GROWTH INHIBITION OF ras-DEPENDENT TUMORS IN NUDE MICEBY A POTENT ras-DISLODGING ANTAGONISTYaakov EGOZI1, Boaz WEISZ1, Mali GANA-WEISZ1, Gilad BEN-BARUCH2 and Yoel KLOOG1*1Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel2Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine,Tel Aviv University, Tel Aviv, Israel

A lipophilic farnesyl moiety attached to the carboxyl termi-nal cysteine of ras proteins structurally supports their mem-brane anchorage, required for ras-dependent growth-factorsignaling and for transforming activity of ras oncoproteins. Ithas been shown that inhibition of ras farnesylation can blocktumor growth in nude mice but that some ras-dependenttumors escape such blockage as a result of prenylation of ras.S-trans-transfarnesylthiosalicylic acid (FTS) is a potent ras-dislodging antagonist that does not affect ras prenylation butrather acts on the mature, membrane-bound ras and facili-tates its degradation. Here we demonstrate that FTS inducesreappearance of stress fibers in H-ras-transformed rat-1 cells(EJ cells) in vitro, inhibits their anchorage-independent growthin vitro, and blocks EJ-tumor growth in nude mice. Theanchorage-independent growth of cells expressing ErbB2(B104), but not that of v-raf-transformed cells, is also inhib-ited by FTS, suggesting specificity towards activated ras. FTStreatment (5 mg/kg i.p. daily) caused inhibition (75–80%) oftumor growth in nude mice implanted with EJ, but not inmice implanted with v-raf-transformed cells, with no evi-dence of systemic toxicity. Moreover, FTS treatment in-creased the survival rate of EJ-tumor-bearing mice from 48 to68 days. Here we demonstrate anti-tumor potency in asynthetic, non-toxic, ras-dislodging antagonist acting indepen-dently of farnesyltransferases. Int. J. Cancer 80:911–918, 1999.r 1999 Wiley-Liss, Inc.

A multitude of intracellular signaling cascades associated withcell growth and differentiation are triggered by the active forms ofras proteins (Marshall, 1995). Activation of wild-type isoforms (H,N and K ras), i.e., their conversion from a GDP-bound to aGTP-bound state, is regulated by various types of cell-surfacereceptors including receptor tyrosine kinases and some types ofG-protein-coupled receptors (Marshall, 1995). The wild-type ras isconverted to its inactive, GDP-bound form by GTPase-activatingproteins (GAPs) that bind the ras-GTP and thereby enhance thelatter’s GTPase activity (Scheffzeket al., 1997). ras oncoproteinsthat are insensitive to GTPase-activating proteins are constitutivelyactive and thus contribute to uncontrolled cell growth and transfor-mation (Barbacid, 1987; Bos, 1988).ras-gene mutations involvingactivation are detected in as many as 30% of human tumors,underscoring their significance in the pathogenesis of cancer(Barbacid, 1987; Bos, 1988). Moreover, many types of humantumors (such as various melanomas and breast, pancreatic andcolon carcinomas), even if they do not harbor an activatedrasgene,may depend on active ras, since they express high levels ofras-activating receptors such as epidermal-growth-factor (EGF)receptors or ErbB2 (Sepp-Lorenzinoet al., 1995). In addition,oncogene products that act upstream of ras, or others that have lostras-inactivation activity (e.g., neurofibromatosis-type-B protein),are important in several types of human tumors (DeClueet al.,1992).

These data suggest that ras is an appropriate target for anti-cancer drugs. The knowledge gained from intensive studies ofpost-translational modifications of ras proteins and their signifi-cance for ras-membrane anchorage and function (Caseyet al.,1989; Hancocket al.,1989) has indeed led to the development of anew generation of potential anti-cancer drugs. These drugs arepotent farnesyltransferase inhibitors (FTIs) (Jameset al., 1993;Kohl et al.,1994; Manneet al.,1995; Nagasuet al.,1995; Sunetal., 1995), a group of synthetic compounds that block the farnesyl-

transferase and thus prevent farnesylation of ras, an absoluterequirement for its subsequent processing, membrane associationand transforming activity. Several FTIs display significant inhibi-tion of both anchorage-dependent and anchorage-independent cellgrowth (in several types of cells that express activated ras) andattenuate tumor growth in animal models (Kohlet al., 1994;Nagasuet al., 1995; Sunet al., 1995). However, a number ofquestions remain unresolved. First, several types of ras proteins,particularly K-ras and N-ras (Whyteet al.,1997), appear to escapethe block imposed by FTIs and instead undergo prenylation by theenzyme geranylgeranyl transferase. Other questions concern theresistance to FTIs of several human tumor cell lines that expressactivated ras, and the apparent lack of correlation between some ofthe inhibitory effects of FTIs in cells and the inhibition of rasprocessing (Sepp-Lorenzinoet al., 1995). Taken together, theseconstraints highlight the desirability of developing additionalapproaches, independent of prenyltransferases, to affect the func-tions of Ras oncoproteins.

We have described a novel way to inhibit ras functions in a studyshowing that the synthetic compound S-trans,trans-farnesylthiosali-cylic acid (FTS) inhibits the anchorage-dependent growth ofhuman H-ras-transformed Rat-1 cells (EJ cells) and ErbB2-transformed fibroblasts but not of v-raf-transformed cells (Mar-cianoet al.,1995; Maromet al.,1995). Although FTS can inhibitras methylation in cell-free systems, it does not inhibit rasmodification in intact cells. FTS inhibits cell growth at concentra-tions (5–25 µM) at which ras farnesylation and ras methylation arenot inhibited (Maromet al.,1995). Thus, unlike FTIs, FTS does notblock ras maturation. Its rather specific effects on the growth of EJcells while not affecting rat-1 cells appear to be associatedprimarily with the dislodgment of the mature ras oncoprotein fromthe plasma membrane, an event accompanied by facilitated degra-dation of ras (Haklaiet al., 1998). Consequently, the raf-1-MEK-ERK mitogen-activated protein kinase (MAP kinase) cascaderequired for cell growth and transformation (Segeret al.,1991) ismarkedly inhibited (Gana-Weiszet al., 1997). Several lines ofevidence point to selectivity and specificity of FTS towards ras.First, farnesyl analogs such as N-acetyl-S-farnesyl-L-cysteine orgeranyl thiosalicylic acid do not inhibit EJ cell growth and have noeffect on ERK activity, whereas other analogs, such as geranylgera-nyl thiosalicylic acid, inhibit both EJ cell growth and ERK activity(Aharonsonet al., 1998). Second, membrane localization of theprenylated Gbg of heterotrimeric G proteins is not affected by FTSin EJ cells (Haklaiet al.,1998). Third, FTS does not inhibit growthof cells transformed by N-myristyl (unfarnesylated) H-ras and doesnot reduce the total amount of the N-myristyl H-ras in these cells(Haklaiet al.,1998).

Grant sponsor: Israel Science Foundation; Grant number: (646/96-16.1);Grant sponsors: Israel Cancer Research Foundation (ICRF); SAFAHOFoundation.

Correspondence to: Department of Neurobiochemistry, George S. WiseFaculty of Life Sciences, Tel Aviv University, 69978 Tel Aviv, Israel. Fax:(972) 3-6407643. E-mail: kloog@ccsg.tau.ac.il

Received 3 August 1998

Int. J. Cancer:80,911–918 (1999)

r 1999 Wiley-Liss, Inc.

Publication of the International Union Against CancerPublication de l’Union Internationale Contre le Cancer

Although FTS and its analogs appeared to alter EJ cell morphol-ogy (Marom et al., 1995), it was not clear whether these com-pounds can also affect ras-dependent cell transformation. In thepresent study we examined whether FTS can interfere withcytoskeleton organization, since cell transformation by ras isaccompanied by alterations in the actin cytoskeleton (Symons,1996) and because these and other rac/rho GTPase pathwaysappear to contribute synergistically with the raf-1-MEK-ERKpathway to ras transformation (Jonesonet al.,1996; Prendergastetal., 1996). To further establish whether FTS can affect thetransforming activity of ras, we examined its effects on theanchorage-independent growth of EJ cells in soft agar and on EJtumor growth in nude mice. We show here that FTS induces thereappearance of stress fibers and suppresses membrane ruffling inEJ cells, inhibits the growth of EJ cells in soft agar, attenuatestumor growth, and increases the survival rate of tumor-bearingnude mice. Thus, FTS may represent a new type of anti-cancer drugthat acts directly on membrane domains responsible for Rasanchorage.

MATERIAL AND METHODS

Material

FTS was prepared, purified and analyzed as described in detailelsewhere (Marcianoet al.,1995). Its purity, as assessed by TLC,[1H] NMR and mass spectral analysis, was.95%. For each set ofexperiments, FTS was prepared in chloroform (0.1 M stocksolution) and maintained at270°C. The chloroform was removedfrom the stock solution by a stream of nitrogen prior to use, andFTS was then dissolved either in DMSO (in vitro experiments) orin ethanol (in vivo experiments). The FTS/DMSO solutions werediluted with DMEM/10% FCS to yield a 1003 drug stock solutioncontaining 10% DMSO. A portion of this solution was applied tothe cells at a dilution of 1:100. The FTS/ethanol solution wasalkalinized by the addition of 1N NaOH, then diluted with PBS toobtain a solution of 1.0 mg FTS/ml (pH 8.0, 0.5% ethanol). Thissolution (100–150 µl per mouse) was used in thein vivo experi-ments. Noble agar, used for soft-agar assays, was from Difco(Detroit, MI). Tissue-culture supplies were from Biological Indus-tries (Beit Haemek, Israel). TRITC-labeled phalloidin was fromSigma (St. Louis, MO).

CellsHuman H-ras-transformed rat-1 cells (EJ cells) rat-1 cells,

v-raf-transformed NIH3T3 cells (Maromet al., 1995) and ratneuroblastoma B104 cells (Peleset al., 1991) were used forsoft-agar assays and for thein vivo experiments, as described ineach section. Cells were routinely grown in DMEM/10% FCS at37°C in a humidified atmosphere of 95% air/5% CO2.

AnimalsNude CD1-numale mice weighing 25–30 g (from the Weizmann

Institute of Science, Rehovot, Israel) were used as indicated in eachexperiment.

Labeling of cells with phalloidin-TRITCCells were plated on glass coverslips placed in 35-mm dishes at a

density of 2500 cells per dish. The cells were incubated for 24 hrand then treated either with 0.1% DMSO or with 25 µM FTS for 24hr. They were then fixed with 3% formaldehyde (20 min), treatedwith 0.2% Triton X-100 (5 min) and stained with 0.1 µg/mlphalloidin (40 min). F-actin was visualized by confocal micros-copy.

Soft-agar assaysCells were seeded at a density of 13 104 cells per well in 6-well

plates. The cells (in DMEM/10% FCS) were mixed with 0.5 ml of0.33% noble agar, and the mixture was poured onto a layer of 1.5ml 0.5% noble agar in DMEM/10% FCS. The upper layer of agarwas covered with 250 µl of medium. All agar layers containedeither 0.1% DMSO (control) or FTS dissolved in 0.1% DMSO. We

estimated the numbers and sizes of colonies on day 21, using a lightmicroscope with an image-analyzer program.

Tumor implantationNude CD1-nu mice (6 weeks old) were housed in barrier

facilities on a 12-hr light/dark cycle. Food and water were suppliedad libitum.On day zero, 23 106 EJ or v-raf/NIH3T3 cells in 0.1 mlPBS were implanted s.c. just above the right femoral joint.Immediately after tumor implantation, FTS-treated groups wereseparated from controls. Starting either on day zero or on days12–14, as described in each experiment, each mouse received0.1–0.15 ml of FTS solution (dissolved in ethanol and PBS) or thecarrier solution. The drug was administered s.c. (in the upper backregion) or i.p., as described in each experiment. Tumor growth wasmeasured on the indicated days. Tumor volume was calculated aslength3 width 3 [(length1 width) 4 2]. Statistical significance(FTS-treatedvs.control) was determined using Student’st-test;pvalues of,0.05 were considered to be of statistical significance.

RESULTS

Cell transformation by ras is accompanied by marked changes inthe actin cytoskeleton (Symons, 1996). To discover whether FTScan affect the actin cytoskeleton, rat-1 and H-ras-transformed rat-1(EJ) cells were grown in DMEM/10% FCS in the absence or in thepresence of 25 µM FTS for 24 hr, then stained with phalloidin,which associates with polymeric F-actin. In agreement with otherreports (Prendergastet al.,1996; Symons, 1996), strong labeling ofactin stress fibers was observed in the untransformed fibroblasts butnot in the ras-transformed fibroblasts (Fig. 1). Labeling at the cellperiphery, indicative of membrane ruffling, was however observedin EJ cells, while FTS treatment, which did not appear to affectstress fibers in rat-1 cells, induced their reappearance in EJ cells.The drug treatment was also accompanied by a marked reduction inmembrane ruffling (Fig. 1). Since reorganization of the actincytoskeleton appears to be an essential component of cell transfor-mation (Prendergastet al., 1996; Symons, 1996), these resultssuggest that FTS may have inhibited ras transformation in EJ cells.

To verify this possibility, we examined the effects of FTS on theanchorage-independent growth of EJ cells, using soft-agar assays.As expected, EJ cells formed relatively large colonies in soft agar(Fig. 2a). Colony formation was strongly inhibited (by approx.70%) in the presence of 100 µM FTS. A small, non-significanteffect was observed in the presence of 50 µM FTS, and 25 µM FTSwas not effective (data not shown). In these conditions, FTS causeda decrease in the size of the colonies (Fig. 2a,b) with no apparentcytotoxic effects. Lack of cytotoxicity was evident from theunaltered number of EJ-cell colonies in the FTS-treated cultures.We did, however, observe a 60% decrease in the number ofcolonies in cultures grown in the presence of 200 µM FTS, possiblyreflecting some cytotoxic activity at this dosage. These resultssuggest that FTS can inhibit the transforming activity of ras. In linewith this suggestion was the finding that FTS inhibited theanchorage-independent growth of B104 cells expressing ErbB2(Fig. 3a), which acts upstream of ras (Peleset al., 1991). Thesecells formed relatively large colonies and appeared to be assensitive as the EJ cells to FTS (see Figs. 2,3a). On the other hand,cells transformed by v-raf were not sensitive to FTS; first, earlierexperiments had shown that FTS did not inhibit the anchorage-dependent growth of cells transformed by v-raf (Maromet al.,1995), a constitutively active raf which, unlike raf-1, acts indepen-dently of ras. Second, FTS did not affect the anchorage-independent cell growth of v-raf-transformed cells (Fig. 3b).Compared with EJ cells (Fig. 2) or B104 cells (Fig. 3a), thev-raf-transformed cells formed relatively small colonies (Fig. 3b).This is consistent with the finding that full oncogenic transforma-tion by ras requires raf-dependent and raf-independent signalingpathways (Khosravi-Faret al.,1995).

Next we examined whether FTS can inhibit tumor growth innude mice. EJ cells were implanted s.c. above the right femoral

912 EGOZIET AL.

FIGURE 1 – FTS induces actin re-arrange-ments in EJ but not in rat-1 cells. EJ and rat-1cells were grown on cover slips, treated for 24hr either with 0.1% DMSO (control) or with25 µM FTS, then fixed and labeled withphalloidin, as described in ‘‘Material andMethods’’. The cells were then subjected tofluorescence confocal microscopy. Typical im-ages of control rat-1 cells(a), FTS-treatedRat-1 cells(b), control EJ cells(c) and FTS-treated EJ cells(d) are shown (scale bar, 10µm). Results similar to those shown wereobtained in 3 independent experiments.

FIGURE 2 – Anchorage-indepen-dent growth of EJ cells in soft agaris inhibited by FTS. Soft-agar as-says were performed in quadrupli-cate, as described in ‘‘Material andMethods’’. Photomicrographs weretaken 21 days after cultures wereseeded.(a) Photomicrographs of acontrol culture and of culturestreated with 100 µM FTS or with200 µM FTS (scale bar, 200 µm).(b) Analysis of the inhibitory effectof FTS on the growth of EJ cells insoft agar. The total area of colonieswas calculated using an image ana-lyzer, as described in ‘‘Material andMethods’’. Data are presented asthe area of colonies in drug-treatedcultures as percent of control(means6 SD, n5 4).

913GROWTH INHIBITION OF ras-DEPENDENT TUMORS BY FTS

joint and the mice were then subjected to FTS treatment. In the firstset of experiments, FTS treatment was administered s.c. onalternate days at dosages of 0.5, 1.6 and 3.2 mg/kg, starting on theday of cell implantation. Significant inhibition of tumor growth wasobserved 2 weeks after commencement of FTS treatment atdosages of 1.6 and 3.2 mg/kg (Fig. 4). Tumor growth was notinhibited at a dosage of 0.5 mg/kg (not shown). These data suggestthat FTS can inhibit tumor growth in a dose-dependent manner(Fig. 4c).

The efficacy of FTS treatment in this experimental paradigm ofs.c. administration on alternate days was however limited. Al-though inhibition of tumor growth at a dosage of 3.2 mg/kg wasstill significant (70–80%,p , 0.05) on days 23–27, no differencebetween control and FTS-treated mice was observed thereafter(Fig. 4b). Interestingly, we nevertheless observed a small butsignificant increase (of 13.7%) in the survival rate of the FTS-treated mice (Table I). In an effort to increase the treatment efficacy,we performed additional experiments in which the mice receiveddaily i.p. injections of 5.0 mg/kg FTS. This treatment protocolresulted in significantly stronger inhibition of tumor growth (Fig.

5), reaching about 80% inhibition 3 weeks after its commencement.Notably, the rate of tumor growth in the FTS-treated mice was farslower than in controls. Moreover, even after cessation of drugtreatment, tumors continued to grow more slowly in the FTS-treated mice (Fig. 5). Another parameter that showed improvementfollowing the daily i.p. treatment was the mean survival rate, whichwas significantly increased, from 48 to 68 days, in the FTS-treatedmice (Table I). In a similar set of experiments using the FTStreatment protocol described above, we examined the effects ofFTS on tumor growth in nude mice xenografted with v-raf-transformed cells. As shown in Figure 6, FTS did not affect tumorgrowth in mice implanted with v-raf-transformed cells. Theseresults support the suggestion that the anti-tumor effects of FTS,observed in mice implanted with EJ cells, reflects mainly itsanti-ras activity.

Next we examined whether FTS can inhibit tumor growth inmice that have already developed tumors. In these experiments,FTS treatment (5 mg/kg i.p.) was started 12 to 14 days after cellimplantation, when the mean tumor volume was 0.05 to 0.1 cm3. Asshown in Figure 7, this treatment was also highly efficient, as

FIGURE 3 – FTS inhibits the anchorage-independent growth of B104 cells but notof v-raf-transformed cells. Soft-agar as-says were performed in quadruplicate, asdescribed in ‘‘Material and Methods’’.Photomicrographs were taken 21 daysafter cultures were seeded (scale bar, 200µm). (a) Photomicrographs of a controlB104 cell culture and of cultures treatedwith 100 µM FTS or with 200 µM FTS.The extent of decrease in colony size,estimated as described in Figure 2, indi-cated 506 18% and 856 15% decreasesin colony size for the 100-µM and 200-µM(n 5 4) FTS treatments respectively.(b)Photomicrographs of a control v-raf-transformed cell culture and of culturestreated with 100 µM FTS or with 200 µMFTS. There were no differences betweenthe size of colonies in control cultures andin FTS-treated cultures, where the size ofthe treated colonies presented values of956 15% of controls (n5 4).

914 EGOZIET AL.

indicated by the dramatic decrease in the rate of tumor growth.Mean tumor volumes in controls increased from 0.06 cm3 on dayzero to 12.42 cm3 on day 37, whereas the corresponding increase inthe FTS-treated mice was from 0.11 cm3 to 4.32 cm3. Thus, the

actual tumor volume in the drug-treated mice was about 30% ofthat in controls (Fig. 7).

DISCUSSION

The results of this study demonstrate that the synthetic rasantagonist FTS (Marcianoet al.,1995; Maromet al.,1995; Haklaiet al., 1998) reverses the morphological transformation of H-ras-transformed EJ cells (Fig. 1), inhibits the anchorage-independentgrowth of these cellsin vitro (Fig. 2), and inhibits the growth ofras-dependent tumors in a nude-mouse model (Figs. 5,6). Theobserved inhibition of tumor growth was dose-dependent. Treat-ment with FTS at a dosage of 3 to 5 mg/kg resulted in an 80%decrease in average tumor size. For at least 1 week after cessationof drug treatment at the dosage of 5 mg/kg, the tumors continued to

FIGURE 4 – Effect of FTS on tumor growth induced by EJ cells. EJcells were injected s.c. into the flank areas of nude mice. Daily FTStreatment was started on day 1 and tumor volume was estimated asdetailed in ‘‘Material and Methods’’.(a) Time-dependent inhibition oftumor growth by 1.6 mg/kg FTS injected s.c. (control, n5 8;FTS-treated, n5 4). Significant inhibition (p , 0.05) was observedonly during the first 2 weeks of follow-up.(b) Time-dependentinhibition of tumor growth by 3.2 mg/kg FTS injected s.c. (control,n 5 10; FTS-treated, n5 4). Significant inhibition (p , 0.05) wasobserved throughout the first 4 weeks of follow-up.(c) Dose-dependentinhibition of tumor growth by FTS. Percent inhibition was estimatedfrom the data presented in(a) and(b) and from additional experimentswith 0.5 mg/kg FTS injected s.c. Results were calculated as percentinhibition after 3 weeks of treatment.

TABLE I – SURVIVAL RATES OF TUMOR-BEARING MICEAFTER FTS TREATMENT (DAYS)

Treatment Control FTS

3.2 mg/kg s.c. 40.66 3.8* 46.26 5.35.0 mg/kg i.p. 48.46 6.2** 68.2 6 8.7

*p 5 0.03; **p 5 0.003.–Mean survival rate of mice treated with 3.2mg/kg FTS s.c. (Fig. 4b) was increased by 6 days (approx. 13%); meansurvival rate of mice treated with FTS 5.0 mg/kg i.p. (Fig. 6) wasincreased by 20 days (approx. 41%).

FIGURE 5 – Effect of FTS, 5.0 mg/kg injected i.p., starting on day 1,on EJ tumor growth in nude mice. Cells were injected s.c. into the flankareas of nude mice and daily FTS treatment (5 mg/kg i.p.) began on day1. Tumor volume was estimated at the times indicated in ‘‘Material andMethods’’ (control, n5 5; FTS-treated, n5 5). Significant inhibition( p , 0.05) was observed as of day 25. FTS treatment was stopped onday 30.

915GROWTH INHIBITION OF ras-DEPENDENT TUMORS BY FTS

grow at a relatively slow rate, and the mean rate of animal survivalwas dramatically increased. It is important to note that FTStreatment also effectively reduced tumor growth in mice that hadalready developed tumors of an average size of 0.08 cm3 (Fig. 7).This is a striking demonstration of a functional ras antagonist thataffects mature ras directly and inhibits tumor growth in an animalmodel.

Several inhibitors that affect ras prenylation have also beenshown to block tumor growth in nude mice. These includelovastatine (an inhibitor of 3-hydroxy-3-methyl-glutatyl co-enzyme-A reductase, which catalyzes the formation of the isopren-oid precursor mevalonic acid), limonene (Crowellet al.,1996), andhighly specific inhibitors of the enzyme farnesyltransferase (Jameset al., 1993; Kohlet al., 1994; Manneet al., 1995; Nagasuet al.,1995; Sunet al., 1995). A common attribute of these inhibitors istheir ability to block ras farnesylation, a process required forras-membrane anchorage and for ras biological activity (Caseyetal., 1989; Hancocket al., 1989). FTS, unlike farnesyltransferaseinhibitors, has a direct effect on membrane-anchored ras but noeffect on ras farnesylation (Maromet al., 1995). We have shownthat FTS dislodges ras from H-ras-transformed EJ cells andincreases its rate of degradation in a dose-dependent manner(Haklai et al., 1998). We also showed that FTS has no effect onN-myristylated ras or on the prenylated Gbg sub-units of heterotri-meric G proteins in rat-1 cells (Haklaiet al., 1998). These datasuggest that FTS acts as a specific ras antagonist. Thus, the mode ofaction of FTS on ras proteins clearly differs from that of farnesyl-transferase inhibitors, though both bring about the depletion ofmembrane-bound ras and consequently the inhibition of rassignaling to the MAP kinase cascade (Jameset al., 1994; Gana-

Weiszet al., 1997; Aharonsonet al., 1998). In addition, farnesyl-transferase inhibitors (Prendergastet al., 1996), as well as FTS(Fig. 1), induce alterations in cytoskeleton organization in H-ras-transformed rat-1 cells, most notably the induction of stress fibers.Interestingly, we find considerably more stress fibers in theFTS-treated ras-transformed rat-1 cells than in rat-1 cells (Fig. 1).This could reflect combined, perhaps synergistic, effects of FTS onras-dependent and on Ras-independent signaling to the actincytoskeleton. According to this suggestion, FTS would affect alsoprenylated proteins other than rase.g.,rho and rac. Moreover, wecannot rule out the possibility of secondary effects associated withthe FTS-induced reduction in the amounts of activated ras. Forexample, FTS may induce over-expression of cytoskeleton-associated proteins such asa-actinin and vinculin. This wouldoppose the reduction in the synthesis of such proteins that isaccompanied by ras transformation (Symons, 1996). The observedreduction in membrane ruffling following FTS treatment (Fig. 1)may also reflect a combined action of the drug on ras and on racwhich controls lamellipodia (Symons, 1996). Additional experi-ments are required to determine whether or not FTS can preventassociation of rac and rho with the cell membrane or inducedegradation of these proteins.

It is likely that both the inhibition of transformation and theanti-tumor effect of FTS are due to its direct action on themembrane-anchored activated ras and the subsequent dislodgmentand accelerated degradation of ras, as demonstratedin vitro (Haklaiet al., 1998). We should, however, point out that relatively high

FIGURE 6 – FTS does not inhibit tumor growth in nude miceimplanted with v-raf-transformed cells. v-raf-transformed cells wereinjected s.c. into the flank areas of nude mice and daily FTS treatment(5 mg/kg i.p.) was started on day one. Tumor volume was estimated asdescribed in ‘‘Material and Methods’’ at the times indicated. (Control,n 5 5; FTS-treated, n5 5.)

FIGURE 7 – FTS inhibits tumor growth in tumor-bearing nude mice.EJ cells were injected s.c. into the flank areas of nude mice. Whentumors (mean size 0.08 cm3) were visible in all mice, the animals wereseparated randomly into control (n5 5) and experimental (n5 7)groups. Controls received daily i.p. injections of the vehicle andexperimental mice received daily i.p. injections of FTS 5 mg/kg. Datarepresent the mean increase in tumor volume in control and inFTS-treated mice. Significant inhibition (p , 0.05) was observed as ofday 21.

916 EGOZIET AL.

concentrations of FTS (100 µM) were required for the inhibition ofcell transformation (Fig. 2) as compared with the concentrations(5–25 µM) required for the inhibition of cell growth (Marcianoetal., 1995; Maromet al.,1995). In the light of these differences, andsince at high concentrations FTS can inhibit ras methylation(Marom et al., 1995), we cannot rule out the possibility thatblockage of ras transformation also requires inhibition of rasmethylation.

Importantly, the action of FTS is not limited to activated H-ras inEJ cells. FTS also inhibits the growth of fibroblasts transformed byK-ras-4B (V12) or by N-ras (V13) (data not shown) and of cellstransformed by erbB2 acting upstream of ras (Maromet al.,1995).Moreover, FTS inhibits the basal activities of raf-1 and erk in EJcells (Gana-Weiszet al.,1997), blocks EGF-stimulated erk activi-ties in rat-1 cells (Gana-Weiszet al., 1997), and inhibits themitogenic stimuli of these growth factors in rat-1 cells (Marometal., 1995). Taken together, these data suggest that FTS is a specificras antagonist that affects various types of ras proteins in theiractive state. Thus, the ability of FTS to affect cell growth andtransformation appears to correlate with the activation state of rasrather than with the status ofras-gene mutations. Interestingly, asimilar lack of correlation betweenras-gene-mutation status andinhibition of cell transformation was observed with farnesyltrans-ferase inhibitors (Sepp-Lorenzinoet al.,1995). The data presentedhere lend additional support to the above notion, since they showthat FTS inhibits the anchorage-independent growth of B104 cellsexpressing erbB2 (Fig. 3). It is clear that in these transformed cellsit is the constitutively active erbB2 protein that activates theendogenous wt ras proteins (Peleset al.,1991).

The observed inhibition of ras-dependent and erbB2-dependentbut not v-raf-dependent transformation (Figs. 2,3) and tumorgrowth (Figs. 6,7) provides additional support for the specificity ofFTS towards ras. In this connection, it is important to note that theras-dependent rac/rho pathways and the ras-dependent raf-erkpathway contribute synergistically to cell transformation (Joneson

et al.,1996). Thus, the observed effects of FTS on erk (Gana-Weiszet al., 1997) and on actin cytoskeleton in EJ cells (Fig. 1) wouldsuggest that FTS may disrupt synergistic effects of these ras-dependent pathways.

The present study also demonstrates that FTS is a remarkablysafe compound. At the effective dosage, FTS caused no adversetoxic effects in nude mice. A 2-week toxicology study indicated nochange in blood-cell counts or in liver, kidney or heart enzymes,and further studies showed that the LD50 of FTS in mice is 75 to100 mg/kg, which is far higher than the effective anti-tumor dose(data not shown). Also, chronic FTS treatment of the tumor-bearingnude mice resulted in a significant increase in their rate of survival(from 48 days in the control group to 68 days in FTS-treatedanimals, a mean increase of 20 days). Taken together, these resultsindicate that FTS exhibits a remarkable therapeutic ratio inanimals. Effects similar to those described here with 3 to 5 mg/kgFTS, namely, 70 to 80% inhibition of ras-dependent tumor growth,have been reported with higher doses (20–100 mg/kg) of farnesyl-transferase inhibitors (Kohlet al.,1994; Nagasuet al.,1995). Thus,FTS appears to be one of the most potent non-cytotoxic functionalras antagonists within vivo anti-tumor activity yet described.Finally, it is noteworthy that the anti-tumor effects of FTS are notlimited to human EJ cells, as shown by our recent finding that FTScan also inhibit tumor growth in nude mice and in SCID miceimplanted with human tumor cell lines such as Panc-1 andmelanomas (data not shown). We suggest that the use of FTS as anon-cytotoxic anti-tumor drug may be advantageous, particularlyfor the treatment of tumors that prove resistant to farnesyltransfer-ase inhibitors due to alternative prenylation of their K- or N-rasproteins (Whyteet al.,1997).

ACKNOWLEDGMENTS

We thank Mrs. S. Smith for editorial assistance.

REFERENCES

AHARONSON, Z., GANA-WEISZ, M., VARSANO, T., HAKLAI , R., MARCIANO, D.and KLOOG, Y., Stringent structural requirements for anti-ras activity ofS-prenyl analogues.Biochem. biophys. Acta,1406,40–50 (1998).

BARBACID, M., rasgenes.Ann. Rev. Biochem.,56,779–827 (1987).

BOS, J.L., Therasgene family and human carcinogenesis.Mutat. Res.,195,255–271 (1988).

CASEY, P.J., SOLSKI, P.A., DER, C.J. and BUSS, J.E., p21ras is modified by afarnesyl isoprenoid.Proc. nat. Acad. Sci. (Wash.),86,8323–8327 (1989).

CROWELL, P.L., SIAR-AYOUBI, A. and BURKE, Y.D., Antitumorigenic effectsof limonene and prillyl alcohol against pancreatic and breast cancer.Advanc. exp. Med. Biol.,401,131–136 (1996).

DECLUE, J.E., PAPAGEORGE, A.G., FLETCHER, J.A., DIEHL, S.R., RATNER, N.,VASS, W.C. and LOWY, D.R., Abnormal regulation of mammalian p21rascontributes to malignant tumor growth in von Recklinghausen (type 1)neurofibromatosis.Cell, 69,265–273 (1992).

GANA-WEISZ, M., HAKLAI , R., MARCIANO, D., EGOZI, Y., BEN-BARUCH, G.and KLOOG, Y., A ras dislodging antagonist interrupts MAPK activation.Biochem. biophys. Res. Comm.,239,900–904 (1997).

HAKLAI , R., GANA-WEISZ, M., ELAD, G., PAZ, A., MARCIANO, D., EGOZI, Y.,BEN-BARUCH, G. and KLOOG, Y., Dislodgment and accelerated degradationof ras.Biochemistry,37,1306–1314 (1998).

HANCOCK, J.F., MAGEE, A.I., CHILDS, J.E. and MARSHALL, C.J., All rasproteins are poly-isoprenylated but only some are palmitoylated.Cell, 57,1167–1177 (1989).

JAMES, G.L., BROWN, M.S., COBB, M.H. and GOLDSTEIN, J.L., Benzodpep-tidomimetic BZA-5B interrupts the MAP-kinase-activation pathway inH-ras-transformed rat-1 cells, but not in untransformed cells.J. biol. Chem.,269,27705–27714 (1994).

JAMES, G.L., GOLDSTEIN, S.L., BROWN, M.S., RAWSON, T.E., SOMERS, T.C.,MCDOWELL, R.S., CROWLEY, C.W., LUCAS, B.K., LEVINSON, A.D. andMARSTERS, J.C. JR., Benzodiazepine peptidomimetics: potent inhibitors ofras farnesylation in animal cells.Science,260,1937–1942 (1993).

JONESON, T., WHITE, M.A., WIGLER, M.H. and BAR-SAGI, D., Stimulation of

membrane ruffling and MAP kinase activation by distinct effectors of ras.Science,271,810–812 (1996).

KHOSRAVI-FAR, R., SOLSKI, P.A., CLARK, G.J., KINCH, M.S. and DER, C.J.,Activation of rac1, rhoA and mitogen-activated protein kinases is requiredfor ras transformation.Mol. cell. Biol.,15,6443–6453 (1995).

KOHL, N.E., WILSON, F.R., MOSSER, S.D., GIULIANI , E., DESOLMS, S.J.,CONNER, M.W., ANTHONY, N.J., HOLTZ, W.J., GOMEZ, R.P. and LEE, T.J.,Protein farnesyltransferase inhibitors block the growth of ras-dependenttumors in nude mice.Proc. nat. Acad. Sci. (Wash.),91,9141–9145 (1994).

MANNE, V. and 16OTHERS, Bisubstrate inhibitors of farnesyltransferase: anovel class of specific inhibitors of ras transformed cells.Oncogene,10,1763–1779 (1995).

MARCIANO, D., BEN-BARUCH, G., MAROM, M., EGOZI, Y., HAKLAI , R. andKLOOG, Y., Farnesyl derivatives of rigid carboxylic acid inhibitors ofras-dependent cell growth.J. med. Chem.,38,1267–1272 (1995).

MAROM, M., HAKLAI , R., BEN-BARUCH, G., MARCIANO, D., EGOZI, Y. andKLOOG, Y., Selective inhibition of ras-dependent cell growth by farnesylthiosalicylic acid.J. biol. Chem.,270,22263–22270 (1995).

MARSHALL, C.J., Specificity of receptor-tyrosine-kinase signaling: transientversussustained extracellular-signal-regulated kinase activation.Cell, 80,179–185 (1995).

NAGASU, Y., YOSHIMATSU, K., ROWELL, C., LEWIS, M.D. and GARCIA, A.M.,Inhibition of human tumor xenograft growth by treatment with farnesyltransferase inhibitor B956.Cancer Res.,55,3510–3514 (1995).

PELES, E., BEN-LEVY, R., OR, E., ULLRICH, A. and YARDEN, Y., Oncogenicforms of the neu/HER2 tyrosine kinase are permanently coupled tophospholipase C gamma.EMBO J.,10,2077–2086 (1991).

PRENDERGAST, G.C., DAVIDE, J.P., LEBOWITZ, P.F., WECHSLER-REAY, R. andKOHL, N., Resistance of a variant ras-transformed cell line to phenotypicreversion by farnesyl-transferase inhibitors.Cancer Res.,56, 2626–2632(1996).

SCHEFFZEK, K., AHMEDIAN, M.R., KABSCH, W., WIESMULLER, L., LAWTWEIN,A., SCHMITZ, F. and WITTINGHOFER, A., The ras-rasgap complex: structural

917GROWTH INHIBITION OF ras-DEPENDENT TUMORS BY FTS

basis for GTPase activation and its loss in oncogenicras mutants.Science,277,333–338 (1997).SEGER, R., AHN, N.G., BOULTON, T.G., YANCOPOULOS, G.D., PANAYOTATOS,N., RADZIEJEWSKA, E., ERICSSON, L., BRATLIEN, R.L., COBB, M.H. andKREBS, E.G., Microtubule associated protein-2 kinases, erk1 and erk2,undergo autophosphorylation on both tyrosine and threonine residues:implications for their mechanism of activation.Proc. nat. Acad. Sci.(Wash.),88,6142–6146 (1991).SEPP-LORENZINO, L., MA, Z., RANDS, E., KOHL, N.E., GIBBS, J.B., OLIFF, A.and ROSEN, N., A peptidomimetic inhibitor of farnesyl protein transferaseblocks the anchorage-dependent and -independent growth of human tumorcell lines.Cancer Res.,55,5302–5309 (1995).

SUN, J., QIAN, Y., HAMILTON , A.D. and SEBTI, S.M., ras CAAX peptidomi-metic FTI 276 selectivity blocks tumor growth in nude mice of human lungcarcinoma with K-ras mutation andp53 deletion. Cancer Res.,55,4243–4247 (1995).

SYMONS, M., rho family GTPases: the cytoskeleton and beyond.TrendsBiochem. Sci.,21,178–181 (1996).

WHYTE, D.B., KIRSCHMEIER, P., HOCKENBERRY, T.N., NUNEZ-OLIVIA , I.,JAMES, L., CATINO, J.J., BISHOP, W.R. and PAI, J.K., K- and N-rasgeranylgeranylated in cells treated with farnesyl protein transferase inhibi-tors.J. biol. Chem.,272,14459–14464 (1997).

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