isotypes undergo farnesylation almost exclusively, but when treated with FTIs, K- and N-Ras, but not H-Ras, can be efficiently geranylgeranylated (31) , providing an alternative pathway for their post-translational processing.

High-grade astrocytomas do not harbor oncogenic/activating Ras mutations (1 , 6 , 14) ; however, we have demonstrated that Ras activation does occur in these tumors, attributable in part to overexpression of receptor tyrosine kinases (14) . Hence, we have hypothesized that these tumors would be sensitive to growth-inhibition by FTIs. Indeed, we have demonstrated recently (22) that the FTI L-744,832 (Merck Research Laboratories, West Point, PA) inhibits the anchorage-dependent proliferation of five established human malignant astrocytoma cells through a combination of p53-independent G₁-S and G₂-M cell cycle arrest and apoptosis. Furthermore, L-744,832 inhibited the secretion of VEGF, suggesting a further effect of FTIs in vivo (16 , 22) . Other investigators (44) have demonstrated similar results using FTI-276 against U87 cells in an intracranial model. Although these studies serve to demonstrate that FTIs appear capable of inhibiting the growth of astrocytoma cell lines, these and other studies using other human tumor types have not established a causal relationship between Ras activation and drug efficacy. In the present study, we have evaluated the candidate clinical compound SCH66336 (45) to determine its efficacy against astrocytomas and to further our understanding of the relationship of Ras activation and FTIs in human cancers.

Consistent with our previous studies (14) with L-744,832, SCH66336 was capable of inhibiting the growth of established human astrocytoma cell lines, none of which harbor oncogenic Ras mutations. However, not all of the cell lines were sensitive, with IC₅₀ ranging from 0.6 µM in SF767 cells to 32.3 µM in U373 cells. Furthermore, SCH66336 was effective in inhibiting the growth of two of the three human GBM explant xenografts grown in NOD-SCID mice. Total Ras·GTP levels were not predictive of drug efficacy in either the cell lines or the xenograft models; however, this should not be meant to imply that Ras is not a therapeutic target, because isotype-specific Ras·GTP levels were highly predictive of FTI efficacy. In malignant astrocytomas, cell lines, and specimens, FTIs preferentially inhibited the viability/growth of cells/tumors, in which a large amount of the aberrant growth-promoting signals was being transduced through activation of H-Ras. This finding is entirely consistent with the known pharmacodynamics of FTIs, as discussed above, and demonstrates that high levels of H-Ras·GTP, when combined with low levels of K-Ras·GTP and N-Ras·GTP, are predictive of drug efficacy, even in the absence of oncogenic Ras mutations.

Levels of RhoB were measured in the astrocytoma lines and GBM xenografts, because this farnesylated small G-protein has been implicated in transformation and appears to be a major therapeutic target of FTIs. Although we did not measure RhoB activity, expression levels measured by a RhoB-specific antibody on Western blot analysis were not predictive of drug efficacy, unlike the isotype-specific Ras·GTP levels (data not shown). In particular, SF767 (sensitive) and U373 (resistant) cell lines express similar levels of RhoB, and XEN01 (resistant) and XEN08 (sensitive) xenografts express similar levels of RhoB (data not shown). Therefore, our findings in astrocytomas provide compelling evidence that H-Ras is a major therapeutic target of FTIs in human malignancies and that isotype-specific Ras·GTP levels may be a promising diagnostic tool in predicting drug efficacy. Such assays would improve on the current practice of evaluating FTI efficacy through evaluation of inhibition of prelamin A farnesylation in buccal mucosa cells (46 , 47) or in peripheral blood mononuclear cells (48) as a surrogate marker of drug efficacy. Finally, our study demonstrates that the lack of activating Ras mutations is not an appropriate exclusion criterion for clinical trials of FTIs and that these agents may be useful in the management of tumors such as malignant human astrocytomas.

	ACKNOWLEDGMENTS

 
We thank Dr. W. Robert Bishop (Schering-Plough Research Institute, Kenilworth, NJ) for providing the SCH66336 agent.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by an operating grant from the National Cancer Institute of Canada (to A. G.). M. M. F. was supported by a Research Fellowship award from National Cancer Institute of Canada (provided in part by funds from the Terry Fox Run) and by a Research Fellowship from the Medical Research Council of Canada. A. G. is supported by a Clinician Scientist Award from the Medical Research Council of Canada.

² To whom requests for reprints should be addressed, at Division of Neurosurgery, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8. Phone: (416) 603-5740; Fax: (416) 603-5298; E-mail: Abhijit.Guha{at}uhn.on.ca

³ The abbreviations used are: GBM, glioblastoma multiforme; FTI, farnesyl transferase inhibitor; VEGF, vascular endothelial growth factor; IHC, immunohistochemical; HPßCD, hydroxypropyl-ß-cyclodextrin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Received 7/ 5/00. Accepted 4/ 3/01.

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