Farnesyl Protein Transferase Inhibitors as Potential ...farnesyl moiety for both normal and oncegenic activity. Inhibitors of the enzyme that catalyzes this reaction, farnesyl protein

Vol. 6, 267-282, April 1997 Cancer Epidemiology, Biomarkers & Prevention 267

Review

Farnesyl Protein Transferase Inhibitors as Potential

Cancer Chemopreventives

Gary J. Kelloff,’ Ronald A. Lubet, Judith R. Fay,Vernon E. Steele, Charles W. Boone, James A. Crowell,and Caroline C. Sigman

Chemoprevention Branch, Division of Cancer Prevention and Control,

National Cancer Institute, Bethesda, Maryland [0. J. K., R. A. L., V. E. S.,

C. W. B., J. A. C.], and CCS Associates, Mountain View, California [I. R. F.,

C. C. S.]

Abstract

Among the most important targets for chemopreventiveintervention and drug development are deregulated signaltransduction pathways. Ras proteins serve as centralconnectors between signals generated at the plasmamembrane and nuclear efTectors; thus, disrupting the Rassignaling pathway could have significant potential as a

cancer chemopreventive strategy. Target organs for Ras-based chemopreventive strategies include those associatedwith activating ras mutations (e.g., colorectum, pancreas,and lung) and those carrying aberrations in upstreamelement(s), such as growth factors and their receptors.Ras proteins require posttranslational modification with afarnesyl moiety for both normal and oncegenic activity.Inhibitors of the enzyme that catalyzes this reaction,

farnesyl protein transferase (FPT) should, therefore,inhibit Ras-dependent proliferative activity in cancerousand precancerous lesions (J. B. Gibbs et aL, Cell, 77:175-178, 1994). Because growth factor networks areredundant, selective inhibition of signaling pathwaysactivated in precancerous and cancerous cells should bepossible.

Requirements for Ras farnesylation inhibitorsinclude: specificity for FPT compared with other prenyltransferases; specificity for FPT compared with otherfarnesyl PP1-utilizing enzymes; ability to specificallyinhibit processing of mutant K-ras (the most commonlymutated ras gene in human cancers); high potency;selective activity in intact cells; activity in vivo; and lackof toxicity. Numerous FPT inhibitors have been identifiedthrough random screening of natural products and byrational design of analogues of the two substrates,farnesyl PP1 and the COOH-terminal CAAX motif of Rastetrapeptides. A possible testing strategy for developingFPT inhibitors as chemopreventive agents includes thefollowing steps: (a) determine FPT inhibitory activityin vitro; (b) evaluate selectivity (relative to other protein

prenyl transferases and FP’f-utiuizing enzymes); (c)determine inhibition of Ras-mediated effects in intactcells; (d) determine inhibition of Ras-mediated effects invivo (e.g., in nude mouse tumor xenografts); and (e)

determine chemopreventive efficacy in vivo (e.g., incarcinogen-induced A/J mouse lung, rat colon, or hamsterpancreas).

Mechanism-based Strategies in the Development ofCancer Chemopreventive Agents: Signal TransductionPathways

This paper is the second in a series on mechanism-based strat-

egies used by the National Cancer Institute’s ChemopreventionBranch for development of cancer chemopreventive drugs. Thereader is referred to the first paper in this series ( I ) and ourprevious reviews (2-4) for a general discussion of chemopre-vention and the rationale for this approach. One of the mech-anism-based strategies that can be targeted for cancer chemo-

preventive drug development is interference with deregulatedcellular growth controls.

Cells respond to signals from extracellular stimuli via acomplicated network of highly regulated events collectively

referred to as signal transduction pathways. Stimulation ofthese pathways results in changes in transcriptional activity (5,6). Whereas normal cells respond appropriately to extracellularstimuli, many precancerous and cancerous cells have lost this

ability and display aberrant signaling (7).Many sites on the deregulated signaling pathways are

potential targets for chemopreventive intervention (8-10).Blocking signal transduction pathways at the cell membrane viainhibition of receptor protein tyrosine kinases, specifically

EGFR,2 was the subject of the previous paper in this series (1).Interference with mediators, specifically activated Ras proteins,is the subject of this paper.

Targeting Specific Cytoplasmic Intracellular Mediators:Ras Proteins

ras genes code for Mr 2] ,000 proteins that belong to a largesuperfamily of GTP-binding proteins that cycle between an

active GTP-bound and an inactive GDP-bound state. Four hu-man ras genes have been identified: H-, K-4A, K-4B, andN-ras ( 1 1 ). Normal Ras proteins serve as molecular switches inthe mitogenic signal transduction pathway and are crucial reg-ulators of many physiological functions including cell growthand differentiation. Activating mutations in ras genes result in

Received 9/4/96; revised 1/9/97; accepted 1/10/97.

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.

I To whom requests for reprints should be addressed, at the Chemoprevention

Branch, National Cancer Institute, Executive Plaza North, Suite 201, 6130 Ex-

ecutive Boulevard, Rockville, MD 20852.

2 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF,

epidermal growth factor; FF1’, farnesyl protein transferase; IGF. insulin-like

growth factor; PDGF, platelet-derived growth factor; MAPK, mitogen-activated

protein kinase; MNU, N-methyl-N’-nitrosourea; AOM, azoxymethane; AML.

acute myeloid leukemia; ACF, aberrant crypt foci; FPP, farnesyl PP; GGTase Iand II, geranylgeranyl protein transferase types I and II, respectively; HMG-CoA,

hydroxymethylglutaryl-CoA.

on February 13, 2020. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

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268 Review: FPT Inhibitors as Cancer Chemopreventives

GF (EGF, PDGF, IGF, GF Ligands)

I Cytokines (IL-i)� � rir�c��

proteins that are “stuck” in the active GTP-bound state andconstitutively transmit growth signals (I 1, 12). Oncogenic ac-

tivity can also result from overexpression of normal Ras pro-teins (13). As discussed below (see “Specific Human Cancers

Associated with Activating ras Lesions”), oncogenic mutationsin ras genes have been identified in a variety of human tumors

(14).Because Ras proteins are central connectors between sig-

nals generated at the plasma membrane and nuclear effectors(see “Ras and Signal Transduction”), disrupting Ras signaling

has significant potential as a cancer chemopreventive strategy.Possible methods include using antisense oligonucleotides that

block mutant ras genes or disrupting interactions of Ras withdownstream effector molecules (15).

Another strategy is based on the observation that Rasproteins require posttranslationa] modification with a farnesylmoiety for oncogenic activity (16-18). Inhibitors of the enzyme

that catalyzes this reaction, FPT, should therefore inhibit Ras-

dependent proliferative activity in cancerous and precancerouslesions (19). The rationale for using Ras antagonists as chemo-preventive agents, analysis of potential clinically relevant target

organs, and identification of inhibitors with specificity toward

FI�T are discussed.

Ras and Signal Transduction

An important role for Ras proteins is as cytoplasmic mediators

of signaling by extracellular stimuli including growth factorssuch as EGF, PDGF, and IGF and cytokines such as theinterleukins (Fig. 1). Ras proteins can be activated by a variety

of signals, including receptor tyrosine kinases (e.g., for EGF,PDGF, and IGF), receptor-associated tyrosine kinases (for cy-tokines), and G-protein-coupled seven-member serpentine re-

ceptor (20-23). The most extensively studied pathways are viaactivation by growth factors and their receptors, particularly

EGFR. Ligand binding to receptor tyrosine kinases causes

receptor dimerization and autophosphorylation. The phospho-rylated tyrosine residue binds growth factor receptor binding

Fig. 1. Signal transduction pathways: Ras-mediation.

Ras proteins can be activated by a variety of signals,

including receptor tyrosine kinases. The most exten-

sively studied pathway is activation by EGFR. Ligand

binding to EGFR causes receptor dimerization and au-

tophosphorylation. The phosphorylated tyrosine residue

of EGFR binds growth factor receptor binding protein

2, which in turn complexes with the RaSGEF, Fl’T.

Translocation of FPT to the plasma membrane in the

vicinity of Ras results in Ras activation. Several poten-

tial downstream targets of Rat have been identified,

most significantly the protein Raf. Activated Ras re-

cruits Raf to the plasma membrane, resulting in a series

of phosphorylation and activation events along theMAPK cascade and leading ultimately to activation of

several transcription factors and their target genes.

However, Raf-mediated responses cannot account for

all of the consequences observed in Ras-activated cells;other possible Ras target proteins include MEKKI

(MAPK/ERK kinase), phosphatidylinositol-3-hydroxy

kinase [P1(3)1(1, p 1200AP, RaIGDS, and protein kinase

C (PKC�) (see text for references). GF, growth factor;

IL-I, interleukin 1 ; RTK, receptor tyrosine kinase; Grb2,

growth factor receptor binding protein 2; GEF, growthenhancing factor; TK, tyrosine kinase; SMSR, seven-

member serpentine receptor; JNK/SAPKs, Janus-acti-

vated kinase/stress-activated protein kinases; NF, neu-

rofibromatosis gene; MAPKK, MAPK kinase; TCF.

ternary complex factor.

protein 2, which in turn complexes with the RasGEF FPT.

Translocation of FPT to the plasma membrane in the vicinity of

Ras results in Ras activation (21).

Several potential downstream targets of Ras have been

identified, most significantly Raf (20-22), phosphatidyli-

nositol-3-hydroxy kinase (22), the neurofibromatosis gene

(5), Pl2Ogap (21, 24, 25), Jun N-terminal kinase/stress-

activated protein kinases (5), and Bcl-2 (21). Other possible

Ras target proteins are Ra1GDS and protein kinase C� (21,

24, 25). These targets become intermediate steps in path-

ways leading to effects on cell growth and proliferation. For

example, in the Raf pathway, activated Ras recruits Raf to

the plasma membrane, resulting in a series of phosphoryla-

tion and activation events along the MAPK cascade and

leading ultimately to activation of several transcription fac-

tors and their target genes (e.g. , ternary complex factor and

c-Fos; Refs. 5 and 20-22). Similarly, transmission along Jun

N-terminal kinaselMAPK pathways leads to activation of

c-Jun (5). The interaction of Ras and Bcl-2 is not yet elu-

cidated but is interesting in light of the role of Bcl-2 in

suppression of apoptosis (21).

Selective inhibition of activated signaling pathways in

precancerous and cancerous cells should be possible based

on the redundancy of growth factor networks. Proliferation

of normal cells is dependent on more than one growth factor,

and one growth factor activates multiple intracellular sig-

naling pathways. Numerous genetic knockout experiments

have established that if a particular growth factor signaling

pathway is inactivated, an alternative pathway takes over (8,

23). Oncogene overexpression or mutation can lead to con-

stitutive activation of a single signaling pathway. In contrast,

inhibition of this specific pathway should not disturb other

pathways necessary for normal cell function. Therefore, Ras

signaling inhibition in precancerous or cancerous cells con-

stitutively overexpressing the ras transduction pathway

should have greater effects on cell growth and proliferation

than in normal cells (23).



Cancer Epidemiology, Biomarkers & Prevention 269

Association of Deregulated Ras Activity with

Carcinogenesis

General Considerations. The association of activated rasgenes with oncogenic transformation in experimental animals

(26) and in humans (14, 26, 27) is well established. The acti-

vation of ras genes is generally associated with mutations at

codons 12, 13, and 61 (27). Although overexpression of normal

Ras proteins also leads to transforming activity (27), mutationalactivation has been observed more consistently in human can-

cers (28), and interpretation of studies examining overexpres-sion has been complicated by methodological problems, lead-

ing to inconclusive results (28).Patterns of ras mutations may vary across both species and

target organs. For example, ras mutations are rare occurrences(<5%) in human breast cancer; however, “'‘90% of MNU-

induced rat mammary cancers carry codon 12 H-ras mutations

and ““‘50% also harbor codon 12 K-ras mutations (29).AOM-induced rat colon tumors (30), N-nitrosobis(2-oxopro-

pyl)amine-induced hamster pancreatic carcinomas (31, 32), and

human cancers at both of these target sites carry a high per-

centage of K-ras mutations (14). Similarly, lung adenocarcino-

mas in humans and similar carcinogen-induced lesions in micehave high frequencies of K-ras mutations (26, 33, 34).

The specific ras gene altered can also vary across species.In general, H-ras oncogenes are activated in numerous animal

cancer models (26) but are infrequently activated in humantumors (14, 26, 27). Furthermore, different target organs within

a species show varied patterns of individual ras oncogeneactivation. In humans, most carcinomas (colon, pancreas, andlung) harbor activated K-ras genes, whereas N-ras mutations

have been associated with myeloid leukemia (35).Another factor implicating ras mutation and overexpres-

sion in carcinogenesis is correlation to cancer prognosis. How-ever, the clinical significance of ras mutation and overexpres-

sion without consideration of other co-occurring molecular and

phenotypic changes is not clear (14). Colon and lung tumorsand melanoma do not show a strong correlation of ras mutation

or overexpression frequency to disease severity (14), although

mutant ras has been associated with a more aggressively trans-formed phenotype in human fibrosarcoma and colon carcinoma

cells (36) and colorectal adenomas (14). The most informative

association of ras mutation to cancer prognosis has been ob-served in leukemia patients. In patients with AML, ras muta-

(ions present during active disease usually cannot be detected

during remission (14, 37) and are sometimes seen in relapse(14, 38). Also, myelodysplastic syndrome patients with ras

mutations appear to have a higher probability of progressing to

AML (14, 39).

Target Organs for Chemopreventive Intervention with RasProcessing Inhibitors: Activated ras Genes in Specific Hu-man Cancers. Although only = 15% of total human cancersharbor oncogenic ras mutations (40), higher mutation rateshave been observed in specific human neoplasms. As discussed

below, targets for chemopreventive intervention include tissuesin which ras activation, generally via mutation, occurs prior to

invasion. It has been noted that the timing of these mutations

may vary during the carcinogenic process in the same tissue.For example, in the colorectum, activating K-ras mutations

often occur early during malignant progression but may also beacquired during the later stages (41, 42). Recent data further

suggest that the frequency and, particularly, the incidence of ras

mutations should be interpreted with some caution as biomar-

kers of disease progression. As will be described in the follow-ing paragraphs under the specific cancer target organs, espe-

cially colon, the incidence of ras mutations is often higher inprecancerous lesions and nearby normal-appearing tissue thanin cancers in these tissues. In this regard, Jen et a!. (43)

evaluated ACF, which are early precursors to colorectal ade-

nomas and carcinomas (43). Although K-ras mutations wereprevalent in nondysplastic ACF (19/19), no ras mutations weredetected in the one dysplastic ACF evaluated. However, obser-vations such as these do not necessarily discount the value of

controlling Ras-mediated proliferative activity in slowing car-cinogenesis. The etiology is not yet well understood, but it ispossible that some early ras lesions are labile or are dysfunc-tional rather than activating. Moreover, high frequencies of

mutated ras, even in normal-appearing tissues, indicate poten-tial for hyperproliferative activity that may be associated withcarcinogenesis and may be amenable to damping by chemo-preventive agents.

The particular Ras inhibitors discussed below affect Ras

activity by inhibiting FPT, the enzyme that catalyzes the criticalstep in Ras posttranslational processing. It has been shown that

the various ras gene products (H-, N-, Ka-, and Kb-) display

distinct affinities for FPT and are inhibited to different degreesby inhibitors of the enzyme (44). Therefore, for chemopreven-tive drug development, it is particularly important to note thespecific ras gene(s) activated in various human cancers.

Target Organs for Ras Processing Inhibitors: Aberrationsin Elements Upstream of Ras in the Signal TransductionPathway. Numerous additional target organs for Ras-basedchemopreventive strategies can also be envisioned based on thecentral role of Ras proteins in the signal transduction pathway.

Activation of upstream element(s), such as growth factors and

their receptors, could lead to deregulated signaling via Rasproteins (21, 45). Blocking signals at the level of Ras proteinmay provide a viable means of inhibiting deregulated upstreamelements. An in-depth review of all these potential target organs

is beyond the scope of this article. However, targets mayinclude, for example, cancers associated with deregulated sig-

naling via EGFR in lung, cervix, and prostate (1), p185 erbB-2

in breast and ovary (23), and PDGF receptor in glioblastomas(23). Importantly, based on this hypothesis, some of the major

target organs that do not harbor ras mutations, such as breastcancers, may be amenable to chemopreventive strategies thatblock Ras activity (45).

Specific Human Cancers Associated with Activatingms Lesions

Colorectum. Since the original reports by Bos et al. (46) andForrester et a!. (47) in 1987, considerable evidence has accu-

mulated for the involvement of ras mutations, particularlyK-ras, in colorectal cancer development. McLellan et a!. (48)

collated results of many studies and found that 40% (377 of940) of sporadic cancers and 37% (20 of 54) of familial

adenomatous polyposis-associated cancers harbor K-ras

mutations. In cancers in ulcerative colitis patients, mutationsare either common or infrequent, depending on the study.Benhattar and Saraga (49) combined the results of five studiesand reported that 30% (17 of 58) carcinomas examined from

ulcerative colitis patients carried K-ras mutations.Mutant K-ras oncogenes are clearly associated with the

precancerous events in the colorectum. This genetic lesion has

been identified in 10-75% of adenomatous polyps, which arewell-established precursors of colorectal cancer (41, 46, 50-

54), and in dysplastic colorectal tissues from ulcerative colitispatients (49). The large variations reported in overall ras mu-tation frequency in adenomatous polyps might result from a




number of factors, including differences in patient populations

reflecting disparate etiological factors, assay sensitivity, or spe-

cific ras mutations analyzed.An important factor contributing to variations in mutation

frequency is the characteristics of the adenomas themselves.

Increased prevalence of ras mutations in adenomas has been

associated with parameters that correlate to increased malignantpotential including severity of dysplasia (5 1 , 52, 55, 56), in-

creased size (41 , S I , 55-57), and increased degree of villous

architecture (55, 57, 58). These observations and the usual earlyoccurrence of the K-ras mutations noted above suggest that thepresence of K-ras mutations in precancerous colorectal adeno-

mas “marks” these lesions with increased risk for malignant

progression. Additionally, K-ras mutations have been detected

in the colonic effluent of patients at high risk for developing

colorectal cancer; in one patient, the mutation was detected 4

years prior to cancer diagnosis (59, 60).However, as suggested above, the role of K-ras mutations

in colorectal cancer development appears complex. For exam-

ple, these mutations have been detected in up to 85% (61-64)

of ACF (the earliest identified putative precursors of colorectal

cancer) in the grossly normal-appearing mucosa of colorectalcancer patients. This incidence is much higher than that in small

adenomas (‘�l0%). Additionally, the incidence of K-ras mu-tations decreased with increasing size in a large number ofACF, although increasing size correlated to increasing adenom-

atous character of the lesion (61).

Pancreas. The incidence of K-ras mutations in pancreatic can-cers far exceeds that in other human cancers. Mutations, usually

in codon 12 of the K-ras gene, have been detected in 75-95%of ductal pancreatic adenocarcinomas (65-68). The very high

frequency of these genetic lesions in pancreatic cancers, their

appearance in a large percentage of small pancreatic adenocar-cinomas (69), and the lack of correlation to other tumor stage

or gradeparameters (66, 70) suggest that they occur during theearly stages of carcinogenesis.

Exploring the contribution of K-ras mutations to the nat-

ural history of pancreatic cancer has been hampered by the lackof clearly defined precancerous lesions. For example, K-ras

mutations have been detected in histological lesions with ques-

tionable malignant potential, such as papillary mucinous duct

hyperplasia without atypia (71), and in ducts from chronic

pancreatitis patients showing papillary hyperplasia in the ab-

sence of carcinoma elsewhere within the pancreas (72). Indeed,it has been proposed that K-ras mutations be used to assess the

precancerous nature of such lesions (73).

It was reported recently that the specific K-ras codon 12mutation identified in pancreatic ductal hyperplasia withoutatypia was not detected in any of the pancreatic carcinomas,

despite detection of other codon 12 mutations in 30 of 30carcinomas. The authors suggested that hyperplasia bearing

these genetic lesions might have low malignant potential (74).This observation is very interesting in light of the recent find-

ings in the colorectum cited above, which suggest that ACF

carrying K-ras mutations have little potential to progress to-

ward malignancy.Because pancreatic tissue is not easily accessible, alterna-

tive means of detecting K-ras mutations in pancreatic cells is

important to potential chemopreventive strategies. K-ras mu-

tations have been detected in pancreatic cancer patients byexamining pancreatic (75) and duodenal juice (76) and stoolspecimens (77). The feasibility of using these techniques toidentify lesions that may be amenable to chemopreventive

intervention is bolstered by the detection of K-ras mutations in

the pancreatic juice of patients who later developed cancer (78).However, K-ras mutations have also been detected in the stool

(77) and in pancreatic juice (79) of patients with chronic pan-creatitis. As noted above, the significance of these observations

is unclear.

Lung. Reynolds et a!. (80) collated results of several studies

and reported that 32% (76 of 237) lung adenocarcinomas con-

tam ras gene mutations; 67 of 76 were in K-ras. Mutations aremost frequently detected at codon 12 (81). Using more sensitive

techniques, an increased prevalence (46%) of K-ras mutationshas been reported recently in adenocarcinomas (82). ras muta-

(ions have also been detected in other non-small cell lung

cancers at lower frequencies but are rarely observed in other

forms of lung cancer (33).In various animal tissues, chemical carcinogens including

asbestos, 7,12-dimethylbenz(a)anthracene, MNU, vinyl chlo-ride, and benzo(a)pyrene have induced ras mutations (34). Theimportance of carcinogen-induced ras mutations are empha-

sized by observations in lung (33). K-ras mutations are much

more prevalent in adenocarcinomas from smokers (30%, 41 of141) than from non-smokers (5%, 2 of 40; Ref. 81). Further-

more, mutations are as common in former as current smokers,even those who had not smoked for � 15 years (83). The most

frequent type of mutation found in tumors from former smokersis a G->T transversion, which is the same type of mutationinduced by the cigarette smoke carcinogen benzo(a)pyrene

(83). K-ras mutations have been detected in the normal-appear-ing bronchial tissue of smokers with non-small cell lung can-

cers that harbored K-ras mutations (84) and in cytologicallynegative stored sputum samples from 7 of 15 patients who later

developed resectable adenocarcinoma (85). These findings im-

plicate tobacco smoke as a causative agent and suggest that ras

mutations may be early genetic lesions during lung adenocar-

cinoma development.However, as in other tissues, it appears that K-ras muta-

tions may also occur later but still prior to invasion. Sugio et a!.

(86) observed K-ras mutations in 4 of 5 areas of carcinoma insitu associated with adenocarcinomas bearing K-ras mutations

but not in earlier stages of premalignancy. Li et a!. (87) alsofailed to detect ras mutations in preinvasive bronchial epithe-hum but found a homogeneous distribution of mutant K-ras in

malignant cells of adenocarcinomas, suggesting that the muta-tions occurred prior to invasion. Larger studies are needed to

clarify these findings.

Leukemias. Leukemias are one of few human cancers associ-ated with mutations in ras genes other than K-ras. Mutations,usually in N-ras, have been observed in 25-60% of chronic

myelomonocytic leukemia and 30% of adult AML. N-ras mu-tations have also been observed in 9-40% of myelodysplasticsyndromes, stem cells disorders which may progress to AML

(35).

Endometrium. Mutations, usually in codon 12 of the K-ras

gene, have been reported in 10-30% of endometrial cancers

(88-90). Mutation frequency has consistently been shown to beabout 10-15% in U. S. women compared with about 20-30%in Japanese women (88, 89, 91), reflecting possible etiological

differences.K-ras mutations have also been identified in precancerous

endometrial hyperplasia with frequencies correlated to severity

of the lesions (88, 89). In one study in which mutations werepresent in 1 8% of cancers, the total percentage of K-ras mu-

tations in endometrial hyperplasia was 16% with mutationspresent in 10% of simple hyperplasia, 14% of complex lesions,and 22% of atypical hyperplastic lesions (88). These results



Cancer Epidemiologj�, Biomarkers & Prevention 271

suggest that although K-ras mutations do not occur in a high

percentage of endometrial cancers, when present, they oftenrepresent early genetic events during malignant transformation.

Cervix. ras mutations appear to be rare in squamous cervical

cancers (27, 92, 93). However, expression of unspecified Rasproteins has been reported to increase in high grade and inva-sive lesions compared with low grade or normal cervical epi-

thelium (94, 95). Based on preliminary results showing pro-gressive increases in Ras expression during lesion progression,

Mitchell et a!. (96) are investigating Ras expression as a sur-

rogate end point for chemoprevention clinical trials in thecervix.

Breast. ras mutations are also rare in human breast cancer

(<5%), but overexpression of H-ras proto-oncogene has been

reported in up to 70% of breast cancers (97, 98). Furthermore,in one study, expression increased during histological stages of

malignant progression and was also significantly higher inpatients (n = 18) with hyperplastic changes, who subsequently

developed breast cancer (99). Recent evidence suggests that

aberrant function of the Ras-related protein TC21R-Ras2 (55%homologous to Ras) may contribute to breast cancer. TC21R-

Ras2 activates the MAPK cascade and other downstream ele-ments of the Ras signaling pathway (100). TC21R-Ras2 over-expression or mutation induces transformation in breastepithelial cell lines, and overexpression is seen in the majority

of human breast tumor cell lines (101). The significance ofthese findings awaits confirmation that TC21R-Ras2 is aber-rantly regulated in primary breast lesions.

Ras Protein Processing

Because of its central role in signal transduction, Ras proteinprocessing has been studied intensely and reviewed frequently(16-19). Ras protein function depends on association with the

inner surface of the plasma membrane. Ras proteins are initiallysynthesized as cytoplasmic, soluble proteins lacking the con-

ventional transmembrane or hydrophobic domains typical ofother membrane-associated proteins. To overcome this, they

are posttranslationally modified with a lipophilic C-l5 farnesyl

moiety in a reaction catalyzed by FPT. Following farnesylation,the Ras COOH-terminal sequence undergoes proteolytic cleav-

age and carboxymethylation.Experiments using mutant proteins that cannot undergo

these various posttranslational processing events demonstrate

that membrane association and farnesylation are critical for Ras

transforming activity. Nonfarnesylated mutants of oncogenicRas are cytosolic and devoid of transforming activity. Further

support for the importance of Ras farnesylation comes fromstudies limiting the availability of FPP, the isoprenyl moietyused in this reaction (17). FPP is a metabolic product of me-valonate, formed via a series of steps from HMG-CoA. The first

and rate-limiting step in this pathway is catalyzed by HMG-CoA reductase. Inhibition of HMG-CoA reductase with com-pactin or lovastatin decreases the availability of FPP, prevents

Ras farnesylation, membrane association, cell transformation,

and tumorigenesis (102, 103). However, the utility of HMG-

CoA reductase inhibitors as Ras famesylation suppressors islimited because the formation of numerous other isoprenoids in

addition to FPP would be affected; additionally, Ras proteinsare only a subset of >40 posttranslationally isoprenylated pro-teins. The majority of these proteins are modified by gera-nylgeranyl groups rather than by farnesylation; however, inhi-

bition at the level of HMG-CoA reductase would influence all

of these isoprenylation reactions (17).

FPT and Related Prenyl Transferases

Two types of cellular prenyl group transfers are the mostcommon and involve transfer of a C-iS farnesyl or a C-20

geranylgeranyl moiety to a cysteine residue via a thioetherlinkage. Prenylated proteins share characteristic COOH-termi-nal sequences, which include the CAAX, XXCC, and XCXC

motifs (where C is cysteine, A is usually an aliphatic aminoacid; X is another amino acid). Three enzymes that catalyze

protein prenylation have been identified: FPT, GGTase I, and

GGTase II (also called Rab GGTase). GGTase II modifiesproteins ending in XXCC and XCXC (19). Until recently, the

CAAX tetrapeptide was believed to be the minimum regionrequired for interaction of protein substrates with FPT or GG-

Tase I, with the last residue of the CAAX motif directing

enzyme specificity. However, newer studies suggest that en-zyme specificity is more complex. Both enzymes form stablenon-covalent complexes with FPP and geranylgeranyl PPwhen a protein acceptor is not present. FPT only transfers the

farnesyl moiety, whereas GGTase will transfer either the far-nesyl or geranylgeranyl moiety, depending on which protein

acceptor in present (104).The four cellular Ras proteins are substrates for FF1’. In

addition, at least eight other cellular proteins undergo farnesy-lation: nuclear lamins a and b, the Ras-related proteins Rap2and RhoB, phosphorylase kinase, rhodopsin kinase, cyclic

GMP phosphodiesterase a, and the -y subunit oftransducin (19).The latter three proteins are involved in vision (105). GGTase

I substrates include the ‘y subunit of mammalian G proteins,Rapl, and CDC42 (106). GGTase H prenylates many Rab

proteins, which are involved in protein secretion and endocy-

tosis (106).

Requirements for Specific Ras Farnesylation Inhibitors

Requirements for Ras farnesylation inhibitors include: speci-ficity for FPT compared with GGTases, particularly, GGTase I;specificity for FPT compared with other FPP-utilizing en-

zymes; ability to specifically inhibit processing of mutant K-ras(the most commonly mutated ras gene in human cancers); highpotency; selective activity in intact cells; activity in vivo; andlack of toxicity.

Ras Farnesylation Inhibitors

Two general approaches for identifying FPT inhibitors have

been used. The first is random screening of microbial or other

products for inhibitory activity. The second is based on rationaldesign of analogues of the two substrates, FPP and the COOH-terminal CAAX motif of Ras tetrapeptides. The discussionbelow is intended to provide a general overview of the types of

structures that have demonstrated inhibitory activity towardFlaT. The inhibitors are divided into four categories accordingto their proposed mechanism of action: (a) FPP competitive

inhibitors; (b) CAAX competitive inhibitors; (c) bisubstrateinhibitors; and (d) inhibitors with unknown mechanism(s) of

action.Based on their activity in in vitro and in vivo screening

assays, some of these drugs may have chemopreventive poten-tial. It should be noted that FPT inhibitors that do not meet all

of the requirements set forth above may still be viable chemo-preventive drugs under appropriate circumstances.

The most current available animal data have been obtainedin models relevant for establishing chemotherapeutic effective-

ness, i.e. , tumor cell growth inhibition in vivo. Because changesin Ras-mediated signaling occur during the process of carcino-



Compound II (hydroxamate analogue of

FPP)

Refs.

Chaetomellic acids

Manumycin

Perillyl alcohol, d-limonene and metabolites

I 56

132, 157

157, 158

157, 158

I 34

159

160, 161

132, 162

1, 163

110, 115, 117-128

164

132, 165


3 B. S. Reddy, unpublished results.

Table 1 FP’F inhibitors competitive with FPP”

Synthetic compounds

(a-Hydroxyfarnesyl)phosphonic acid

Compound I (amide analogue of FPP)

Compound III (pivolyboxymethyl ester

analogue of FPP, prodrug of compound II)

FPPA1

Fluorinated FPP analogues

Natural products

Actinoplanic acids

RPR1 13228 (Chrysoporium lobatum,

secondary metabolite)

Zaragozic acids

Characteristics

Cell-free enzyme: Selective cf. GGTases I, II Noncompetitive

with Rat tetrapeptide

Cell-free enzyme: More potent than (a-hydroxy-

farnesyl)phosphonic acid. Highly selective cf GGTases I, II

Whole cells: Inactive in H-ras-transformed cells

Cell-free enzyme: More potent than (a-hydroxy-farnesyl)phosphonic acid. Highly selective cf GGTases I, II

Whole cells: Inactive in H-ras-transformed cells

Inhibits geranylgeranylation (<farnesylation)

Whole cells: Inhibits H-ras-dependent transformation

Inhibits geranylgeranylation (<farnesylation)

Cell-free enzyme: Selective cf. 55

Cell-free enzyme: a-Fluorination increased inhibitory potency

Cell-free enzyme: Selective cf. GGTase and 55. Acyclic more

potent than cyclic form

Whole cells: Inactive in H-ras-transformed NH-I-3T3 cells

Cell-free enzyme: Selective cf. GGTase I and 55

Whole cells: A isomer inactive in H-ras transformed NIH-

3T3 cells

Cell-free enzyme: Selective cf. GGTase

Whole cells: Inhibited growth of K-ras-transformed

fibrosarcoma cells (not clear that effect was due to inhibition

of Rat processing)

Cell-free enzyme: Inhibit both FPT and GGTase (more potent

toward GGTase)

Chemopreventive activity: Perillyl alcohol inhibited induction

of rat colon and liver, and hamster pancreas tumors

Tumor growth inhibition and regression: Perillyl alcoholreduced growth of established hamster pancreatic tumors,

regression of established rat mammary gland tumors, andretarded growth of a prostate tumor cell xenograft in athymic

nude mice. d-Limonene inhibited growth of mouse lung and

skin tumors, rat mammary gland tumors induced by MNU,

DMBA, and direct in situ transfer of v-Ha-ras, and rat liver

tumorigenesis, as well as regressed established mammarytumors

Cell-free enzyme: Selective cf. GGTase and 55

Cell-free enzyme: Also inhibits 55 (more selective for 55)

Whole cells: Inactive in H-ras-transformed NLH-3T3 cells

a See Figs. 2 and 3 for representative structures. 55, squalene synthase; DMBA, 7, 12-dimethyl benz(a)anthracene.

genesis, (in)effectiveness toward established tumors may not

accurately predict (in)activity toward precancerous lesiongrowth and development. This differential effectiveness has

been demonstrated, for example, with retinoids, which are wellknown chemopreventive agents but have generally been inef-

fectual against established tumors in animal models (107).

FPP Competitive Inhibitors. FPP competitive inhibitors(see Table 1) bind to FPT at the FPP binding site and have

been identified through both rational drug design (Fig. 2) andrandom screening (Fig. 3). The most promising inhibitors in

this class are expected to be those specific for FPT comparedwith other FPP-utilizing enzymes, most notably squalene

synthase.Although they are not highly specific for FPT, perillyl

alcohol, d-limonene and related metabolites are noteworthybecause of the chemopreventive and tumor-shrinking potentialthey have demonstrated. Perillyl alcohol is a hydroxylatedderivative of d-limonene. Both monoterpenes have demon-strated preclinical chemopreventive and chemotherapeutic ac-tivity possibly through metabolism to perillic and dihydroper-illic acids (108, 109). Both metabolites inhibit FPT and

GGTases directly (I 10-I 13) or by selectively decreasing ras

levels (1 14). Perillyl alcohol significantly inhibited AOM-in-

duced colon and small intestine tumor development.3 In

published chemoprevention studies, perillyl alcohol inhib-

ited rat liver (1 15) and hamster pancreatic (1 16) tumor

development; in chemotherapeutic studies (1 10, 1 17, 1 18), it

significantly reduced the growth of established hamster pan-

creatic tumors, caused regression of established rat mam-

mary gland tumors, and retarded growth of a prostate tumor

cell xenograft in athymic nude mice. The parent compound

d-limonene inhibited the growth of mouse lung (119) and

skin tumors (120), rat mammary gland tumors induced by

MNU, 7,12-dimethylbenz(a)anthracene, and direct in situ

transfer of v-Ha-ras (121-124), and rat liver tumorigenesis

(125, 126), as well as causing established mammary tumors

to regress (127, 128).



H..N I0 )‘�P-ONa

0 0 0 0 0

Compound Ill


o#{149}o#{149}I I

II II

0 0

FPP

OH

� �OH

O�”OH0 0

FPPA I

Fig. 2. FPP competitive inhibi-

tors: synthetic compounds.

Difluorinated �-Ketophosphonic Acid

Compound II

0

� �II ONaH CO2Na 0

Compound I

CAAX Competitive Inhibitors. Numerous tetrapeptides thatconform to the CAAX consensus sequence act as alternative

substrates for FPT and result in competitive inhibition of Rasfarnesylation in vitro. Some tetrapeptides inhibit FPT withoutserving as alternative substrates and are thus true inhibitors ofthe enzyme. Structure activity analyses showed that nonfarne-

sylated tetrapeptides containing an aromatic residue in the A2position of the CA1A2X sequence (129) and a positive charge

on the cysteine amino group (130) are the most potent inhibi-tors. However, these tetrapeptides are inactive in whole cells,probably because they are unable to enter cells efficiently or are

easily degraded, or both (19). Because of limited utility in vivo,these tetrapeptide inhibitors are not discussed further here.

Several research groups have developed promising CAAX mi-metics, which are listed in Table 2 (Fig. 4). Bisubstrate inhib-itors are also being developed (Table 3 and Fig. 5).

Inhibitors with Unknown Mechanism(s) of Action. A num-ber of natural product FPT inhibitors have been identified, themechanism(s) of action of which have not been determined.These are described in Table 4; their structures appear inFig. 6.

Approach to Developing Ras Farnesylation Inhibitors asChemopreventive Agents

A number of in vitro and in vivo tests may be used in thedevelopment of Ras farnesylation inhibitors as potential che-

mopreventive agents. Based on differences in the affinities ofthe Ras proteins for FF1’, it is particularly important to establishinhibitory activity toward K-Ras, the form of Ras most oftenmutated in human cancers. A possible testing strategy is out-

lined below (Table 5). Priorities for further development wouldbe based on results at each step in the order that follows.

Determine Inhibition of FPT Activity. Inhibitory activity can

be determined by measuring incorporation of [3H]FPP into Ras

proteins or Ras-related peptides in a reaction catalyzed by

isolated or recombinant FPT (131).

Evaluate Selectivity for FPT. The selectivity of the inhibitortoward FF1’ relative to GGTases can be measured in vitro using

GGTases I and II isolated from several sources, such as bovine

brain (132); recombinant human GGTase I (133) can also be

used. Inhibition ofGGTase I and H activity can be measured via

incorporation of [3H]GGT into Ras-CAIL (Cys-Ala-Ile-Leu)

and Ypt-GGCC, respectively (132). If the drug is competitive

with respect to FPT, selectivity toward FPT relative to squalene

synthase should also be determined (134).

Determine Inhibition of Ras-mediated Effects in Intact

Cells. Selective inhibition of Ras processing and effects on

Ras-mediated proliferation and transformation should be exam-

med in whole cells. Specificity for FPT in intact cells involves

establishing inhibition of prenylation of farnesylated as com-

pared with geranylgeranylated proteins upon incubation with

[3H]mevalonate. Often these experiments are performed in the

presence of an HMG-CoA reductase inhibitor to prevent iso-

topic dilution of [3H]mevalonate. Because cells are relatively

impermeable to mevalonate, Met-18b-2 cells (CHO cells with

efficient mevalonate uptake) or cells transfected with cloned

mutant Mev cDNA, which facilitates cellular uptake, can be

used. Inhibition of ras-mediated cellular effects, such as inhi-

bition of anchorage-independent and -dependent growth, and

reversal of morphological transformation should be established

(135).

Determine Inhibition of Ras-mediated Effects in Vivo. Ac-

tivity of FPT inhibitors on the growth of Ras-dependent tumors



H#{176} 0

OH

Chaetomellic Acid A

CH2OH

(L1

H3XH2Perillyl Alcohol

OHHO � OH

�

H3C��’� �CO�H

CH3

RPR 113228

Manumycin

j HO

QC�O\�OOH

Zaragozic Acid


0 C0,H

OH 0

Fig. 3. FPP competitive inhibitors: natural products.

can be evaluated in nude mice injected with transformed rodent

or human cells carrying mutant ras genes (133).

Determine Chemopreventive Efficacy in Vivo. Potentially,

mouse lung is the most efficient model for evaluating FPT

inhibitors. In A/J (or A/J F1) mice, virtually all chemically

induced tumors have mutated K-ras (136). Using 4-(methylni-

trosamino)-1-(3-pyridyl)-1-butanone as the carcinogen, >90%

of lung tumors have K-ras, and approximately “50% of liver

tumors have Ha-ras mutations. The relatively small size of the

mice and high tumor multiplicity allow testing with small

amounts of drug. Alternative models are AOM-induced rat

colon tumors (30) and N-nitrosobis(2-oxopropyl)amine-in-

duced hamster pancreatic carcinomas (31, 32). Both are con-

sidered good models for human cancers at these target sites and

carry high percentages of K-ras mutations. Further, K-ras mu-

tations have clearly been associated with the development of

human cancers at these target sites (41, 44, 50-60, 65-70). A

caveat is that K-ras is preferentially activated by geranylgera-

nylation, not farnesylation. Tests for chemopreventive efficacy

in other established animal tumor models where high levels of

proliferation are particularly important to carcinogenesis, e.g.,

rat and mouse bladder, may logically follow. See Steele et a!.

(137) for a description of the animal models currently used in

the National Cancer Institute Chemoprevention Branch drug

development program.

Actinoplanic Acid B

Discussion and Conclusions

The use of FI�T inhibitors represents a rational, targeted ap-

proach for the development of mechanism-based chemopreven-

tive drugs. Activated ras genes have been associated with the

early stages of carcinogenesis in several organs of high interest

to the Chemoprevention Branch, including colon, pancreas, and

lung. Advances in analytical techniques are allowing detection

of these molecular alterations in increasingly smaller numbers

of cells, thus facilitating detection at earlier stages of carcino-

genesis (138). Of practical importance, mutant K-ras genes

have been detected in stool and sputum samples of patients

whose cancerous and precancerous lesions harbor ras muta-

tions. This suggests that such mutations would be detectable by

noninvasive techniques at an early stage when the probability

for successful chemopreventive intervention is high.

In light of the role of Ras proteins as central connectors

between signals generated at the plasma membrane and nuclear

effectors, Ras-based prevention strategies could have broad-

based clinical potential. Thus, besides targeting activated Ras

proteins, target organs may include those in which Ras signal-

ing pathways are constitutively activated but which do not bear

activating ras lesions per se. This could result from aberrations

in elements upstream of Ras in the signal transduction pathway,

such as receptor tyrosine kinases. One of the most important

examples for chemopreventive purposes is breast cancer. Al-




Table 2 CAAX competitive inhibitors”

Characteristics Refs.

BZA-2B Cell-free enzyme: Highly selective cf. GGTases I and II 166

Whole cells: Reversed morphologic phenotype of H-ras-transformed

cells

BZA-5B (carboxymethylated prodrug for BZA-2B) Cell-free enzyme: Highly selective cf. GOTases I and II 166

Whole cells: Reversed morphological phenotype of H-ras-transformed

cells (>BZA-2B)

B58l Cell-free enzyme: Selective cf. GGTase I 167, 168

Whole cells: Inhibited ras farnesylation (and not geranylgeranylation)

in H-ms-transformed NIH-3T3 cells. Selectively inhibited soft agar

growth of cells dependent on H-Ras farnesylation for transformation

B956 Cell-free enzyme: Selectivity cf. GGTases not reported 151

Whole cells: Inhibited soft agar growth of ras-transformed cell lines

(H-ras > N-ras > K-ras)

Nude mouse xenografts: Inhibited growth of H-ras-transformed NIH-3T3 fibroblasts; inhibition correlated to inhibition of Ras membrane

localization in tumors

B1086 (methyl ester of B956) Cell-free enzyme: Selectivity cf. GGTases not reported 151

Whole cells: Inhibited soft agar growth of ras-transfonned cell lines(H-ras > N-ras > K-ras)

Nude mouse xenografts: Inhibited growth of ms-transformed NIH-3T3

fibroblasts (H-ras > N-ras > K-ras); inhibition correlated toinhibition of Ras membrane localization in tumors

FTI-276 Cell-free enzyme: Highly potent and selective cf. GCiTase I 145, 146

Whole cells: Inhibited growth of H-ras-transformed NIH-3T3

fibr#{244}blasts

Nude mouse xenografts: Inhibited growth of human lung cancer cell

line carrying K-ras mutation and p53 deletion; no effect on lung

cancer cell line with wild-type ras; inhibition of ras-transformed cell

growth correlated to Ras processing inhibition in tumor tissue

F1’I-277 (methyl ester of Ffl-276) Cell-free enzyme: Highly potent and selective cf. GGTase I 145, 146

Whole cells: Inhibited growth of H-ms-transformed NIH-3T3

fibroblasts (lOX > FTI-276)

Nude mouse xenografts: Inhibited growth of human lung cancer cell

line carrying K-ras mutation and p53 deletion (�FT1-276); no effect

on lung cancer cell line with wild-type ras; inhibition of ras-

transformed cell growth correlated to inhibition of Ras processing in

tumor tissue

L-739,749 (methyl ester of L-739,750) Cell-free enzyme: Highly selective cf. GGTase I 133, 169

Whole cells: Potent and highly selective for Ras processing relative togeranylgeranylation; inhibited anchorage-dependent growth of RatI

cells transformed with H-, K-, or N-ras and ineffective toward cells

transformed with v-ras �r v-mos (neither dependent on famesylation

for transforming activity)

Nude mouse xenografts: Inhibited growth of ras-transformed Ratl

cells and not v-rafor v-mos-transformed cells

Toxicity: No gross toxicity in rapidly dividing tissues or where

farnesylation required for normal function (e.g., retina, skeletal

muscle)

L-739,750 Cell-free enzyme: Highly selective cf. GGTase I 169

L-744,832 (isopropyl ester of L-739,750) Cell-free enzyme: Highly selective cf. GGTase I 142, 170

Whole cells: Potent inhibitor of Ras processing and anchorage-

dependent growth of ras-transformed cells; equally effective against

cells with wild-type ras

Nude mouse xenografts: Inhibited growth of H-ras-dependent tumors

Animal studies: Caused regression of mammary and salivary gland

adenocarcinomas in MMTV-v-H-ras transgenic mice (tumorsreappeared after treatment stopped); important for chemoprevention;

no new tumors appeared during the 70-day treatment, and no toxicity

was observed

PD 083 176 (pentapeptide) and related dipeptides Cell-free enzyme: PD 08 1376 and dipeptides inhibited 17 1 , I 72

Whole cells: PD 083176 not cell permeable, but dipeptides had anti-

Ras activity

Pseudopeptide amides (no carboxyl moiety) Cell-free enzyme: Potent inhibitors and highly selective cf GOTase I 173

Whole cells: Most amides cytotoxic, but a dimethyl derivative

inhibited Ras processing noncytotoxic concentrations (additional

analogues might be designed that retain FPT inhibitory activity with

less toxicity)

SCH 44342 (not peptide) Cell-free enzyme: Selective cf. GGTase I for both rat and human 174

enzyme using both H- and K-ras peptide substrates

Whole cells: Selective for inhibiting H-Ras processing relative to

geranylgeranylation; inhibited H-ras transformation

a See Fig. 4 for representative structures.



CYIM (C-terminus of K-Ras)

SCH 44342

L-739,749, R=CH,

L-739,750, R=HL-744-832, R=CH(CH3)2

B-956, R=HB-1086, R=CH,

Ffl-276, R=O-

FTI-277, R=OCH,

276 Review: FF1� Inhibitors as Cancer Chemopreventives

HS � � 0

HSN�NLN�(N�OR H

0 N 0

BZA-2B, R=HBZA-SB, R=CR,

HS

:ui� �H2N N

Compound 32

-� 0

B-581

Fig. 4. CAAX competitive inhibitors.

Table 3 Bisubstrate inhibitor”

Characteristics Refs

BMS-l865l 1

a 5� Fig. 5 for re

Cell-free enzyme: Selective cf. GGTase I

Whole cells: Selective inhibition of Rasprocessing cf. geranylgeranylation and

myristoylation; selective growth

inhibition and morphological expression

inhibition in ras-transformed cf. normal

cells and cells transformed withgeranylgeranylation- or myrisotylation-

dependent ras mutants; blocksneurofibromatosis type I malignant

phenotype (associated with up-regulation

of wild-type Ras)

presentative structures.

16, 175, 176

though only ““5% of human breast cancers harbor mutant rasgenes, deregulated signaling mediated by the upstream ele-

ments EGFR (139, 140) and p185cerbB2 receptor tyrosinekinases have been associated with breast cancer development(141). The observation that many tumor cell lines harboring

activated tyrosine kinases and wild-type ras are very sensitiveto growth inhibition with FPT inhibitors (142) lends credence tothe feasibility of this approach. Demonstrating activity of theseinhibitors in experimental animals with tumors carrying wild-

type ras, but with aberrations in upstream elements, will furtherestablish the utility of this approach.

A possible pitfall to the use of FPT inhibitors is toxicity

0 �

� � H o

HO�fl�fl�(

BMS-186511

Fig. 5. Bisubstrate inhibitor.

associated with inhibition of constitutive, wild-type Ras (143).However, chemoprevention may not require total Ras blockade,and lower doses permitting activity sufficient for normal cel-

lular processes while damping carcinogenesis-associated hy-peractivity may be possible. Moreover, the potential utility of

FPT inhibitors as chemopreventive agents is supported by theirapparent lack of toxicity. They have shown remarkable selec-tivity toward inhibiting the growth of transformed cells inculture as well as in animal studies. Explanations for these

effects have been suggested: (a) nonfarnesylated oncogenic Rasproteins exhibit a dominant negative phenotype that couldcontribute to specificity. When activated Ras proteins are cy-

tosolic they can act as inhibitors of membrane-bound Ras (19,144). This could explain the observation that incomplete mlii-bition of Ras farnesylation can result in complete inhibition of



Gliotoxin, RHAcetyigliotoxin,

R=COCH,

1ik�pHO

SCH 58450

OCH3

H3C� 0� OR

RO/�5�X5Fusidienol, RHFusidienolDiacetate,

R=COCH3

0’ �O�H,

�:f�5�0 � 0

��Ii�1I

0

I 00 �-

OH

Patulin

H3COyL��

H2N’�(�1 N COOH

0

1O’-Desmethoxystreptonigrin

Preussomerin G

Pepticinnamin E


Tab le 4 Inhibitors with unknown mechanisms of action”

Characteristics Refs.

Barceloneic acid (from Phoma) Cell-free enzyme: Selectivity unknown 177

Cylindrol A (from Cylindrocarpon lucidum) Cell-free enzyme: Selective cf. GGTases and SS; noncompetitive with

both FPP and Ras-CVLS peptide178

Fusidienol Cell-free enzyme: Selective cf. GGTases and 55; noncompetitive with

both FPP and Ras-CVLS peptide

179

Gliotoxins (gliotoxin and acetylgliotoxin) Cell-free enzyme: Inhibited partially purified FF1’, not selective for FF1’

Toxicity: Immunosuppressive and other effects which have prevented

therapeutic antimicrobial use

106, 180

Nonadrides (CP 225,917 and CP 263,1 14 Cell-free enzyme: Also inhibits 55 181from unidentified fungus in juniper)

Patulin Cell-free enzyme: Inhibits partially purified FF1’ 182, 183

Pepticinnamins (from Streptomyces OH-4652) Cell-free enzyme: Inhibits partially purified FPT 184

Preussomerins Cell-free enzyme: Inhibits partially purified FF1’

Mechanism: May be true inhibitor or nonspecific Michael substrate for

Ras-CVLS

185

185

SCH 58450 (from Streptomyces) Cell-free enzyme: Selective cf. GGTase I 186

Streptonigrmns (l0’-desmethoxy-streptonigrmn, Cell-free enzyme: Inhibited partially purified FPT (lO’-desmethoxy 3X 187streptonigrmn) > streptonigrin and 5 X < acutely toxic)

a See Fig. 6 for representative structures.

C0�H CH2OH

H0�yL�0�

� HO��OCH3

CH3

Barceloneic Acid

Fig. 6. Inhibitors with unknown mechanisms of action.

0’ ‘�O CH3 CH3

H��A(�

H3C OH

L..CH3Cylindrol A

Ras signaling (145); (b) the redundancy of signaling pathways

predicts that inhibition of a specific deregulated pathway in

cancerous or precancerous lesions should have minimal effectson normal cell function. Support for this hypothesis comes from

studies in which FPT inhibitors diminished growth factor-

induced MAPK activation in transformed, but not in normal,cells (146, 147).

Redundancy may be due to continued signaling along



278 Review: FFl� Inhibitors as Cancer Chemopreventives

Table 5 Steps in precinical evaluation of Fl’T inhibitors as chemopreventive

drugs

I. In vitro inhibition of FF1’ activity

2. In vitro assays with other prenyl transferases (GGTase I and II)

and other FPP-utilizlng enzymes to determine specificity

3. Inhibition of Ras processing in intact cells

4. Inhibition of Rat-mediated proliferation and transformation in

intact cells

5. in vivo inhibition of Ras processing in tumor system dependent on

Rat [e.g., xenografts of human cell lines carrying mutant rasgenes (particularly K-ras) in nude mice]

6. in vivo demonstration of chemopreventive activity in animalmodels of carcinogenesis dependent on mutant K-ms (e.g.. rat

colon, hamster pancreas).

non-Ras-mediated, parallel pathways, such as the Jak/Stat path-

way (148). Signaling via kas-related proteins may also be

involved. For example, the Ras-related protein TC21R-Ras2 is

able to activate the MAPK cascade and other downstreamelements of the Ras signaling pathway (100). Interestingly,

these proteins are not sensitive to FPT inhibitors, perhapsbecause they can be posttranslationally modified by either

farnesylation or geranylgeranylation (149). Moreover, recentevidence suggests that FF1’ inhibitors are capable of differen-tially inhibiting the four cellular Ras proteins. The K-Ras4bprotein has a 50-fold higher affinity for FF1’ than H-Ras in

vitro, and K-Ras4B farnesylation is inhibited by FPT inhibitorsonly at concentrations 8-fold higher than those active towardH-Ras (44). Thus, selective inhibition of H- versus K-Rasmediated signaling may allow continued growth of normal

cells.However, the latter observations also have significance for

chemopreventive strategies, because most human cancers with

ras mutations selectively harbor them in the K-ras4B gene(150). The majority of early work with FPT inhibitors usedH-ras-transformed cells and xenografts. More recent studieshave shown that K-ras-transformed cell lines are generallymuch less sensitive to growth inhibition by FF1’ inhibitors than

cell lines transformed with other ras genes (15, 15 1 ); indeed, inone study, K-ras-transformed cell lines were not more sensitivethan those with wild-type ras (151). These observations under-

score the necessity of using K-ras4B-transformed cells fortesting potential inhibitors both in vitro and in vivo.

Recent studies also suggest that FF1’ inhibitors are notabsolutely selective for Ras but may also affect other transfor-

mation-associated proteins. Phenotypic reversions of ras-trans-formed cells treated with the CAAX mimetic L-734,749 did not

correlate to the state of Ras processing but rather to regulationof action stress fiber formation (152). One non-Ras protein thatmay be affected by FPT inhibitors is RhoB, which is usuallygeranylgeranylated but may also be farnesylated in vivo (153).Dominant inhibitory mutants of RhoB mimic FPT inhibitorability to block Ras transformation (154), and the effects of

L-734,749 can be suppressed by ectopic expression of fame-sylation-independent forms of RhoB (153).

Additionally, data have been presented that K-Ras4B maybe resistant to FF1’ inhibitors because it is posttranslationallymodified by geranylgeranylation rather than farnesylation.

K-Ras4B, not H-Ras, is a substrate for CAAX GGTase I invitro (44); in fact, oncogenic K-Ras4B processing and consti-

tutive activation of MAPK were potently inhibited by a GO-Tase I-selective inhibitor, GGTI-286, but not by the FPT se-lective inhibitor VFI-277. On the other hand, oncogenic H-Raswas very sensitive to FFI-277 and highly resistant to GGTI-286

(150). These results may be explained by the higher affinity ofK-Ras4B compared with H-Ras for FPP. However, the authors

suggested that K-Ras4B is geranylgeranylated in cultured cellsbased on the observation that GGTI-286 inhibited oncogenicK-Ras4B processing and MAPK activation at concentrations

that did not affect farnesylation-dependent processing (150).Demonstration that K-Ras4B is geranylgeranylated in vivo,

especially under circumstances in which FPT is inhibited, could

have a major impact on the design of Ras processing inhibitors.Increasing knowledge of signaling pathways downstream

of Ras suggests other strategies for improving the specificity

and selectivity of the Ras processing blockade in preventingcarcinogenesis. Particularly, specific inhibitors of downstreamcarcinogenesis-associated c-Jun or c-Fos activation (see Fig. 1)could be designed to supplement or replace FPT inhibition incertain high-risk tissues. For example, a combination with aninhibitor of downstream pathways may allow lower doses of

FF1’ inhibitor to be used with concomitantly less toxicity to

normal cell functions requiring Ras mediation.Because precancerous and cancerous lesions generally

harbor more than one genetic abnormality, drugs showing in

vivo efficacy against lesions carrying multiple genetic changeswould be especially promising for further clinical development.The FPT inhibitor FTI-276 exhibits antitumor activity against ahuman xenograft harboring both a K-ras mutation and a p53deletion (146). This observation, together with their apparent

lack of toxicity, suggests that FPT inhibitors indeed have prac-tical chemopreventive potential. Detailed studies to determine

the mechanism(s) of action of these drugs is critical for deter-mining further clinical development.

A new picture of Ras processing appears to be emergingthat could significantly impact the design of Ras posttransla-

tional processing inhibitors. However, it is clear that tremen-dous progress has been made in understanding the biochemical

processes involved in Ras-mediated signal transduction. Thehigh frequency of ras mutations in selected cancers and pre-

cancers, as well as the crucial role of Ras proteins as centralcomponents of signal transduction pathways, makes disruption

of Ras-mediated signaling a very promising target for chemo-preventive drug development.

Weinstein has described carcinogenesis as a progressivedisorder in signal transduction (155). In this regard, the signif-icance to chemoprevention of these early and promising studieswith FPT inhibitors, as well as those described previously with

EGFR inhibitors (1), extends well beyond the individual mech-

anistic classes. These studies exemplify the drug design strat-egies that are becoming possible as our understanding of themolecular pathways and elements controlling cell fate in-

creases. Particularly important for chemoprevention are thenewly identified opportunities to block early proliferative

changes, making use of the signal transduction mechanisms

before they are destroyed.

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1997;6:267-282. Cancer Epidemiol Biomarkers Prev G J Kelloff, R A Lubet, J R Fay, et al. chemopreventives.Farnesyl protein transferase inhibitors as potential cancer

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