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Experimental Therapeutics |
Greenebaum Cancer Center, University of Maryland Medical System, Baltimore, Maryland 21201 [B. Z., R. G. F.], and Glenolden Laboratory, DuPont Pharmaceuticals Co., Glenolden, Pennsylvania 19036 [G. C. P.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumor necrosis factor 
. This is manifested by an increase in fas mRNA, Fas cell surface expression, and Fas-induced apoptosis. Whereas FTI up-regulates expression of FAS in Ras-transformed cells, it inhibits the expression of vascular endothelial growth factor. Culture of Ras-transformed cells in the presence of the histone deacetylase inhibitor trichostatin A resulted in morphological reversion and G1 arrest (as observed with FTI); however, no induction of Fas was observed. Furthermore, the effects of FTI on Fas-induced death were shown to be independent of RhoB. Therefore, inhibition of oncogenic Ras by FTI can result in two events that alter host-tumor interactions: up-regulation of Fas, rendering tumors more sensitive to immune cytotoxic effector cells, and down-reglation of VEGF, which may inhibit tumor angiogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cellular transformation by the ras oncogene involves activation of signal transduction pathways that promote deregulated cell cycle progression and enhance cell survival (8) . Oncogenic Ras induces constitutive activation of the mitogen-activated protein kinase pathway, culminating in activation of the ERK12 and ERK2 serine/threonine kinases (9) . ERK targets include other cytoplasmic kinases, such a p90Rsk (10) , and nuclear transcription factors such as members of the serum response factor family (11) . Activated Ras promotes cell cycle progression by cooperation with other proto-oncogenes such as Myc, leading to increases in cyclin D1 levels and Cdk2/cyclin E activity, and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 (12 , 13) . Another direct target of Ras is PI3K, the activation of which results in increases in plasma membrane 3,4- and 3,4,5-phosphatydylinositol triphosphate, which provide docking sites for pleckstrin homology domain-containing proteins, including the ser/thr kinase Akt (14) . Akt promotes cell survival by regulating a number of apoptotic pathways (15, 16, 17, 18, 19) . Other cellular pathways are required for Ras transformation, including those regulated by the Ras-related small GTPase proteins of the Rho family (20) .
In addition to regulating genes involved in cell autonomous processes such as proliferation and cell survival, the ras oncogene product regulates the expression of genes that play important roles in regulating host-tumor interactions, such as those encoding VEGF and matrix metalloproteinases (21
, 22)
. Previous studies from our laboratory demonstrated that oncogenic Ras inhibits the expression of the Fas (CD95) death receptor, thus rendering cells resistant to Fas-mediated apoptosis (23)
. Oncogenic Ras also inhibited the up-regulation of Fas by the inflammatory cytokines TNF- and IFN-. Inhibition of Fas-induced death by oncogenic Ras was attributable to inhibition of fas expression from the endogenous fas promoter, because the Ras-induced block could be overcome by ectopic expression of Fas via retroviral transduction (23)
. These observations were subsequently confirmed by others (24)
.
Fas is a type I transmembrane glycoprotein that contains a cytoplasmic death domain homologous to that encoded by members of the TNF receptor supergene family (25) . Trimerization of the Fas receptor by Fas ligand expressed on T or natural killer cells, by soluble Fas ligand, or anti-Fas antibodies, results in the formation of a membrane-proximal signaling complex associated with the Fas death domain. Recruited to this complex through death domain interactions is the adaptor protein FADD/MORT1 (26 , 27) , the death effector domain of which subsequently associates with caspase 8 (28 , 29) . Caspase 8 is activated through autocleavage of the clustered proenzyme, and the caspase cascade is activated, resulting in cell death. The ability of tumor cells to down-regulate death receptor expression could enhance their chances of evading host immunosurveillance mechanisms.
An important goal of cancer therapeutics is to target specifically the genetic lesions responsible for the malignant phenotype (30) . One of the first cases of oncogene-targeted therapy is the development of FTIs that prevent the function of Ras proteins by blocking post-translational attachment of prenyl-moieties to the COOH terminus of Ras, thereby inhibiting membrane localization and function (31 , 32) . This concept has been validated by in vitro studies demonstrating relative specificity for transformed cells (33) , and by studies demonstrating the ability of FTI to regress spontaneously arising tumors in oncogene-transgenic mice (34) . Recently, however, studies have raised questions as to whether Ras is the real target responsible for the antitumor activity of FTI, or whether other farnesylated proteins (such as RhoB, nuclear lamins, or the chaperone protein HDS-2) may be the true intracellular targets that mediate growth inhibition (35, 36, 37) . In addition, few studies have examined how FTI treatment may change key regulators of the host-tumor interaction. Because FTIs have entered into Phase I clinical studies, understanding the mechanisms by which these drugs function is of great importance (36 , 38) . Therefore, we have tested the effects of FTI in our system in which oncogenic Ras blocks the expression of Fas. We find that treating Ras-transformed cells with FTI results in reversal of the inhibition of fas gene expression and renders FTI-treated cancer cells susceptible to Fas-induced apoptosis. These data have implications for host-tumor interactions in patients treated with FTI.
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MATERIALS AND METHODS |
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and IFN- in culture media. Cells were grown in DMEM containing 5% FCS. For some experiments, cells were grown overnight in medium containing 100 units/ml murine IFN- (Genzyme, Cambridge, MA) and/or murine TNF- (PeproTech, Inc., Rocky Hill, NJ). The addition of TNF- and IFN- to the cultures had no adverse effects on the growth or survival of any of the cell lines used in this study. Trichostatin A (Wako Chemicals, Inc., Richmond, VA) was stored as a 1.5 mM stock in DMSO, and was used at 150 nM.
FTI LB42722 and GGTI 286.
The farnesyltransferase inhibitor LB42722, referred to subsequently as FTI, was kindly provided by LG Chemical Ltd. (Taejon, Korea). LB42722 has the following biochemical properties: the IC50 for H-ras farnesylation is 1.2 nM; the IC50 for Rat2/H-ras soft agar colony formation is 5.0 nM; and the IC50 for GGTase I is > 100 µM. A stock solution containing 10 mM FTI was prepared in DMSO and stored at -20°C. FTI was added to cell cultures at a final concentration of 1 µM. This dose of FTI resulted in a >75% inhibition of H-Ras processing (see Fig. 5
). GGTI 286 (Calbiochem, La Jolla, CA) was used at a final concentration of 10 µM and stored as a 10 mM stock in DMSO.
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Northern Blot Analysis.
RNA was isolated from cells grown in the presence or absence of FTI under conditions described in the figure legends. Total cell RNA was isolated using the Trizol reagent (Invitrogen) as suggested by the manufacturer. RNA (15 µg) was size fractionated on 1% denaturing formaldehyde gels and transferred onto nylon membranes (Hybond-N+; Amersham Biosciences, Piscataway, NJ). cDNA probes used for the detection of fas and vegf RNAs and Northern blot analysis were as described previously (23)
. For determination of the effect of FTI on fas mRNA half-life, C3HpBRas cells were grown in the presence or absence of FTI for 48 h, and then actinomycin D was added to a final concentration of 2 µg/ml. Total cellular RNA was harvested at 0, 6, 12, and 24 h after the addition of actinomycin D.
Flow Cytometric Analysis.
Cells were grown in the presence or absence of 1 µM FTI for 48 h. IFN- and TNF- were included in the culture medium for the final 18 h. Cells were then harvested and stained with phycoerythrin-conjugated hamster antimurine Fas MoAb Jo2 (BD PharMingen, San Diego, CA) or with an isotype-matched control for 45 min on ice. Cells were washed, fixed in 1% paraformaldehyde, and analyzed using a FACScan Flow Cytometer and the Cell Quest program.
Ras Assay and Western Blot Analysis.
The plasmid Raf1 Gst-RBD (Ras binding domain; kindly provided by J. L. Bos, Utrecht, The Netherlands) contains an in-frame fusion protein between GST and amino acids 51–131 of Raf1, which encodes a domain that binds only to the activated, GTP-bound form of Ras (40)
. Gst-RBD expression was induced with 0.1 mM isopropyl-1-thio-ß-D-galactopyranoside, and bacteria were lysed by use of a French press. The lysate was clarified by centrifugation, glycerol was added to 10%, and aliquots were stored at -80°C. For preparation of Gst-RBD bound to Sepharose beads, 50 µl of 20% glutathione-Sepharose 4B (Pharmacia, Piscataway, NJ) was mixed with 50 µl of Gst-Raf-RBD bacterial lysate, incubated for 30 min at room temperature with occasional mixing, and washed three times with RIPA buffer. Cell lysates were prepared in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EGTA, 0.25% sodium deoxycholate, 1% NP40, 0.1% SDS, proteinase inhibitors leupeptin, aprotinin, and PMSF]. Cell lysate at 250 µg in 400 µl of RIPA buffer was added to Gst-Raf-RBD beads and rotated at 4°C for 1 h. The complexes were then washed, and the final pellet was resuspended in 20 µl of SDS-PAGE loading buffer and boiled to elute bound Ras. Proteins were size-fractionated 10% SDS-3-(N-morpholino) propane sulfonic acid NuPAGE gels (Novex, San Diego, CA), transferred to Immobilon-P membranes (Millipore, Bedford, MA), and probed with Ras MoAb Y13-259 (Santa Cruz Biotechnology, Santa Cruz, CA). Ras protein was visualized by enhanced chemiluminescence (Amersham Biosciences).
Morphological Analysis of Apoptosis.
Cells were cultured at 1 x 105 cells/well in six-well plates with or without FTI for 48 h. In some cultures, IFN- and TNF- (100 units/ml) were added for the final 18 h. Anti-Fas MoAb Jo2 or isotype-matched control antibody was added to wells at the concentration of 1 µg/ml for an additional 6 h. In indicated cultures, the broad specificity apoptosis inhibitor BOC (25 µM; Enzyme Systems Products, Livermore, CA) was added 30 min before the addition of antibody. For determination of the percentage of apoptotic cells, photomicroscopy was performed using an Olympus IX70 microscope. These photographs were used to enumerate the cells with normal or apoptotic morphology, and the percentage of apoptotic cells was determined. At least 200 cells were counted for each data point.
Analysis of Cell Death by Trypan Blue Exclusion.
Cells were cultured with or without FTI for 48 h, and in the presence or absence of cytokine during the final 18 h. The anti-Fas MoAb Jo2 or an isotype-matched control antibody control was added at 1 µg/ml. After an additional 6 h of culture, adherent and floating cells were collected and counted after a 1:1 dilution with trypan blue. Experiments were performed twice, and duplicate wells were analyzed in each experiment. Data are expressed as the percentage of dead (apoptotic) cells in the total cell population.
Caspase Assays.
Cells were cultured with or without FTI for 48 h, and in the presence or absence of cytokine for the final 18 h. Anti-Fas MoAb Jo2 isotype control was added at 1 µg/ml. After 6 h incubation, cells were pelleted and lysed in 50 mM HEPES (pH 7.9), 10 mM EDTA, 1% Triton X-100, 4 mM NaPPi, 10 mM NaF, 2 mM NaVO4, 1 mM PMSF, 100 mM NaCl, 2 µg/ml aprotinin, 2 µg/ml leupeptin. Lysates were clarified by centrifugation for 10 min at 12,000 rpm, 4°C, and extracts were aliquoted and stored at -80°C. Protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). Caspase assays were performed in 96-well plates. Each well contained 200 µl of caspase assay buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM DTT] to which 10 µg of cell extract and 10 µM Ac-DEVD-AMC fluorogenic substrate (BD PharMingen) were added. The reactions were incubated at 37°C for 2 h. Measurement of the AMC cleavage product was performed using a CytoFluor II Microplate Fluorescence Reader (PerSeptive Biosystems, Inc., Bedford, MA). Experiments were performed in triplicate, and results were expressed as the mean ± SE.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TNF- (Fig. 1
, Lanes 1 and 2). Pools of Ras-transformed fibroblasts were generated by infection of C3H10T1/2 with the retrovirus pBabe-Ras, which encodes an activated H-ras (position 12 valine). These cells exhibit undetectable basal levels of fas mRNA and much lower levels of cytokine-induced fas mRNA expression (Fig. 1
, Lanes 5 and 6) compared with C3HpBpuro control cells. Similar results were obtained with a Ras-transformed clone of C3H10T1/2 (61LpBpuro; Fig.1
, Lanes 9 and 10). To determine the effect of FTI on fas expression by these cell lines, cells were cultured for 48 h in the presence of 1 µM FTI. IFN- and TNF- were added to some cultures for the final 18 h of the incubation period, and mRNA was analyzed. FTI had modest effects on C3HpBpuro cells, inducing an increase in fas mRNA levels in the absence of cytokines (compare Fig. 1
Lanes 1 and 3). FTI treatment of C3HpBRas also resulted in a significant increase in basal levels of fas mRNA expression (Fig. 1
, Lane 5 versus Lane 7) and a notable increase in cytokine-induced levels (Fig. 1
, Lane 6 versus Lane 8). Similar FTI-induced increases in fas mRNA were obtained with the 61LpBpuro clone (Fig. 1
, Lane 9 versus Lane 11). Of note, cytokine-induced levels of fas mRNA in the Ras-transformed cell lines increased to control levels after treatment with FTI (compare in Fig. 1
, Lanes 8 and 12 with the control Lane 4). These data indicate that FTI can reverse the Ras-mediated inhibition of fas mRNA expression under basal and cytokine-induced conditions.
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, DR5). Expression of the TNF- type I receptor mRNA was also not regulated by Ras transformation (data not shown). Therefore, down-regulation of death receptor gene expression is not a general property of Ras transformation.
The secretion of vascular endothelial growth factor (VEGF) has been shown to be increased by oncogenic Ras (22)
. The effect of FTI treatment on vegf gene expression was determined. The up-regulation of vegf RNA expression by oncogenic Ras was demonstrated (Fig. 1
; compare Lane 1 with Lanes 5 and 9). VEGF expression in the untransformed C3HpBpuro control cell line was up-regulated by cytokines, FTI, and the combination of these treatments. The opposite effects were observed in the C3HpBRas and 61LpBpuro lines: incubation in the presence of inflammatory cytokines, FTI, or the combination resulted in an inhibition of vegf mRNA compared with cultures with no additions (Fig. 1
; compare Lane 5 with Lanes 6–8, and Lane 9 with Lanes 10–12). The data demonstrate that effects on cellular mRNA induced by Ras transformation can be reversed by FTI treatment, resulting in either an increase (Fas) or a decrease (VEGF) in the expression of specific genes.
Induction of Fas Plasma Membrane Expression by FTI.
Experiments were performed to determine whether up-regulation of fas mRNA by FTI correlated with an increase in plasma membrane expression. Cells were cultured in 1 µM FTI for 48 h with the addition of IFN- and TNF- for the final 18 h, and stained with the anti-Fas MoAb Jo2 or isotype-matched control. Under these growth conditions, there is significant Fas cell surface expression by C3HpBpuro; this is unchanged by culture in the presence of FTI (Fig. 2B)
. In contrast, FTI treatment of Ras-transformed cells induces levels of cell surface Fas expression (Fig. 2, C and D)
to nearly that observed in the control cell line (Fig. 2B)
. Fig. 2E
shows data from the 61LpBFas27 clonal cell line, which was derived by infection of C3H-Ras61L-transformed cells with a retrovirus encoding murine Fas. These cells express Fas under the regulation of the retroviral LTR promoter/enhancer; and, hence, Fas expression is not inhibited by oncogenic Ras (clone 27 expresses Fas levels equal to that of cytokine-treated C3HpBpuro parental lines). As expected, culture in FTI has no effect on Fas expression in this clone, consistent with the hypothesis that oncogenic Ras targets fas expression from the endogenous chromosomal fas gene. These data confirm that FTI can reverse the inhibition of Fas expression at both the RNA and protein levels.
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and TNF- were added for the final 18 h of the culture period. Cross-linking of Fas was accomplished by the addition of anti-Fas MoAb Jo2 or isotype-matched control antibody. As shown in Table 1
, Fas-mediated killing of C3HpBpuro was greatly increased by culture in the presence of cytokines, consistent with the effects of these cytokines on Fas expression. Note that IFN- and TNF-, alone or in combination, are not toxic to any of the cell lines used in this study. Addition of FTI had no significant effect on killing in the presence of cytokines (68.8% versus 72.0%) but did increase Fas-mediated death in the absence of cytokines (11.9% versus 37.6%). This correlates with FTI-induced up-regulation of fas mRNA in C3HpBpuro cells (Fig. 1
, Lane 1 versus Lane 3). The pattern of Fas-induced cell death was quite different for the C3HpBRas line. Fas-induced death was equal to control levels when C3HpBRas cells were grown in the absence of cytokines (7.0% versus 6.8%). Addition of cytokines increases this to 13.8%, much less than the 68.8% observed with C3HpBpuro. When FTI is added to C3HpBRas cultures without cytokines, killing increases modestly to 13.4%. Addition of FTI to C3HpBRas cultures containing cytokines increases the frequency of Fas-induced death to 64.5%, nearly equal to levels observed with C3HpBpuro under the same conditions. In contrast, in the Ras-transformed line 61LpBFas27, which constitutively expresses Fas via the retroviral LTR, Fas-mediated death is independent of cytokines, oncogenic Ras, and treatment with FTI. Together, these data confirm the inhibition of Fas-mediated killing by ras oncogenes and demonstrate that Ras-transformed cells can be rendered sensitive to Fas-induced death by treatment with FTI.
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demonstrate that the Fas-mediated death was significantly inhibited by BOC, both in the presence and absence of FTI. No Fas-induced death occurred in the C3HpBRas cell line in the absence of FTI, but this was triggered in the presence of FTI. Importantly, this death was also inhibited by BOC (Fig. 3B)
. Together, these data are consistent with the notion that death in this system is apoptotic. To further prove this point, the levels of caspase 3 (i.e., DEVD-cleaving) activation were studied. As shown in Fig. 4
, high levels of caspase activity were observed when C3HpBpuro cells were exposed to Jo2; FTI treatment had minimal effects on this activity. In contrast, C3HpBRas cells were resistant to Jo2 antibody-induced caspase activation unless cultured in the presence of FTI; under these conditions, caspase activity was induced to levels similar to those of C3HpBpuro cells. Taken together, these data demonstrate that FTI reverses oncogenic Ras-mediated inhibition of Fas-induced apoptosis.
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, there are very low levels of Ras-GTP present in growing C3HpBpuro cells (undetectable levels in this experiment; Fig. 5
, Lane 1). Under normal growth conditions, C3HpBRas and 61LpBpuro cells express readily detectable levels of Ras-GTP (Fig. 5
, Lanes 2 and 4, respectively). After culture for 48 h in FTI, Ras is still detectable, but for both cell lines the levels of full-length Ras have declined significantly (Fig. 5
, Lanes 3 and 5). Furthermore, a faint, slower migrating band is observed (Fig. 5
, arrow) that corresponds to unprocessed Ras. In addition, two smaller bands are observed that represent Ras degradation products (Fig. 5
, arrowheads).
Oncogenic Ras Does Not Alter the Stability of the Fas mRNA.
The Ras-mediated inhibition of Fas expression appears to occur at the mRNA level. A direct role for Ras in regulation of fas gene transcription could explain this; however, effects on the half-life of fas mRNA are also possible. To explore the latter possibility, we cultured C3HpBRas cells with or without FTI for 48 h. RNA was prepared from untreated control cultures, and then actinomycin D was added (2 µg/ml) to block further transcription. RNA was prepared from cultures at 6, 12, and 24 h after addition of actinomycin D, and Northern blot analysis was performed. As demonstrated in Fig. 6
, levels of fas mRNA decreased during the culture period in actinomycin D. Although cells grown in FTI expressed higher levels of fas mRNA as expected, densitometric analysis of the rate of mRNA decay (Fig. 6B)
showed that the half-life of fas mRNA under the different growth conditions was very similar. Therefore, the increase in fas mRNA levels induced by culture in the presence of FTI cannot be ascribed to increased stability of the fas mRNA.
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and AML1-ETO) alter the pattern of gene transcription by the recruitment of histone deacetylase inhibitors to the promoters of genes required for cell cycle progression or for lineage-specific differentiation (42, 43, 44)
. We hypothesized that oncogenic Ras could alter tumor cell gene expression through downstream kinase pathways that effect the activities of multiple transcription factors; in the case of the fas gene, this could lead to the assembly of a repressor complex that blocks transcription of the gene. To test this hypothesis, we treated C3HpBRas cells with TSA, a HDAC inhibitor that has been shown to cause morphological reversion and G1 arrest of Ras-transformed cells (45)
. As shown in Fig. 7
, cells cultured in the presence of TSA for 48 h assume a flat morphology similar to that observed in FTI-treated cells (Fig. 7, E and B
, respectively). Both drugs induce a significant arrest in G1 of the cell cycle (Fig. 7G)
. When the anti-Fas antibody Jo2 is added to these cultures, >50% of FTI-treated cells undergo rapid induction of apoptosis (Fig. 7C)
, whereas TSA-treated cells remain resistant (Fig. 7F)
. This suggested that despite its effects on cell growth and morphology, TSA did not overcome the block to fas gene expression. This was shown to be true by culturing C3HpBRas cells in FTI or TSA and analyzing the effects on fas mRNA levels. Consistent with the data in Fig. 1
, C3HpBRas cells express very low levels of fas mRNA, which is minimally induced by inflammatory cytokines (Fig. 8
, Lanes 1 and 2); addition of FTI to cultures dramatically increases the levels of fas mRNA in the presence of cytokines (Fig. 8
, Lane 4). In contrast, there is no enhancement of fas mRNA levels by culture in the presence of TSA (Fig. 8
, Lanes 5 and 6); there is low-level induction by cytokines, but no synergistic effect of TSA. Interestingly, although TSA did not effect fas gene expression, it did decrease the level of vegf mRNA in Ras-transformed cells (Fig. 8
; compare Lanes 1 and 5). These studies demonstrate that inhibition of fas gene expression by oncogenic Ras is not mediated by HDAC activity, and that morphological reversion and G1 arrest per se are not sufficient to change the pattern of fas gene expression.
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, the RhoB-Dead vector had no effects on cell viability. In these experiments, parallel cultures received either no treatment (Fig. 9A; C)
, or the addition of FTI (Fig. 9A; F)
, GGTI (Fig. 9A; G)
, or FTI plus GGTI (Fig. 9A; B)
during the final 18 h of culture. These drugs had no effect on viability of RhoB-Dead-transfected cells. In contrast, RhoB-GG-transfected cells demonstrated >50% death of transfected cells; this was unaffected by FTI, but the toxicity was blocked when GGTI was added to the cultures. Of importance, in the presence of both drugs the RhoB-GG-induced cell death was blocked. Similar results were obtained with RhoB-V14, confirming that the farnesylated form of RhoB is not toxic but, rather, the RhoB-GG mediates cell death. (Note that in the presence of FTI, RhoB is preferentially GG modified, which is toxic; in the presence of GGTI, RhoB is farnesylated, but not GG modified, and no toxicity ensues.) In these transient assays, the expression of all three HA-tagged isoforms of RhoB was confirmed by anti-HA Western blots (data not shown). We surmise that in these experiments, a significant portion of RhoB-V14 is GG modified, and the toxic effects of this RhoB isoform are inhibited by GGTI.
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. As a control, cells were cultured in FTI and cytokines, and Jo2 was added for 6 h (approximately 50% cell death). In contrast to Jo2-treated cells, there is virtually no induction of caspase activity during the death induced by RhoB overexpression. This makes it unlikely that RhoB-induced death in this system works through the Fas death receptor and indicates that death occurs through a caspase-independent pathway. A second experiment directly asked whether inhibition of RhoB-GG formation using GGTI (which is a highly effective inhibitor, as shown in Fig. 9A
) would inhibit the effects of FTI on Fas-mediated killing. C3HpBRas cells were cultured in the presence of FTI, GGTI, or both for 48 h, followed by the addition of Jo2 (all cultures received cytokines for the final 18 h). Neither controls nor GGTI-treated cells were sensitive to Jo2 (Fig. 9C
, Lanes 1–4). FTI-treated cells were susceptible to Fas-induced death when cytokines were present (Fig. 9C
, Lane 6), and the addition of GGTI to FTI-treated cultures did not inhibit FTI-mediated Fas up-regulation (Fig. 9C
, Lane 8). These data indicate that although GGTI is an effective inhibitor of RhoB-GG-mediated death (Fig. 9A)
, it does not prevent FTI-mediated, Fas-induced cell death. Therefore, FTI up-regulation of Fas is independent of RhoB. |
DISCUSSION |
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Our previous work showed that Ras transformation can induce down-regulation of fas gene expression, thus rendering tumor cells resistant to Fas-induced apoptosis (23)
. Oncogenic Ras inhibited fas gene expression under normal growth conditions and those that mimic an inflammatory tumor milieu, in which the inflammatory cytokines IFN- and TNF- normally up-regulate Fas expression and render cells sensitive to cross-linking by FasL-expressing T cells or natural killer cells. Killing of Ras-transformed cells was inhibited under these conditions. We now confirm and extend these results by demonstrating that treatment of Ras-transformed cells with FTI reverses the block to fas gene expression. FTI treatment induces an increase in fas mRNA expression under basal and cytokine-induced conditions, and an increase in Fas plasma membrane expression that was sufficient to render the FTI-treated tumor cells sensitive to Fas-induced death. Although the changes in the level of cell surface Fas expression were not dramatic, data in other systems demonstrate that similar degrees of change in Fas expression can alter the sensitivity of target cells to death induction (47
, 48)
. We show that the increased levels of fas mRNA after FTI treatment are most likely attributable to increased transcription, because the half-life of fas mRNA is identical before and after treatment with FTI. This is an important observation, because the fas mRNA contains multiple AU-rich elements that could confer differential regulation of the mRNA half-life under different physiological conditions (49)
. Changes in the rate of fas mRNA processing or nuclear transport have not been excluded.
Oncogenic Ras has opposing influences on the regulation of two critical genes involved in host-tumor interactions: up-regulation of VEGF expression that should promote tumor growth through increased angiogenesis, and inhibition of Fas expression that could enable the tumor to escape immunosurveillance. Our data demonstrate that FTI treatment can reverse both of these events. This may help to explain the efficacy observed for FTI treatment in murine animal models in vivo (34)
. We speculate that the differential effects of oncogenic Ras on fas and vegf gene expression occur through opposing actions at the respective promoters. The mechanisms by which this occurs are unknown, although changes in the pattern of gene transcription are often regulated at the level of chromatin structure, such as the status of histone acetylation (50, 51, 52)
. Accordingly, we examined the effects of the histone deacetylase inhibitor TSA on the expression of the fas and vegf genes in our system. TSA induced morphological reversion and growth arrest of Ras-transformed cells as described by others (45)
; however, unlike FTI, it did not up-regulate fas gene expression (Fig. 8)
. With respect to regulation of fas gene expression by oncogenic Ras, we conclude that (a) Ras inhibition of fas expression is not mediated by histone deacetylation; and (b) phenotypic reversion and growth arrest per se are not sufficient to up-regulate fas gene expression. It is likely that the negative effect of oncogenic Ras on fas gene expression occurs at the level of the Fas promoter through mechanisms that do not involve recruitment of co-repressor/HDAC complexes (53)
.
In contrast, both TSA and FTI led to a down-regulation of vegf mRNA in Ras-transformed cells. Oncogenic Ras is known to up-regulate expression of VEGF, acting via either the Raf/MAPK or the PI3K pathway (54
, 55)
. Hence, inhibition of VEGF expression by FTI is not surprising and has been observed previously (22)
. The mechanism by which TSA inhibits VEGF expression in Ras-transformed cells is less clear. Others have recently shown that increased VEGF expression by cancer cells is attributable to increased HDAC activity, which leads to transcriptional repression of the p53 and VHL tumor suppressor genes (56)
. It was demonstrated that TSA inhibited tumor VEGF expression by up-regulating p53 and VHL, and causing repression of the transcription factor HIF-1, a key positive regulator of VEGF gene expression (56)
. A model can be proposed in which oncogenic Ras up-regulates VEGF expression through induction of HDAC activity; this could act to repress p53 and VHL, stabilizing HIF-1, and leading to increase vegf gene expression (57)
. This model is currently being tested. These studies represent the first demonstration that up-regulation of vegf gene expression by oncogenic Ras can be inhibited by HDAC inhibitors.
FTIs were initially developed as a class of drugs for which the main mechanism of antiproliferative action was thought to be inhibition of Ras function (32)
. However, a variety of studies have led to the concept that other farnesylated cellular proteins may be the true targets of FTI, and most data implicate RhoB (46
, 58)
. As put forth by G. C. Prendergast and colleagues, the FTI-Rho hypothesis states that in the presence of FTI, RhoB prenylation shifts to RhoB-GG modification, and these authors have demonstrated that RhoB-GG mediates growth inhibition and apoptosis when expressed ectopically (39)
. Furthermore, the requirement for RhoB in FTI-mediated antiproliferative events has recently been demonstrated using murine embryo fibroblasts from RhoB knockout mice (59)
. We examined the possibility that the effects of FTI on Fas expression observed in our system were mediated by RhoB-GG. The data in Fig. 9
demonstrate that RhoB-GG is toxic when overexpressed in Ras-transformed cells and kills by a caspase-independent mechanism. Interestingly, the morphology of cells appears apoptotic, and the mechanism of RhoB-GG-induced death remains unclear. When GGTI is added to cells cultured in FTI, there is no inhibition of FTI-induced up-regulation of fas gene expression or Fas-induced apoptosis. From these data we conclude that RhoB-GG is not required for changes in the pattern of fas gene expression mediated by FTI and must occur through a different molecular pathway.
Under what clinical circumstances might down-reglation of Fas be of significance? The potential role of the Fas/FasL death signal in immune-mediated tumor killing has been discussed above. The role of the Fas/FasL system in chemotherapy-induced apoptosis remains controversial (60) . This model postulates that chemotherapy induces expression of FasL on tumor cells, with subsequent ligation of Fas causing tumor cell death. Although it appears clear that many chemotherapeutic agents induce cancer cell death through apoptosis, in most cases this seems to occur through the mitochondrial pathway with the generation of the Apaf-1/caspase 9/cytochrome c complex (60) . However, in the case of 5-FU treatment of colon cancer cell lines, it appears that there may be a critical role for the Fas/FasL system, because death is inhibited by blocking antibodies directed against Fas or FasL (61, 62, 63) . In the context of the data presented here, it will be of interest to determine whether oncogenic Ras can render colon cancer cells resistant to 5-FU by inhibition of Fas expression. If this does occur, then one may expect to see a synergy between FTI and 5-FU in the treatment of colorectal cancers.
At least five different FTIs have entered clinical testing. Correlative laboratory studies are in progress to better define the precise intracellular target(s) of FTI action and to develop clinical markers of FT inhibition in patient samples (36 , 38) . We suggest that clinical assays should also be developed to monitor effects of FTI on the expression of genes involved in host-tumor interaction, such as Fas and VEGF. It should be noted that the work presented in this study used the H-Ras oncogene, and that targeting the K-Ras and N-ras oncogenes by FTI may prove more difficult.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at University of Maryland Greenebaum Cancer Center, Bressler Research Building, Room 7-023, 655 West Baltimore Street, Baltimore, MD 21201. Phone: (410) 328-0372; Fax: (410) 328-6559; E-mail: rfent001{at}umaryland.edu 
2 The abbreviations used are: ERK, extracellular signal-related kinase; VEGF, vascular endothelial growth factor; TNF-, tumor necrosis factor ; FTI, farnesyltransferase inhibitor; LTR, long terminal repeat; HA, hemagglutinin; RIPA, radioimmunoprecipitation assay; PMSF, phenylmethylsulfonyl fluoride; MoAb, monoclonal antibody; GGTI, geranylgeranyl-transferase inhibitor; TSA, trichostatin A; HDAC, histone deacetylase inhibitor; RhoB-GG, geranylgeranyl-modified RhoB; EGFP, enhanced green fluorescent protein; GGT-I, geranylgeranyltransferase type I; 5-FU, 5-fluorouracil.
Received 6/ 6/01. Accepted 11/14/01.
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