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The role of Raf kinases in malignant transformation

Walter Kolch, Ashwin Kotwaliwale, Keith Vass and Petra Janosch

Expert Reviews in Molecular Medicine © Cambridge University Press ISSN 1462-3994

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Structure and function of the Raf family
Raf-1 is ubiquitously expressed, whereas A-Raf is predominantly found in urogenital tissue and B-Raf shows the highest expression in neural tissue and testis (Ref. 2). The three Raf proteins share a common structure consisting of an N-terminal regulatory domain and a C-terminal kinase domain (Fig. 1). There are three conserved regions (CR1–3) of homology: in the regulatory domain, CR1 harbours a Ras-binding domain and a cysteine-rich domain, and CR2 is a serine/threonine-rich domain; CR3 is sited in the kinase domain and is required for Raf activity. The removal of the regulatory domain generates an oncogenic kinase, which in the case of Raf-1 is often referred to as BXB. All three Raf proteins also share common mechanisms of activation and downstream effectors. They are all activated by Ras and in turn activate the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway by phosphorylating the MAPK/ERK kinase (MEK), which activates MAPK/ERK (recently reviewed in Refs 3, 4). In this review, MAPK/ERK is referred to simply as ERK, and the MAPK/ERK pathway as the ERK pathway (or, when emphasising its constituents, the Ras–Raf–MEK–ERK pathway).

Raf regulation and signalling
Raf-1 is activated by a wide range of growth factors and hormones, and is a point of integration for numerous external signals. Hence, it is not surprising that the activation of Raf kinases is complex and still incompletely understood. Most of the stimuli that activate Raf-1 also activate Ras, a small G-protein whose gene was the first oncogene to be isolated from human cancer cells (recently reviewed in Refs 3, 4). The model presented in Figure 2 summarises the current consensus of the organisation and function of the Ras–Raf–MEK–ERK pathway using epidermal growth factor (EGF) as a paradigm (animated version of Fig. 2).

New functions of Raf kinases?
Although the Raf protein members share Ras as an upstream activator and MEK as a downstream substrate, they fulfil distinct functions, which is most convincingly demonstrated by the different phenotypes of c-raf-1-, A-raf- and B-raf-knockout mice (reviewed in Ref. 4). A-raf-deficient mice are born alive, but are growth retarded and present with intestinal and subtle neurological defects. B-raf-knockout mice have defects in neuroepithelial differentiation and in the maturation and maintenance of endothelial cells. These mice die in utero from massive vascular haemorrhage because their endothelial cells succumb to apoptosis. In addition, sensory and motoneurons explanted from these embryos completely fail to respond to neuronal survival factors and die rapidly. c-raf-1-deficient mice die between midgestation and shortly after birth, dependent on the genetic background (Refs 18, 19). These mice show growth retardation and general developmental defects, especially affecting the liver and the placenta. The most prominent phenotype is the propensity of c-raf-1^–/– cells to undergo apoptosis. But surprisingly, mice embryo fibroblasts (MEFs) derived from these embryos proliferated normally and ERK activation was also completely normal. The rescue of ERK regulation might reflect compensation for the defect by other Raf isoforms, although these isoforms could not save the cells from apoptosis.

These observations fit the phenotype of the c-raf-1^–/– mice quite well. With regard to differentiation, an activated Raf-1 mutant, but not an activated MEK mutant, could drive the differentiation of hippocampal cells (Ref. 24). Moreover, a mutant Raf-1 that could not activate MEK was still able to induce differentiation of PC12 cells (Ref. 25). These findings argue for a branching of the signalling pathway at the level of Raf-1. Thus, the ability of Raf-1 to induce neuronal cell differentiation seems to be executed not only by MEK and ERK, but also by novel pathway(s) that involve new substrates of Raf-1. Raf-1 often is perceived as a relay station that funnels input from various different sources into the ERK pathway, but these findings add a role of Raf-1 as a switchboard that distributes signals into different pathways. The identity of these pathways is still uncertain, but, as discussed below, several candidates are emerging.

Raf, the cell cycle and tumour suppressor genes
Checkpoint controls in the cell cycle
The cell-cycle machinery is required to drive cell proliferation, and it is no surprise that it is the target of both oncogenes and tumour suppressor genes. At the heart of the cell cycle are the cyclins, a family of proteins whose levels oscillate congruent with the cell cycle, and the cyclin-dependent kinases (CDKs), which need cyclins for activation (reviewed in Refs 26, 27). Different cyclin–CDK combinations are expressed in different phases of the cell cycle: cyclinD–CDK4/6 in G1; cyclinE–CDK2 later in G1 and at the G1–S-phase transition; cyclinA–CDK2 during S and G2 phase; and cyclinB–CDK1 at the G2–M-phase transition (Fig. 3). These cyclin–CDK complexes are needed to transit the various checkpoints that ensure the licensed and ordered progression through the cell cycle. If the cell fails to pass a checkpoint, cell-cycle arrest is enforced, typically through the expression of CDK inhibitors (CKIs), which inhibit the cyclin–CDK complexes.

Role of the Rb and p53 tumour suppressors
The first checkpoint to overcome in G1 is imposed by the retinoblastoma (Rb) tumour suppressor. Its name belies the ubiquitous expression and function of this protein as gatekeeper between mitogen signalling and the cell-cycle machine (reviewed in Ref. 28). Mitogens induce the expression of cyclinD, which associates with and activates CDK4/6. Activated cyclinD–CDK4/6 phosphorylates and initiates the inactivation of the Rb protein, causing it to release E2F transcription factors. E2F induces a variety of genes needed for cell-cycle progression and DNA synthesis, but is sequestered by Rb in quiescent cells. One of the genes induced by E2F is cyclin E, which recruits CDK2 to complete Rb inactivation in late G1. From now on, cell-cycle progression occurs independently of mitogens. This point where a cell commits itself to go through a complete cell cycle is called the restriction point and has its molecular correlate in Rb inactivation. The restriction point couples proliferation to external cues, and hence is frequently inactivated in tumours that grow autonomously. This can be achieved by deletion or mutation of Rb itself, overexpression of cyclinD, or mutation of p16^ink-4, the CKI responsible for inhibiting cyclinD–CDK4/6. Expression of p16^ink-4 can induce senescence and is regularly lost in cells adapted to grow permanently in culture, re-emphasising the importance of this checkpoint (reviewed in Ref. 26).

The Rb pathway also cross-regulates another major tumour suppressor, p53 (reviewed in Ref. 29) (Fig. 4). The p53 protein is short-lived, because an associated protein, Mdm2, induces its ubiquitination and degradation by the proteasome. In response to genetic insults, the p53 protein becomes stabilised and can cause cell-cycle arrest or apoptosis. Cell cycle arrest is in part due to the transcriptional activation by p53 of a CKI, p21^waf/cip, which inhibits cyclinE–CDK2 complexes. p53 causes apoptosis by the induction of pro-apoptotic proteins, such as Bax and PUMA, and other, as yet unknown, mechanisms (Ref. 30). The expression of Mdm2 is enhanced by p53 itself, constituting a self-regulatory feedback loop that keeps the levels of p53 and Mdm2 in balance. However, p53 stability is also regulated by other mechanisms, including a protein called ARF, which can prevent the interaction between Mdm2 and p53, and increase the p53 levels as a consequence. ARF transcription is induced by E2F released from Rb. Thus, the inactivation of Rb not only allows the cell to pass the restriction point, but also arms the G1–S phase checkpoint controls by favouring p53 stabilisation. If this exceeds a certain threshold, p53 can induce p21^waf/cip, which, via inhibition of CDK2, interferes with Rb inactivation and can halt the cell cycle at the G1–S-phase boundary. Such mutual feedback controls are encountered quite frequently in growth control. Curiously, ARF is encoded by the same genetic locus as p16^ink-4, but is transcribed from a different promoter and uses an alternative reading frame (which is where the acronym ARF comes from). Therefore, the loss of this locus eliminates two tumour suppressor genes and might compromise both the Rb and p53 pathway.

Role of the ERK pathway
Raf impinges on cell-cycle control circuitry on a number of levels (reviewed in Refs 27, 29). CyclinD induction requires multiple signals provided by the ERK and the phosphoinositide 3-kinase (PI-3K) pathways. Furthermore, ERK can phosphorylate and induce the degradation of p27^kip, another CKI that inhibits G1 cyclin–CDKs. ERK can also induce the transcription of both Mdm2 and ARF. The activation of these two functionally opposing genes might seem counterintuitive, but this sensitises the system to react to other signals that converge on the same regulatory circuit (Fig. 4). For instance, DNA replication also incurs an increased risk of DNA damage, which would trigger p53 stabilisation. In this scenario, the ERK signal would enhance p53 stabilisation via ARF and the damage response. By contrast, when ARF is lost, ERK increases p53 degradation and helps to override p53 checkpoints.

Apart from cyclinD, the best-documented cell-cycle target for the ERK pathway is p21^waf/cip (reviewed in Refs 27, 29). This CKI inhibits cyclinE–CDK2 and also (at high concentrations) cyclinD–CDK4 complexes. However, at low concentrations, it also promotes the assembly of cyclinD–CDK4. Work with artificial Raf-1 constructs, whose activity can be regulated by drugs in a dose-dependent fashion, has shown that high-intensity activation of ERK results in a cell-cycle block that is caused by the expression of p21^waf/cip and, in some cells, p27^kip. In primary cells, constitutive high-intensity Ras or Raf-1 signalling results in the induction of senescence, and it has been argued that this provides intrinsic protection against transformation caused by Ras mutations. In primary murine keratinocytes, activation of Raf-1 induces a p21^waf/cip cell-cycle block and differentiation. In keratinocytes lacking p53 or p21^waf/cip, Raf-1 induces differentiation markers as well as proliferation (Ref. 33). In some cell types, but not others, p21^waf/cip induction can be mediated by p53. The exact molecular mechanisms are unclear, but an important corollary of these findings is that the strength of a signal can determine specificity in terms of the biological outcome. Put into the context of tumour development, on the one hand this adds another level of complexity, but on the other hand it might open new therapeutic opportunities. For instance, small-cell lung cancer (SCLC) cell lines are very susceptible to Raf-1-triggered growth inhibition that is mediated by p21^waf/cip and p27^kip. Indeed, it has been suggested that SCLC could be treated by Raf-1 stimulation rather than by its inhibition (Ref. 34).

Transformation by the v- oncogene
v-Raf and oncogenesis
The v-Raf oncogene product is a truncated version of murine Raf-1, where the regulatory domain is deleted and the kinase domain is fused to retroviral Gag sequences. The v-raf transducing retrovirus, 3611-MSV, was isolated from a mouse with lung carcinoma and peritoneal tumours (Ref. 35). This fact has inspired many of the later investigations to look for a connection between Raf and lung tumours. However, although the v-raf oncogene can transform a variety of established cell lines in vitro, including epithelial cells, its main target is fibroblasts, and the predominant lesions observed in mice are fibrosarcomas and erythroid hyperplasia (Ref. 36).

Oncogene co-operation
The experiments described above involved the constitutive expression of exogenous genes. By their very nature, such experiments can unveil the full range of what these genes are capable of doing, but not necessarily what they do under physiological conditions. Despite these limitations, such experiments reinforced the concept of ‘collaborating oncogenes’. Most established cell lines are efficiently transformed by a single oncogene. However, the ability of established cell lines to multiply indefinitely in cell culture indicates that they at least have overcome the crucial restriction of mortality and probably have acquired other genetic changes favourable for unrestricted proliferation. A seminal finding was that at least two oncogenes, such as ras and myc, are needed to transform primary rodent cells (Ref. 40). Human cells, which are much more resilient to transformation, require two oncogenes in addition to an immortalising event, such as telomerase expression (Ref. 41). In hindsight, the early studies demonstrated that oncogenic transformation is a co-operative process that has to overcome multiple restrictions.

Unravelling the molecular basis of oncogene co-operation has lagged far behind its conceptual realisation. But, with a more-detailed knowledge about the signalling pathways that are influenced by oncogenes, this question is rapidly becoming a focus again. For instance, it has been shown that high-intensity Raf signalling induces cell-cycle inhibitors, such as p21^waf/cip and p21^kip, causing cell-cycle arrest (reviewed in Refs 27 and 29). Myc has been shown to counteract these cell-cycle inhibitors, in particular p27^kip (Ref. 42), and, in addition, to induce the transcription of the CDK4 gene (Ref. 43). Thus, in the presence of activated Myc, the safeguard against high Raf activity fails, and the deranged cell can proliferate. ERK can contribute to this process by phosphorylating and stabilising the Myc protein (Ref. 44). By contrast, Myc has been shown to induce apoptosis in the absence of survival signals (Ref. 45). Consequently, cells with activated Myc are doomed unless rescued by a survival signal. This signal can be provided by the AKT pathway (Ref. 46), a major anti-apoptotic signalling network, or the Raf pathway (Ref. 20), probably depending on the cell type. Again, this would allow unlicensed proliferation. In fact, an elegant study with cells in which one allele of c-myc was removed by somatic gene knockout demonstrated that even the reduction of endogenous Myc expression by 50% resulted in a pronounced resistance to ras and raf transformation (Ref. 47). Thus, attractive and testable hypotheses are emerging that have apoptosis protection and cell-cycle progression as common denominators for oncogene co-operation.

Raf in tumours
The ERK pathway features the most complete ensemble of proto-oncogenes performing in one signal transduction pathway. It is stimulated by all classical growth factor ligand and receptor oncogenes (proto-oncogenes) – for example, by platelet-derived growth factor B (PDGF-B; v-Sis), EGFR/Erb-b1 (v-Erb), Erb-2/Her-2 (v-Neu) and Met (v-Met). The pathway contains Ras and Raf as intracellular transducers, and Fos and Myc as nuclear effectors (Refs 10, 11). Thus, it is no surprise that this pathway is hyperactive in about 30% of all human cancers (Ref. 48). Although this number is impressive, the caveat applies that many results were obtained from tumour cell lines rather than from primary tumour tissue. The most frequent lesions seem to be ras mutations, myc amplification, and EGF and Neu receptor overexpression. ras gene mutations occur in up to 90% of pancreatic carcinomas, 50% of colon carcinomas, 40% of lung carcinomas, 20% of leukaemias and at a lower rate in various other tumours. Her-2 overexpression is common in breast cancer and colon cancer. myc amplifications are found in a variety of tumours, in particular gliomas and colon cancer (reviewed in Ref. 49).

The ERK pathway and cancer: clinical implications
Hanahan and Weinberg recently summarised the hallmarks of a cancer cell in a review (Ref. 54). The ERK pathway can contribute to any single one of these criteria (Table 1); it can induce immortalisation, growth-factor-independent growth, insensitivity to growth-inhibitory signals, ability to invade and metastasise, ability to secure nutrients by stimulating angiogenesis, and avoidance of apoptosis. An altered response to chemotherapeutic drugs could also be added to this list. The potential of the ERK pathway to affect these aspects of the cancer cell might not be realised in full in all types of cells or tumours, and will depend on the other genetic lesions and the microenvironment of the tumour. However, the high frequency of activation of this pathway indicates that some aspects are essential for cancers to develop.

Immortalisation
DNA polymerase is unable to replicate the ends of linear DNA. Thus, the ends of chromosomes, the telomeres, are shortened during each cell cycle; it has been suggested that this is the internal clock that limits the number of cell divisions that primary cells can undergo. However, the majority of established cell lines and tumour cells escape this problem by expressing telomerase, an enzyme that replenishes telomeres (reviewed in Refs 55, 56). The induction of telomerase expression appears to be a key event in immortalisation. Little is known about the pathways that regulate telomerase expression, but a recent paper suggests that it is induced by the ERK pathway (Ref. 57).

Growth-factor-independent growth
Oncogenes are ‘internal mitogens’ that can substitute for growth factors and uncouple their host cells from the dependence on external cues to proliferate. raf oncogenes accomplish this by the usurpation of signalling pathways that stimulate cell-cycle progression, as discussed above.

Insensitivity to growth-inhibitory signals
The best-understood growth-inhibitory mechanisms emanate from the p53 and Rb tumour suppressor genes and involve CKIs ‘braking’ the cell cycle. The intricate interactions between p53, Rb and the ERK pathway have been outlined above. It is important to stress that the regulation of CKIs is dependent on the strength of Raf signalling. High-intensity signalling induces p21^waf/cip and/or p27^kip expression, leading to a G1 arrest. However, lower signal strength avoids the transcriptional induction of these CKIs and even promotes their degradation, possibly triggered by ERK phosphorylating p27^kip. The induction of these CKIs by Raf can also be inhibited by Rho signalling, which might explain the strong synergism between the Rho and ERK pathways in transformation assays (Refs 27, 58). Rho is a small G-protein belonging to the Ras superfamily that plays a major role in mitogen-controlled remodelling of the cytoskeleton (Ref. 58). It has been estimated that a cell has to accumulate three to six genetic aberrations to become a cancer cell (Ref. 59). Because of the high selection pressure a cancer cell has to overcome, it can be safely assumed that the activation of synergistic pathways is favoured. This might provide a window of opportunity for therapy, because a modest, and therefore tolerable, inhibition of several pathways might abrogate their synergistic effects, and thus harm the cancer cell while leaving normal cells unscathed.

Ability to invade and metastasise
Most cancer patients do not die from the primary tumour, but from the metastases (Ref. 60). The ability to invade adjacent tissues and eventually spread into remote parts of the body epitomises the malignancy of a cancer cell. The mechanisms underlying these traits are incompletely understood, but proteases that degrade the extracellular matrix and basal membrane play a key role. Several studies have shown that the ERK pathway induces proteases known to be involved in invasion, such as urokinase-plasminogen activator, matrix metalloproteases 1, 2 and 9, and cathepsinL (Ref. 61). About 50% of metastatic tumours feature activating Ras mutations. Using NIH 3T3 fibroblasts transfected with Ras mutants that selectively activate different downstream effectors, it was shown that all Ras mutants formed tumours in mice, but only a Ras mutant able to activate Raf supported metastasis. Cells re-isolated from the tumours and metastases exhibited higher levels of active ERK than did the cells originally injected, suggesting that a selection for higher ERK activity had occurred in vivo (Ref. 62).

Stimulation of angiogenesis
A tumour exceeding 2 mm in diameter requires a blood supply to grow further (reviewed in Ref. 64). This means that virtually all clinically relevant tumours have succeeded in attracting blood vessels. Angiogenesis is stimulated by growth factors produced by tumour cells, most notably vascular endothelial growth factor (VEGF), as well as by changes in the extracellular stroma that prompts blood vessels to sprout into the tumour mass. Most tumour centres are hypoxic, and hypoxia is the most potent angiogenic stimulus, inducing a burst of VEGF expression. The activation of VEGF expression in response to hypoxia has been reported to involve a signalling pathway comprising Src and Raf-1 (Ref. 65), but other data suggested that Raf-1 was not involved and that a PI-3K-mediated pathway was responsible (Ref. 66). Hypoxia increases VEGF expression by both transcriptional induction and post-transcriptional mechanisms, and the contribution of different signals to these processes might vary between cell types. However, the activation of the VEGF promoter by the ERK pathway is well documented (reviewed in Ref. 67). In addition, this pathway also stimulates the expression of other factors that promote angiogenesis (Ref. 67), such as tissue factor (TF). By triggering blood coagulation, TF helps to create a stroma permissive for capillary sprouting. Angiogenesis is now the focus of anti-cancer targets and the inhibition of its inducers appears an attractive way to fight tumour growth.

Avoidance of apoptosis
Data from raf-knockout mice testify to the important role of Raf kinases in ensuring the survival of different cell types. Notably, these experiments point to an ERK-independent role of Raf-1 (Refs 18, 19). Several mechanisms have been proposed to explain how Raf can prevent apoptosis (reviewed in Refs 68, 69): inactivation of the pro-apoptotic protein BAD; activation of the NF-kB survival pathway; induction of caspase inhibitors; and inhibition of ASK-1 (Ref. 23). This list will probably grow further, suggesting that Raf can interfere with apoptosis at various levels and that this might be a prime target that could be exploited for tumour therapy. ERK also can prevent apoptosis via its substrate Rsk, which mediates BAD inactivation and activation of the protective transcription factor CREB (Ref. 70).

Radiation resistance and multidrug resistance
Several studies have connected Raf expression with radiation resistance (reviewed in Ref. 71). Most showed that Raf is protective, but some also found an inverse correlation. Although, the reasons for these discrepancies are unclear, these studies used different cell lines and looked at Raf-1 protein expression levels and not activity levels, which might be misleading. The acquisition of drug resistance is a common problem in tumour chemotherapy, and is often related to the expression of transporter proteins, such as multidrug resistance 1 (MDR1), which expedite drug clearance from the cancer cells. MDR1 expression is enhanced by Raf-1 (Ref. 72) and might help tumours to fend off a chemotherapeutic attack.

Clinical applications
Currently, three approaches are under investigation to inhibit the Raf–MEK–ERK pathway. The first is the downregulation of Raf-1 protein levels using the expression of antisense RNA. ISIS 5132 is an antisense oligonucleotide directed to the 3' untranslated region of the raf-1 mRNA, which could inhibit the growth of human tumour cell lines in vitro and in xenograft assays. In clinical Phase I trials, ISIS 5132 reduced Raf-1 mRNA levels in the blood cells of treated patients (Ref. 73). The side effects, mainly fever and fatigue, were not dose limiting. Phase II trials have ensued and the results are to be expected soon. In addition, Raf-1 downregulation by antisense RNA sensitises tumours against ionising radiation, leading to enhanced regression.

Research in progress and outstanding research questions
The involvement of Raf in so many different aspects of the cancer cell phenotype makes it a plausible target for tumour therapy. The attractiveness of this target lies in the fact that a single inhibitor could block several cancer-promoting elements at once, and thereby cause the collapse of synergistic processes that sustain the cancer cell without the necessity to inhibit any single element to completion. This could open a therapeutic window where high efficacy is achievable with few side effects. Further results of the ongoing clinical trials are eagerly awaited. These data will also help to test the hypothesis of a therapeutic window by revealing which processes are affected in cancer cells in response to Raf inhibition.

Results from the raf-knockout mice have substantiated the idea that Raf kinases have other targets in addition to MEK, and have inspired the quest to find them. Their identification will contribute to explaining the biological differences between the Raf isoforms and will broaden the scope of potential targets that can be exploited for tumour therapy and diagnosis.

Clearly, one would like to have reagents available that allow evaluation of the activity status of Raf kinases in clinical specimens. The development of such reagents is hampered by the fact that, despite 15 years of worldwide research, Raf activation is still incompletely understood. However, new concepts are being explored that hopefully will break the deadlock. In particular, the role of protein–protein associations in the adjustment of signalling strength and duration is beginning to be appreciated. These dynamic parameters can determine the biological outcome and, as the intricacies of Raf signalling unfold, we might learn how to selectively manipulate the diverse biological functions of this network.

Acknowledgements and funding
We thank Drs Vaughn Cleghon, Margaret Frame and Paul Harrison (The Beatson Institute for Cancer Research, Glasgow, UK) for critical reading and comments. The authors’ work is supported by grants from Cancer Research UK, the Association for International Cancer Research and the European Union.

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