regulation of the ras superfamily of gtpases as anticancer ...dipbsf.uninsubria.it/monti/bfpn...

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals into intracellular changes through second-messenger cascades1. G proteins include the heterotrimeric G proteins (large G proteins) that are activated by membrane G-protein-coupled receptors (GPCRs) and the monomeric small G proteins (also referred to as small GTPases)2. Of the small G proteins, the RAS superfamily of GTPases is the most studied and comprises over one hundred small (20–40 kDa) monomeric G proteins3. This superfamily is structur-ally classified into five major subfamilies: the RAS, RHO, RAB, RAN and ARF families4–8 (BOX 1). These proteins function as regulators of important biological processes, including transmembrane signal transduction (RAS), cytoskeletal reorganization (RHO), gene expression (RAS, RHO), intracellular vesicle trafficking (RAB, ARF), microtubule organization (RAN) and nucleocyto-plasmic transport (RAN). Despite their functional and structural diversity, they all possess GDP/GTP-binding and intrinsic GTPase activities that enable them to switch between biologically active (GTP-bound) and inactive (GDP-bound) conformations9. Each cycle of activation and inactivation is coupled with the transduction of an upstream signal to downstream effectors.

The molecular switch mechanism of GDP/GTP exchange is tightly controlled in vivo by a complex regulatory network consisting of several other classes of proteins (mainly GTPase activating proteins (GAPs)10, guanine nucleotide exchange factors (GEFs)11 and

guanine nucleotide dissociation inhibitors (GDIs))12. Furthermore, RAS superfamily GTPases undergo exten-sive post-translational modifications that regulate protein–protein interactions, protect them from proteolytic degradation and most importantly, facilitate membrane attachment and determine their subcellular localization and function13.

The RAS superfamily of GTPases in cancer

The role of the RAS superfamily of GTPases in carcino-genesis is well established (TABLE 1). Mutational activa-tion of the RAS subfamily occurs in ~20% of human cancers; members of the RAS subfamily that have been reported to be mutated in different types of cancer include KRAS in pancreatic cancer, non-small-cell lung cancer, colorectal cancer and seminoma; NRAS in melanoma, hepatocellular cancer, myelodysplastic syn-drome and acute myelogenous leukaemia; and HRAS in follicular and undifferentiated papillary thyroid cancer, bladder cancer and renal cell cancer14,15. All these muta-tions stabilize RAS in a constitutively active GTP-bound conformation15. Moreover, several other human cancers harbour alterations in factors that lie upstream of RAS, leading to overexpression (in ovarian and breast cancers) or mutational activation (in glioblastomas and non-small-cell lung cancer) of growth factor receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptors (EGFR and ERBB2), or downstream of RAS, such as mutations of BRAF in melanomas,

*Department of Biological Chemistry, Medical School, University of Athens, 75 Mikras Asias, Athens 11527, Greece. ‡Division of Hematology–Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.Correspondence to A.G.P. e-mail: [email protected]:10.1038/nrd2221

Published online 22 June 2007

Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targetsPanagiotis A. Konstantinopoulos*‡, Michalis V. Karamouzis* and Athanasios G. Papavassiliou*

Abstract | The involvement of the RAS superfamily of monomeric GTPases in carcinogenesis

is increasingly being appreciated. A complex array of post-translational modifications and

a highly sophisticated protein network regulate the spatio-temporal activation of these

GTPases. Previous attempts to pharmacologically target this family have focused on the

development of farnesyltransferase inhibitors, but the performance of such agents in cancer

clinical trials has not been as good as hoped. Here, we review emerging druggable

targets and novel therapeutic approaches targeting prenylation and post-prenylation

modifications and the functional regulation of GDP/GTP exchange as exciting alternatives

for anticancer therapy.

NATURE REVIEWS | DRUG DISCOVERY VOLUME 6 | JULY 2007 | 541

REVIEWS

© 2007 Nature Publishing Group

amplification of p110 in ovarian cancer, amplification of AKT2 in ovarian and breast cancers and deletion of PTEN in 30–40% of all human malignancies16,17.

Unlike the RAS subfamily, no mutated, constitutively active forms of the RHO subfamily of small GTPases have been documented in tumours18. Only RHOH is genetically altered (rearranged) in multiple myeloma and non-Hodgkin’s lymphoma, and mutations in the 5′-untranslated region of RHOH (independent of chro-mosomal translocations) have been shown to affect its expression in B-cell lymphomas19,20. Overexpression of RHOA (in breast, colon, bladder and testicular germ cell tumours); RHOC (in melanoma, pancreatic ductal adeno-carcinoma bladder and inflammatory breast cancer); RAC1 and RAC2 (in breast, colon, bladder and head and neck cancers); and CDC42 (in breast cancer) has been documented and frequently correlates with clini-cal outcome18,21–23. Although overexpression alone does not necessarily imply a functional role in carcinogenesis, the fact that increased levels of these GTPases consist-ently correlate with aggressive histological features and clinical behaviour suggests an important role in tumori-genesis22,23. The absence of mutational activation and the prevalence of overexpression of RHO proteins in carcinogenesis indicate that the rapid cycling of RHO proteins between inactive (GDP-bound) and active (GTP-bound) conformations is necessary for the bio-logical processes that they regulate (such as cytoskeletal

remodelling and vesicle transport), which are important for malignant transformation24,25. Finally, it is important to emphasize that many regulatory RHO GTPase pro-teins are genetically altered in carcinogenesis; this issue is discussed in detail later.

Aberrant expression of the RAB subfamily of GTPases has also been documented. RAB25, located in chromo-some 1q22, is amplified at the DNA level and overex-pressed at the RNA level in ovarian and breast cancer26. RNA microarray analyses demonstrated that approxi-mately 50% of the RAB genes are overexpressed in ovarian cancer. RAB25 is also upregulated in prostate cancer and transitional-cell bladder cancer26. Overexpression of RAB5A and RAB7 has been documented in thyroid adenomas, and RAB1B, RAB4B, RAB10, RAB22A, RAB24 and RAB25 are upregulated in hepatocellular carcinomas and cholangiohepatomas27,28.

Of the ARF family, ARL5, SARA1 (also known as SAR1A) and SARA2 have been shown to be overexpressed in hepatocellular carcinoma, whereas the levels of ARF6 correlate with breast-cancer-cell invasiveness28,29. ARLTS1, another member of the ARF family (also known as ARL11), functions as a tumour suppressor gene in humans, and a nonsense ARLTS1 polymorphism predisposes patients to familial cancer30.

The central role of the RAS superfamily of GTPases in carcinogenesis has stimulated efforts to develop novel therapeutic strategies that inhibit their actions. In this Review, we focus on targeting the post-translational modifications, which determine their localization and mediate their attachment to membranes, and on target-ing the complex regulatory network that controls the activation of the RAS superfamily GTPases as potential strategies for anticancer drug development.

Post-translational modifications

RAS GTPases are the founding members of another class of proteins (often referred to as CAAX proteins), which contain a CAAX motif (C denotes cysteine, A repre-sents any aliphatic amino acid and X may be any amino acid) in their carboxyl terminus. The CAAX motif serves as a substrate for a series of post-translational modifications that create a lipidated hydrophobic domain, which mediates attachment to specific proteins (for example, the interaction of RHO with GDI) as well as membranes. These modifications include the covalent attachment of a non-sterol isoprenoid (either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP)) to the cysteine residue of the CAAX motif by prenylation (farnesylation and geranylgeranylation, respectively)31. FPP is an intermediate product of the mevalonate pathway and can be converted to choles-terol or GGPP (FIG. 1). Farnesylation occurs when the CAAX sequence ends in any amino acid other than leucine and is catalysed by farnesyltransferase (FTase), whereas geranylgeranylation occurs when the CAAX sequence ends in leucine (as in the case of RHOB) and is catalysed by geranylgeranyltransferase I (GGTase I)32 (FIG. 2). It is important to note that HRAS is only far-nesylated, whereas KRAS4A, KRAS4B and NRAS can be farnesylated and geranylgeranylated.

Box 1 | The RAS, RHO, RAB and ARF subfamilies

Three RAS genes are translated into four RAS proteins: HRAS, NRAS, KRAS4A and KRAS4B (KRAS4A and KRAS4B are splice variants of a single gene). RAS proteins activate the RAF/MEK/ERK pathway, which mediates cell growth and cell-cycle entry by phosphorylation of transcription factors (such as c-FOS, ELK1 and MYC); phosphorylation of the RSK (ribosomal protein S6 kinase) and MNK (MAPK-interacting serine/threonine kinase) family of kinases; and the PI3K/AKT pathway, which controls cell survival, cell growth and metabolism.

The RHO subfamily includes 18 members that are subdivided into several subgroups according to their sequence and functions. Important subgroups include proteins that are similar to RHOA, those that are similar to RAC1 and those similar to CDC42. The classic biological functions of RHO GTPases are organization of the actin cytoskeleton, cell adhesion and cell motility. RHO GTPases promote cell-cycle progression through G1 by regulating cyclin D1 and cyclin-dependent inhibitors p21 and p27. Furthermore, they are involved in epithelial to mesenchymal transition, promote invasion and metastasis, and, like the RAS subfamily, mediate resistance to important chemotherapeutic drugs such as cisplatin.

Approximately 60 RAB proteins are encoded by the human genome and additional RAB proteins are generated by alternative splicing. They regulate receptor internalization, vesicle formation and trafficking to various cellular sites, including the nucleus, lysosome and plasma membrane. Through regulation of endocytic trafficking, they integrate multiple signalling pathways that are involved in cellular proliferation, apoptosis and migration.

Six mammalian ARF proteins have been identified and are categorized into three classes. Class I ARFs (ARF1, 2 and 3) are involved in the assembly of different coat proteins onto budding vesicles and activate lipid-modifying enzymes. Class II ARFs (ARF 4 and 5) are involved in Golgi transport, and the class III ARF (ARF6) is involved in the organization of the cell surface. ARF1 and ARF6 are implicated in carcinogenesis as they regulate cellular adhesion, migration and tumour invasion.

For more information regarding RAS, RHO, RAB and ARF GTPases, their signalling pathways and their implication in carcinogenesis, the reader is referred to several excellent reviews3–5,8,14,18.

R E V I E W S

542 | JULY 2007 | VOLUME 6 www.nature.com/reviews/drugdisc


Prenylation targets these proteins to the endoplasmic reticulum, where the three terminal amino-acid residues (AAX) are removed by the endoprotease RCE1 (RAS con-verting enzyme 1) and the carboxyl group of the terminal cysteine is methyl esterified by ICMT (isoprenylcysteine carboxymethyltransferase)33. NRAS, HRAS and KRAS4A are then palmitoylated (at another cysteine residue) and transferred to the plasma membrane. KRAS4B does not require this modification and is sent directly to the plasma membrane. NRAS, HRAS and KRAS4A attach to the plasma membrane through their farnesyl and palmitoyl moieties, whereas KRAS4B attaches to the plasma mem-brane through its farnesyl moiety and a polybasic, lysine-rich sequence located near the terminal cysteine34 (FIG. 2).

RHO GTPases have CAAX sequences and undergo similar post-translational modifications (prenylation-RCE1-ICMT). However, they differ from RAS in that they are mostly geranylgeranylated and in that, after they are fully processed, they bind to RHO GDIs, which keep them in a soluble state in the cytosol and deliver them to various membrane locations to exert their functions35 (FIG. 3).

Most RAB GTPases have either CXC (RAB3A) or CC (RAB1) sequences in their carboxy termini. Both cysteines in CXC or CC are geranylgeranylated by RAB geranylgeranyltransferase (RAB GGTase; also known as GGTase II). Unprenylated RAB GTPases are presented by a REP1 (RAB escort protein 1; also known as CHM) to RAB GGTase36. After prenylation, only RAB GTPases ending in CXC undergo carboxymethylation by ICMT, whereas those ending in CC do not (obviously there is no RCE1 step in either case). RAB GTPases interact with the plasma membrane through their two prenyl moieties (FIG. 3). ARF proteins undergo myristoylation and interact with membranes via their myristoylated and amphipathic amino terminus37.

Functional regulation

Activation of the RAS superfamily of GTPases requires dissociation of GDP from the inactive GDP-bound conformation and binding of GTP to adopt the active GTP-bound conformation that facilitates the interaction of RAS with its downstream effectors (FIG. 4). The rate-limiting step of this activation process is usually the dissociation of GDP from the inactive conformation, which is stimulated by GEFs. Similar to other regulator proteins, GEFs may be specific for each individual protein member, or group, within the RAS superfamily. SOS, the most well-studied RAS GEF, is recruited to the plasma membrane by growth factor receptor-bound protein 2, an adaptor protein that binds to phosphory-lated tyrosine residues of activated RTKs.

Another class of regulator proteins known as GAPs prevents prolonged activation of RAS GTPases by stimu-lating the intrinsic GTPase activity of RAS38. This is an important aspect of regulation of RAS activity that is fre-quently deregulated in tumorigenesis. All oncogenic RAS mutations compromise its GTPase activity by preventing GAPs from stimulating the hydrolysis of GTP or by affect-ing GAP action, thereby maintaining RAS constitutively in the active GTP-bound conformation. Besides RAS mutations, prolonged activation of RAS in carcinogenesis may also occur from inactivation of RAS GAPs. Of the 13 known RAS GAPs, neurofibromin 1 (NF1) is reportedly inactivated in carcinogenesis (TABLE 2). Deletion of one NF1 allele is associated with neurofibromatosis type I, which is characterized by numerous benign or malignant tumours of neural-crest origin39. Malignant tumours associated with neurofibromatosis type I have lost both copies of NF1. Tuberin (also known as TSC2) functions as a GAP for RHEB (RAS homologue enriched in brain) and is regulated by hamartin (or TSC1). Mutations in TSC2 or TSC1 lead to enhanced RHEB signalling, downstream mTOR activation and many of the severe manifestations and benign tumours of the tuberous sclerosis complex40.

Similar to RAS GTPase, RHO GTPase cycling between active GTP-bound and inactive GDP-bound states is controlled by RHO GEFs that catalyse the exchange of GDP for GTP and GAPs that stimulate the intrinsic RHO GTPase activity, leading to inactivation. An additional level of regulation is exerted by GDIs, which form soluble cytoplasmic complexes with prenylated, GDP-bound RHO GTPases and regulate their delivery or extraction to and from their sites of action41. RHO GDIs were named after their ability to block RHO GEF-stimulated dissocia-tion of GDP (thereby preventing activation of GTPases) and extract inactive GDP-bound RHO proteins from membranes (FIG. 4). Complexes of RHO proteins attached to GDIs contain all the information that is necessary for accurate membrane delivery. Another class of regulator protein known as GDI displacement factors (GDFs) recognize specific RHO proteins and catalyse their dissoci-ation from GDIs and their subsequent delivery to specific subcellular membranes. Candidate RHO GDFs are the ezrin, radixin and moesin proteins, which crosslink the actin cytoskeleton to the plasma membrane, and merlin, a product of the neurofibromatosis 2 (NF2) tumour suppressor gene, which is an antagonist of ezrin42,43.

Table 1 | Implication of RAS superfamily GTPases in carcinogenesis

RAS superfamily GTPase

Implication Malignancies

KRAS Mutations Pancreatic, non-small-cell lung, colorectal cancers, seminoma

NRAS Mutations Melanoma, hepatocellular cancer, myelodysplasia, acute myelogenous leukaemia

HRAS Mutations Follicular and papillary thyroid, bladder, renal cell cancer

RHOA Overexpression Breast, colon, testicular germ-cell tumours

RHOC Overexpression Melanoma, pancreatic adenocarcinoma, inflammatory breast cancer

RAC1/2 Overexpression Breast, colon, head and neck cancers

CDC42 Overexpression Breast cancer

RAB25 Amplification or overexpression

Breast, ovarian, prostate and transitional cell cancer

RAB5a, RAB7 Overexpression Thyroid adenoma

RAB1B, RAB4B, RAB10, RAB22A, RAB24

Overexpression Hepatocellular carcinoma and cholangiohepatoma

ARL5, SARA1, SARA2 Overexpression Hepatocellular carcinoma

ARF6 Overexpression Invasive breast cancer

R E V I E W S



Acetyl-CoA

HMG-CoA

Mevalonate

Mevalonate-PP

Isopentyl-PPDimethylallyl-PP

Isopentyl-PPisomerase

HMG-CoA reductase

Farensyl-PP synthase

GGTaseFTase

Geranyl-PP

Farnesyl-PP Exogenousisoprenoids

Cholesterol

Membrane andsteroid synthesis

Dolichyl-P

N-glycosylationof growth-factorreceptors

Geranylgeranyl-PP

Prenylation ofCAAX proteins

Deletion of NF2 and/or overexpression of ezrin gives rise to highly metastatic tumours.

Many regulatory RHO GTPases are genetically altered in carcinogenesis (TABLE 2). The RHO GEFs LARG (also known as ARHGEF12) and BCR have been isolated as fusion partners of MLL and ABL, respectively, in acute myeloid leukaemia (AML) 44,45. TIAM1 (T-cell invasion and metastasis 1) is a RAC-specific GEF that was identi-fied in a screen for genes that increase the invasiveness of T-lymphoma cell lines and has been found mutated in

renal cell carcinomas46. RHO GAPs, which downregulate RHO activity, have been suggested to act as tumour sup-pressors in human malignancies. Accordingly, the gene encoding p190RHO GAP is located at 19q13.3 of the human chromosome, a locus that is deleted in 50–80% of oligodendrogliomas47,48. DLC2 (deleted in liver cancer 2) is another RHO GAP (specific for RHOA and CDC42) that is frequently underexpressed in hepatocellular carcinomas; its chromosomal region shows frequent deletion49. Aberant GDI expression in cancer cells has also been documented. RHO GDIs are overexpressed in non-small-cell lung and ovarian cancers, and underex-pressed in breast cancer cells. These conflicting results probably reflect different stages (early versus metastatic) or mechanisms of carcinogenesis50–52.

Functional regulation of RAB GTPases is analogous to RHO and involves GAPs, GEFs and GDIs53 (FIG. 4). It is important to note that heat-shock protein 90 (HSP90) may be involved in the extraction of RAB GTPases (from the membranes) by enhancing the ability of GDIs to bind and retrieve RAB GTPases from their membrane locations. No genetic alterations in RAB regulatory pro-teins have been specifically linked with tumorigenesis, although TSC2 protein (associated with tuberous sclerosis complex) may also function as RAB5 GAP. Genetic defects in RAB GDIα have been linked with X-linked non-specific mental retardation. Last, the functional regulation of ARF GTPases is similar to that of RAS54.

Targeting post-translational modifications

Inhibitors of the post-translational modifications of the RAS superfamily of GTPases (FIG. 5) can be classi-fied as those that interfere with the mevalonate pathway (TABLE 3), with prenylation (TABLE 4) or post-prenylation modifications.

Inhibitors of the mevalonate pathway. Several enzymes within the mevalonate pathway (FIG. 1) have been targeted for anticancer drug development. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme at the apex of the mevalonate pathway55. By inhibiting the synthesis of mevalonate, statins also inhibit the formation of down-stream isoprenoids FPP and GGPP, which are used as substrates for prenylation, and inhibit the formation of cholesterol and biosynthesis of dolichyl phosphate, which is used for N-glycosylation of growth-factor receptors such as the insulin-like growth factor56. Inhibition of geranylgeranylation of RHO proteins (rather than far-nesylation of RAS) seems to be an important anticancer effect of statins57. Statins may also exert anticancer effects independently of the mevalonate pathway as they inhibit the interaction between the integrin LFA1 and intercell-ular adhesion molecule 1 (ICAM1) thereby affecting invasion, migration and cell adhesion58. Furthermore, statins inhibit the proteasome degradation machinery, leading to apoptosis and inhibition of proliferation59.

Results from clinical trials of statins as monotherapy against various human malignancies were only modest, primarily because of dose-limiting toxicities including gastrointestinal toxicity, myelotoxicity, elevation of creatine

Figure 1 | Biosynthesis of isoprenoids through the mevalonate pathway. Fungi, mammals and archaebacteria exclusively use the mevalonate pathway for

biosynthesis of isoprenoids. 3-hydroxy 3-methylglutaryl-CoA (HMG-CoA) is converted

to mevalonate by HMG-CoA reductase, the rate-limiting enzyme at the apex of the

mevalonate pathway. Mevalonate is then converted to isopentenyl pyrophosphate

(isopentenyl-PP; the 5-carbon basic isoprene unit), which is subsequently converted

to farnesyl pyrophosphate (farnesyl-PP; a 15-carbon isoprenoid) through a series of

enzymatic reactions. After addition of isopentenyl-PP, farnesyl-PP can be converted to

geranylgeranyl pyrophosphate (geranylgernayl-PP; a 20-carbon isoprenoid),

or alternatively farnesyl-PP can be converted to cholesterol or dolichyl phosphate

(dolichyl-P), which is used for N-glycosylation of growth factor receptors such as

insulin-like growth factor receptor. HMG-CoA reductase is the target of the cholesterol-

lowering statins, whereas isopentenyl-PP isomerase and farnesyl-PP synthase are

targets of bisphosphonates. Importantly, in normal cells, cholesterol and isoprenoid

products suppress HMG-CoA reductase via post-translational downregulation.

Conversely, tumour cells are resistant to cholesterol-mediated suppression, although

they remain sensitive to isoprenoid-mediated suppression. Addition of isoprenoids to

statins may prevent upregulation of HMG-CoA reductase, which might occur as a

resistance mechanism to statin therapy. This explains the synergistic activity of statins

and isoprenoids. CAAX, C denotes cysteine, A represents any aliphatic amino acid and

X may be any amino acid; FTase, farnesyltransferase; GGTase, geranylgeranyltransferase.

R E V I E W S



RAS COCH3

OCH3

F or GG

CRCE1

+FPPor GGPP

+FPPor GGPP

FTaseor GGTase I

–AAX

O–

F or GG

Plasma membrane

Endoplasmic reticulum

RAS C C

RAS C C

OCH3Palmitoyl

transferase

ICMT

+SAM

OCH3

F or GG

F or GG

F or GG Palmitoyl

RAS C COCH3

ICMT

+SAM

RAS C CRCE1

HRAS: FTase onlyNRAS, KRAS4A: FTase or GGTase I

–AAX

O–

F or GG

RAS C C A A X

RAS

NRASHRASKRAS4A

KRAS4B

C C A A X

F or GG

Plasma membrane

Golgi


+RAS +

CRAS

+

CAAX

F or GG

RAS +

CAAXRAS +

F or GG

(KRAS4B)

C A A XRAS +

RAS Ca

(NRAS, HRAS, KRAS4A)

C A A X

RAS

Palmitoyl

b

RhabdomyolysisThe breakdown of muscle

fibres, resulting in the release

of muscle-fibre contents into

the circulation. Some of these

are toxic to the kidney.

phosphokinase (CPK), hepatotoxicity and rhabdomyolysis60. Combinations of statins with conventional chemo-therapeutic agents have also been evaluated in several cancers60. In a randomized trial of 91 patients with hepatocellular cancer, addition of pravastatin to standard chemotherapy was associated with a statistically signifi-cant increase in survival (18 months in the pravastatin

group compared with 9 months in the control group, p=0.006)61. However, there are no ongoing clinical trials of statins as single agents or in combinations for anti-cancer therapy, although there are a few Phase I and II trials that are evaluating various statins (simvastatin, fluvastatin and atorvastatin) as chemopreventing agents against breast and colorectal cancers.

Figure 2 | Prenylation and post-prenylation reactions of RAS GTPases. a | HRAS, NRAS and KRAS4A are prenylated

(HRAS is only farnesylated, whereas NRAS and KRAS4A can be farnesylated or geranylgeranylated) before undergoing

proteolytic removal of the AAX tripeptide by RAS converting enzyme 1 (RCE1) and carboxymethylation by

isoprenylcysteine carboxymethyltransferase (ICMT) in the endoplasmic reticulum (ER). Subsequently, they are

palmitoylated in the Golgi and transferred to the plasma membrane (PM) to which they attach through their farnesyl (F)

or geranylgeranyl (GG), and palmitoyl moieties. b | KRAS4B can be farnesylated or geranylgeranylated and then

undergoes proteolytic removal of the AAX tripeptide by RCE1 and carboxymethylation by ICMT in the ER. It does

not undergo palmitoylation, but attaches to the PM through its farnesyl moiety and a polybasic, lysine-rich sequence

located near the terminal cysteine. FPP, farnesyl pyrophosphate; FTase, farnesyltransferase; GGPP, geranylgeranyl

pyrophosphate; GGTase I, geranylgeranyltransferase I; SAM, S-adenyosyl methionine.

R E V I E W S



+FPP if X = Not Leu/Phe

or +GGPPif X = Leu/Phe

(more frequentthan FPP)

RHO CICMT

+SAM

OCH3

F or GG

RHO

Delivery tomembranes

C

OCH3

F or GG GDI

RHO CRCE1

FTaseor GGTase I

–AAX

O–

F or GG

RHO C A A X

RHORHO

C( )A A X

RHO C A A X

F or GG


a

Delivery tomembranes

+2GGPP

+GDI –RPE1

GGTase II

RABRAB

C( )X C RAB C Cor

ICMT

+SAM

OCH3O–

RAB C X C

GG GG

b

REP1

RAB C X C

GG GG

REP1

RAB C X C

REP1

+RPE1

RAB C X C

RAB C X C

GG GG

GDI

OsteoclastBone cell that has a key role in

bone resorption.

Bisphosphonates inhibit two critical enzymes in the mevalonate pathway: isopentenyl diphosphate (IPP) isomerase and FPP synthase, which are required for the synthesis of FPP and GGPP and subsequent prenylation62. Aminobisphosphonates (such as pamidronate, ibandro-nate and zoledronic acid) are newer bisphosphonates that

have more potent clinical activity. Aminobisphosphonates inhibit farnesylation and geranylgeranylation of KRAS, NRAS and HRAS, leading to a downregulation of down-stream ERK and AKT1 activity and an increase of p21 and p27 expression63. Inhibition of RAS signalling in osteoclasts results in the aberrant formation of the tight-sealing zones or ruffled borders required for bone resorption64. Inhibition of geranylgeranylation of RHOA decreases expression of pro-invasive molecules such as matrix metalloproteinase 7 (MMP7), tissue inhibitor of metalloproteinase 2 (TIMP2) and u-PA (plasminogen activator urokinase; also known as PLAU)65. A newer aminobisphosphonate, NE10790, can selectively inhibit geranylgeranylation of RAB, without affecting prenyla-tion of RAS or RHO, and suppress bone resorption by inhibition of osteoclast intracellular trafficking66. Aminobisphosphonates exhibit anti-angiogenic effects, independently of the mevalonate pathway, by downregu-lating the expression of angiogenic factors — vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) — and antimigratory effects, by reducing avβ3 expression65. Aminobisphosphonates are used routinely for treating patients with malignant bone disease. Nevertheless, despite in vitro data supporting their antitumour effects, clinical evaluation of aminobi-sphosphonates as anticancer agents outside the context of metastatic bone disease is limited.

It is important to note that there is no evidence of inhi-bition of RAS prenylation by bisphosphonates in patient samples (this has never been looked at in clinical studies). Design of future clinical trials of bisphosphonates with inclusion of appropriate pharmacodynamic markers could provide valuable information about the usefulness of this clinical approach. In this regard, if bisphosphonates do not inhibit prenylation of marker proteins (such as HDJ2, also known as DNAJA1, in peripheral blood mononuclear cells (PBMCs)) in patients, this approach may not be as useful as suggested by some preclinical work.

Prenylation inhibitors. FTase inhibitors (FTIs) can be FPP analogues that compete with FPP for binding to FTase, CAAX peptidomimetics that compete with RAS-CAAX motif for FTase, or both (FPP analogues and CAAX peptidomimetics)67. FTIs prevent HRAS far-nesylation and reverse HRAS-induced transformation68. By contrast, KRAS and NRAS are geranylgeranylated (cross-prenylation) by GGTase I in FTI-treated cells, resulting in persistent membrane association of KRAS and NRAS and persistent downstream activation of MAPK/ERK69,70. Inhibition of farnesylation of RHOB seems to have an important role in the antitumour activity of FTIs. RHOB is both farnesylated and geranylgeran-ylated in non-FTI-treated cells and the increased ratio of geranylgeranylated RHOB to farnesylated RHOB in FTI-treated cells leads to growth inhibition71. Accordingly, several studies suggest that geranylgeranylated RHOB has antigrowth properties, whereas farnesylated RHOB is tumorigenic72. However, it is important to note that the role of RHOB still remains controversial because overexpression of a mutant form of this protein that can only be farnesylated also inhibits cell growth73.

Figure 3 | Prenylation and post-prenylation reactions of RHO and RAB GTPases. a | RHO GTPases are prenylated (farnesylated if X is neither leucine (Leu) nor

phenylalanine (Phe), or geranylgeranylated if X is either Leu or Phe). They then undergo

proteolytic removal of the AAX tripeptide by RAS converting enzyme 1 (RCE1) and

carboxymethylation by isoprenylcysteine carboxymethyltransferase (ICMT).

Subsequently, they bind to RHO guanine nucleotide dissociation inhibitors (GDIs), which

keep them in a soluble state (by recognizing their prenylation moiety) in the cytosol and

deliver them to various membrane locations where they exert their functions. b | Most

RAB GTPases end in C-X-C or C-C residues (where C denotes cysteine and X denotes

another amino acid). They are geranylgeranylated in both C residues by RAB geranylger-

anyltransferase II (GGTase II). Unprenylated RAB GTPases are presented by a REP1 (RAB

escort protein 1) to GGTase II. Subsequently only RAB GTPases ending in C-X-C undergo

carboxymethylation by ICMT. REP1 is not required for RAB methylation, but it has a

prenyl-binding site, like GDIs, and can bind to prenylated RABs to keep them soluble

in the cytosol. Last, these RAB GTPases bind to RAB GDIs, which recognize their two

geranylgeranyl (GG) moieties and deliver them to various membrane locations

where they exert their functions. They attach to various membranes through their

two GG moieties. F, farnesyl; FPP, farnesyl pyrophosphate; FTase, farnesyltransferase;

GGPP, geranylgeranyl pyrophosphate; SAM, S-adenosylmethionine.

R E V I E W S



QT prolongation The QT interval represents

the time for electrical

activation and inactivation of

the ventricles, the pumping

chambers of the heart.

Prolongation of the QT interval

can result in potentially lethal

arrhythmias (some of which are

known as torsades de pointes).

ParaesthesiaA sensation of tingling, pricking

or numbness of the skin with

no apparent physical cause.

FTIs inhibit farnesylation of RHEB1 and RHEB2 GTPases (which cannot be alternatively prenylated) and so may have a role in the treatment of benign tumours in patients with tuberous sclerosis, which demonstrate sustained RHEB-mTOR signalling74,75. Certain FTIs are potent inhibitors of GGTase II (RAB GTPase)76, and the inhibition of several other farnesylated proteins (such as lamins, HDJ2, PxF and the centromeric proteins CENPE and CENPF) may contribute to the antitumour effects of FTIs77. Importantly, HDJ2 (a HSP40 member that func-tions as a chaperone that regulates HSP70 activity) seems to be a good biomarker of FTI activity78.

Although several FTIs were tested in early-stage trials, only tipifarnib and lonafarnib were tested in Phase III trials67. True clinical outcome was not tested for the others because of several issues (QT-prolongation for example) that were identified in Phase I trials. Phase III trials were negative for both tipifarnib (in combination with gem-citabine in pancreatic cancer) and lonafarnib (in combi-nation with carboplatin and paclitaxel in non-small-cell lung cancer)67,79. Overall, the antitumour activity of FTIs as single agents in solid tumours was far less than anti-cipated80,81. Alternative prenylation (cross-prenylation) of KRAS and NRAS (as opposed to HRAS, which is only farnesylated) has been the most consistently cited reason for this clinical outcome. FTIs may prove more effective against solid tumours in combinations with cytotoxic or hormonal agents, such as taxanes and anti-oestrogens.

However, in certain haematological malignancies (AML, chronic myelomonocytic leukaemia and myelo-dysplastic syndromes) promising response rates to FTI monotherapy were observed82. In a Phase II study of tipi-farnib in older patients (median age, 74 years) with previ-ously untreated AML, complete remission was achieved in 14% of the patients and partial remission or haemato-logical improvement occurred in 9% of them. The median duration of complete remission was 7.3 months and the median survival of complete responders was 18 months. Inhibition of farnesylation of the surrogate protein HDJ2 occurred in most of marrow samples tested83.

The side-effect profile of FTIs in Phase I studies was diverse. Lonafarnib demonstrated primarily gastrointes-tinal toxicities (diarrhoea, nausea and vomiting), whereas tipifarnib’s dose-limiting toxicities in Phase I studies included nausea, vomiting, fatigue, myelosuppression and neurotoxicity. The FTI BMS-214662, when administered intravenously (oral formulations showed dose-dependent gastrointestinal side-effects that prohibited oral dosing) demonstrated schedule-dependent toxicity that included nausea, vomiting, diarrhoea, increased liver enzymes, elevated creatinine levels, acute pancreatitis, electrolyte abnormalities and cardiovascular problems.

It is important to note that no trial with FTIs has been successfully completed in patients with tumours that harbour HRAS mutations (such as follicular and undifferentiated papillary thyroid cancer, and renal cell cancers). Even though these mutations are rare clini-cally, this population is attractive for proof-of-concept that inhibition of the RAS pathway is of clinical use. This proof-of-concept still remains elusive. In that regard, design of clinical trials of FTIs in tumours with

HRAS mutations could provide valuable clinical and biological information.

GGPP analogues and CAAL (L denotes leucine) peptidomimetics or bi-substrate analogues have been designed as GGTase inhibitors (GGTIs)84. Despite their potent antiproliferative and pro-apoptotic properties in both in vitro and in vivo models, clinical development of GGTIs has been problematic owing to toxicity85. Specifically, a 72-hour infusion of GGTIs at doses suf-ficient to block KRAS prenylation in the presence of FTIs was lethal to mice86. To overcome the toxicity of GGTIs, compounds that simultaneously inhibit GGTase I and FTase (dual prenylation inhibitors, DPIs) have been developed. This strategy takes into consideration several lines of evidence that suggest a synergistic antigrowth activity of combined FTase and GGTase inhibition on tumour cell lines87. The CAAX mimetic FTI L778123 has been shown to bear significant inhibitory activity against GGTase I (besides FTase)88. L778123 was well tolerated in Phase I and II clinical trials and was able to reduce prenylation of RAP1a and HDJ2 but not KRAS89. Unfortunately, in all L778123 trials, QT prolongation was observed in at least one patient and although this was not dose limiting, and did not recur after dose reduction, further development of this agent was aborted. It is important to emphasize that a molecular marker that can be used as an appropriate pharmacodynamic end point for these agents is missing. In this regard, total elimination of KRAS prenylation, which has guided dosing of GGTIs to induce excessive toxicity, may correlate less with anti-tumour activity than prenylation of other proteins (that is, RAP1a)89. AZD3409, another DPI that inhibits FTase and GGTase I, has been well tolerated in in vivo models and demonstrated significant inhibition of tumour growth90. In healthy human volunteers, there were no clinically significant changes in laboratory parameters, heart rate, blood pressure or electro-cardiogram recordings. Adverse events considered possibly related to AZD3409 were orthostatic hypotension, paraesthesia, nausea, diarrhoea, abdominal pain and dizziness. Diarrhoea was established

as the dose-limiting toxicity, but it was of short duration and resolved spontaneously. Clinical evaluation of GGTIs and DPIs is set to begin soon.

Post-prenylation inhibitors. Lack of single-agent activity of FTIs in tumour types known to harbour high frequencies of KRAS or NRAS mutations prompted the development of novel strategies for targeting the post-translational modifications of the RAS superfamily of GTPases. Inhibition of the two post-prenylation enzymes RCE1 and ICMT has attracted considerable attention as post-prenylation reactions are shared by both far-nesylated and geranylgeranylated proteins. Therefore, even alternatively prenylated proteins are sensitive to inhibition of either the ICMT or the RCE1 enzyme.

Studies in cells and mice in which the Rce1 and Icmt genes were disrupted have offered significant informa-tion regarding the potential of post-prenylation inhibi-tion. In these models, abrogation of RCE1 activity alone had only modest effects in preventing RAS-induced oncogenic transformation and was less effective than

R E V I E W S



Extr

actio

n

Att

achm

ent

GDPGTP

GDI

GDFGAP

GAP

GEF

GEF

RHO/RAB

RAS/ARF

RHO/RAB RHO/RAB

b

a

RHO/RABHSP90

GTPGDP

GDP

GDP

RAB(Gapcena,RAB3GAP)

RHO(P190GAP,DLC2)

Phosphate

Phosphate

RAB(RAB3GEP,RABEX-5)

RHO(TIAM1,BCR, LARG)

RAB(RABGDIa,b,c)

RHO(RHOGDI1,2,3)

RHO/RAB

GDPRAS/ARF

GTP

GDP

GTPRAS/ARF

GDI

GDI

RAS(NF1, P120, TSC2(RhebGAP))

ARF(ARD1, CEND1 (centaurin δ-1))(both ARF, RHO)

RAS(SOS, CDC42)

ARF(ARNO, GRP1)

Effectors

Inac

tivat

ion

Act

ivat

ion

FTI monotherapy91. However, RCE1 blockade sensitizes tumour cells to FTI treatment, as FTIs inhibited cell growth more potently in fibroblasts and skin carcinoma cells that were deficient in RCE1 activity.91.

In contrast to blocking RCE1, blocking ICMT activity has more profound biological consequences. Targeted deletion of Icmt is embryonically lethal in mice, whereas inhibition of ICMT activity blocks oncogenic KRAS-induced transformation by decreasing the methylation

of KRAS, HRAS and NRAS92. Deletion of the Icmt gene in immortalized mouse embryonic fibroblasts effectively blocked BRAF-induced transformation by reducing the levels of RHOA and upregulating p21CIP1 (REF. 92). Inhibition of ICMT affects multiple pathways besides RAS. Specifically, several CAAX proteins (including RAS superfamily GTPases and non-RAS superfamily GTPases) and RAS superfamily members that regulate proliferation, cell division (CENPE, CENPF and lamin B), apoptosis (RAC and RHO), angiogenesis (RHO), and metastasis (PTP4A3) can be affected when post-prenylation modi-fications are blocked93. These observations suggest that ICMT may be a good target for anticancer therapy.

Both prenylated CAAX peptidomimetics and non-peptidic, non-prenylic compounds have been devel-oped as inhibitors of RCE1 (REFS 93,94). Additionally, chloromethylketone protease inhibitors have also been shown to inhibit RCE1, and this has led to the design of prenylcysteine chloromethylketone derivatives as RCE1 inhibitors95. Inhibition of ICMT has followed two general approaches. First, compounds that are analogues of S-adenosylhomocysteine (AdoHcy), or that increase cellular levels of AdoHcy by inhibition of AdoHcy hydrolase, competitively inhibit nearly all methyltransferases, including ICMT. These agents have demonstrated both antitumour and antiviral activities, but lack selectivity, and their specific target is unclear96. In this regard, even methotrexate,which elevates AdoHcy levels, is postulated to exert some of its anticancer effects by inhibiting ICMT97. Second, analogues of the substrate prenylcysteine (that is, N-acetyl-S-farnesyl-cysteine and N-acetyl-S-geranylgeranyl-cysteine) have been investi-gated as potential ICMT inhibitors. An indole-based small-molecule inhibitor of ICMT has also been devel-oped with good in vitro activity98.

Although inhibitors of RCE1 and ICMT are undergoing preclinical testing, there are two concerns in anticipation of their clinical evaluation. ICMT and RCE1 act on more targets than FTase or GGTase, so the potential for toxicity may be higher. However, studies in Rce1-null mice, and the finding that inhibition of ICMT does not cause mislocalization of geranylgeranylated RHO proteins (it only reduces their levels) suggest that the toxicity of these agents may not be excessive92,99. An important toxic effect that may be associated with ICMT inhibition is atherosclerotic vascular injury. Elevated homocysteine levels in vivo have been associated with atherosclerotic vascular injury in humans by promoting endothelial cell apoptosis. Similarly, it has been shown that inhibition of ICMT causes endothelial cell apoptosis by attenuation of RAS GTPase methylation and activation of downstream anti-apoptotic-signalling pathways100.

Second, although RCE1 and ICMT inhibition cause mislocalization of RAS, it has been shown that farnesylated or geranylgeranylated RAS (specifically NRAS and HRAS) can still signal from other locations besides the plasma membrane (such as the endoplasmic reticulum or Golgi)101. Therefore, inhibition of ICMT or RCE1 alone may not completely abrogate downstream RAS signalling; this may have evolved as an escape mechanism against ICMT or RCE1 inhibition.

Figure 4 | Functional regulation of the RAS superfamily of GTPases. a | RAS and

ARF GTPases are regulated by guanine nucleotide exchange factors (GEFs), which

catalyse the exchange of GDP for GTP and mediate activation, and GTPase activating

proteins (GAPs), which stimulate the intrinsic RAS GTPase activity, leading to

inactivation. Each cycle of activation and inactivation is coupled to the transduction of

an upstream signal to downstream effectors. b | Cycling of RHO and RAB GTPases

between active and inactive states is controlled similarly by GEFs and GAPs. Guanine

nucleotide dissociation inhibitors (GDIs) bind prenylated inactive RAB and RHO GTPases

and regulate their delivery or extraction to and from their sites of action. Another class

of regulator proteins known as GDI displacement factors (GDFs) recognize specific

RAB and RHO proteins and catalyse their dissociation from GDIs and their subsequent

delivery to specific subcellular membranes. In the case of RAB GTPases, heat-shock

protein 90 (HSP90) may be involved in their extraction from membranes by enhancing

the ability of GDIs to bind and retrieve these GTPases from their membrane locations.

Again, cycling of RHO and RAB GTPases between active and inactive states is coupled to

the engagement of downstream effectors. Examples of specific GAPs and GEFs for RAS,

ARF, RHO and RAB GTPases and of GDIs for RHO and RAB are shown in the figure.

R E V I E W S



Median effect isobologram analysisA method for evaluating drug

interactions such as synergism,

additive effects or antagonism.

Development of ICMT and RCE1 inhibitors remains challenging. Most agents have demonstrated limited specificity or activity, and substrate analogues with good pharmaceutical properties are hard to find. However, the distinct molecular structure and mode of action of the post-prenylation enzymes continues to stimulate efforts to identify specific and effective inhibitors102,103.

Synergistic activity of mevalonate and prenylation inhibitors. Data indicate that there may be a synergistic activity in combinations of statins with FTIs, with iso-prenoids or with bisphosphonates (TABLE 5). Statin/FTI combinations can induce a more effective inhibition of prenylation modifications by both decreasing isopre-noid levels and by inhibiting geranylgeranylation and farnesylation. In multiple myeloma cells, FTIs potenti-ate the ability of lovastatin to inhibit RHO, KRAS and NRAS prenylation and MEK/MAPK activation, thereby decreasing cell migration and promoting apoptosis104.

This combination may enable similar efficacy with reduced doses of statins or FTIs thereby avoiding exces-sive doses of statins or FTIs104. Furthermore, statin/iso-prenoid combinations induce synergistic inhibition of growth of human DU145 and LNCaP prostate carci-noma and murine B16 melanoma cells105. In normal cells, HMG-CoA reductase is negatively regulated by transcriptional, translational and post-translational feedback signalling from sterol and non-sterol products of the mevalonate pathway106. In this regard, isoprenoids can counteract the statin-induced upregulation of HMG-CoA reductase levels, leading to a more potent inhibition of the mevalonate pathway. Finally, bisphosphonate/FTI and bisphosphonates/statin combinations have report-edly synergistic anticancer properties. Alendronate and the FTI R115777 synergistically inhibit prenylation-dependent membrane association and migratory func-tion of RHO proteins, leading to suppression of in vitro tumour cell invasiveness and in vivo metastasis107. Zoledronic acid enhances the cytotoxic effects of fluv-astatin as measured by median effect isobologram analysis and apoptosis assays in vitro108.

Given that an increasing number of patients (with and without cancer) are currently on statins and/or bisphosphonates for cardiovascular disease therapy and osteoporosis prevention, respectively, it would be feasible to evaluate whether the aforementioned combinations of these agents exhibit clinical anticancer activity. In this regard, trials of these agents in non-cancer patients may also include specific cancer prevention end points, or, alternatively, trials of FTIs, isoprenoids or other agents in patients with cancer who are also on statins or bisphosphonates may be controls for these agents or their combinations.

Synergistic activity with conventional chemotherapy and radiation therapy. Several lines of evidence indi-cate a synergistic activity of prenylation inhibitors with conventional chemotherapeutics and radiation therapy. Lonafarnib enhances both the growth inhibition and apoptotic response of BCR–ABL transformed cells to imatinib67,109. Furthermore, lonafarnib has been shown to reverse the resistance of BCL–ABL transformed cells to imatinib110. The FTI L-744,832 has been shown to enhance taxane-induced mitotic arrest and apoptosis in breast cancer cell lines111. Lonafarnib exhibits synergism with paclitaxel and docetaxel both in cell cultures and xenograft models, and FTIs have been shown to reverse taxane resistance112,113. The molecular mechanism under-lying the synergism between FTIs and taxanes may be that FTIs lead to G2/M cell arrest (because of decreased farnesylation of important mitotic proteins such as CENPs), thereby sensitizing cells to taxane therapy, which acts in the M phase by inhibiting microtubule function. Moreover, FTIs increase taxane binding to microtubules, leading to enhanced microtubule stability and tubulin acetylation, even in paclitaxel-resistant cells. This mecha-nism is dependent on the function of the tubulin deacety-lase, HDAC6 (REF. 113). Synergistic activity has also been documented with platinum agents, fluoropyrimidines, tamoxifen and cyclin-dependent kinase inhibitors114–117.

Similar to FTIs, bisphosphonates have been shown to exhibit synergistic activity with various chemothera-peutics. Zolendronic acid exhibits synergism with pacli-taxel on the induction of apoptosis of breast cancer cells in vitro118. This synergistic interaction is drug-sequence dependent, with maximal levels of apoptosis achieved when cells are treated with paclitaxel followed by zoledronic acid. The synergistic induction of apoptosis is attributed to zoledronic-acid-mediated inhibition of the mevalonate pathway. In prostate cancer cells, treatment with zoledronic acid and docetaxel has synergistic cyto-static effects119. Zoledronic acid has also shown synergism with gemcitabine in several cancer cell lines108. Moreover, synergistic anticancer activity between bisphosphonates and imatinib, cyclooxygenase 2 inhibitors and tyrosine kinase inhibitors has been documented120,121.

Resistance to radiation therapy has been linked with RAS. Several preclinical and Phase I clinical studies combining inhibition of activated RAS with radiother-apy have demonstrated that prenylation inhibitors are radiation-sensitizing agents122. Inhibiting RAS prenyla-tion has been shown to decrease clonogenic survival of

Table 2 | Implication of regulatory proteins in carcinogenesis

Regulatory protein Implication Malignancies

NF1 (RAS GAP) Deletion Neurofibromatosis 1

TSC2 (RHEB GAP) Mutations Tuberous sclerosis

LARG (RHO GEF) Translocation (fusion partner)

Acute myeloid leukaemia

BCR (RHO GEF) Translocation (fusion partner)

Acute myeloid leukaemia

TIAM1 (RAC GEF) Mutation Renal cell cancer

p190 (RHO GAP) Deletion Oligodendrogliomas

DLC2 (RHOA GAP, CDC42 GAP)

Deletion or underexpression

Hepatocellular carcinoma

RHO GDIs Overexpression Non-small-cell lung, ovarian cancers

RHO GDIs Underexpression Breast cancer

GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor.

R E V I E W S



Prenylation

Mevalonate pathway

Prenylationinhibitors

(FTIs, GGTIs, DPIs)

Post-prenylation

GDP/GTP exchange

RAS superfamilyGTPase

RAS superfamilyGTPase

Activation ofeffector proteins

Regulatoryproteins

(GAP, GEF,GDI)

Post-prenylationinhibitors (RCE1,ICMT inhibitors)

Interaction inhibitors(interfacial inhibitors)

GTPase inhibitors(bacterial toxins,GTP analogues,siRNA inhibitors)

Mevalonate inhibitors(statins, bisphosphonates,isoprenoids)

Regulatoryprotein inhibitors(siRNA inhibition)

tumour cells exposed to radiation therapy both in vitro and in vivo123. Even in tumours without RAS mutations, FTIs have proved to be potent radiation sensitizers. Increased oxygenation after FTI therapy and decreased prenylation of RHOB and RAS have been implicated in the radiosensitizing effects of FTIs.

As a proof-of-concept, a Phase I study of the dual FTase and GGTase I inhibitor L-778,123 in combina-tion with radiotherapy was carried out in patients with locally advanced pancreatic cancer. L-778,123 was given by continuous intravenous infusion with concomitant radiotherapy to 59.4 Gy in standard fractions. Reversible inhibition of HDJ2 farnesylation and radiosensitization were demonstrated in a patient-derived cell line. The combination of L-778,123 at a dose of 280 mg per m2 per day over weeks 1, 2, 4 and 5 with radiotherapy showed acceptable toxicity; one out of eight patients showed a partial response of 6 months in duration124.

Targeting functional regulation

Several strategies have been developed for targeting the functional regulation of the RAS superfamily of GTPases (FIG. 5). These strategies focus either on compounds that

inhibit the interaction between the RAS superfamily of GTPases and regulatory proteins (that is, GEFs, GAPs and GDIs) or on development of drugs that target indi-vidual RAS superfamily GTPases or regulatory proteins, thereby blocking GDP/GTP exchange and inhibiting the activation of downstream effectors.

Targeting interactions between the RAS superfamily of GTPases and regulatory proteins. The exact role of the GAP, GEF and GDI regulatory proteins in carcino-genesis depends on the type of cancer, tumour stage (early versus late/metastatic) and, most importantly, on the RAS superfamily of GTPases that they regulate. Although in most circumstances GEFs and GDIs act as oncogenes and GAPs act as tumour suppressor genes, there are many exceptions. For example, AMAP1/PAG2 is an ARF-GAP that is overexpressed in highly invasive breast cancer cells and its short-interfering RNA (siRNA)-mediated silencing effectively blocks invasion125. Furthermore, various GDIs may be over- or underexpressed in human malignancies correspond-ing to different types of tumours and different stages of disease. Last, regulatory proteins may regulate RAS superfamily GTPases that may be tumorigenic or not (RAS and RHOA, for instance, are tumorigenic, whereas RHOB has growth inhibitory activity)70. In light of the complex roles of regulatory proteins in the carcinogenic process, a deep understanding of the role of individual regulatory proteins in different tumour types and stages of the disease is required before targeting each regulatory protein for anticancer therapy.

There are several problems with identifying com-petitive inhibitors of interactions between regulatory proteins and RAS superfamily GTPases. First, both regulatory proteins (that is, GEFs, GAPs and GDIs) and RAS superfamily GTPases exist as families with highly similar members, making the discovery of specific inhibi-tors challenging. Second, many regulatory proteins have more than one RAS superfamily GTPase as targets, and conversely many RAS superfamily GTPases may be regu-lated by more than one regulatory protein. Therefore, competitive inhibitors might non-selectively interfere with multiple pathways, inducing prohibitive toxicity.

A promising way to overcome these challenges is the development of interfacial inhibitors that bind at the interface of the RAS-superfamily-GTPase–regulatory-protein macromolecular complex as it undergoes structural transitions from one stable conformation to another126,127. These transitions frequently generate struc-tural and energetic ‘hot spots’ (usually contributed by a few amino-acid residues) that constitute excellent targets for the development of small-molecule inhibitors. These inhibitors bind to the hot spot of a transient intermediate RAS-superfamily-GTPase–regulatory-protein complex, preventing it from carrying out its biological function. An important characteristic of interfacial inhibitors is that they recognize both components of the complex (the RAS superfamily GTPase and the regulatory protein), resulting in superior specificity. Additionally, interfacial inhibitors target the two components only when they interact in a complex and not the RAS superfamily

Figure 5 | Possible strategies for targeting post-translational modifications and functional regulation of the RAS superfamily of GTPases. Statins, biphosphonates

and isoprenoids, alone or in combinations, target the mevalonate pathway, whereas

farnesyltransferase inhibitors (FTIs), geranylgeranyltransferase inhibitors (GGTIs) and

dual prenylation inhibitors (DPIs) target the prenylation process. Several strategies have

been developed for targeting the functional regulation of the RAS superfamily of

GTPases. These focus either on compounds that inhibit the interaction between the RAS

superfamily of GTPases and regulatory proteins (for example, interfacial inhibitors), or on

drugs that target individual RAS superfamily GTPases (for example, bacterial toxins,

GTP analogues, small-interfering RNA (siRNA) inhibitors), or regulatory proteins (siRNA

inhibition), thereby blocking GDP/GTP exchange and inhibiting activation of

downstream effectors. GAP, GTPase-activating protein; GDI, guanine nucleotide

dissociation inhibitor; GEF, guanine nucleotide exchange factor; ICMT, isoprenylcysteine

carboxymethyltransferase; RCE1, RAS converting enzyme.

R E V I E W S



GTPase or regulatory protein individually, thereby selecting for a specific signalling pathway and avoiding non-selective inhibition128.

This type of inhibition of a RAS-superfamily-GTPase–regulatory-protein complex is exemplified by the natural product brefeldin A (BFA)129. BFA binds at the interface between ARF GDP and the catalytic Sec7 domain of ARF GEFs, and stabilizes this complex just before GDP dis-sociation. Sensitivity to BFA inhibition depends on both ARF protein (ARF1 and ARF5 are BFA-sensitive, whereas ARF6 is not) and the Sec7 domain of ARF GEFs (BIG1 GEF is BFA-sensitive, whereas ARNO GEF is not). A par-ticular amino-acid residue (Tyr190) in the Sec7 domain is essential and sufficient to confer sensitivity to BFA129.

Following the paradigm of BFA, several inhibitors that target the RAS-superfamily-GTPase–regulatory-protein interactions have been developed. NSC23766 was identi-fied by structure-based virtual screening of compounds that fit into the GEF-recognition groove centred on Trp56 of RAC1 (REF. 130). Importantly, NSC23766 targets RAC1 activation by RAC-specific GEFs: TIAM-1 and TRIO, but not VAV, and LBC GEFs. Furthermore, NSC23766 selec-tively affects RAC1 and not the closely related CDC42 or RHOA binding to their respective GEFs. The 50% inhibitory dose along with the inhibitory potency of the binding interaction of NSC23766 was approximately 50 μM. NSC23766 demonstrated growth inhibitory and anti-invasive properties in PC-3 cells. Another inhibitor, TRIAPα, which was identified by a genetic screen of pep-tide aptamers in yeast, specifically targets the RHO–GEF TRIO interaction with RHOA131.

The effects of NSC23766 in haematopoietic stem cells have important clinical implications132. RAC GTPases are major regulators of haematopoiesis. Specifically, RAC1 is involved in short-term engraftment and proliferation after growth-factor stimulation, whereas RAC2 is involved in defective growth-factor rescue of apoptosis in vitro. Administration of NSC23766 in a single intraperitoneal dose of 2.5 mg per kg induced a doubling of circulating haematopoietic stem cell/progenitor cells in the mobili-zation-resistant C57Bl/6 mouse strain. The same group reported that continuous administration of NSC23766 over a period of 60 days was not associated with either changes in peripheral haematological parameters or his-tological evidence of toxicity in bone, brain, heart, lungs, bladder, stomach, liver, spleen, intestine or kidneys132.

BFA, NSC23766 and TRIAPα validate the concept that RAS superfamily GTPases can be inhibited by interfacial inhibition of their interactions with regulatory proteins. To improve the inhibitory effect of NSC23766 on RAC1, modification of its structure to achieve a better fit into the groove on the RAC1 surface and to increase its docking affinity is necessary130. This approach has also been used to inhibit the interaction between RAS and its downstream effector RAF, and to inhibit RAS-dependent transforma-tion in vitro133. These compounds could reverse the trans-formation phenotypes induced by RAS in several cancer cell lines at a concentration of approximately 20 μM.

Targeting individual RAS superfamily GTPases or regulatory proteins. Bacterial toxins have been demon-strated to target individual RAS superfamily GTPases.

Table 3 | Important clinical trials of mevalonate-pathway inhibitors

Agent Design/cancer Number of patients

Results Refs

Apomine (SR-45023A)

Phase II/melanoma

42 • SD was achieved in 2 patients (5%)• No complete or partial responses were observed• Median overall survival was 6.1 months• Apomine was well tolerated• Failed to produce necessary benefit for further

development

140

Pravastatin and standard therapy versus placebo and standard therapy

Phase III/ hepatocellular cancer

91 • Median survival was 18 months in pravastatin group versus 9 months in placebo (p = 0.006)

• Cox proportional hazards model showed that pravastatin significantly contributed to survival

61

Lovastatin Phase II/gastric adenocarcinoma

16 • No responses were documented• Median number of cycles was 2• Anorexia was the most common toxicity• Two patients developed myalgia with elevated

muscle enzyme

141

Lovastatin Phase I/squamous cell cancer of head, neck and cervix

26 • DLT consisted of reversible muscle toxicity• MTD was determined to be 7.5 mg per kg per day,

administered for 21 days every 28 days• No objective responses were seen• Median survival of patients on study was 7.5 months• SD for more than 3 months was seen in 23% of patients

142

Simvastatin and standard chemotherapy

Phase I/myeloma, lymphoma

28 • MTD was 15 mg per kg per day of simvastatin• Side effects were fatigue, gastrointestinal toxicity

and neutropaenic fever• DLT was neutropaenic sepsis and grade 3

gastrointestinal side effects

143

DLT, dose-limiting toxicity; MTD, maximum-tolerated dose ; SD, stable disease.

R E V I E W S



Specifically, Clostridium difficile (the aetiological agent of pseudomembranous colitis) toxins A and B glycosylate RHO, RAC and CDC42 GTPases and inhibit downstream signalling while stimulating RHOB expression134,135.

Furthermore, bacterial C3-like ADP-ribosyltransferase induces ADP-ribosylation of RHOA. Nevertheless, no toxin-based compounds have been evaluated as inhibi-tors of RAS superfamily GTPases so far.

Inhibition of the RAS superfamily of GTPases using siRNA technology may represent an alternative strategy. Anti-RHOA and anti-RHOC siRNAs have been shown to inhibit cell proliferation and invasion more effectively than GGTIs and statins in breast cancer cell lines, as well as in a mouse model136. This approach has also been used to knockdown regulatory proteins. Accordingly, RHO GDIα and RHO GDIβ have been inhibited by siRNAs in breast cancer cell lines137.

GTP analogues, such as 6-thioGTP (SGTP), which is a metabolite of azathioprine, can directly inhibit RAS superfamily GTPases. SGTP causes immunosuppression by blockade of GTPase activation in T lymphocytes138. SGTP specifically blocks the activation of RAC1 and RAC2 (but not of CDC42 and RHOA) in primary T cells after stimulation with fibronectin. This happens because after hydrolysis of RAC-SGTP into RAC-SGDP, SGDP cannot be exchanged for GTP by VAV1 GEF, leading to accumulation of SGDP-loaded, inactive RAC proteins.

So, azathioprine-generated SGTP disrupts RAC activity by blocking VAV GEF activity on RAC proteins.

Conclusions and perspectives

The role of the RAS superfamily of GTPases in carcino-genesis is increasingly being recognized. Current pharmacological strategies have mainly focused on targeting the prenylation enzyme FTase or factors downstream (mTOR, ROCK, RAF and MEK) of RAS GTPases. FTIs, which have progressed to late-stage clinical trials, seem to have limited single-agent activity against most human cancers with the exception of certain haematological malignancies.

Nevertheless, several lines of evidence suggest that there may be a synergistic activity between FTIs and other molecularly targeted agents, including inhibitors of the mevalonate pathway (that is, statins or bisphos-phonates) or other prenylation inhibitors (such as GGTIs). This prospect needs to be evaluated further, especially as a large number of patients are currently on statins and/or bisphosphononates. The major challenge in the case of GGTI/FTI combinations is toxicity, which could be overcome by the development of DPIs, which are better tolerated. Development of GGTase I inhibi-tors that demonstrate selectivity for monomeric versus heterotrimeric G proteins (such as GGTI-DU40) is another particularly promising therapeutic strategy139.

Table 4 | Important clinical trials of prenylation inhibitors

Agent Design/ Cancer

Number of patients

Results Refs

Tipifarnib and gemcitabine versus gemcitabine and placebo

Phase III/ pancreatic cancer

688 • Median overall survival for tipifarnib and gemcitabine was 193 versus 182 days for the placebo arm (p = 0.75)

• 6-month and 1-year survival rates were 53% and 27% for tipifarnib versus 49% and 24% for the placebo arm

• Comparable and acceptable toxicity in both arms

79

Tipifarnib versus placebo

Phase III/ colon cancer

368 • Median overall survival for tipifarnib was 174 days versus 185 days for placebo (p = 0.376)

• One patient achieved a partial response in the tipifarnib arm

• Tipifarnib was well tolerated

80

Lonafarnib, and carboplatin and paclitaxel versus carboplatin, and paclitaxel and placebo

Phase III/ non-small cell-lung cancer

675 • Trial discontinued after a planned interim analysis of 616 patients showed insufficient activity

• Overall survival was 144 days for patients treated with lonafarnib and 168 days for patients who received placebo

144

Tipifarnib Phase II/ poor risk AML

158 (older patients; median age 74 years)

• CR was achieved in 22 patients (14%); partial remission or haematological improvement occurred in 9%

• Median duration of CR was 7.3 months and survival was 18 months

• Inhibition of farnesylation of the surrogate protein HDJ2 was noted

• Tipifarnib was well tolerated

83

L-778,123 and radiotherapy

Phase I/ pancreatic cancer

128 (level 1); 4 (level 2)

• There were no dose-limiting toxicities observed in dose level 1

• At dose level 2, grade 3 diarrhoea and gastrointestinal haemorrhage associated with grade 3 thrombocytopaenia and neutropaenia

• One patient on dose level 1 showed a partial response of 6 months in duration

• Reversible inhibition of HDJ2 farnesylation and radiosensitization of a study patient-derived cell line were demonstrated

124

AML, acute myeloid leukaemia; CR, complete remission.

R E V I E W S



The prospect of targeting the post-prenylation enzymes ICMT and RCE1 is an exciting alternative strategy. This approach offers the advantage of inhibiting both far-nesylated and geranylgeranylated RAS GTPases, thereby avoiding the phenomenon of cross-prenylation, which confers resistance to FTIs. In the case of RCE1 inhibitors, preclinical evidence suggests that RCE1 inhibition alone will not be sufficient for anticancer therapy, but indicates a synergistic activity with FTIs. ICMT inhibition may be more efficacious, but may also be less selective (ICMT has more RAS superfamily GTPase targets than FTase), result-ing in excessive toxicity. Pharmaceutical considerations might lead to the development of new classes of inhibitors of ICMT, as S-adenosyl-Met analogues or prenylcysteine analogues may be difficult to develop.

Interference with the interactions between RAS superfamily GTPases and regulatory proteins, or inhibi-tion of individual RAS superfamily GTPases or regula-tory proteins, offers the advantage of higher specificity. Interfacial inhibitors, designed on the basis of a strategy used commonly in nature (that is, the topoisomerase I inhibitor camptothecin and the tubulin inhibitor col-chicine), recognize both regulatory proteins and RAS superfamily GTPases and inhibit them only when they

interact, thereby targeting a specific downstream pathway. Design or identification of interfacial inhibitors is easier than competitive inhibitors as small molecules are more likely to bind at the concave surfaces of macromolecular interfaces than the flatter surfaces of unbound molecules (that is, competitive inhibitors)99.

It is important to note that the agents that we refer to here demonstrate different specificities against RAS superfamily GTPases. In this regard, interfacial inhibitors targeting the interaction between regulatory proteins and RAS superfamily GTPases have the highest specificity for individual pathways, whereas prenylation and post-prenylation inhibitors are the most ‘unspecific’ drugs in this category, as they are capable of targeting several RAS superfamily GTPases. Furthermore, the combina-tions referred to earlier demonstrate low specificity for individual RAS superfamily GTPases. Higher specificity is associated with fewer side effects, but it is currently unclear whether it is also related with greater clinical benefit. Conversely, it is possible that the most unspecific drugs are associated with higher efficacy simply because of simultaneous targeting of several pathways implicated in metastasis, invasion, angiogenesis, drug resistance, cellular proliferation and apoptosis. Unfortunately, it is currently unclear which specific RAS superfamily GTPases are important in different malignancies and whether selectively targeting one RAS superfamily GTPase in certain malignancies is better than simultane-ous targeting multiple RAS superfamily GTPases. In this respect, the idea that the most unspecific drugs are more effective might just reflect the fact that we do not know what the relevant targets in each malignancy are, and so we cannot effectively and selectively target them.

A major challenge for future development of these agents is the identification of molecular markers that are predictive of response. These markers will enable us to improve the selection of patients that could benefit from these drugs or their combinations, thereby avoiding tox-icity in patients who would otherwise derive no benefit from them. Furthermore, identification of appropri-ate pharmacodynamic molecular or radiographic end points that would guide dose escalation and help to assess response is also important for the development of these drugs. In addition to HDJ2, other potential molecular end points may include RAS GTP levels, determination of CENPE, CENPF and RHEB farnesyla-tion and RAP1a geranylgeranylation in tumour cells or PBMCs. Last, these molecularly targeted agents may not necessarily induce tumour regression but rather only growth inhibition, and so standard response criteria might not be applicable.

Table 5 | Synergistic activity between mevalonate and prenylation inhibitors

Combination Rationale Refs

Statins and isoprenoids

• Inhibition of mevalonate pathway• Prevent upregulation of HMG-CoA reductase that may

occur after decreased production of cholesterol and isoprenoids

• Tumour cells are sensitive to isoprenoid post-transcriptional downregulation of HMG-CoA

105,106

Statins and FTIs

• Effective reduction of prenylation by reduction of FPP availability and enzymatic inhibition of FTase

• Inhibition of FTase by FTIs may lead to increased synthesis of cholesterol and dolichyl-phosphate because of FPP availability; statins prevent this possibility

104

Statins and BPs

• Sequential action on the mevalonate pathway (inhibition of HMG-CoA reductase by statins, and FPP synthase and IPP isomerase by BPs

• Synergistic antiangiogenic and anti-invasive properties independently of mevalonate pathway

108

BPs and FTIs • More effective reduction of prenylation by reduction of FPP availability and enzymatic inhibition of FTase

• Anti-angiogenic and anti-invasive properties of BPs independently of mevalonate pathway

107

FTIs and GGTIs • Prevent alternative prenylation of KRAS, NRAS and RHO GTPases

87

BP, bisphosphonate; FTase, farnesyltransferase; FTI, farnesyltransferase inhibitor; FPP, farnesyl pyrophosphate; GGTI, geranylgeranyltransferase inhibitor; HMG-CoA, 3-hydroxy3-methylglutaryl-CoA; IPP, isopentenyl pyrophosphate.

1. Gilman, A. G. G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615–649 (1987).

2. Oldham, W. M., Van Eps, N., Preininger, A. M., Hubbell, W. L. & Hamm, H. E. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nature Struct. Mol. Biol. 9, 772–777 (2006).

3. Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81, 153–208 (2001).

4. Etienne-Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature 420, 629–635 (2002).

5. Pfeffer, S. & Aivazian, D. Targeting Rab GTPases to distinct membrane compartments. Nature Rev. Mol. Cell Biol. 5, 886–896 (2004).

6. Rocks, O., Peyker, A. & Bastiaens, P. I. Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors. Curr. Opin. Cell Biol. 18, 351–357 (2006).

7. Quimby, B. B. & Dasso, M. The small GTPase Ran: nterpreting the signs. Curr. Opin, Cell Biol. 15, 338–344 (2003).

8. D’Souza-Schorey, C. & Chavrier, P. ARF proteins: roles in membrane traffic and beyond. Nature Rev. Mol. Cell Biol. 7, 347–358 (2006).

9. Manser, E. Small GTPases take the stage. Dev. Cell 3, 323–328 (2002).

10. Bernards, A. GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim. Biophys. Acta 1603, 47–82 (2003).

11. Rossman K. L., Der C. J. & Sondek J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nature Rev. Mol. Cell Biol. 6, 167–180 (2005).

12. DerMardirossian, C. & Bokoch, G. M. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 15, 356–363 (2005).

R E V I E W S



13. Radhika, V. & Dhanasekaran, N. Transforming G proteins. Oncogene 20, 1607–1614 (2001).

14. Shaw, R. J. & Cantley, L. C. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441, 424–430 (2006).

15. Bos, J. L. Ras oncogenes in human cancer: a review. Cancer Res. 49, 4682–4689 (1989).

16. Downward, J. Targeting RAS signalling pathways in cancer therapy. Nature Rev. Cancer 3, 11–22 (2003).

17. Daub, H., Specht, K. & Ullrich, A. Strategies to overcome resistance to targeted protein kinase inhibitors. Nature Rev. Drug Discov. 3, 1001–1110 (2004).

18. Sahai, E. & Marshall, C. J. RHO-GTPases and cancer. Nature Rev. Cancer 2, 133–142 (2002).

19. Preudhomme, C. Non-random 4p13 rearrangements of the RhoH/TTF gene, encoding a GTP-binding protein, in non-Hodgkin’s lymphoma and multiple myeloma. Oncogene 19, 2023–2032 (2000).

20. Pasqualucci, L. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature 412, 341–346 (2001).

21. Ridley, A. J. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol. 16, 522–529 (2006).

22. Kleer, C. G. et al. RhoC-GTPase is a novel tissue biomarker associated with biologically aggressive carcinomas of the breast. Breast Cancer Res. Treat. 93, 101–110 (2005).

23. Kamai, T. et al. Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin. Cancer Res. 9, 2632–2641 (2003).

24. Fidyk, N., Wang, J. B. & Cerione, R. A. Influencing cellular transformation by modulating the rates of GTP hydrolysis by Cdc42. Biochemistry 45, 7750–7762 (2006).

25. Lin, R., Cerione, R. A. & Manor, D. Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation. J. Biol. Chem. 274, 23633–23641 (1999).

26. Cheng, K. W. et al. The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers. Nature Med. 10, 1251–1256 (2004).

27. Croizet-Berger, K., Daumerie, C., Couvreur, M., Courtoy, P. J. & van den Hove, M. F. The endocytic catalysts, Rab5a and Rab7, are tandem regulators of thyroid hormone production. Proc. Natl Acad. Sci. USA 99, 8277–8282 (2002).

28. He, H. et al. Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues. Gene Expr. 10, 231–242 (2002).

29. Hashimoto, S. et al. Requirement for ARF6 in breast cancer invasive activities. Proc. Natl Acad. Sci. USA. 101, 6647–6645 (2004).

30. Calin, G. A. et al. Familial cancer associated with a polymorphism in ARLTS1. N Engl J Med. 352, 1667–76 (2005).

31. Zhang, F. L. & Casey, P. J. Protein prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65, 241–269 (1996).

32. Reid, T. S., Terry, K. L., Casey, P. J. & Beese L. S. Crystallographic analysis of CAAX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J. Mol. Biol. 343, 417–433 (2004).

33. Ashby, M. N. CaaX converting enzymes. Curr. Opin Lipidol. 9, 99–102 (1998).

34. Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 57, 1167–1177 (1989).

35. Hoffman, G. R., Nassar, N. & Cerione, R. A. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100, 345–356 (2000).

36. McTaggart, S. J. Isoprenylated proteins. Cell Mol. Life Sci. 63, 255–267 (2006).

37. Amor, J. C., Harrison, D. H., Kahn, R. A. & Ringe, D. Structure of the human ADP-ribosylation factor 1 complexed with GDP. Nature 372, 704–708 (1994).

38. Donovan, S., Shannon, K. M. & Bollag, G. GTPase activating proteins: critical regulators of intracellular signaling. Biochim. Biophys. Acta 1602, 23–45 (2002).

39. Weiss, B., Bollag, G. & Shannon, K. Hyperactive Ras as a therapeutic target in neurofibromatosis type 1. Am. J. Med. Genet. 89, 14–22 (1999).

40. Crino, P. B., Nathanson, K. L. & Henske, E. P. The tuberous sclerosis complex. N. Engl. J. Med. 355, 1345–1355 (2006).

An excellent review on the role of RHEB/mTOR in

the pathogenesis of tuberous sclerosis.

41. Dransart, E., Olofsson, B. & Cherfils, J. RhoGDIs revisited: novel roles in Rho regulation. Traffic 6, 957–966 (2005).

42. Ivetic, A. & Ridley, A. J. Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes. Immunology 112, 165–176 (2004).

43. Maeda, M., Matsui, T., Imamura, M., Tsukita, S. & Tsukita, S. Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with Ezrin/Radixin/Moesin proteins. Oncogene 18, 4788–4797 (1999).

44. Kourlas, P. J. et al. Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: evidence for its fusion with MLL in acute myeloid leukemia. Proc. Natl Acad. Sci. USA 97, 2145–2150 (2000).

45. Kin, Y., Li, G, Shibuya, M. & Maru, Y. The Dbl homology domain of BCR is not a simple spacer in P210BCR-ABL of the Philadelphia chromosome. J. Biol. Chem. 276, 39462–39468 (2001).

46. Engers, R. et al. TIAM1 mutations in human renal-cell carcinomas. Int. J. Cancer 88, 369–376 (2000).

47. Wolf, R. M. et al. p190RhoGAP can act to inhibit PDGF-induced gliomas in mice: a putative tumor suppressor encoded on human chromosome 19q13.3. Genes Dev. 17, 476–487 (2003).

48. Peck, J., Douglas, G., Wu, C. H. & Burbelo, P. D. Human RhoGAP domain-containing proteins: structure, function and evolutionary relationships. FEBS Lett. 528, 27–34 (2002).

49. Leung T. H. et al. Deleted in liver cancer 2 (DLC2) suppresses cell transformation by means of inhibition of RhoA activity. Proc. Natl Acad. Sci. USA 102, 15207–15212 (2005).

50. MacKeigan, J. P. et al. Proteomic profiling drug-induced apoptosis in non-small cell lung carcinoma: identification of RS/DJ-1 and RhoGDIα. Cancer Res. 63, 6928–6934 (2003).

51. Tapper, J. et al. Changes in gene expression during progression of ovarian carcinoma. Cancer Genet. Cytogenet. 128, 1–6 (2001).

52. Jiang, W. G. et al. Prognostic value of rho GTPases and rho guanine nucleotide dissociation inhibitors in human breast cancers. Clin. Cancer Res. 9, 6432–6440 (2003).

53. Stein, M. P., Dong, J. & Wandinger-Ness, A. Rab proteins and endocytic trafficking: potential targets for therapeutic intervention. Adv. Drug Deliv. Rev. 55, 1421–1437 (2003).

54. Randazzo, P. A. & Hirsch, D. S. Arf GAPs: multi-functional proteins that regulate membrane traffic and actin remodelling. Cell Signal. 16, 401–413 (2004).

55. Demierre, M. F., Higgins, P. D., Gruber, S. B., Hawk, E. & Lippman, S. M. Statins and cancer prevention. Nature Rev. Cancer 5, 930–942 (2005).

56. Mo, H. & Elson, C. E. Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention. Exp. Biol. Med. (Maywood) 229, 567–585 (2004).

57. Wong, W. W., Dimitroulakos, J., Minden, M. D. & Penn, L. Z. HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis. Leukemia 16, 508–511 (2002).

58. Weitz-Schmidt, G. et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nature Med. 7, 687–692 (2001).

59. Rao, S. et al. Lovastatin-mediated G1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase. Proc. Natl Acad. Sci. USA. 96, 7797–7802 (1999).

60. Hindler, K. et al. The role of statins in cancer therapy. Oncologist 11, 306–315 (2006).An overview of the clinical trials evaluating the

activity of statins as anticancer agents.

61. Kawata, S. et al. Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial. Br. J. Cancer 84, 886–891 (2001).

62. van Beek, E. et al. Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo. Biochem. Biophys. Res Commun. 255, 491–494 (1999).

63. Santini, D. et al. Mechanisms of disease: preclinical reports of antineoplastic synergistic action of bisphosphonates. Nature Clin. Pract. Oncol. 3, 325–338 (2006).

64. Alakangas, A. et al. Alendronate disturbs vesicular trafficking in osteoclasts. Calcif. Tissue Int. 70, 40–47 (2002).

65. Caraglia, M. et al. Emerging anti-cancer molecular mechanisms of aminobisphosphonates. Endocr. Relat. Cancer 13, 7–26 (2006).

66. Coxon, F. P. et al. Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo. Bone 37, 349–358 (2005).

67. Basso, A. D., Kirschmeier, P. & Bishop, W. R. Lipid post-translational modifications. Farnesyl transferase inhibitors. J. Lipid Res. 47, 15–31 (2006).

68. Kohl, N. E. Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 260, 1934–1937 (1993).

69. Law, B. K., Norgaard, P. & Moses, H. L. Farnesyltransferase inhibitor induces rapid growth arrest and blocks p70s6k activation by multiple stimuli. J. Biol. Chem. 275, 10796–10801 (2000).

70. Whyte, D. B. et al. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J. Biol. Chem. 272, 14459–14464 (1997).

71. Lebowitz, P. F., Casey, P. J., Prendergast, G. C. & Thissen, J. A. Farnesyltransferase inhibitors alter the prenylation and growth-stimulating function of RhoB. J. Biol. Chem. 272, 15591–15594 (1997).

72. Du, W., Lebowitz, P. F. & Prendergast, G. C. Cell growth inhibition by farnesyltransferase inhibitors is mediated by gain of geranylgeranylated RhoB. Mol. Cell Biol. 19, 1831–1840 (1999).One of the most important studies that

demonstrate that geranylgeranylated RHOB has

antigrowth properties, while farnesylated RHOB

can be tumorigenic.

73. Chen, Z. et al. Both farnesylated and geranylgeranylated RhoB inhibit malignant transformation and suppress human tumour growth in nude mice. J. Biol. Chem. 275, 17974–17978 (2000).

74. Basso, A. D. et al. The farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits Rheb farnesylation and mTOR signaling. Role in FTI enhancement of taxane and tamoxifen anti-tumor activity. J. Biol. Chem. 280, 31101–31108 (2005).

75. Tabancay, A. P. et al. Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 278, 39921–39930 (2003).

76. Lackner, M. R. et al. Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 7, 325–336 (2005).

77. Appels, N. M., Beijnen, J. H. & Schellens, J. H. Development of farnesyl transferase inhibitors: a review. Oncologist 10, 565–578 (2005).

78. Adjei, A. A., Davis, J. N., Erlichman, C., Svingen, P. A. & Kaufmann, S. H. Comparison of potential markers of farnesyltransferase inhibition. Clin. Cancer Res. 6, 2318–2325 (2000).A useful discussion of potential biomarkers of

FTase inhibition.

79. Van Cutsem, E. et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J. Clin. Oncol. 22, 1430–1438 (2004).

80. Rao, S. et al. Phase III double-blind placebo-controlled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer. J. Clin. Oncol. 22, 3950–3957 (2004).

81. McDonald, J. S. et al. A phase II study of farnesyl transferase inhibitor R115777 in pancreatic cancer: a Southwest Oncology Group (SWOG 9924) study. Invest. New Drugs 23, 485–487 (2005).

82. Zimmerman, T. M. et al. Dose-ranging pharmacodynamic study of tipifarnib (R115777) in patients with relapsed and refractory hematologic malignancies. J. Clin. Oncol. 22, 4816–4822 (2004).

83. Lancet, J. E. et al. A Phase II study of the farnesyltransferase inhibitor tipifarnib in poor-risk and elderly patients with previously untreated acute myelogenous leukemia. Blood 109, 1387–1394 (2007).

84. Caraglia, M., Budillon, A., Tagliaferri, P., Marra, M., Abbruzzese, A. & Caponigro, F. Isoprenylation of intracellular proteins as a new target for the therapy of human neoplasms: preclinical and clinical implications. Curr. Drug Targets 6, 301–323 (2005).

R E V I E W S



85. Di Paolo, A. et al. Inhibition of protein farnesylation enhances the chemotherapeutic efficacy of the novel geranylgeranyltransferase inhibitor BAL9611 in human colon cancer cells. Br. J. Cancer. 84, 1535–1543 (2001).

86. Lobell, R. B. et al. Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models. Cancer Res. 61, 8758–8768 (2001).

87. Morgan, M. A., Wegner, J., Aydilek, E., Ganser, A. & Reuter, C. W. Synergistic cytotoxic effects in myeloid leukemia cells upon cotreatment with farnesyltransferase and geranylgeranyl transferase-I inhibitors. Leukemia 17, 1508–1520 (2003).

88. Reid, T. S. et al. Crystallographic analysis reveals that anticancer clinical candidate L-778,123 inhibits protein farnesyltransferase and geranygeranyltransferase-I by different binding modes. Biochemistry 43, 9000–9008 (2004).

89. Lobell, R. B. et al. Preclinical and clinical pharmacodynamic assessment of L-778,123, a dual inhibitor of farnesyl:protein transferase and geranylgeranyl: protein transferase type-I. Mol. Cancer Ther. 1, 747–758 (2002).

90. Kelly, J. et al. The prenyltransferase inhibitor AZD3409 has anti-tumour activity in preclinical models of urothelial carcinoma. Proc. Am. Assoc. Cancer Res. 46, 5962 (2005).

91. Bergo, M. O. et al. Absence of the CAAX endoprotease RCE1: effects on cell growth and transformation. Mol. Cell Biol. 22, 171–181 (2002).

92. Bergo, M. O. et al. Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf. J. Clin. Invest. 113, 539–550 (2004).A pivotal paper showing that the inhibition of ICMT

activity blocks oncogenic KRAS-induced

transformation by decreasing the methylation of

KRAS, HRAS and NRAS.

93. Winter-Vann, A. M. & Casey, P. J. Post-prenylation-processing enzymes as new targets in oncogenesis. Nature Rev. Cancer. 5, 405–412 (2005).

94. Schlitzer, M., Winter-Vann, A. & Casey, P. J. Non-peptidic, non-prenylic inhibitors of the prenyl protein-specific protease Rce1. Bioorg. Med. Chem. Lett. 11, 425–427 (2001).

95. Chen, Y. Selective inhibition of ras-transformed cell growth by a novel fatty acid-based chloromethyl ketone designed to target Ras endoprotease. Ann. NY Acad. Sci. 886, 103–108 (1999).

96. Wnuk, S. F. et al. Anticancer and antiviral effects and inactivation of S-adenosyl-L-homocysteine hydrolase with 5′-carboxaldehydes and oximes synthesized from adenosine and sugar-modified analogues. J. Med. Chem. 40, 1608–1618 (1997).

97. Winter-Vann, A. M. et al. Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate. Proc. Natl Acad. Sci. USA 100, 6529–6534 (2003).

98. Winter-Vann, A. M. et al. A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells. Proc. Natl Acad. Sci. USA 102, 4336–4341 (2005).

99. Michaelson, D. et al. Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases. Mol. Biol. Cell 16, 1606–1616 (2005).

100. Kramer, K., et al. Isoprenylcysteine carboxyl methyltransferase activity modulates endothelial cell apoptosis. Mol. Biol. Cell 14, 848–857 (2003).

101. Chiu, V. K. et al. Ras signalling on the endoplasmic reticulum and the Golgi. Nature Cell Biol. 4, 343–350 (2002).An important study that provides evidence that

mislocalized farnesylated or geranylgeranylated

RAS (specifically NRAS and HRAS) can still signal

from other locations besides the plasma membrane.

102. Leow, J. L., Baron, R., Casey, P. J. & Go, M. L. Quantitative structure–activity relationship (QSAR) of indoloacetamides as inhibitors of human isoprenylcysteine carboxyl methyltransferase. Bioorg. Med. Chem. Lett. 17, 1025-1032 (2006).

103. Roberts, M. J. et al. Hydrophilic anilinogeranyl diphosphate prenyl analogues are ras function inhibitors. Biochemistry 45, 15862–15872 (2006).

104. Morgan, M. A. Combining prenylation inhibitors causes synergistic cytotoxicity, apoptosis and disruption of RAS-to-MAP kinase signalling in multiple myeloma cells. Br. J. Haematol. 130, 912–925 (2005).

105. Mo, H. & Elson, C. E. Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention. Exp. Biol. Med. (Maywood) 229, 567–585 (2004).

106. Elson, C. E. et al. Isoprenoid-mediated inhibition of mevalonate synthesis: potential application to cancer. Proc. Soc. Exp. Biol. Med. 221, 294–311 (1999).

107. Andela, V. B. Synergism of aminobisphosphonates and farnesyl transferase inhibitors on tumour metastasis. Clin. Orthop. Relat. Res. 397, 228–239 (2002).

108. Budman, D. R. & Calabro, A. Zoledronic acid (Zometa) enhances the cytotoxic effect of gemcitabine and fluvastatin: in vitro isobologram studies with conventional and nonconventional cytotoxic agents. Oncology 70, 147–153 (2006).

109. Hoover, R. R., Mahon, F. X., Melo, J. V. & Daley, G. Q. Overcoming STI571 resistance with the farnesyl transferase inhibitor SCH66336. Blood 100, 1068–1071 (2002).

110. Jorgensen, H. G. et al. Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro. Leukemia 19, 1184–1191 (2005).

111. Moasser, M. M. et al. Farnesyl transferase inhibitors cause enhanced mitotic sensitivity to taxol and epothilones. Proc. Natl Acad. Sci. USA 95, 1369–1374 (1998).

112. Shi, B. et al. The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumour activity in vivo. Cancer Chemother. Pharmacol. 46, 387–393 (2000).

113. Marcus, A. I. et al. Farnesyltransferase inhibitors reverse taxane resistance. Cancer Res. 66, 8838–8846 (2006).

114. Adjei, A. A., Davis, J. N., Bruzek, L. M., Erlichman, C. & Kaufmann, S. H. Synergy of the protein farnesyltransferase inhibitor SCH66336 and cisplatin in human cancer cell lines. Clin. Cancer Res. 7, 1438–1445 (2001).

115. Doisneau-Sixou, S. F., Cestac, P., Faye, J. C., Favre, G., Sutherland, R. L. Additive effects of tamoxifen and the farnesyl transferase inhibitor FTI-277 on inhibition of MCF-7 breast cancer cell-cycle progression. Int. J. Cancer 106: 789–798 (2003).

116. Edamatsu, H., Gau, C. L., Nemoto, T., Guo, L., & Tamanoi, F. Cdk inhibitors, roscovitine and olomoucine, synergize with farnesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines. Oncogene 19, 3059–3068 (2000).

117. Russo, P., Malacarne, D., Falugi, C., Trombino, S. & O’Connor, P. M. RPR-115135, a farnesyltransferase inhibitor, increases 5-FU cytotoxicity in ten human colon cancer cell lines: role of p53. Int. J. Cancer 100, 266–275 (2002).

118. Neville-Webbe, H. L., Evans, C. A., Coleman, R. E. & Holen, I. Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro. Tumour Biol. 27, 92–103 (2006).

119. Ullen, A. et al. Additive/synergistic antitumoural effects on prostate cancer cells in vitro following treatment with a combination of docetaxel and zoledronic acid. Acta Oncol. 44, 644–650 (2005).

120. Melisi, D. et al. Zoledronic acid cooperates with a cyclooxygenase-2 inhibitor and gefitinib in inhibiting breast and prostate cancer. Endocr. Relat. Cancer 12, 1051–1058 (2005).

121. Segawa, H. et al. Zoledronate synergises with imatinib mesylate to inhibit Ph primary leukaemic cell growth. Br. J. Haematol. 130, 558–560 (2005).

122. Brunner, T. B., Hahn, S. M., McKenna, W. G. & Bernhard, E. J. Radiation sensitization by inhibition of activated Ras. Strahlenther Onkol. 180, 731–740 (2004).

123. Brunner, T. B. et al. Farnesyltransferase inhibitors as radiation sensitizers. Int. J. Radiat. Biol. 79, 569–576 (2003).

124. Martin, N. E. et al. A Phase I trial of the dual farnesyltransferase and geranylgeranyltransferase inhibitor L-778,123 and radiotherapy for locally advanced pancreatic cancer. Clin. Cancer Res. 10, 5447–5454 (2004).

125. Onodera, Y. et al. Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities. EMBO J. 24, 963–973 (2005)

126. Tesmer, J. J. Hitting the hot spots of cell signaling cascades. Science. 312, 377–378 (2006).An excellent review that discusses the concept of

interfacial inhibition and its potential as a strategy

for drug discovery.

127. Pommier, Y. & Cherfils, J. Interfacial inhibition of macromolecular interactions: nature’s paradigm for drug discovery. Trends Pharmacol. Sci. 26, 138–145 (2005).

128. Zeghouf, M., Guibert, B., Zeeh, J. C. & Cherfils, J. Arf, Sec7 and Brefeldin A: a model towards the therapeutic inhibition of guanine nucleotide-exchange factors. Biochem. Soc. Trans. 33, 1265–1268 (2005).

129. Zeeh, J. et al. Dual specificity of the interfacial inhibitor brefeldin A for arf proteins and sec7 domains. J. Biol. Chem. 281, 11805–11814 (2006).

130. Gao, Y., Dickerson, J. B., Guo, F., Zheng, J. & Zheng Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc. Natl Acad. Sci. USA. 101, 7618–7622 (2004).A seminal paper describing the development of

NSC23766 by a structure-based virtual screening

of compounds that fit into the GEF-recognition

groove centring on Trp56 of RAC1.

131. Schmidt, S., Diriong, S., Mery, J., Fabbrizio, E. & Debant, A. Identification of the first Rho GEF inhibitor, TRIPα, which targets the RhoA-specific GEF domain of Trio. FEBS Lett. 523, 35–44 (2002).

132. Cancelas, J. A. et al. Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization. Nature Med. 11, 886–891 (2005).

133. Kato-Stankiewicz, J. et al. Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells. Proc. Natl Acad. Sci. USA. 99, 14398–14403 (2002).

134. Fritz, G. & Kaina, B. Rho GTPases: promising cellular targets for novel anticancer drugs. Curr. Cancer Drug Targets 6, 1–14 (2006).

135. Just, I. et al. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500–503 (1995).

136. Pille, J. Y. et al. Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo. Mol. Ther. 11, 267–274 (2005).

137. Zhang, B. Rho GDP dissociation inhibitors as potential targets for anticancer treatment. Drug Resist. Updat. 9, 134–141 (2006).

138. Poppe, D. et al. Azathioprine suppresses ezrin-radixin-moesin-dependent T cell-APC conjugation through inhibition of Vav guanosine exchange activity on Rac proteins. J. Immunol. 176, 640–651 (2006).

139. Peterson, Y. K., Kelly, P., Weinbaum, C. A. & Casey, P. J. A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity. J. Biol. Chem. 281, 12445–12450 (2006).This paper highlights the development of GGTase I

inhibitors (GGTIs) that demonstrate selectivity for

monomeric versus heterotrimeric G proteins (such

as GGTI DU40).

140. Lewis, K. D. et al. A Phase II open-label trial of apomine (SR-45023A) in patients with refractory melanoma. Invest. New Drugs 24, 89–94 (2006).

141. Kim, W. S. et al. Phase II study of high-dose lovastatin in patients with advanced gastric adenocarcinoma. Invest. New Drugs 19, 81–83 (2001).

142. Knoxx, J. J. et al. A Phase I trial of prolonged administration of lovastatin in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or of the cervix. Eur. J. Cancer 41,523–530 (2005).

143. van der Spek, E. et al. Dose-finding study of high-dose simvastatin combined with standard chemotherapy in patients with relapsed or refractory myeloma or lymphoma. Haematologica 91,542–545 (2006).

144. Blumenschein, G. et al. O-082. A randomized Phase III trial comparing lonafarnib/carboplatin/paclitaxel versus carboplatin/paclitaxel (CP) in chemotherapy-naive patients with advanced or metastatic non-small cell lung cancer (NSCLC). Lung Cancer 49 (Suppl. 2), 30 (2005).

AcknowledgementsThe authors would like to thank A. Konstantinopoulou for her invaluable help with the figures.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:UniProtKB: http://ca.expasy.org/sprot

BRAF | CDC42 | CENPE | CENPF | DNAJA1 | HRAS | ICMT |

KRAS | NRAS | RAC1 | RAC2 | RCE1 | RHOA | RHOC | RHOH

Access to this links box is available online.

R E V I E W S



regulation of the ras superfamily of gtpases as anticancer ...dipbsf.uninsubria.it/monti/bfpn...

Documents