RAS: Target for Cancer Therapy
ABSTRACT
The RAS protein controls signaling pathway are major player in cell growth, its regulation and malignant transformation. Any activation in RAS brings alteration in upstream or downstream signaling component. Activating mutation in RAS is found in approximately 30% of human cancer. RAS plays essential role in tumor maintenance and is therefore an appropriate target for anticancer therapy. Among the anti-RAS strategies that are under evaluation in the clinic are pharmacologic inhibitors designed to prevent: (1) association with the plasma membrane (prenylation and post prenylation inhibitors). (2) Downstream signaling (kinase inhibitor), (3) upstream pathway (kinase inhibitor and monoclonal antibody), (4) Expression of RAS or other component of pathway (siRNA and antisense oligonucleotide). Several of these new therapeutic agents are showing promising result in the clinic and many more are on the way. Here, we review the current status and new hopes for targeting RAS as an anticancer drug.
THE RAS FAMILY
Any change in the cellular genome affecting the expression or function of genes controlling cell growth and differentiation are considered to be the main cause of cancer. Cancer research aims at identifying the genes that are altered in the various tumor types and elucidating the role of these genes in carcinogenesis. A family of genes that is frequently found to harbor a mutation in human tumors is that of the RAS genes. The elucidation of RAS synthesis, posttranslational modification, signaling pathway at the molecular level allows the design of rational, mechanism- based therapeutic agents
that can antagonize the activity of RAS (1).
RAS protein are guanine nucleotide binding protein that is key intermediate in signal transduction pathways that mediate proliferative and other types of signal largely from upstream of receptor tyrosine kinases to a downstream cascade of pro- tein kinases, which control wide variety of cellular processes, including growth, differentiation, apoptosis, cytoskeletal orga- nization and membrane trafficking (2, 3). After stimulation by various growth factors and cytokine, RAS activate several down-stream effectors, including the Raf-1, MAP kinase and Rac/Rho pathway. RAS is mutationally activated in 30% of all cancers, with pancreas (90%), colon (50%), thyroid (50%), lung (30%) and melanoma (25%) having the highest prevalence (4). In all these cancers, mutated RAS genes produce mutated protein that remain locked in an active state, thereby relaying uncontrolled proliferative signals (5, 6).
Mammalian genome encodes three RAS genes that give rise to four ubiquitously expressed gene products: H-RAS, N-RAS, K-RAS 4A and K-RAS 4B (K-RAS 4A and K-RAS 4B are splice variants of single gene). H-RAS gene (homologous to the oncogene of the Harvey murine sarcoma virus) the K-RAS- (homologous to oncogene of Kirsten murine sarcoma virus) and the N-RAS gene (which does not have a retroviral homolog and was first isolated from a neuroblastoma cell lines) (7). The RAS oncogenes encode 21-kd proteins, called p21RAS or RAS, that are localized to the inner surface of the plasma membrane in mammalian cells.
The normal function of RAS proteins require them to be posttranslational modified. The purpose of this is primarily to localize them to correct subcellular compartment, principally the inner face of plasma membrane. RAS proteins that are mis- localized at other sites in the cell are inactive, probably, because they cannot recruit their target enzymes. The fact that correct, posttranslational modification of RAS is required for its biologi- cal activity has made the enzyme involve in this processing very attractive target for therapeutic interventions.
Posttranslational modification of RAS
Posttranslational modification of RAS includes prenylation, proteolysis, carboxymethylation and palmitoylation (Fig. 1) (8).RAS GTPases contain a CAAX motif (C denotes cysteine, A represents any aliphatic amino acid, and X may be any amino acid) in their carboxy terminus. The CAAX motif serves as a substrate for a series of posttranslational modification that cre- ate lapidated hydrophobic domain, which mediates attachment to specific proteins 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 preny- lation (farnesylation and geranylgernyalation respectively) (9). After prenylation protein moves to the endoplasmic reticulum, where the three terminal amino-acid residue AAX are removed by the endoprotease named RAS converting enzyme 1(RCE1), and the carboxyl group of the terminal cysteine is methyl ester- ified by isoprenylcysteine carboxylmethyltransferase (ICMT). N-RAS, H-RAS and K-RAS 4A are then palmitoylated and transferred to the plasma membrane. K-RAS 4B does not require this modification and is sent directly to the plasma membrane. N-RAS, H-RAS and K-RAS 4A attach to the plasma membrane through their farnesyl and palmitoyl moieties, whereas K-RAS 4B attaches to the plasma membrane through its farnesyl moiety and a polybasic lysine rich sequence located near the terminal cysteine (10, 11).
RAS effector pathway
The activation state of RAS depends on whether they bound to GTP or GDP. In case of when they bound to GTP, they are able to initiate downstream signaling pathway, and when they bound to GDP, they are inactive and fail to start downstream pathway. GTP to GDP hydrolysis is controlled by GTPase acti- vating protein (GAPs), and GDP to GTP process is catalyzed by guanine nucleotide exchange factor (GEF) (12). It is the balance between these two proteins, which determines the activation sta- tus of RAS and its down stream pathways (Fig. 2).
GAPs act as a regulator which prevents prolonged activation of RAS by stimulating the intrinsic GTPase activity of RAS. This is an important aspect of regulation of RAS activity that is frequently deregulated in tumorogenesis. All oncogenic RAS mutations compromise its GTPase activity by preventing GAPs from stimulating the hydrolysis of GTP or by affecting GAP action thereby preventing RAS constitutively in the active GTP bound conformation. Besides RAS mutations, prolonged activa- tion of RAS in carcinogenesis may also occur from inactivation of RAS GAPs. There are 13 known RAS GAPs. Inactivation of these GAPs showed benign or malignant tumors (13, 14).
After RAS activation, first downstream effector molecule of RAS is the protein serine/threonin kinase Raf. There are three closely related Raf proteins, C-Raf, B-Raf and A-Raf. Raf phophorylates mitogen activated proteins kinase kinase 1 and 2 that are capable of activating the ERK1 and ERK2. Substrate for ERK1/2 includes cytosolic and nuclear proteins. ERK phos- phorylates ETS family transcription factors, such as ELK-1, that regulate the expression of c-fos and c-jun. This leads to activation of AP-1 transcription factor, which is made up of FOS-JUN heterodimer (15). As a result of stimulating these transcription regulators, key cell cycle regulatory proteins, such as D-type cy- clins, are expressed, which enables the cell to progress through G1 phase of the cell cycle (16).
In addition to this, RAS can interact directly with the phosphatidylinositol-3–kinases (PI3-Ks) as a result of it translo- cation to the membrane and conformational changes. PI3-Kinase leading to activation of the lipid kinase. PI3 kinase phospho- rylate phosphotidylinositol 4,5-bisphosphate to produce phos- phatidylinositol −3, 4, 5 triphosphate (17). PI3-Kinase controls the activity of a large number of downstream enzymes. Much attention has been paid to the activation of the kinase 3 phospho inositide-dependant protein kinase-1(PDK-1) and Akt kinase. PDK-1 is important for the activation of a large number of pro- tein kinase of the AGC family. Akt has a strong anti apoptotic effect by phosphorylating various targets and seems to be an im- portant part of the survival signal that is generated by RAS (18).
RAS-GTP also activates the G proteins Rac and Rho through an activation pathway often referred to as the cell morphology pathway (19, 20). Rho protein play an important role in reg- ulation of the actin cytoskeleton, affecting processes as mem- brane ruffling and formation of stress fiber, focal adhesion and filopodia (21). This may also lead to morphological changes that increase the invasive properties of transformed cells. So con- stant activation of RAS will cause extensive actin polymeriza- tion membrane ruffling and increased number of focal adhesion (17, 22).
In addition to this, RAS also stimulate Ral (RAS related protein) proteins. This further activates phospholipase D1 and CDC42. This pathway along with Akt pathway inhibit FORK- HEAD transcription factor of the Fox O family. This path- way is implicated in promoting cell cycle arrest through in- duction of the cyclin dependant kinase inhibitor KIP1 or P 27 and apoptosis through the expression of FAS and FAS ligand (23).
RAS–GTP can also activate phospholipase Cξ (PLCξ ) by di- rectly binding and then hydrolyze in to PIP2 and diacylglycerol, which facilitate to release calcium and activate protein kinase C (24, 25). Finally, RAS is a key downstream effector of the epidermal growth factor (EGFR), which is mutationally activated and/or overexpressed in a wide variety of human cancers (26). Another link between RAS and EGFR is ERK activation. ERK, which is downstream of RAS, also promotes upregulated expression of EGFR ligand that is transforming growth factor alpha (TGF- α), which promotes an autocrine growth loop critical for tumor development (27).
Role of RAS in cancer
The role of RAS super family GTPases in carcinogenesis is well established. Mutational activation of the RAS occurs in 30% of human cancer. Members of the RAS that have been reported to be mutated in different types of cancer include K-RAS in pancreatic cancer, non small cell lung cancer, colorectal can- cer and seminoma. N-RAS in melanoma, hepatocellular cancer, myelodysplastic syndrome, and acute myelogenous leukemia, and H-RAS in follicular and undifferentiated papillary thyroid cancer, bladder cancer and renal cell cancer (28). All these muta- tions stabilizes RAS in a constitutively active GTP bound con- formation (5). Moreover, several other human cancer harbors alteration in factors that lie upstream of RAS, leading to over expression or mutational activation of growth factor receptor tyrosine kinase, such as epidermal growth factor (EGFR and ERBB2), or downstream of RAS, such as mutation of B-Raf in melanomas (5, 28).
The majority of studies analyzing a role for effectors in RAS- mediated oncogenesis have been performed in cultured cells. Experiments in human cell lines ectopically expressing onco- genic RAS have shown that activities of Raf, PI3 kinase, and Ral-GEF effector pathways are required for tumor growth. Us- ing RNA interference, it was demonstrated that Ral-GEF-Ral pathway has major role to play in tumoriogenesis, invasion, and metastasis in pancreatic cancer (29).
Various animal studies have also proved the role of RAS effectors in cancer. Mice deficient for the effectors Ral–GDS showed impaired tumor initiation in dimethylbenzanthracine in- duced skin tumorogenesis, where H-RAS activation is frequent (30). Genetic ablation of other RAS effectors, like p110α and p110β, results in defective development of RAS-mediated cancer (31).
Activated mutant RAS in cells can promote several of the characteristics of malignant transformation. These include in- creased proliferation due to induction of cell cycle regulators such as cyclin D1, which leads to inactivation of the retinoblas- toma pathway, and suppression of cell cycle inhibitors, such as KIPI. In addition, cells become desensitized to apoptosis through Akt signaling and less well defined mechanism that are downstream of Raf (32). RAS effector pathway also lead to the induction of angiogenesis, mainly by means of ERK me- diated transcriptional upregulation of angiogenic factors and matrix metallo proteinases. It was shown that genetic inacti- vation of K-RAS or N-RAS leads to increased radiosensitiv- ity in human tumor cells and reintroduction of activated RAS gene resulted in increase radiation resistance. This study further suggest that activated RAS can contribute to intrinsic radiation resistance in human tumor cell line (33) RAS signaling path- way are also commonly activated in tumors in which growth factor receptor tyrosine kinase have been over expressed. The most common examples are EGFR and ERBB2, which are fre- quently activated by their over expression in many types of cancer, including breast, ovarian and stomach carcinoma. B- Raf is frequently activated by mutation in human tumors. In melanoma (70%) and colon carcinoma (15%) mutations in B- Raf occur in the kinase domain, all of which result in kinase activation. Targeting RAS and its effector pathway could, there- fore, have a potential impact on several different aspects of malignancy.
RAS AS A THERAPEUTIC TARGET
RAS is mutated in human cancer more frequently than any other oncogene. Accordingly, RAS has been a center of focus for cancer biologists seeking to develop rationally designed anti-cancer drug. For RAS as a therapeutic agent, we can target the post translational modifications, which determine their localization and mediate their attachment to membrane, and targeting the complex regulatory network that controls the activation of the RAS super family GTPases is another potential strategies for anticancer drug development. Elimination of RAS function by homologous gene recombination or antisense RNA has demonstrated that expression of activated RAS is necessary for maintaining the transformed phenotype of tumor cells (Fig. 3) (34, 35).
Farnesyl transfeRASe inhibitors (FTIs)
The covalent attachment of the farnesyl isoprenoid group to the H-RAS, K-RAS and N-RAS proteins is the first step in the carboxy terminal post translational modification of these proteins. Because this processing, which results in the stable localization of RAS to the plasma membrane, is essential for the biological activity of RAS. It was an obvious and early target for the design of new rational therapies against the RAS pathway.
Several strategies have been developed to inhibit the farnesy- lation of RAS, the most common being the design of compounds that mimic the carboxy terminal CAAX motif of RAS and com- pete for binding to farnesyltransfeRASe. The earliest of these compounds were peptides, which were subsequently modified to give better drug peptide (CAAX peptidomimetics) that compete with RAS CAAX motifs for FTAse or combination of both (36).
RAS: Target for Cancer Therapy 951
Number of highly effective FTIs has been identified, mostly through high-throughput screening of compound libraries and developed as potential cancer therapies. These were shown to efficiently inhibit the farnesylation of H-RAS in cell culture. The prototypical tricycilc FTase inhibitor SCH 44342 actively competes with the CAAX substrate. This agent inhibits human FTase and RAS processing in Cos-7 monkey kidney cells that transiently express H-RAS (37–40). Lovastatin or prenyltrans- ferase inhibitors showed reduced radiation survival in cells ex- pressing activated RAS (41, 42). In an early experiment, trans- geneic mouse expressing activated H-RAS under the MMTV promoter were treated with a peptidomimetic FTIs. These mice frequently develop mammary carcinomas, which showed very impressive reversal by the FTIs. Even after successful result in in vitro, these results have not been repeated in human patients. Tipifarnib and lonafarnib were tested in phase III trials. Phase III trials were negative for tipifarnib in pancreatic cancer and lonafarnib in non small cell lung cancer (36, 43). The root of the problem lies in the fact that, although H-RAS is exclusively modified by farnesyltransfeRASe, K-RAS and, to a lesser ex- tent, N-RAS can also be modified by gernylgeranyltransfeRASe (GGT). That is still able to support the biological activity of RAS. Geranylgeranylation of K-RAS and N-RAS becomes im- portant only when farnesylation is blocked. As the vast majority of RAS mutations in human are in K-RAS, followed by N-RAS, with very few in H-RAS. It is likely that inhibition of mutant RAS farnesylation is not responsible for any antitumour effects of FTIs. Attempting to inhibit the function of K-RAS and N- RAS by using FTIs and GGTIs together has failed because of the very high toxicity that is associated with this combination (44).
The FTIs that have been developed to target RAS also in- hibit the farnesylation of great many other proteins. Despite the rebranding of FTIs as signal-transduction, rather than RAS spe- cific, inhibitors, they are yet to show great promise in the clinic, particularly in phase II and Phase III trials on common can- cers. However, some more encouraging results have emerged from early trials on leukemia. It is certainly possible that they could eventually prove to be beneficial in certain tumor types or in combination with other agents, but it seems unlikely that they will live up to the early high expectations generated by the pre-clinical models.
Inhibition of the two post prenylated enzymes RCE1 and ICMT can be a good target for RAS inhibition. Post prenylation reaction are shared by both farnesylated and geranylgeranylated proteins. Therefore, even alternatively prenylated proteins are sensitive to inhibition of either the ICMT or the RCE1 enzyme. Rce1 and Icmt genes disruption studies was done in in vitro and in vivo condition. In these studies, RCE1 activity alone had very moderate effect in preventing RAS induced oncogenesis (45). Interestingly, targeted disruption of ICMT activity blocks oncogenic K-RAS induced transformation by decreasing methy- lation of K-RAS, H-RAS, and N-RAS (46). But inhibition of ICMT affects multiple pathways besides RAS. Specifically, sev- eral CAAX proteins, RAS superfamily GTPase, and non RAS superfamily GTPase that regulate cell division, cell prolifera-
tion, apoptosis, angiogenesis, and metastasis (47). It has been shown that inhibition of ICMT causes endothelial cell apopto- sis by attenuation of RAS GTPase methylation and activation of downstream anti apoptotic-signaling pathway. An important toxic effect of this ICMT inhibition is atherosclerotic vascular injury (48). FTIs and post prenylation inhibitors prevent RAS lo- calization to plasma membrane, but they can signal from other endoplasmic reticulum or Golgi. Therefore, inhibition of post translational modification of RAS can not stop RAS mediated signaling (49).
Kinase inhibitors targeting RAS effector pathways
In recent years, the pharmaceutical industry has become in- creasingly adept at developing effective inhibitors of protein kinases. The most impressive example of such inhibitors in the clinic has been imatinib. The inhibitor of BCR-ABL has pro- vided a great leap forward in the treatment of chronic myeloid leukemia. Inhibitors of kinases that are involved in several RAS signaling pathway have been under development for some time and now entered in clinical trials. These include inhibitors that target both upstream regulators of RAS, such as growth factor receptors, and downstream effectors, such as the components of the Raf -MAPK pathway.
In RAS MAPK pathway, Raf-MAPK pathway is activated in 30% of human tumors, as determined by the phosphorylated status of the MAPKs and ERK-1 and ERK-2. Sixty percent of cutaneous melanomas have an activating mutation in the cat- alytic domain of the serine threonine kinase B-raf. PD98059 and U0126 are fairly specific, non ATP competitive MEK in- hibitors that have been widely used for research purpose (50, 51). They effectively inhibit ERK activation and can inhibit pro- liferation, survival, and motility of some tumor cell lines under certain condition. MEK inhibitors have proved to be effective in inhibiting the growth of tumors in immunodeficient mice; for example, the related inhibitor PD 184352 has been used in large study of colon carcinomas. PD 184352 is also known as CI 1040 is a highly potent, orally active selective MEK inhibitor (52). Treatment with this inhibitors reduced tumor growth up to 80% in mice. CI-1040 showed promising results in phase I clin- ical trials in patients with advanced cancer (53). In contrast to this, phase II trails in patients with breast cancer, colorectal can- cer and pancreatic cancer showed negative results. PD 0325901 is a derivative of CI-1040 showed far better result then CI-1040. It has entered in Phase I/II trials with tumors expected to have activated ERK-MAPK signaling (54, 55).
Targeting the ERK-MAPK pathway that has reached clinical trials is BAY 43–9006. BAY 43–9006 received FDA approval in 2005 for the use in advanced renal carcinoma. It is orally active inhibitors of c-Raf that targets the ATP binding sites. It is also active against B-Raf, so it is likely to be effective at reversing ERK activation that is caused by RAS or B-Raf mutation, which is evident in most melanomas. This provide an important advan- tages over the exclusive targeting of c-Raf that is achieved by the antisense approach. In in vitro and mouse models representing a wide range of tumor cell types, Bay 43–9006 showed broad antitumor activity and was associated with reduced MRK and ERK activation (56). Bay 43–9006 is well tolerated at doses that results in inhibition of phorbol-ester induced ERK phosphoryla- tion in patients peripheral blood lymphocytes. As with CI-1040, the performance of this drug in phase II and phase III trial will be of considerable importance.
RAF 265 is another orally bioavailable Raf inhibitor cur- rently being investigated in phase I clinical trials in local or advanced or metastatic melanoma. It inhibits all three Raf iso- forms as well as mutated B-Raf. Its antiangiogenic activity through inhibition of VEGFR2 is comparable to Bay 43-9006 (http://www.clinicaltrials.gov/ct/show/NCT00304525).
Kinase inhibitors targeting pathways upstream of RAS
Even in tumors in which RAS in not mutationally activated, RAS might be well stimulated by aberrant activation of upstream signaling pathways. The components of the regulatory system for RAS that have proved most amenable to therapeutic inter- ventions are the growth factor receptor tyrosine kinase. In par- ticular, the EGFR and its close relative ERBB are able to stim- ulate RAS through GRB2 and SOS (GTPase). Targeting EGFR or ERBB2 might be effective in the types of tumors that over express them, resulting in failure to activate endogenous wild type RAS and several other downstream proliferative and anti- apoptotic pathway. Several approaches have been used to target the ERBB family, but by far, the most progress has been made in two areas: small molecule tyrosine kinase inhibitors and hu- manized antibodies against the receptor intercellular domains. At least six small molecule inhibitor of EGFR tyrosine kinase activity are now in clinical trials. These all effectively block the kinase activity of this receptor and might also inhibit other members of the ERBB family to some degree. The two drugs in this class that are at the most advanced stage of development are ZD 1839 from Astra Zeneca and OSI -774 (Tarceva) from OSI / Geneteck.
The other important class of therapeutic agent that is directed against ERBB family receptors are humanized monoclonal an- tibodies. These agents bind to the extracellular domain of the receptors, inhibit their activation by ligand and promote receptor internalization and downregulation. In addition, they are thought to induce a cytotoxic immune response against the tumor cells. The most advanced of this drug type against EGFR is a chimeric antibody IMC-C225, which is developed by Imclone Systems and Bristol Myers Squibb. Phase I and II trial of these drugs showed promising results. Generic name of this antibody is ce- tuximab, a chimeric IgG1 monoclonal antibody approved for EGFR-detectable metastatic colorectal cancer (57) and squa- mous cell cancer of the head and neck (58). After this two hu- manized (matazumab and nimotuzumab) and two fully human mAbs (panitumumab and zalutumumab) were developed. Phase I trial of matuzumab is well tolerated with RASh and diarrhea in colorectal, cervical, and esophageal cancers and in squamous cell cancer of the head and neck (59–62). Phase II trials include cervical and gastric cancer, and NSCLC. Nimotuzumab and Za- lutumumab have reached phase III trial for treatment of various solid tumors with special reference to non small lung cell can- cer (NSCLC). Panitumumab has already approved from FDA for CRC.
Antisense oligonucleotide(ASO) approach to the therapeutic targeting of the RAS pathways is to inhibit the expression of H-RAS and its downstream target c-RAF; this strategy has al- ready reached clinical trials. This inhibition has been achieved by using short antisense synthetic oliginucleotides that are spe- cific for sequences in the mRNAs for these proteins. On binding to cRNA, these oligonucleotide can inhibit protein production by several mechanism, one is to promote degradation of the mRNA by directing RNAse H to the RNA-DNA duplex; an- other is to interfere with translation. Pharmaceutical companies have been successful in developing several stabilized phospho- rothioate derivatives of oliginucleotides that effectively reduce the expression of H-RAS or c-Raf when added to the cells. But very high level of specificity of these agents exist, which could mean that they do not target the most important proteins in the tumors: for example, mutation of H-RAS is very rare in tumors so removing its expression is likely to be less effective than tar- geting K-RAS. ASO against K-RAS that are effective in cell culture have been developed but no one has been taken in to clinical trials. Similarly, c-RAF might not be the most impor- tant mediator of RAS induced ERK/MAPK activation, as this function is effectively provided by B-Raf. Another problem of effective delivery to tumors cell only not to normal cells. In ad- dition, several problems with nonspecific toxicity of ASO have been encountered.
CONCLUSION
The role of RAS family in carcinogenesis is now well estab- lished and well studied. Current pharmacological strategies are mainly focused on post translational modification mainly FTIs and GGTIs. Targeting the post prenylation enzymes ICMT and RCE 1 is another strategy. Because multiple pathways are in- volved in proliferation, invasion, and metastases of malignant cells and because combination therapy is more effective than are single-agent regimens, we should try combination of FTase inhibitor or RCE1 with anticancer agent. The issue of the degree of the therapeutic window that will be provided by drugs that target the RAS pathways is a crucial one. All cell use RAS sig- naling pathway to some extent, so there is danger that inhibitors will have severe effects on normal cells as well as tumor cells. Blocking RAS at translation level is a nonspecific mechanism. These non specific drugs are associated with lots of side ef- fects. Potent inhibition of RAS function through the expression of dominant negative mutants or microinjection of neutralizing antibodies has long been known to block normal cell prolifera- tion. Although, ultimately, each drug target has to be validated experimentally for its differential effect on tumor versus normal cells, there are conceptual reasons for believing that certain types of signaling inhibitors,ARS-853 in particular those that inhibit survival pathway, might selectively disadvantage tumor cells.