Farnesyltransferase inhibitors and cancer treatment: targeting simply Ras?

Ž .Biochimica et Biophysica Acta 1333 1997 F51–F71

Farnesyltransferase inhibitors and cancer treatment: targeting simplyRas?

Adrienne D. Cox a,b,c,), Channing J. Der b,c,d

a UniÕersity of North Carolina at Chapel Hill, Department of Radiation Oncology, Chapel Hill, NC 27599, USAb UniÕersity of North Carolina at Chapel Hill, Department of Pharmacology, Chapel Hill, NC 27599, USA

c UniÕersity of North Carolina at Chapel Hill, The Lineberger ComprehensiÕe Cancer Center, Chapel Hill, NC 27599, USAd UniÕersity of North Carolina at Chapel Hill, Curriculum in Genetics and Molecular Biology, Chapel Hill, NC 27599, USA

Received 13 January 1997; accepted 11 March 1997

Contents

1. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F522. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F523. The role of aberrant Ras function in humancarcinogenesis . . . . . . . . . . . . . . . . . . . . . . F524. The role of Ras proteins in signal transduction, normalproliferation and cell cycle progression:

establishing avenues fordrug discovery efforts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F545. Generation of prenylation inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F576. Initial characterization of FTIs: potency andselectivity . . . . . . . . . . . . . . . . . . . . . . . . . F587. Inhibition of Ras processing in H-Ras transformed rodentfibroblasts . . . . . . . . . . . . . . . . F598. Inhibition of H-Ras signaling and transformation invitro and in vivo . . . . . . . . . . . . . . . . F599. Lack of toxicity to normal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F61

10. FTI action against K-Ras and human tumors: is theunderstanding of mechanism important? . . F6211. K-Ras processing and inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F6212. Lack of correlation between Ras mutation status and FTIsensitivity . . . . . . . . . . . . . . . . . F6313. Non-Ras targets: is there a ‘‘target X’’? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F6414. Candidates for ‘‘target X’’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F6515. Adjuvant therapy with FTIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F6716. Development of tumor resistance to FTIs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F6817. Conclusions and future questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F68Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F68References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F69

) Corresponding author. Fax: q1 919 9667681; E-mail: [email protected]

0304-419Xr97r$32.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0304-419X 97 00011-5

( )A.D. Cox, C.J. DerrBiochimica et Biophysica Acta 1333 1997 F51–F71F52

1. Summary

The high prevalence of mutated ras genes inhuman cancers makes them an attractive target fordrug design. A promising method of interfering inRas function involves inhibiting the enzyme farnesyl-

Ž .transferase FTase , which modifies Ras and otherproteins with the farnesyl isoprenoid lipid that isrequired for their correct cellular localization andbiological activity. FTase inhibitors, or FTIs, havedemonstrated the potent and specific ability to blockRas processing, signaling and transformation in trans-formed and tumor cell lines both in vitro and inanimal models. During the development of FTIs aspharmaceutical agents for clinical use, several intrigu-ing questions about their activity have arisen. Forexample, despite the requirement for intact Ras func-tion for normal cell growth, FTIs have turned out tobe surprisingly nontoxic to normal cells. In addition,K-Ras, the most commonly mutated form of Ras inhuman cancers, is far more resistant to FTI actionthan H-Ras. Nevertheless, FTIs show potent activityin reducing the tumorigenicity of some human tumorcell lines containing activated K-Ras mutants. Indeed,a simple determination of the ras mutation status of agiven tumor is not a predictor of the ability of FTIs toinhibit its growth. These findings have led to thelikelihood that inhibition of mutated Ras proteinfunction is not the sole means by which FTIs evoketheir anti-tumor activity. Thus, in addition to thenecessary improvements in pharmacokinetics of leadcompounds required to bring drugs to clinic, a con-siderable amount of attention is now being devoted toattempts to understand the mechanism of biologicalaction of FTIs. What are the biological consequencesof the recently discovered ability of K-Ras4B toserve as a substrate for geranylgeranyltransferaseŽ .GGTase I in addition to FTase? In addition toinhibition of Ras processing, what other farnesylatedproteins are affected? How important are these otherproteins in the final determination of FTI responsive-ness? What can be done to optimize the efficacy ofFTIs? This review will summarize our current under-standing of the biological consequences of FTI treat-ment of normal and transformed cells, and presentour own speculations as to where future researchefforts in these areas might usefully be directed.

2. Introduction

Since 1982, when mutated ras genes were firstdetected in human cancers, extensive research studieshave determined the function of Ras proteins andhave established an important contribution of aber-rant Ras function to human carcinogenesis. Conse-quently, there is considerable interest in the develop-ment of inhibitors of Ras function for use in cancertreatment. In particular, significant progress has beenmade in the development of farnesyltransferase in-hibitors as potential anti-Ras drugs. While there issignificant evidence to support the anti-tumor actionof these inhibitors, much remains to be understoodabout how these inhibitors may target Ras functionand how effective they will be for cancer treatment.This review will provide an assessment of the currentstatus of these efforts.

3. The role of aberrant Ras function in humancarcinogenesis

Mutated versions of the three human ras geneshave been detected in 30% of all human cancers,implying an important role for aberrant Ras function

w xin carcinogenesis 1–3 . The frequency is not uniformwith respect to tumor type, suggesting that ras muta-tions contribute to the development of some, but notall, tumors. For example, ras mutations are highly

Ž . Ž .prevalent in pancreatic 90% , lung 40% and col-Ž .orectal 50% carcinomas. Thus, aberrant Ras func-

tion is believed to contribute to the development of atleast a major subset of these neoplasms. In contrast,mutated ras genes are rarely associated with thedevelopment of breast, ovarian and cervical carcino-

Ž .mas )5% . Therefore, aberrant Ras function maynot be important in promoting or maintaining themalignant and invasive properties of these tumors.Alternatively, since Ras proteins are regulators ofdiverse extracellular signaling pathways that regulate

w xcell growth, differentiation and apoptosis 4,5 , thederegulated function of other cellular components cancause aberrant Ras function in the absence of muta-

w xtions in Ras itself 6 . For example, overexpression ofthe HER2rNeurErbB2 or epidermal growth factor

( )A.D. Cox, C.J. DerrBiochimica et Biophysica Acta 1333 1997 F51–F71 F53

Ž . Ž .EGF cell surface receptor tyrosine kinases RTKsis common in breast cancers, and their transformingactions are dependent on signaling through Ras pro-

w xteins 7 . Similarly, the loss of function of negativeRas regulators, such as the protein encoded by the

Žtumor suppressor gene NF1-GAPrneurofibromin;.see below lost in type 1 neurofibromatosis-associ-

ated malignancies such as malignant schwannomas,w xcan cause aberrant upregulation of Ras function 8,9 .

Therefore, the importance of aberrant Ras function inhuman cancers may be greater than expected andextend beyond those tumors that harbor mutated rasalleles.

The frequency of mutations is also not uniformw xwith respect to the three human ras genes 1–3 .

Thus, although mutated H-ras was the first to beidentified, and consequently has been the one mostheavily studied, mutations in K- and N-ras are muchmore frequently seen in human tumors. Mutated K-rasis seen predominantly in pancreatic, lung and colorec-tal carcinomas, whereas mutated N-ras is most com-monly associated with hematopoietic neoplasms.Since the proteins encoded by the three ras genes

Ž .share strong amino acid identity 90% , and exhibitthe same potent transforming action in experimentalcell culture and animal studies, it has been assumedthat the biochemical and biological properties of H-

Ž .Ras, N-Ras and K-Ras 4A and 4B isoforms arew xessentially identical 1–3 . However, there is emerg-

ing evidence that potentially important differencesmay exist. Therefore, much of our understanding ofRas function, based on the study of H-Ras, may notbe completely generalizable to N-Ras and K-Rasproteins.

The frequent association of mutated ras geneswith human tumors implicates such mutations asimportant genetic lesions that promote human onco-genesis. Although such a causal role is based primar-ily on guilt by association, the extensive body ofevidence from experimental cell culture and animalcarcinogenesis studies provides a compelling argu-ment that aberrant Ras function must contribute sig-nificantly to some aspect of the malignant phenotype

w xof tumor cells 1–3 . However, direct evidence forthis is limited, and the precise contribution of aber-rant Ras to the malignant and invasive growth proper-ties of tumor cells is presently unresolved. The pre-vailing evidence suggests that aberrant Ras function

contributes to different aspects of the transformedand malignant phenotype in different tumors. Forexample, whereas aberrant Ras function may promotethe uncontrolled proliferation of some tumor cells, itmay dictate the invasive or metastatic properties ofother tumor cells. An important additional considera-tion is that ras mutations alone are not sufficient to

w xconvert fully normal cells to full malignancy 10–12 .Instead, this requires the combined action of mutatedoncogenes such as ras together with other genetic

Žlesions e.g., the loss of tumor suppressor gene func-.tion . This demonstrates the multi-step nature of tu-

mor progression and supports the idea that the conse-quences of ras mutations may differ depending oncontext.

Since mutated ras alone is but one genetic lesionessential for the complete conversion of a normal cellto the fully malignant state, this raises the question ofwhether pharmacologic approaches to correct the rasdefect alone will have any significant anti-tumoractivity. Instead, will effective cancer treatment re-quire the combined correction of ras and other de-fects? Fortunately, limited experimental evidencesuggests that correction of just a single defect such asthe ras mutation can significantly impair the aberrantgrowth of tumor cells. Strong support for this hascome from the analyses of genetic variants of humantumor cell lines that differ only in their ras mutationstatus. For example, in one study, homologousrecombination was utilized to isolate variants of two

Žhuman colorectal carcinoma cell lines HCT 116 and.DLD-1 that had lost either the normal or mutated

w xK-ras allele 13 . In another study, a genetic variantof the HT1080 human fibrosarcoma cell line that had

w xlost its mutated N-ras allele was isolated 14 . Al-though loss of the mutated ras alleles did not com-pletely revert the aberrant growth properties of thesetumor cell lines, a significant impairment in theirtransformed and tumorigenic properties was ob-served. Since the variants that lacked the mutated rasgene still harbored the original mutations in the other

Ž .genes e.g., loss of the p53 tumor suppressor gene ,these observations suggested that correction of theras defect, in the continued presence of other geneticlesions, is sufficient to cause a significant impairmentin the growth of tumor cells. Thus, despite the com-plex multitude of genetic defects that drive the malig-nant phenotype, it is believed that correction of the


ras defect alone can have a significant impact ontumor growth.

4. The role of Ras proteins in signal transduction,normal proliferation and cell cycle progression:establishing avenues for drug discovery efforts

The use of anti-sense oligonucleotides or catalyticŽ .RNAs ribozymes to block the expression, rather

than the function, of Ras proteins has met with somesuccess, although these technologies are still being

w xrefined 15,16 . The development of more specificallytargeted means of blocking Ras protein function re-quires that we understand how Ras proteins function,define their role in normal cell physiology and deter-mine how mutated Ras proteins are altered in func-tion and biology. Extensive research studies over the

past decade have accomplished many of these goalsand have identified at least four possible means ofcorrecting aberrant Ras protein function in tumor

w x Ž .cells, as will be described below 17–19 Fig. 1 .The three ras genes encode four closely related 21

w xkDa proteins 1 . K-ras encodes two proteins, desig-nated K-Ras4A and K-Ras4B, that differ only in theirCOOH-terminal 25 amino acids. The transcript for

Žthe 4B variant was the predominant version 10- to.20-fold expressed when evaluated in one mouse and

w xseveral human cell lines 20,21 and it is assumed thatK-Ras4B is the predominant protein species ex-pressed in mammalian cells. All four Ras proteinsfunction as biologic switches that are regulated by

Žtheir association with guanine nucleotides GDP and. w xGTP 22–24 . Normal Ras proteins typically reside

in the inactive, GDP-bound state. Upon stimulationinitiated at the cell surface by a variety of extracellu-

Fig. 1. Approaches to targeting Ras function for cancer treatment. At least four distinct approaches have been considered for thedevelopment of pharmacologic agents to antagonize the transforming actions of mutated Ras proteins. First, since oncogenic mutants ofRas contain mutations at residues 12, 13 or 61 that impair their sensitivity to Ras GAPs, the generation of novel GAPs that can stimulateintrinsic GTPase activity may be possible. Second, since the activated, GTP-complexed form of Ras exhibits a distinct conformation fromthat of its inactive, GDP-complexed counterpart, compounds that preferentially recognize Ras-GTP may selectively antagonize mutatedRas proteins. Third, targeting components important for Ras signaling and function is currently an active area of investigation. Finally,

Ž .approaches that prevent Ras membrane association, such as FTase inhibitors FTIs , represent the direction that has met with the greatestcurrent success. Whether specific membrane components are important for Ras membrane association is not known but such componentsmay also represent targets for blocking Ras function.


lar ligands, Ras guanine nucleotide exchange factorsŽ .SOS1r2 and RasGRFrCDC25 are activated tostimulate the exchange of bound GDP for GTP to

w xform the active, GTP-complexed Ras protein 24 .This active state is transient, and Ras GTPase activat-

Ž .ing proteins Ras GAPs; p120- and NF1-GAP stimu-late the intrinsic GTP hydrolysis of Ras to cycle it

w xback to the inactive GDP-complexed state 23 . Themutated ras genes present in human tumors encode

Ž .single amino acid mutations at residues 12, 13 or 61that render these oncogenic Ras mutant proteins in-sensitive to GAP stimulation. Hence, they remainconstitutively active in the absence of external stim-uli. The fact that Ras function is regulated byGDPrGTP cycling suggests two possible approachesfor antagonizing Ras protein function. First, the gen-eration of variant ‘‘super’’ Ras GAPs that can stimu-late the GTPase activity of oncogenic Ras proteinsand thus convert them to inactive proteins has beenattempted, but with no success reported. Second,since the inactive and active forms of Ras differ in

w xprotein conformation 25,26 , compounds that prefer-entially recognize the active, GTP-bound form mayspecifically target mutated Ras proteins. Similarly,GTP analogs have also been considered. The identifi-cation of peptide sequences that show strong, prefer-ential binding to Ras-GTP suggests the possibilitythat such compounds might be developed into clini-

w xcally useful agents in the future 27,28 .A third approach to correcting aberrant Ras activ-

ity comes from understanding its modes of action.Ras is involved in a wide diversity of signalingpathways, as evidenced by its transient activation bygrowth factors, hormones, neurotransmitters and cy-

w x Ž .tokines 4,5 Fig. 2 . Upon activation, Ras relays thestimulus by activating a cascade of protein kinasesŽ Ž .e.g., Raf and mitogen activated protein MAP ki-

.nases that in turn initiates further cytoplasmic andnuclear events to alter cell growth and differentiationw x29–31 . This key role in modulating such diversepathways accounts for the dramatic consequences ofoncogenic Ras proteins on cell behavior. Thus, athird approach to targeting Ras protein function wouldbe to prevent activated Ras from relaying its down-stream signal by interfering at different points in thecascade. In this regard, the discovery of specificantagonists of the Raf, MEK and MAPK kinases thatplay an important role in Ras signaling are current

Fig. 2. Diverse extracellular signals activate Ras. The involve-ment of normal Ras proteins in signaling pathways initiated bydiverse extracellular ligands is well-established. These include

Ž . Ž .growth factors e.g., EGF , hormones e.g., insulin , cytokinesŽ . Ž .e.g., interleukins and neurotransmitters e.g., carbachol . Rasfunctions as a membrane-associated biologic switch that relaysthe signal from ligand-stimulated receptors to cytoplasmic signal-ing cascades. These include G protein-coupled serpentine recep-

Ž . Ž .tors SR , receptor tyrosine kinase receptors RTK and receptorsthat cause stimulation of associated nonreceptor tyrosine kinasesŽ .NRTK such as Src. Thus normal Ras may be constitutivelyactivated by upregulation of receptor stimulation, and conse-quently, be a target for FTI inhibition of transformation even incells that lack oncogenic Ras mutations.

goals of ongoing anti-Ras drug discovery programsw x32 . The potential application of such kinase in-hibitors as anti-Ras drugs is supported by observa-tions that kinase-deficient mutants of Raf-1, MEKand MAP kinases are potent inhibitors of Ras trans-

w xformation 33–38 . Further, recent observations thatRas may cause transformation by activating multiplesignaling pathw ays, in addition to the

w xRafrMEKrMAPK cascade 39–42 , suggest thatother components important for Ras signaling mayalso serve as key targets for blocking Ras function.


For example, Ras activates a second signaling path-way that involves members of the Rho family of

Ž . w xRas-related proteins e.g., Rac1 and RhoA 43–46 ,which in turn are activators of components of the

Žstress-activated protein kinase SAPK; also Jun. w xNH -terminal kinase, or JNK pathway 47–49 . The2

ability to block Ras transformation in vitro by block-ing Rac1, RhoA, RhoB or JNK activity using domi-

w xnant negative gene constructs 43–46,50 suggeststhat pharmacologic inhibition of this second Raspathway may also be a viable approach to cancertreatment.

A fourth approach to blocking Ras function issuggested by the fact that Ras proteins require lipidmodification and tight association with the plasma

w x Ž .membrane for full biologic activity 51–53 Fig. 3 .Ras proteins are initially synthesized as inactive,cytoplasmic proteins. However, a series of rapid post-translational modifications, signaled by a consensusCOOH-terminal tetrapeptide sequence present on allRas proteins, then causes the translocation of Ras tothe inner surface of the plasma membrane. Thistetrapeptide sequence is generally referred to as the

CAAX box or CAAX motif and consists of cysteinefollowed by two aliphatic amino acids and a terminalserine or methionine. Within minutes of synthesis,the cytoplasmic FTase enzyme catalyzes the covalentaddition of a C15 farnesyl isoprenoid moiety, from a

Ž .farnesyl pyrophosphate FPP donor, to the cysteineresidue of the Ras CAAX motif. This modification isfollowed rapidly by proteolytic removal of the AAXresidues and carboxylmethylation of the now farnesy-lated cysteine residue. H-Ras, N-Ras and K-Ras4Aundergo additional modification of upstream cysteine

Ž .residue s by the fatty acid palmitate, whereas K-Ras4B contains a lysine-rich sequence directly up-stream of the CAAX motif. Although the CAAX-sig-naled modifications are essential to promote Rasmembrane association, the palmitate modification orthe lysine-rich sequence serves as a second mem-brane-targeting signal to facilitate full plasma mem-

w xbrane association 54–56 , along with additional se-w xquence information 57 . However, while each

COOH-terminal modification increases protein hy-drophobicity and contributes to Ras membrane asso-ciation, the initial farnesylation step alone is suffi-

Fig. 3. Ras processing and membrane association is critical for Ras transforming activity. Ras proteins are initially synthesized ascytosolic, inactive proteins. Within minutes, Ras proteins undergo a series of CAAX-signaled posttranslational modifications that promotetranslocation and stable association with the inner surface of the plasma membrane. The first modification is catalyzed by farnesyltrans-

Ž .ferase to cause covalent addition of the farnesol group from farnesylpyrophosphate FPP onto the cysteine residue of the CAAXŽsequence. Proteolytic degradation to remove the AAX residues, followed by carboxylmethylation of the farnesyl-cysteine residue donor:

.S-adenosyl methionine, SAM , complete the CAAX-signaled modifications. FTI inhibition of Ras farnesylation renders Ras completelycytosolic and nontransforming.


cient to promote significant membrane associationw xand transforming potential 58 . Therefore, inhibitors

of FTase may serve as potent anti-Ras drugs.

5. Generation of prenylation inhibitors

Both the screening of natural compounds and ra-tional drug design have resulted in the identificationof compounds that inhibit protein prenylation, al-though their mechanisms of action vary widely. In-

Ž .hibitors of hydroxymethylglutaryl HMG CoA re-Žductase such as compactin, and the statins lovastatin,

.mevastatin, simvastatin, etc. and of mevalonate ki-nase inhibit all prenylation by preventing the synthe-sis of the isoprenoid precursors farnesyl pyrophos-

Ž .phate FPP and geranylgeranyl pyrophosphateŽ . Ž .GGPP Fig. 4 . Limonene, a plant byproduct of themevalonate pathway, has been shown to impair Ras

w xprenylation, possibly by competition for FPP 59 andw xto exhibit anti-tumor activity 60 .

Several general approaches have been employed inthe identification and development of specific FTase

Ž . w xinhibitors FTIs 61,62 . First, high throughput ran-dom screens have been used identify natural or li-brary compounds that inhibit the ability of FTase tocatalyze the addition of farnesol to recombinant H-Rasin vitro. Random screening of microbial and naturalproducts using a yeast genetic screen for cell-permea-ble Ras farnesylation inhibitors has identified themicrobial product manumycin and its relatives as

w xhaving FTI activity 63 . Second, Ras CAAX tetra-peptide sequences alone are sufficient to signal modi-fication by farnesylation and can serve as potent

w xinhibitors of FTase activity in vitro 64,65 . There-fore, a variety of CAAX-based peptidomimetic ornonpeptidic compounds have been synthesized ac-cording to principles of rational drug design. Conse-

Fig. 4. Farnesylpyrophosphate is a critical substrate for protein prenylation and other cellular metabolites. Mevalonate is the essentialŽ .precursor for all isoprenoid biosynthesis, including farnesyl pyrophosphate FPP . FPP is required for production of GGPP, and both FPP

and GGPP are used by three protein prenyltransferases for protein prenylation. FPP is also used for the biosynthesis of cholesterol andother byproducts of the mevalonate biosynthetic pathways. Whereas compactin and lovastatin block all isoprenoid biosynthesis, FTIs

Ž . Žselectively block the farnesylation of Ras and other CAAX where XsS, M and C terminating proteins. GGTase I CAAX, where. Ž .XsL and F and GGTase II CC, CXC and CCXX terminating members of the Rab family of Ras-related proteins catalyze the

geranylgeranylation of proteins.


quently, the potent and selective FTase inhibitorsŽ .FTIs identified to date include a diverse collectionof apparently unrelated structures discovered by ran-dom screening of both natural and synthetic com-pounds, as well as FPP analogs, peptidomimetic in-hibitors that are patterned after the CAAX motif, andeven bisubstrate compounds that include both FPPand peptidomimetic structures. For biologically ori-ented reviews of the structures and activity of FTaseinhibitors described to date, the reader is referred to

w xreferences 61,62,66 . Those inhibitors with demon-strated activity in cells are listed in Table 1.

6. Initial characterization of FTIs: potency andselectivity

In each case, the first screen is usually the abilityof a given agent to inhibit the incorporation in vitro

w xof 3H -prenyl phosphate into recombinant full lengthRas protein or a peptide containing the CAAX se-quence of interest. Over time, the reported IC50s forinhibition of Ras processing in vitro have dropped tosubnanomolar concentrations. The next step, criticalfrom the perspective of drug design, is to overcomethe poor cell permeability and instability that can beassociated with many of the compounds that performwell in vitro processing assays. Compounds fre-quently lose 2 or 3 logs of potency in whole cells dueto these issues, such that even subnanomolar com-

pounds in vitro become submicromolar compounds inwhole cells. Early reports of efficacy in inhibition ofRas processing in intact cells described IC50s of upto 100 mM, but newer compounds are effective at0.01 to 1 mM. The derivation of nonpeptidic, non-thiol compounds has also been pursued vigorously

w xand has been discussed elsewhere 61,62 .Developers of FTIs have sought to identify com-

pounds with a high degree of selectivity for FTaseover the related protein prenyltransferase, geranylger-

Ž .anyltransferase I GGTase I , with which FTase sharesw xa common a subunit 67 . Whether a CAAX-

terminating protein is recognized by FTase or GGTaseI appears to be determined largely, but not exclu-sively, by the nature of the terminal X residue, suchthat GGTase I recognizes Xs leucine whereas FTase

wrecognizes Xsserine, methionine and cysteine 68–x Ž70 . Whereas substrates of FTase are limited ;50

.mammalian proteins , a far greater number of pro-wteins are known to be substrates for GGTase I 51–

x53 . Included among these are Ras-related proteinsthat control actin cytoskeletal organization and gene

Ž .expression Rho family proteins and g subunits ofheterotrimeric G proteins that mediate the signalinginitiated from a wide diversity of serpentine recep-tors. Thus, it has been assumed by most investigatorsthat a nonselective FTI that impairs the processingand function of GGTase I-modified substrates inaddition to FTase-modified substrates will have seri-ous, nonspecific toxic side-effects.

Table 1Cell permeable inhibitors of Ras prenylation

FPP Inhibitors CAAX inhibitors FPPrCAAX inhibitors

Ž . w x w x w xa- hydroxyfarnesyl phosphonic acid 133 B1086 100 BMS-186511 75,76,85w x w xBMS Inhibitor III 134 B581 73,74

w x w xManumycin 135 B956 100,138w xBMS tetrahydroisoquinoline 91

w xBZA-5B 71,108w xFTI-265 136w xFTI-276 101w xFTI-277 77,81,84,114

w xGGTI-286 110w xGGTI-298 84w xL-731,734 72w xL-739,749 88,89,103,131w xL-744,832 90,102w xSCH44342 86,112


Selectivity of the compounds for inhibition ofFTase I vs. GGTase I is then often demonstrated bycomparing the effects of those compounds on the

w xability to transfer 3H -FPP onto H-Ras, but not ontoŽ .an H-Ras mutant terminating in leucine XsL or

onto the authentic substrate Rap1a, which is naturallygeranylgeranylated by GGTase I; generally, only themost selective compounds have been chosen for fur-ther study. Reported selectivity levels have rangedfrom a hundred fold or less to several thousand fold,with considerable debate as to how much selectivity

Ž .is necessary or desirable see below .

7. Inhibition of Ras processing in H-Ras trans-formed rodent fibroblasts

The first demonstrations of specific inhibition ofRas processing in whole cells were reported in rodentfibroblast model systems such as Rat-1 or NIH 3T3

w xcells transformed by oncogenic H-Ras 71–77 . Ad-Ž .vantages of using such model systems include 1Ž .wide availability of reagents to study H-Ras; 2

simple assessment of the processed vs. unprocessedstate of the overexpressed, mutated Ras by elec-

Ž .trophoretic mobility shift assays; 3 an absolute re-quirement for the function of the oncogenic Ras

w xprotein for the transformation of those cells 78–80 ,thereby providing an easily interpretable system inwhich reduction of Ras function reliably results in a

Ž .reduction in transformation; 4 the availability ofwell-characterized signaling pathways involved in Rasfunction whose disruption as a consequence of FTI

Ž .activity can be tested; 5 the availability of matchedsets of control cells transformed either by mutantforms of Ras proteins modified with different lipids

Ž .or by other oncogenes; and 6 not insignificantly,ease of cultureruse. Although there are also disad-vantages, including a nonhuman genetic backgroundand a nonepithelial tissue origin, the use of thesemodel cells clearly provides an excellent startingpoint for the characterization both of FTIs as a classand of the properties of individual compounds.

Characterization of H-Ras processing in such cellsclearly indicated the ability of FTIs to potently andspecifically inhibit H-Ras farnesylation, without af-fecting the modification of geranylgeranylated pro-teins such as Rap1a or genetically-engineered H-Ras-

w xGG mutant. Indeed, 3H MVA labeling of wholecells demonstrated very little effect on overall preny-lation, presumably due to the low proportion of farne-sylated proteins among all prenylated cellular pro-teins.

8. Inhibition of H-Ras signaling and transforma-tion in vitro and in vivo

With compounds that successfully made the transi-tion from in vitro to in vivo activity against process-ing, experiments could be performed that more di-rectly addressed the issue of potential anticancer ther-apy: do the agents that block Ras processing alsoblock Ras function and Ras-dependent transforma-tion?

At about the time that FTIs were first developed,Ras signaling pathways were also being worked out,such that disruption of elements of the newly charac-terized Ras-Raf-MAPK kinase cascade served as aconvenient readout for FTI activity. For example, FTItreatment of NIH 3T3 cells transformed by oncogenicH-Ras resulted in the accumulation of inactiveRasrRaf complexes in the cytosol, whereas in un-treated cells, Ras and Raf interaction at the plasmamembrane is productive and results in a downstream

w xsignal 81 . Several groups showed that FTI treatmentreduced constitutive MAPK activation in Ras-trans-

w xformed rodent cells 74,81,82 , although clearly notall mechanisms for MAPK activation were affected.Neither serum-stimulated nor platelet-derived growth

w xfactor signaling was disrupted by FTIs 82–84 , im-plying a lack of dependence on H-Ras activity inthese events. Further downstream of these cytosolickinases are nuclear events including transcriptional

Ž .activity from Ras-responsive elements RREs foundin the promoters of many genes important to prolifer-ation, carcinogenesis, invasion and metastasis. FTItreatment was found to reduce the transcriptionalactivity from reporters driven by the etsrAP-1 RREand of the transcription factors that stimulate these

Ž . w xelements Elk-1 and c-Jun 74 .These studies were done in parallel with those

attempting to determine the consequences of FTIactivity on Ras transformation. Characteristics of thetransformed phenotype include alterations in mor-


phology of individual cells, a decrease in adherenceto substrate, decreased dependence on growth factors,an ability to grow in an anchorage-independent man-ner, and tumorigenicity in appropriate animal hostsw x80 . All of these features have been addressed in avariety of studies.

The easiest and earliest studies involved the simpleobservation of transformed cells to determine whetherthey reverted to a normal morphology following FTItreatment. In several studies, FTI-treated cells didindeed show such a reversion following 24–48 h of

w xtreatment 71,74–76,85,86 . In some cases, Ras- butnot Raf-transformed Rat-1 cells flattened and spread,in apparent specificity for Ras inhibition. In othercases, however, the alterations in morphology werenot so tightly correlated with inhibition of Ras pro-cessing, and these observations have led to consider-

Ž .able speculation on the nature of the target s in-volved in these events. For example, reminiscent ofthe lovastatin study indicating clearly that its growthinhibitory effects were not due to inhibition of Ras

Žfarnesylation by the demonstration that cells express-ing the genetically-engineered myristylated H-Ras

. w xmutant were equally affected 87 , one FTI studyshowed that morphological reversion occurred inRas-transformed NIH 3T3 cells regardless of the lipid

w xmodification that enabled Ras to transform 74 . Simi-larly, morphologic reversion of Ras-transformed Rat-1cells was found to occur more rapidly than inhibition

w xof Ras processing 88 . These results imply that there

is a target downstream of Ras that controls the mor-Ž .phological reversion in those cells. Candidate target s

are listed in Table 2 and some possibilities are dis-cussed below.

FTIs could also inhibit the growth of Ras-trans-w xformed cells in soft agar 72,74–76,85,89–91 , a

characteristic often cited as the best in vitro growthw xparameter predictive of tumorigenicity in vivo 92 .

Controls for these experiments included Src, Raf, andMos-transformed cells, as well as the lipid mutants ofH-Ras. In general, the preferential inhibition of farne-sylated Ras provided strong support for the notionthat FTIs acted by inhibiting Ras farnesylation andbiological function. Additionally, the failure of FTIsto revert Raf-mediated transformation supported the

w xselectivity of these compounds 72,74,89,93 , sinceRaf transformation has been shown to be Ras-inde-

w xpendent 94,95 .Some cytosolic mutants of oncogenic Ras have

been shown to function as dominant negative mutantsw xof Ras 96–98 . Thus, it has been suggested that FTI

inhibition of Ras transformation may be mediated bya combination of inhibition of most Ras processingand a dominant negative effect of FTI-induced unpro-cessed Ras on any remaining processed Ras. Whetherall forms of oncogenic Ras exhibit this dominantnegative activity when cytosolic and whether this isan important component of the mechanism of FTIinhibition of Ras transformation remain to be deter-mined.

Table 2Farnesylated mammalian proteins

Ras-H, -N, -K4A, -K4B Signaling for growth, differentiation and apoptosisLamins A and B Nuclear membrane structureRhoB, RhoE, others Cytoskeletal organization, gene expression, cell cycle controlRap2 Platelet functionRheb Antagonist of RasPhosphorylase kinase a and b Skeletal muscle functionRhodopsin kinase Visual signal transductionTransducin g Visual signal transductioncGMP phosphodiesterase a Visual signal transductionIFN-induced guanylate-binding protein 1 Cytokine mediatorIP 5-phosphatase Lipid phosphatase, calcium signaling3

PxF Peroxisomal locationPRL-1rPTPCAAX1 and 2 Protein tyrosine phosphatase

and 20–30 additional unidentified proteins of unknown function


These in vitro studies were extended with dramaticresults to in vivo tumor models using both Ras-trans-formed rodent cells grown as subcutaneous

w xxenografted tumors in nude mice 89,99–101 , and inviral H-Ras transgenic mice in which salivary andmammary tumors occur stochastically in a high per-

w xcentage of mice carrying the transgene 102 . Espe-cially in the early studies, FTIs were delivered mostoften by the intraperitoneal route and the animalswere dosed frequently, sometimes as often as fourtimes per day, with up to 40 mgrkgrday of drug.Although few studies actually demonstrated the cor-relation between inhibition of tumor growth and inhi-

Žbition of Ras processing an exception being thew x.work of Nagasu et al. 100 , the sometimes impres-

sive reductions in tumor growth rate and volumeprovided additional evidence that FTIs had the poten-tial for use as anticancer agents.

Even so, it was still not clear from the promisingresults in the tumor xenograft models that thesecompounds would prove to be clinically useful. First,FTIs impaired tumor development without regressionof established tumor masses in nude mouse xenograftsw x89,100,101 , and are believed to act by preventingthe formation of new, fully biologically competentRas proteins, rather than by depleting existing Rasproteins. These observations suggest that the cyto-static nature of FTI action will require chronic, long-term application for efficient tumor inhibition in hu-man cancer patients. Whether long-term treatmentwould result in deleterious side-effects not seen inthese short-term experiments, whether tumor cell re-sistance to FTIs would be likely to occur, and whethercombination therapies will be required for effectiveFTI treatment are questions that are currently beingexplored.

Also, cancer patients generally present with a sub-stantial tumor burden, so a more realistic assessmentof the potential utility of FTIs in clinical situationsrequires that they be tested for their ability to revert,rather than simply to prevent, tumor growth. To thepleasant surprise of many, FTIs appear to be capableof doing just that: a study from the Merck group

w xshowed 102 that the FTI L744,832 caused a dra-matic and rapid reduction in tumor size, nearly ablat-ing large tumors, in the H-Ras transgenic mouse.However, residual tumor cells were able to regrowfollowing withdrawal of the compound, although

many of the recurrent tumors remained sensitive tothe additional drug treatment. The mechanism bywhich the rapid loss of tumor is accomplished isunder investigation, but may involve induction ofapoptosis, as has been shown for FTI-treated Ras-transformed rodent fibroblasts grown under anchor-

w xage-independent conditions 103 . The potential ofFTIs to cause tumor regression in patients remainsunresolved.

9. Lack of toxicity to normal cells

An unexpected, but desired, aspect of FTIs is theirapparent lack of growth inhibitory activity against

w xnormal rodent fibroblast cells 71–77 . This lack oftoxicity at concentrations that prevent H-Ras process-ing was not anticipated and has been a particularlypleasant surprise in light of the original predictionsthat, since farnesylation is also required for normalRas function, which is critical for normal cell viabil-

w xity 94,95 , FTIs might prevent transit through thecell cycle in normal cells as well, thereby providingonly the slimmest of therapeutic windows. Unexpect-edly, however, FTIs not only inhibit the growth oftransformed cells in culture far more than they inhibitthe growth of normal cells, but have also been foundto be surprisingly benign in treated tumor-bearinganimals, with no obvious toxicity reported in anynormal tissues following treatments of up to sixweeks. One possible explanation is that some ger-anylgeranylated Ras-related proteins, such asTC21rR-Ras2, share functional overlap with Rasproteins and can compensate for the loss of Ras

w xfunction 76,104,105 . Another possibility, addressedbelow, is that the endogenous Ras proteins expressedin rodent fibroblasts are K-Ras4B or N-Ras, whoseprenylation and function are not as effectively blocked

w xby FTIs as are those of H-Ras 106 . A third explana-tion is that FTIs can reduce oncogenic Ras functionto below a critical threshold required to cause trans-

w xformation 107 , yet allow sufficient Ras function tobe spared for maintenance of essential normal Rasfunctions. There is evidence for each of these possi-bilities and it is likely that all three may contribute tothe lack of FTI toxicity in normal cells at concentra-tions that impair H-Ras transforming activity.


10. FTI action against K-Ras and human tumors:is the understanding of mechanism important?

Because H-ras mutations are relatively rare inhuman cancers compared to mutated K-ras or N-ras,a key question then became whether FTIs would alsobe effective against the other Ras proteins. Althoughthe first studies using H-Ras transformed rodent fi-broblasts yielded very promising evidence that FTIscould be effective for cancer treatment, the subse-quent extension of these analyses to K-Ras trans-formed fibroblasts and to human tumors resulted insome unexpected complications. One study showedthat the FTI L-739,749 had activity against all threeforms of Ras proteins in a nude mouse xenograft

w xmodel using Ras-transformed Rat-1 cells 89 , but itquickly became apparent that in general the fortu-itously selected H-Ras tumors tested during the earlyattempts were far more sensitive than K-Ras-trans-formed cells. In fact, at least a ten- to 100-folddifference in IC50s was predictable, regardless ofwhich FTI was used. Although relatively few studieshave examined the effectiveness of FTIs on N-Ras-transformed rodent cell lines, study after study hasshown that K-Ras-transformed cells were relativelyresistant. At first glance, this was not surprising,since K-Ras was known to be a better substrate for

w xthe FTase enzyme itself 64 , and therefore its pro-cessing would be predicted to be more difficult toinhibit. However, subsequent experiments showedthat inhibition of K-Ras transformation could be un-linked from inhibition of K-Ras processing. Theseobservations suggested the possibility that K-Ras4Bmay not be farnesylated in vivo or that FTI inhibitionof transformation might be a consequence of inhibit-ing the function of other farnesylated proteins that arerequired for Ras transformation. Determining the va-lidity of either of these scenarios has important con-sequences for the optimal design of effective FTaseinhibitors.

11. K-Ras processing and inhibition

Although it has been relatively simple to demon-strate inhibition of H-Ras processing in whole cells,

demonstrating prenylation inhibition of K-Ras hasproven more troublesome. Because the K-Ras4B pro-tein, the form of Ras most frequently mutated in

Ž .human tumors, terminates in a CAAX motif CVIMthat is a better substrate for FTase than is H-RasŽ .CVLS , it was not considered surprising that itshould be more difficult to block. Indeed, the originalpurification of FTase on a K-Ras4B hexapeptideaffinity column took advantage of the tighter bindingof FTase to K-Ras, and initially, it was believed thatK-Ras4B processing was less sensitive to FTIs sim-ply because of the 20-fold higher affinity of the

w xhexapeptide for FTase 64 . Support for this possibil-ity is suggested by observations that significantlyhigher concentrations of FTIs are required to blocknuclear lamin A and B versus H-Ras farnesylation in

w xvivo 71,73,108 . Like K-Ras4B, the lamins alsoterminate in Xsmethionine. In addition to confer-ring specificity for FTase vs. GGTase I, the terminalX residue also influences affinity for FTase. Thus,K-Ras4B farnesylation may simply require signifi-cantly higher concentrations of FTIs.

Alternatively, recent studies showed that K-Ras4Bwas a substrate for either FTase or GGTase I in vitrow x109,137 , suggesting that FTI insensitivity may bedue to alternative K-Ras4B processing by GGTase Iin vivo. The ability of K-Ras4B to become geranyl-geranylated by GGTase I in vitro requires both theCVIM motif and the upstream polybasic regionw x109,137 , a combination which appears in no otherRas protein. Less clear is what happens to K-Ras4Bin vivo. Support for the possibility that K-Ras4Bmight be geranylgeranylated in vivo was provided bythe rather unexpected observation that a GGTase I

w xinhibitor blocked K-Ras4B transformation 110 . It ispossible that a more selective GGTase inhibitor maynot retain this ability, but this remains to be deter-mined. Oncogenic Ras transforming activity can bepromoted with equivalent efficiency by either farne-

w xsylation or geranylgeranylation 69 . While this issueis presently unresolved, the bulk of the current exper-imental data suggests that K-Ras4B is normally far-

w xnesylated 111 , but can undergo modification byw xGGTase I when cells are treated with FTIs 138 .

Thus, studies are ongoing to determine whether theŽ .coordinate use of GGTase I inhibitors GGTIs and

FTIs will be more effective against cells harboringmutated K-ras.


12. Lack of correlation between Ras mutationstatus and FTI sensitivity

Because of previous findings that FTIs were capa-ble of inhibiting Ras transformation in animal modelsystems, and because of a widespread tendency toview FTIs as Ras inhibitors, rather than as FTaseinhibitors, many investigators assumed that humantumor cells that contained mutated Ras proteins wouldbe more sensitive to FTI action than those that didnot. Further, most human tumors with Ras mutationshave activated K- or N-ras alleles rather than H-ras,and it also seemed reasonable to suppose that therelative sensitivity to FTIs could be predicted by therelative sensitivity of the particular ras allele thatwas mutated. Indeed, one series found just that:tumors could be grouped both according to H-, N- orK-ras mutation status and their relative FTI sensitiv-

w xity decreased in that order 100 . However, in a largerstudy of human tumor cell lines, ras mutation status

w xwas not predictive of FTI sensitivity 90 . To thecontrary, although the overall efficacy of the FTIL744,832 was quite high, )60%, all possible combi-nations were seen, such that some tumor cell lines

containing only wild type Ras were sensitive whereassome lines containing mutant Ras were resistant.

The apparent discordance between FTI sensitivityand ras mutation status of human tumors may beexplained in part because the precise contribution ofmutated ras to the malignant phenotype may varyconsiderably among different tumor cells. Fig. 5shows a number of possibilities for the biologicalconsequences of Ras mutations and summarizes whyRas dependence may be a more important predictiveparameter of FTI sensitivity than Ras mutation status.For example, some tumors may exhibit aberrant Raspathway function due to alterations not in Ras itselfbut in Ras-regulating components, including the

w xHER2rNeurErbB2 or BcrrAbl tyrosine kinases 4,5Ž . w xor neurofibromin NF1-GAP 8,9 . Therefore, FTI

inhibition of Ras function would still have a signifi-cant impact on the aberrant growth of such rasmutation negative tumor cells because they are stillRas-dependent. For example, FTI treatment inhibitedthe transformed growth properties of a malignantschwannoma cell line where Ras activation was aconsequence of the loss of neurofibromin function

w xrather than a mutation in Ras itself 85 . Alterna-

Ž).Fig. 5. Sensitivity of human tumors to FTIs may be dictated by Ras dependency rather than ras mutation status. Mutations in RasŽ . Ž .constitute but one of multiple genetic steps that convert normal N cells to malignant and invasive tumor T cells. The sensitivity of

human tumors to FTI growth inhibition may correlate with whether transformed and tumorigenic growth potential is Ras-dependent.Ž .Thus, constitutive upregulation of receptor tyrosine kinase RTK function may also cause constitutive upregulation of Ras in the absence

of mutations in Ras itself.


tively, a tumor cell may have acquired a mutated rasallele late in tumor progression, such that other ge-netic lesions are responsible for promoting uncon-trolled growth in vitro and tumorigenicity in animals.Hence, these tumors are Ras-independent and un-likely to be impaired by FTI treatment. Finally, undersome circumstances, mutated Ras may directly orindirectly promote tumor cell invasion and metastaticpotential, rather than uncontrolled proliferation. Con-sequently, FTI treatment may still have a significantimpact on the growth of such tumors in patients.However, these inhibitory effects will not be detectedin a soft agar colony formation or nude mousexenograft tumor formation assay. A better determina-tion of the predictive value of Ras dependence vs.mutation status awaits the characterization of the FTIsensitivity of pairs of human tumor cell lines inwhich both the dependence of transformation on mu-tated Ras is known and in which mutated Ras has

w xbeen either removed or replaced 13,14 . Finally, it isalso possible that Ras is not the most important targetof FTIs, such that the presence or absence of another

Ž .target protein s would be predictive of FTI sensitiv-ity.

13. Non-Ras targets: is there a ‘‘target X’’?

Despite the tendency to view FTIs as anti-Rascompounds, it is clear that they are instead successfulanti-FTase compounds that also necessarily interferewith the processing of other farnesylated proteins in

Ž .addition to Ras Fig. 6 . Table 2 shows the mam-malian proteins currently known to be farnesylated.Indeed, original concerns about potential toxicity ofFTIs in vivo included fears that inhibition of severalsignal transducing proteins in the retina would causeblindness during treatment. Although there is no evi-dence of this particular concern at present judgingfrom the mouse studies, it could still pose a problemonce FTIs go to clinic, and the ability of FTIs tocross the blood:brain barrier will be critical in thisregard.

In addition to interference in Ras processing, thereis now evidence that FTIs may block Ras transforma-tion, in part, by blocking the function of other farne-sylated proteins, one or more of which may represent‘‘target X’’ and be critical for FTI action. First, as

Fig. 6. FTIs may block Ras transformation by blocking thefunction of Ras and other farnesylated proteins. FTIs may blockRas transformation by blocking the farnesylation not only of Ras

Ž .but also of other farnesylated proteins designated by circled F .Rho family proteins are activators of the Jun NH -terminal2

Ž .kinase JNK; also referred to as SAPK and are important for RasŽtransformation. As yet undefined farnesylated proteins desig-

.nated X may modulate Ras transformation, either as a down-stream component of Ras signaling or as a regulator or effectorof distinct pathways that can influence Ras transformation. Alter-

Ž .native prenylation of K-Ras4B by geranylgeranylation GG inthe presence of FTIs has been proposed as one mechanism ofK-Ras resistance to FTI inhibition of processing and transforma-tion.

discussed above, morphological consequences includ-ing reversion of the transformed phenotype and flat-tening of rounded cells sometimes occur under condi-tions where Ras processing and function are notinhibited. These results suggest that there may be afarnesylated protein, such as a Rho family member,downstream of Ras that is responsible for the mor-phological phenotype. This idea is discussed furtherbelow.

Second, the unlinking of inhibition of K-Ras pro-cessing and transformation suggests the possibilitythat target X is a farnesylated protein downstream ofK-Ras which is required for its transforming ability.If this protein has a lower affinity for FTase than


does K-Ras, then it would be expected to be inhibitedat lower concentrations of FTI than would K-Rasitself.

Third, the time course of biological inhibitionsometimes appears to be much different than theexpected diminution in processed Ras available. Forexample, the half-life of Ras proteins is ;20–23 hw x113 , such that after 48 h of treatment, approxi-mately 25% of the original total processed Ras wouldbe predicted to be present compared to the beginningof treatment. In addition, immediately upon removalof FTI, newly synthesized Ras becomes processedagain whereas morphological re-transformation re-

w xquires five days or more 108 , indicating a muchlonger recovery period than would be expected fromthe recovery of Ras processing. As another example,FTI treatment of Ras-transformed irradiated cells re-sults in a significant decrease in the survival of suchcells following irradiation, but the length of treatment

w xrequired is only a matter of hours 114 . Thus, eitherŽ .another protein s is important for these effects, or

Ras processing and metabolism are controlled differ-ently than currently thought. It will therefore beimportant to analyze the time course of Ras process-ing inhibition in the presence and absence of FTIsand compare these to the biological effects of FTItreatment. Our preliminary data suggest that FTItreatment can significantly alter the processing andturnover of Ras proteins in FTI-treated Ras-trans-

Žformed rodent fibroblasts and epithelial cells Cox et.al., unpublished data .

14. Candidates for ‘‘target X’’

What kinds of proteins might fit the bill for ‘‘targetX’’? First, such a protein should be farnesylated andits function should be dependent on this lipid modifi-cation. Second, its loss of function should antagonizeRas transformation, either directly or indirectly. Cur-rently, the identities of many farnesylated cellularproteins remain unknown. Candidates for target Xmay or may not turn out to be among those proteinsalready identified as listed in Table 2. Additionaltargets will no doubt be discovered by expressioncloning screens for farnesylated proteins in vitro orby molecular cloning of the genes encoding farnesy-lated proteins in vivo.

One obvious class of proteins includes members ofthe Ras superfamily, as they provide upstream anddownstream targets for the same pathways that FTIinhibition of Ras would block. Particularly attractivecandidates include members of the Rho family, asthey have been shown to be important in controllingcell shape and motility as well as gene expression

w xand cell cycle progression 49,115–117 . Experimen-tal studies showing that Rho protein function was

w xrequired for Ras transformation 43–46 is consistentwith the possibility that FTIs block Ras transforma-

w xtion, in part, by blocking Rho farnesylation 88,93 .In particular, there is some evidence that an immedi-

Ž .ate-early Rho protein RhoB that is both farnesylatedw xand geranylgeranylated 118 may be a target of FTI

w xactivity 88,93 . For example, whereas Rat-1 cellstransformed by myristylated Ras were still suscepti-

Žble to growth inhibition by L739,749 implying a.non-Ras-dependent susceptibility , transformation by

myristylated RhoB conferred resistance. This resultsuggests that RhoB, directly or indirectly, might bethe target for growth inhibition. However, the local-ization data for a genetically-engineered myristylated,nonprenylated, variant of RhoB in the absence andpresence of FTI are confusing. Further, RhoB can

w xbecome farnesylated by the action by GGTase-I 119 ,adding another layer of confusion to the conse-quences of FTI action to RhoB function. Hence, therole of RhoB remains unresolved at present.

Another candidate Rho family target of FTIs is aw xnovel Rho family member, RhoE 120 , which is

exclusively farnesylated and constitutively GTP-bound, suggesting that its function must be regulatedby a process distinct from GTPrGDP cycling. Therole of RhoE in normal growth and transformationhas not yet been determined.

ŽFinally, a novel Ras-related protein, Rheb Ras-. w xhomologous, enriched in brain 121 , has recently

been shown to act as an antagonist of Ras transforma-tion. Rheb is farnesylated and its processing andmembrane localization can be inhibited by FTI-277w x139 . Whether FTI action causes Rheb to become asuper-repressor of Ras, and the role of Rheb innormal cellular proliferation has yet to be established.

Another class of target X candidate includes aŽ .farnesylated tyrosine phosphatase PTPase , PRL-1,

that has the capacity to cause cellular transformationin rodent fibroblasts. First described as an


immediate-early component found in regeneratingŽliver and in mitogen-stimulated cells, PRL-1 protein

.in regenerating liver represents a novel class ofproteins containing a consensus tyrosine phosphatase

w xactive site 122 . At the time, the presence of aŽ .consensus CAAX motif CCIQ on PRL-1 was not

widely appreciated. Subsequently, a search for novelsubstrates of FTase by expression cloning techniquesuncovered both PRL-1 and two highly homologous

Žproteins, named hPTPrCAAX1 human protein.tyrosine phosphatase-CAAX and rCAAX2, respec-

w xtively 123 . These proteins were determined to befarnesylated as shown by their incorporation of a

w x w xproduct of 3H MVA into 3H FPP. Although theirphysiologic substrates are unknown, overexpressionof both PRL-1 and hPTPrCAAX1 in NIH 3T3 cellswas reported to confer increased doubling times andsaturation densities, as well as anchorage independentcell growth. These findings suggest a potential posi-tive role for PRL-1 in modulating cellular prolifera-tion. However, high levels of PRL-1 expression maybe toxic, in that only 10% as many neomycin-re-sistant colonies could be selected from PRL-1 trans-

w xfected cells as from vector-transfected cells 122 .Similarly, clones containing the highest level of ex-pression of PRL-1 exhibited the lowest rate of cloningin soft agar. These apparently contradictory resultsmight indicate that PRL-1 is tightly regulated in orderto promote growth.

Since PRL-1rhPTPCAAX1 has been shown to befarnesylated in vitro, it is likely to be farnesylated invivo. However, it is also remotely possible that itcould be a substrate for GGTase II, since it contains adouble cysteine within the CAAX motif. The rele-vance of PRL-1rhPTPCAAX1 or 2 as physio-logically important substrates for FTase, as well aspotentially important target X proteins for FTIs, willawait the demonstrations that FTI treatment of cellsresults in inhibition of PRL-1 processing and thatsuch inhibition results in alterations in cellular mor-phology, growth or transformation.

Another farnesylated protein that may represent aphysiologically relevant ‘‘target X’’ is type I inositol

Ž .phosphate 3 5-phosphatase IP3 5-PTPase . This pro-tein was farnesylated, but not geranylgeranylated, atits C-terminal CCVVQ motif in vitro, and a Sermutation of the CAAX Cys residue failed to localizeto the plasma membrane or other particulate struc-

w xtures in transiently transfected COS cells 124 . TypeI IP3 5-PTPase activity results in a reduction in the

Ž .amount of IP 1,4,5 3 available to mobilize intra-cellular calcium, and in a reduction in the amount ofŽ . IP4BPIP 1,3,4,5 4 available for binding to GAP1 , an

IP4 receptor protein which exhibits GAP activity forw xsome Ras family members 124 . Whether this PT-

Pase is farnesylated in vivo, and whether it is impor-tant for the response of normal or transformed cellsto FTIs, remains to be determined.

Finally, in addition to consequences for the pro-cessing and biological function of farnesylated pro-teins, FTIs may also alter indirectly the activity ofother molecules that are not themselves farnesylatedbut that depend on available pools of farnesol or itsmetabolites. Since farnesyl pyrophosphate is the lastcommon precursor for all branches of the mevalonate

Ž .pathway Fig. 4 , disruption of FPP pools mightaffect a large number of biochemical processes insubtle or not-so-subtle ways. Indeed, because proteinfarnesylation is a critical and therefore highly pro-tected metabolic pathway, in that it occurs preferen-tially under conditions of low FPP pools, it is likelythat other pathways will be affected at concentrationsof FTIs even lower than those that alter proteinfarnesylation. The different types of FTIs thus maybe predicted to differentially affect such pathways,depending on whether they are competitive with re-spect to FPP or the CAAX motif and on whether theyare substrates for FTase or not. These pathways mayinclude such disparate endpoints as the synthesis ofcholesterol, dolichols, steroids, retinoids, farnesylated

w xand geranylgeranylated proteins, etc. 125 .A recently identified nuclear hormone orphan re-

ceptor called FXR, for farnesoid X-activated recep-tor, may also be an unintended and indirect target of

w xFTI activity 126 . The FXR forms a heterodimerictranscription factor complex with the retinoid X re-

Ž .ceptor RXR and this complex can be activatedŽ .specifically by juvenile hormone JH III, which is a

metabolic derivative of FPP and a physiologicallyimportant target in insects for compounds with FTaseinhibitor activity. However, analogs of JHIII do notactivate FXR-RXR, so whether this connection mayprove to be an artifact is unknown at present.

As indicated above, delineation of the proteins thatinteract with FTase might be expected to provideimportant clues to physiologically relevant targets of


FTI activity. In addition to expression cloning, an-other approach to this question includes the yeasttwo-hybrid interaction technique. This method hasbeen applied successfully in many systems to dis-cover binding partners for the protein of interest.However, it has also resulted in the discovery ofmany proteins that interact in the two-hybrid formatbut appear to have little or no physiological relevancein vivo.

In the case of TGFb , the FTase a subunit wasreported by two groups to specifically interact withthe activated type I receptor and to serve as a sub-

w xstrate for phosphorylation by it 127,128 . This led tothe hypothesis that the biological activities of TGFbŽand activin type I, which also bound the FTase a

.subunit in the two hybrid system might involveregulation of farnesylation and even of Ras function.However, direct examination showed that the a sub-units do not associate with TGFb receptor in thecontext of the intact FTase enzyme, that FTase activ-ity is dispensable for TGFb function, and converselythat TGFb activity does not alter prenyltransferase

w xactivity 129 . Thus, the evidence for a functionalconsequence of FTase arTGFb receptor interactionremains controversial, despite its repeated appearancein the two hybrid system.

15. Adjuvant therapy with FTIs

Because FTIs are presently considered likely to becytostatic rather than cytotoxic in patients, there isinterest in determining their potential as adjuvanttherapeutic compounds. By linking the use of FTIswith other treatment modalities, greater efficacy maybe achieved than with a single regimen alone. To datethere is evidence that these studies are worth furtherpursuit in at least three areas.

First, there is evidence that FTIs may act as ra-diosensitizers. Oncogenic Ras confers increased resis-tance to cell killing induced by ionizing radiation inat least some cell types, suggesting the possibilitythat a reduction in Ras activity could increase theradiosensitivity of tumors expressing oncogenic Ras.Because radiation therapy is administered as fraction-ated doses, small differences in radiosensitivity aregreatly enhanced over the course of the treatment. In

w xone study 114 , FTI-277 treatment of H-Ras trans-formed REF rat embryo fibroblasts significantly de-creased clonogenic survival and increased the per-centage of cells undergoing apoptosis in response toionizing radiation at clinically relevant doses. Theincreased radiosensitivity was shown to be dependenton inhibition of Ras farnesylation, as REFs express-ing a geranylgeranylated mutant form of oncogenicH-Ras were not affected. Most importantly, theparental REF cells were not affected. Other FTIsshow similar ability to selectively target rodent fi-

Žbroblasts transformed by farnesylated H-Ras Cox et.al., unpublished data . If these promising initial find-

ings can be generalized to other cell types includinghuman tumor cells, and particularly to tumors withmutated K-Ras, it would suggest that FTIs may in-deed find clinical use as radiosensitizers.

Second, FTIs can function as angiogenesis in-hibitors. Oncogenic Ras is known to drive pathways

Ž .such as vascular endothelial growth factor VEGFsignaling that culminate in increased angiogenesisw x130 . Since growing tumors must develop their ownblood supply, decreasing the activation of angiogenicpathways is an indirect but effective way of reducingtumor growth. One study used L739,749 to blockRas-induced VEGF production in a rat intestinal ep-

w xithelial cell model 131 . If FTIs reduce angiogenicactivity in animals, then this activity could have asgreat an impact on tumor growth as direct inhibitionof tumor cell proliferation.

Finally, FTIs may enhance the effectiveness ofother approaches to antagonize Ras transformation.Because multiple pathways are important for theproliferation, invasion and metastasis of malignantcells, and because combination therapies are fre-quently far more effective than single agent regimens,it seems likely that FTIs might complement agentsboth in and out of Ras-mediated pathways. For exam-ple, we recently showed that Ras transformation ofRIE-1 rat intestinal epithelial cells was mediated by

w xupregulation of transforming growth factor a 42and that inhibition of its stimulation of the EGF

Žreceptor blocked Ras transforming activity Oldham.et al., unpublished . Therefore, inhibitors of EGF

receptor stimulation may synergistically enhance theability of FTIs to block Ras transformation of these

w xcells 132 . Similarly, FTIs together with inhibitors ofthe RafrMEKrMAPK pathway may also be useful.


Ongoing and future studies will address the value ofsuch combination approaches with FTIs.

16. Development of tumor resistance to FTIs

As with any drug, the possibility of tumor cellescape by the development of resistance may also betrue for FTIs. The relative frequency of this happen-ing is not yet clear. To date, two types of studiesaddressing this problem have been reported. First,FTIs have been withdrawn from successfully treatedtumor bearing mice and the tumors allowed to regrowin the absence of drug. Upon a second round oftreatment with FTIs, most but not all of the tumorswere still sensitive to FTI treatment, indicating thatsome tumors have developed the capability to evade

w xFTIs 102 . No indication of the manner in which thisresistance occurs has been reported. Second, an FTI-resistant H-Ras-transformed Rat-1 cell line has beenselected based on the growth of a focus of trans-formed cells that escaped morphological reversion in

w xthe presence of the drug L-739,749 103 . Uponexamination of the resistant cells, the ability of theFTase enzyme to process Ras or lamin B was re-tained in the presence of the drug, but this was not

Ž .due to increased multiple drug resistance MDRgene activity, decreased FTI accumulation in thecells, or mutations in either subunit of FTase. Thus,at present the mechanism for the development ofresistance in these cells is unclear. The alteration wasstable, because resistance was maintained over thecourse of several weeks of culture in the absence ofdrug. The development of resistance was also a rela-tively rare event, as a single focus of resistant cellsgrew from a starting culture of 2=108 cells. How-ever, a detectable tumor mass can already contain 109

cells, so the potential for resistance via this or anymechanism must be considered seriously.

17. Conclusions and future questions

The variety of FTIs described to date reflects therelative ease with which such inhibitors can be identi-fied and optimized for in vitro use, and the growthinhibitory and anti-tumor action of FTIs in cell cul-ture and animal studies support the promise of FTIsas anti-neoplastic agents. The relative lack of non-

specific toxicity seen in treated cells in culture and intissues of treated animals contrasts dramatically withthe highly toxic nature of currently availablechemotherapeutic drugs. When taken together, theseobservations support the potential for FTIs to beimportant cancer drugs if sufficient bioavailable dosescan be delivered in vivo. However, the possibilitythat FTIs act partially, or not at all, as anti-Ras drugsreflects our current ignorance of the mechanistic ba-sis for the effects of FTI action on normal and tumorcell physiology. Questions regarding the identity ofother growth regulatory, farnesylated proteins that areblocked by FTIs, and whether inhibition of K-Ras4Btransformation inhibition will require the use ofGGTIs, remain to be answered. Whether FTIs aremerely cytostatic agents, and whether their usefulnessas anti-tumor drugs will require combination ap-proaches with other treatments are other importantquestions to resolve. If chronic treatment is required,will non-specific toxicity been seen with long-termapplication and will tumor resistance become a con-cern? Will complications of FTI delivery, pharma-cokinetics and bioavailability provide further hurdlesto getting FTIs to the clinic successfully? Despite theexistence of these unanswered questions, there isconsiderable optimism that FTIs will prove to beeffective for cancer treatment. If successful, the useof FTIs will not only provide a boon to cancer patienttreatment but will also provide evidence that target-ing Ras and other signaling components represents apromising and exciting new direction for additionaltypes of cancer drug development. Also, if success-ful, is this support for the value of rational drugdesign? The original intention of FTIs was to blockRas function. Whether Ras, or another farnesylated

Ž .protein s , is indeed the target responsible for FTIinhibition of transformation still provides evidencethat inhibitors of the farnesyltransferase enzyme mayserve as anti-Ras transformation antagonists. Thus,the notion that rational drug design will be an impor-tant direction for future anti-cancer drug discoveryefforts may well be supported by the success of FTIs.

Acknowledgements

We thank Jennifer Parrish for excellent preparationof figures and references. Our research studies were


Ž .supported by grants from the NIH to ADC CA61951Ž .and CJD CA42978, CA52072 and CA55008 and by

Ža National Cooperative Drug Discovery Grant U19.CA67771 .

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Farnesyltransferase inhibitors and cancer treatment: targeting simply Ras?

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