Targeting Ras and Rho GTPases as opportunities for cancer therapeutics

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The Ras and Rho GTPases contribute to the initiation and progression of cancer by subverting the normal regulation of specific intracellular signalling pathways. As a result, Ras and Rho play significant roles in the development of numerous aspects of the malignant phenotype by promoting cell cycle progression, resistance to apoptotic stimuli, neo-vascularisation and tumour cell motility, invasiveness and metastasis. With these GTPases contributing at so many levels, they are appealing targets for the development of cancer chemotherapeutic agents.

Introduction

The Ras superfamily of low molecular weight GTP-binding proteins includes the 21 members of the Rho subfamily — the most well-characterised being RhoA, Rac1 and Cdc42 — and the Ras subfamily, which consists of 18 members including H-Ras, N-Ras and K-Ras; proteins that are mutated in over 10% of human cancers [1]. These proteins alternate between an inactive GDP-bound state and an active GTP-bound state (Figure 1). This cycling between states allows the GTP-binding proteins to act as a molecular switch; normally in the ‘off’ position until information arriving from upstream signalling pathways promotes GDP–GTP exchange and a change to the ‘on’ position. Eventually, GTP is hydrolysed by the intrinsic GTPase activity of the protein and the switch is returned to the basal ‘off’ position. The switching between these two states is influenced by two classes of proteins, guanine nucleotide exchange factors (GEFs), which promote GDP–GTP exchange and activation of the protein, and GTPase activating proteins (GAPs) which promote GTP hydrolysis to GDP plus phosphate and consequent inactivation of the protein. When the protein is in the active GTP-bound conformation it interacts with effector proteins that propagate further signalling events that lead ultimately to the desired biological responses.

Ras and Rho GTPases promote cancer as a result of increased intensity and/or duration of signalling, which may be achieved by several means 2., 3.. In the case of Ras, direct mutation results in reduced GTP-hydrolysing activity and, consequently, the protein becomes locked in the ‘on’ state. Ras proteins can also become hyper-activated as a result of elevated expression or activating mutation of growth factor receptors that act upstream of Ras-GEFs or, in the specific case of neurofibromatosis, through the loss of the Ras-GAP NF1 [4]. For Rho GTPases, there are no known examples of activating mutations; however, there are numerous examples of overexpression in malignant tissue relative to normal tissue, and it is believed that the elevated Rho expression results in enhanced signalling [2]. Like Ras proteins, elevated expression or activating mutation of growth factor receptors may also result in Rho hyper-activation. In addition, specific examples of mutation of the Rho-GEFs LARG [5] and TIAM1 [6] have been described — by chromosomal rearrangement in acute myeloid leukaemia and by point mutation in renal cell carcinoma, respectively — that are believed to contribute to these forms of cancer.

Given the significant contributions that elevated Ras and Rho signalling make to tumour growth and progression, targeting these signalling pathways has become a major endeavour in the fight against cancer. One strategy has focused on inhibiting the activities of downstream effectors. However, another strategy is to directly target the Ras and Rho proteins themselves. In support of their anti-cancer therapeutic potential, numerous in vitro and in vivo experiments using neutralising antibodies, dominant negative proteins, bacterial toxins, antisense oligonucleotides or small interfering RNA have demonstrated that inhibition of Ras and Rho has negative effects on tumour cell proliferation and/or survival.

In this review, we discuss various strategies that have been used to target Ras and Rho proteins, and some novel approaches that may well prove to be powerful means for discovering effective therapeutic entities.

Section snippets

Inhibiting post-translational modifications

When newly synthesised, Ras and Rho GTPases are soluble cytosolic proteins that must undergo a series of post-translational modifications to enable them to associate with appropriate lipid membranes (Figure 2). These modifications occur at the protein carboxyl terminus, at a sequence called the ‘CAAX box’ (denoting the amino acid sequence Cys–aliphatic residue–aliphatic residue–X residue [usually Met, Ser, Gln or Leu]). The first step is the covalent attachment of either a 15-carbon farnesyl or

FTase and GGTase I inhibitors

Considerable effort has been made at developing FTase and GGTase I inhibitors to block Ras and Rho modification 7., 8.. These inhibitors principally either compete for binding to the CAAX substrate or to the isoprenoid pyrophosphate donor. In experimental systems, FTase inhibitors and GGTase I inhibitors have shown great promise as potential therapeutic agents and several CAAX-mimetic FTase inhibitors have progressed to clinical trials [9], including Zarnestra® (Tipifarnib/R115777; Phase III),

Statins

One advantage for the strategy of inhibiting farnesyl pyrophosphate (FPP) and GGPP production is that well-tolerated bioactive compounds, created for other indications, are already either in trials or even approved for routine clinical use. Inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, also know as statins, were originally designed to block a critical step in the biosynthesis of cholesterol (Figure 3), thereby reducing cholesterol levels in patients at risk of

Bisphosphonates

Another class of therapeutic compounds that may work by inhibiting Ras and Rho modifications are bisphosphonates, which are used clinically to combat bone resorption, one application being to treat patients with bone metastases. Simple bisphosphonates are metabolised into non-hydrolysable cytotoxic ATP analogues and inhibit osteolysis by blocking proliferation and/or inducing apoptosis of osteoclasts. Second generation nitrogen-containing bisphosphonates also inhibit FPP synthase [25] and

Endopeptidase inhibition

After isoprenylation by FTase or GGTase I, the three carboxyl terminal amino acids of the CAAX box of Ras and Rho proteins are digested by the endopeptidase Rce1 (Figure 2). Therefore, an alternative approach to blocking the cancer-promoting activities of Ras and Rho would be to inhibit Rce1. Deletion of the mouse Rce1 gene results in defective endoproteolysis and methylation, and aberrant subcellular localization of Ras proteins [35]. In addition, the Rce1 deletion reduced the growth rate of

Isoprenylcysteine carboxyl methyltransferase inhibitors

The final step in Ras and Rho post-translational modification is methylation of the isoprenylated carboxyl terminal Cys residue by ICMT (Figure 2c). Disruption of the ICMT gene results in mislocalisation of K-Ras [39], and chemical inhibition of ICMT also reduces the carboxyl methylation, localisation and/or function of H-Ras, K-Ras, N-Ras and RhoA 40., 41., 42.. Finally, transformation by oncogenic K-Ras, or by the Ras effector B-Raf, of immortalised mouse embryo fibroblasts deleted for ICMT

Novel strategies for inhibiting Ras and Rho

As an alternative to blocking the post-translational modifications of Ras and Rho proteins, it might be possible to inhibit their contributions to cancer by interfering with their capacity to achieve or maintain the active GTP-bound state. The first step towards a direct competitive inhibition is the generation of guanine-mimetic analogues [44•]. However, this approach might not work because of the high intracellular concentration of GTP and the high affinities of Ras and Rho proteins for both

Conclusions

Given the compelling in vitro and in vivo experimental data, in addition to the clinical evidence linking elevated Ras and Rho signalling to tumour growth and progression, targeting these signalling pathways has become a major endeavour in the fight against cancer. Therapeutic strategies have been largely of two types: focused on the discovery of small molecule inhibitors of Ras and Rho post-translational modifications, or inhibitors of the downstream effectors of these pathways. Although FTase

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported in part by funds from the National Cancer Institute (R01CA30721) to MFO and Cancer Research UK.

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