Review

Oncogene (2011) 30, 3477–3488; doi:10.1038/onc.2011.160; published online 16 May 2011

Raf kinases in cancer–roles and therapeutic opportunities

G Maurer1, B Tarkowski1 and M Baccarini1

1Max F Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria

Correspondence: Professor M Baccarini, Max F Perutz Laboratories, Center for Molecular Biology, University of Vienna, Dr Bohr Gasse 9, Vienna A-1030, Austria. E-mail: manuela.baccarini@univie.ac.at

Received 15 February 2011; Revised 3 April 2011; Accepted 3 April 2011; Published online 16 May 2011.

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Abstract

Raf are conserved, ubiquitous serine/protein kinases discovered as the cellular elements hijacked by transforming retroviruses. The three mammalian RAF proteins (A, B and CRAF) can be activated by the human oncogene RAS, downstream from which they exert both kinase-dependent and kinase-independent, tumor-promoting functions. The kinase-dependent functions are mediated chiefly by the MEK/ERK pathway, whose activation is associated with proliferation in a broad range of human tumors. Almost 10 years ago, activating BRAF mutations were discovered in a subset of human tumors, and in the past year treatment with small-molecule RAF inhibitors has yielded unprecedented response rates in melanoma patients. Thus, Raf qualifies as an excellent molecular target for anticancer therapy. This review focuses on the role of BRAF and CRAF in different aspects of carcinogenesis, on the success of molecular therapies targeting Raf and the challenges they present.

Keywords:

Raf; Ras; ERK pathway; hallmarks of cancer; kinase inhibitors

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Raf proteins and their effectors

The first member of the Raf family, C-Raf-1 (also known as Raf-1), was identified in a oncogene capture experiment in which its catalytic domain was found fused to the retroviral Gag protein, resulting in the constitutive activation of the serine/threonine kinase activity of C-Raf (Rapp et al., 1983; Moelling et al., 1984); 4 years later, B-Raf was discovered in a similar experiment (Marx et al., 1988). Within 10 years of its discovery, C-Raf was identified both as an interaction partner and activator of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), the dual-specificity kinase responsible for activation of extracellular signal-regulated kinase (ERK), and an effector of Ras, which was reported to recruit C-Raf to the membrane and stimulate its activation by mechanisms, which, roughly 18 years later, are still incompletely understood. Both the history and the regulation of Raf have been reviewed recently (Wellbrock et al., 2004; Niault and Baccarini, 2010). Suffice it to say here that a wealth of studies have led to a widely accepted model in which Raf activation primarily consists in the relief of the inhibition imposed on the Raf catalytic domain by an N-terminal regulatory domain, featuring both a Ras-binding domain and a cysteine-rich domain responsible for interaction with the kinase domain and for Raf autoinhibition (Figure 1a). This basic mechanism applies to all three Raf proteins (A-Raf, B-Raf and C-Raf), although both A-Raf and C-Raf need additional steps, such as phosphorylation of activating residues and dephosphorylation of negative regulatory residues, to reach maximal activation. Thus, B-Raf is the family member most easily activated by Ras (Wellbrock et al., 2004; Niault and Baccarini, 2010). In addition, the basal kinase activity of B-Raf is higher than that of C-Raf and, likely, A-Raf (Pritchard et al., 1995; Emuss et al., 2005). This provides a potential rationale for the frequent mutational activation of BRAF (for example by the prominent BRAFV600E mutation; (Davies et al., 2002)), but not CRAF or ARAF, observed in human tumors. A major advance of the past few years was the discovery that Raf kinases can homo- and heterodimerize (Garnett et al., 2005; Rushworth et al., 2006), and that, in fact, the structure of an active Raf kinase is that of a side-to-side dimer in which only one partner must have catalytic activity (Rajakulendran et al., 2009). Dimerization is enhanced by Ras (Weber et al., 2001) and is subject to negative feedback regulation by ERK (Rushworth et al., 2006; Ritt et al., 2010) (Figure 1b).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The structure of Raf and interactions within the Ras/Raf/MEK/ERK pathway. (a) A schematic view of Raf. All three Raf proteins consist of a regulatory and a kinase domain. In quiescent cells, the interaction between these two domains inhibits catalytic activity. The cysteine-rich domain (CRD) is necessary for this inhibition (indicated by the red blunt arrow), which is relieved by the binding of Ras to the Ras-binding domain (RBD). A-Raf and C-Raf need additional steps for full-fledged activation. (b) Regulation of the ERK pathway. Raf kinases can be activated by homo- and heterodimerization. Dimerization is induced by Ras and can occur in different combinations including not only the Raf kinases but also the pseudokinase KSR. Phosphorylation of Raf residues by activated ERK counteracts dimerization, allowing negative feedback control of the pathway (red blunt arrow). RKIP is an inhibitory protein whose expression is often lost in cancer and which can regulate pathway output at the level of Raf as well as MEK activation. ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; RKIP, Raf kinase-inhibitory protein.

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In the Raf/Mek/Erk pathway, dimerization can be used to exert tight temporal control of the signal, in cases in which one dimer subunit is more prone to negative feedback regulation than the other (C-Raf<B-Raf (Dougherty et al., 2005; Ritt et al., 2010) and Mek1<Mek2 (Catalanotti et al., 2009); reviewed by Wimmer and Baccarini (2010)). A further level of control is exerted by the interaction with inhibitory proteins (Kolch, 2005). In the context of cancer, the most relevant of these is the Raf kinase-inhibitory protein, RKIP (Zeng et al., 2008) (Figure 1b). In addition, a high degree of spatial control is provided by the interaction of pathway components with scaffolds that direct them to distinct subcellular compartments (Kolch, 2005; McKay and Morrison, 2007).

Overexpression of full-length Raf or the truncated catalytic domain leads to the activation of the ERK pathway and increases proliferation in cultured cells and in vivo. Thus, MEK/ERK is undoubtedly a target of activated Raf in tumorigenesis. In the case of C-Raf, other targets potentially contributing to cell transformation have been proposed, such as the nuclear factor-κB pathway (Baumann et al., 2000), Rb (Kinkade et al., 2008) and BAD (Polzien et al., 2009), all reviewed by Niault and Baccarini (2010). In addition, C-Raf can inhibit apoptosis by binding to, and inhibiting, the stress-induced kinase ASK-1 (Chen et al., 2001) and the homolog of Drosophila's Hippo, the MST-2 kinase (O’Neill et al., 2004; Matallanas et al., 2007); and finally, C-Raf interferes, by direct binding, with the activity of the cytoskeleton-based Rho effector Rok-α (also known as ROCK2), resulting in defects in cell migration, apoptosis and differentiation (Ehrenreiter et al., 2005, 2009; Piazzolla et al., 2005) (Figure 2).

Figure 2.
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Functions of Raf. Gene ablation experiments have shown that B-Raf is essential for MEK–ERK activation in most systems. A-Raf and C-Raf heterodimerize with B-Raf and can participate in ERK activation (double-headed arrows). It is unclear whether A-Raf has functions outside the MEK/ERK pathway; C-Raf, however, can promote nuclear factor-κB activation and can inhibit (blunt-headed arrow) signal transducers involved in motility (Rok-α), apoptosis (ASK-1 and MST-2), proliferation and angiogenesis (Rb). ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.

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Raf and the hallmarks of cancer

Six hallmarks of cancer, describing the acquired cell-autonomous capabilities of a cancer cell, were outlined in a legendary review by Hanahan and Weinberg (2000) more than 10 years ago. More recently, the list has been revised to include other features of cancer cells related to their interaction with the environment, such as avoidance of immunosurveillance (Dunn et al., 2004; Smyth et al., 2006; Zitvogel et al., 2006) and the stress phenotypes of cancer (Luo et al., 2009), as well as genomic instability (Negrini et al., 2010).

In the following section, we will highlight the contribution of Raf and of the Raf-dependent pathways to the hallmarks and states of cancer (Figure 3).

Figure 3.
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Contribution of Raf to the hallmarks and phenotypes of cancer. The hallmarks of cancer are depicted in black and the stress phenotypes associated with cancer in dark gray (adapted from Negrini et al. (2010)). The contributions of various Raf isoforms to each hallmark/phenotype and the downstream pathway mediating them are indicated. Raf* represents activated B-Raf or C-Raf; the red arrows indicate kinase-dependent functions of Raf; the red blunt arrows represent kinase-dependent inhibition of downstream pathways and the green blunt arrows represent kinase-independent inhibition processes. For clarity, only the hallmarks/phenotypes in which Raf has been implicated are depicted; mitotic stress and proteotoxic stress have been omitted.

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Genomic instability is a feature of almost all human cancers (Negrini et al., 2010). In hereditary cancers, germline mutations in caretaker genes (DNA-repair genes and mitotic checkpoint genes) promote tumor development by increasing the mutational rate and leading to chromosomal instability. In sporadic cancer, the caretaker genes are not mutational targets, and chromosomal instability is rather a consequence of the DNA replication stress induced by the activation of oncogenes, notably Ras.

Germline Raf mutations do not appear to contribute to cancer; instead, mutation in both BRAF and CRAF have been found in human genetic syndromes defined as ‘Rasopathies’ because they are caused by mutations in components of the Ras/ERK pathway (Tidyman and Rauen, 2009). The observed mutations cause activation of BRAF or CRAF, but the two kinases are not interchangeable in this context: mutations in the regulatory domain of CRAF are associated with the development of Noonan Syndrome, also caused by mutations in SOS1 and KRAS, and Leopard syndrome. By contrast, BRAF mutations are associated with Cardio-facio-cutaneous syndrome (CFC), also initiated by activating mutations of MEK (reviewed by Tidyman and Rauen, 2009).

In addition to the mutations identified in Noonan and Leopard syndrome, two weakly transforming germline mutations in the kinase domain of CRAF have been described in patients with therapy-related acute myeloid leukemia, which arises from concomitant loss of the Raf-inhibitory protein, RKIP (Zebisch et al., 2006, 2009). In general, the frequency of mutational changes of CRAF in human cancers is low (1%; http://www.sanger.ac.uk/genetics/CGP/cosmic). However, amplification of CRAF and other members of the ERK pathway have been observed during hormone escape in androgen-independent prostate cancer (Edwards et al., 2003), and both CRAF amplifications (4%) and deletions (2.2%) are strongly associated with tumor progression and an overall poorer survival in bladder cancer (Simon et al., 2001). Similarly, activation of the ERK pathway owing to BRAF gene duplication or mutation has emerged as a mechanism in the pathogenesis of low-grade astrocytomas (Pfister et al., 2008). Besides alterations in copy number, chromosomal translocations involving CRAF are found in certain human cancer sub-types such as stomach cancer (Shimizu et al., 1986) and pilocytic astrocytomas (Jones et al., 2009). The latter tumors also harbor chromosomal translocations involving BRAF activation (Jones et al., 2008); although seldom, such alterations have also been observed in nevi (Dessars et al., 2007) and radiation-induced thyroid cancer (Ciampi et al., 2005). In all cases, the alterations lead to constitutive RAF activation through loss of the autoinhibitory N-terminal domain.

More recently, chromosomal translocations yielding gene fusion transcripts containing the C-terminal kinase domain of CRAF or BRAF have been identified at low frequency in prostate cancer, gastric cancer and melanoma. Both fusion proteins promoted MEK/ERK-dependent cell proliferation, migration and anchorage-independent growth in human prostate cells, but whereas expression of the BRAF fusion protein in NIH 3T3 cells induced tumor formation in nude mice, the CRAF fusion protein failed to do so (Palanisamy et al., 2010), implying crucial signaling differences between the BRAF and the CRAF fusion proteins. Interestingly, prostate cancer also harbored the reciprocal CRAF fusion, containing the CRAF-regulatory domain; this protein, however, has not been investigated in detail.

Besides being the target of chromosomal rearrangements, RAF has also been implicated in the induction of genomic instability. Two types of mutations have been associated with increased genomic instability thus far: BRAFV600E, the activating mutation observed with the highest frequency in melanoma and other cancers, induces genomic instability in a thyroid cell line (Mitsutake et al., 2005); in addition, expression of B-RafD594A, a transforming B-Raf mutant with impaired MEK kinase activity, can promote aneuploidy in a C-Raf-dependent, MEK-independent manner in mouse splenocytes and embryonic fibroblasts (Kamata et al., 2010).

C-Raf, too, has been implicated in promoting genomic instability, albeit indirectly. A balance between C-Raf and RKIP, the Raf inhibitor often lost in breast, prostate and melanoma tumors (Granovsky and Rosner, 2008), is necessary to guarantee fidelity of chromosome segregation. Loss of RKIP or C-Raf overexpression lowers the activity of the Aurora-B kinase, allowing cells to bypass the spindle assembly checkpoint and potentially resulting in genomic instability (Eves et al., 2006).

Self-sufficiency in proliferative signals is a crucial step on the road to transformation. In healthy tissues, soluble mitogenic growth factors are produced by one cell type and stimulate the proliferation of another. Many cancer cells are able to produce and respond to their own growth factors, resulting in a positive feedback signaling loop (autocrine stimulation), which makes them independent from their tissue environment. These proliferative signals include production of growth factors, overexpression/constitutive activation of growth factor receptors and alterations in downstream signaling cascades. The first two changes are likely to activate the Raf pathway, although the degree to which the resulting proliferation may depend on it may vary. In the context of alterations in signaling components, apart from mutational activation of RAF itself, the most direct connection is that between RAF and the members of the RAS gene family, which are activated in 33% of human cancers, particularly in those of epithelial origin (http://www.sanger.ac.uk/genetics/CGP/cosmic).

Several RAF mutations driving the proliferation of cancer cells have been described. The most frequent BRAF mutation, BRAFV600E, causes constitutive activation of the kinase as well as insensitivity to negative feedback mechanisms (Davies et al., 2002; Pratilas et al., 2009). In addition, less frequent BRAF mutations have been described that can stimulate the MEK/ERK pathway by activating wild-type CRAF in the context of a heterodimer (Davies et al., 2002; Garnett et al., 2005; Kamata et al., 2010). Mutations in CRAF itself are extremely rare, but overexpression has been reported at high frequency in subsets of human cancers, including hepatocellular carcinoma and squamous cell carcinoma of the head and neck (Riva et al., 1995; Hwang et al., 2004). CRAF overexpression is regarded as an early tumor marker for human lung adenocarcinoma (Cekanova et al., 2007); consistent with this, lung-restricted overexpression of full-length CRAF or of its truncated kinase domain causes the MEK-dependent formation of lung adenomas (Kerkhoff et al., 2000; Kramer et al., 2004). Similarly, elevated BRAF and CRAF expression and kinase activity have been observed in human glioblastomas, and a constitutive active CRAF mutant contributes to glioma formation in mice (Lyustikman et al., 2008). Cumulatively, these results imply that most alterations in Raf drive proliferation through stimulation of the MEK/ERK pathway. In addition to the dominant role played by BRAF oncogenic mutants, endogenous, wild-type BRAF mediates ERK activation and proliferation in uveal melanoma cells lacking RAS/RAF mutations (Calipel et al., 2006); conversely, CRAF, but not BRAF, is required for these process downstream from mutated NRAS in melanoma cell lines (Dumaz et al., 2006) or from mutated KRAS in non-small cell lung cancer cell lines (Takezawa et al., 2009). Studies in cultured cells and in vivo studies suggest that autocrine/paracrine factors resulting from ERK activation play a role in the self-sufficiency of cells harboring activating Raf mutations (Troppmair et al., 1998; Schulze et al., 2001, 2004; Vale et al., 2001), generating a feed-forward loop and promoting the concomitant activation of parallel proliferative pathways.

In addition to generating their own proliferative signals, either in a cell-autonomous or in a paracrine manner, cancer cells must develop insensitivity to antiproliferative signals that maintain tissue homeostasis. A crucial inducer of antiproliferative signals is transforming growth factor-β (TGFβ) (Seoane, 2008). Many tumors disable TGFβ signaling by downregulation or mutation of the TGFβ receptor, or through inactivation of its downstream targets SMAD4, p15INK4B and the retinoblastoma protein Rb (Hanahan and Weinberg, 2000). Activation of Raf and ERK induces TGFβ production but at the same time protects cells from differentiation and apoptosis (Lehmann et al., 2000; Park et al., 2000; Schulze et al., 2001, 2004; Wang et al., 2004; Riesco-Eizaguirre et al., 2009), enabling them to draw on the pro-tumorigenic effects of this cytokine such as promotion of proliferation, invasiveness, radioresistance and immunosuppression. In addition, C-Raf interacts with, and phosphorylates, Rb. This interaction results in the recruitment of the Rb/C-Raf complex to proliferative promoters and increases E2F1-dependent transcriptional activity, counteracting the antiproliferative function of Rb (Wang et al., 1998; Dasgupta et al., 2006; Kinkade et al., 2008).

Induction of differentiation is a powerful obstacle to proliferative signals. As long as the initiated stem cells or early progenitor cells giving rise to a tumor have not lost sensitivity to differentiating signals, these can be exploited in therapy. A particularly good illustration of this is the introduction of a combination of chemotherapy and differentiation therapy, which has revolutionized the treatment of leukemia (Wang and Chen, 2008). Recently, we have shown that endogenous C-Raf is essential to maintain an undifferentiated status in Ras-driven epidermal tumors. Conditional ablation of C-Raf results in rapid regression of established tumors through MEK/ERK-independent activation of a differentiation program induced by hyper-activation of the cytoskeleton-based kinase Rok-α (Ehrenreiter et al., 2009). These data show that Ras-driven tumors are addicted to non-oncogenic C-Raf, and offer proof of principle that differentiation (co)therapy may be feasible in solid tumors.

Confirming the importance of Raf in the maintenance of an undifferentiated state, recent work has shown that amplification of CRAF leads to the ERK-dependent activation of β-catenin, and to the expansion of breast tumor-initiating cells in culture and cancer progression in xenografts (Chang et al., 2011). Thus, both MEK/ERK-dependent and -independent mechanisms can contribute to the maintenance of an undifferentiated state in tumor cells.

Evasion of senescence and apoptosis

From the above, it is clear that overexpression of full-length RAF or the truncated catalytic domain leads to the activation of the ERK pathway in cultured cells and in vivo. In both situations, strong activation of the pathway correlates with the induction of senescence, which has to be bypassed before hyper-proliferation ensues (Sewing et al., 1997; Woods et al., 1997; Ravi et al., 1998; Zhu et al., 1998; Roper et al., 2001). Thus, senescence is the Achilles’ heel of the Ras/Raf/Erk pathway. Possible bypass mechanisms include direct regulation of the activity of the B-RafV600E mutant, to lower it to a level that would not induce senescence. In this context, candidates are Akt3, which can decrease the activity by phosphorylating negative-regulatory residues on BRAF (Cheung et al., 2008), and endogenous C-Raf, which has been shown to restrain B-RafV600E activity in the context of a heterodimer (Karreth et al., 2009).

Typically, however, senescence is disabled when tumor suppressors such as p16INK4a, p19ARF, p53 or PTEN are lost (Fedorov et al., 2003; Michaloglou et al., 2005; Goel et al., 2006, 2009; Gray-Schopfer et al., 2006; Dankort et al., 2007, 2009; Lyustikman et al., 2008; Dhomen et al., 2009; Yu et al., 2009; Carragher et al., 2010), or cooperating proto-oncogenes such as c-myc or Rac1b are expressed (Matos et al., 2008; Zhuang et al., 2008).

While RAF activation does not contribute to senescence evasion, it does have multiple, in part isoform-specific, roles in counteracting apoptosis. Downstream from activated Raf and Ras, but also from other oncogenes, the ERK pathway restrains apoptosis by regulating the expression and/or the activity of BCL-2 family members (Balmanno and Cook, 2009). In addition, MEK-independent pro-survival mechanisms, such as activation of MEKK1 and the nuclear factor-κB pathway (Baumann et al., 2000) and inactivation of the BH3-only BCL-2 family member BAD (Polzien et al., 2009), have been proposed for C-Raf (all reviewed by Niault and Baccarini, 2010). Reinforcing the connection between C-Raf and the Bcl2 family, Bcl2 deletion hinders the development of lung adenomas induced by the truncated, oncogenic form of C-Raf (Fedorov et al., 2002). In addition, endogenous C-Raf can restrain apoptosis in a kinase-independent manner by binding to, and inhibiting, the stress-induced, mitochondria-based kinase ASK-1 (Chen et al., 2001) as well as the homolog of Drosophila's Hippo, the MST-2 kinase (O’Neill et al., 2004; Matallanas et al., 2007), and by regulating Fas trafficking through its interaction with the cytoskeleton-based kinase Rok-α (Piazzolla et al., 2005). By conferring a survival advantage, any of these events might potentially promote tumorigenesis, although their significance in this context has not yet been shown in vivo.

Limitless replicative potential can be achieved through avoidance of telomere shortening, which causes a DNA-damage response mediated by p53 and p21, and finally senescence. This senescent program, induced by telomere attrition, differs from the fast, oncogene-induced proliferation barrier observed, for instance, in BRAFV600E-expressing premalignant nevi (Michaloglou et al., 2005; Gray-Schopfer et al., 2006). An 85–90% portion of all cancer cells escape telomere attrition by upregulating telomerase, a reverse transcriptase that restores telomeric repeats after every cell division (Chan and Blackburn, 2004). Ets transcription factors, well-established targets of activated ERK, can stimulate the transcriptional activation of the telomerase catalytic subunit gene downstream from oncogenic growth factor receptor, Ras and Raf (Goueli and Janknecht, 2004; Dwyer et al., 2007), thereby potentially antagonizing telomere shortening and supporting the replicative potential of the mutated cells.

Sustained angiogenesis is absolutely required for growth of solid tumors beyond a size of about 3mm3. Tumor cells are able to initiate an angiogenic shift toward angiogenic initiating signals (for example, vascular endothelial growth factor (VEGF) and fibroblast growth factor-1 and 2 and suppress inhibitory signals (thrombospondin-1 and interferon-β). The impact of Raf on angiogenesis in vivo has been established by the delivery of a kinase-dead C-Raf construct to the tumor-associated vasculature in mice. The kinase-dead protein induced the apoptosis of both endothelial and tumor cells, leading to tumor regression (Hood et al., 2002). C-Raf can promote endothelial cell survival by either MEK-dependent or -independent pathways, including ASK-1 inhibition (Alavi et al., 2003, 2007). In addition, selective disruption of the interaction between C-Raf and Rb inhibits the development of tumor-associated microvessels and suppresses the growth of tumor xenografts (Dasgupta et al., 2004; Kinkade et al., 2008). Thus, several C-Raf-dependent pathways can contribute to angiogenesis. By contrast, induction of angiogenesis by B-RAFV600E, involving expression of hypoxia-inducible factor-1α (Kumar et al., 2007) and VEGF (Sharma et al., 2005, 2006; Sumimoto et al., 2006), is entirely MEK-dependent. Conversely, conditional ablation of endogenous B-Raf prevents the angiogenic switch in a mouse model of pancreatic islet carcinoma driven by loss of function of the tumor suppressors p53 and Rb. B-Raf-deficient tumor cells proliferate normally despite decreased ERK activation, but produce insufficient amounts of the proangiogenic factors VEGF and TGFβ, resulting in reduced blood vessel density and tumor proliferation, and delayed tumor progression (Sobczak et al., 2008).

Tissue invasion and metastasis depends upon all the other hallmarks acquired during the process of tumor formation as well as on changes in proteins tethering cells to their surroundings. Changes in the expression of cell–cell adhesion molecules and/or in the binding specificities of integrins, as well as upregulation and activation of extracellular proteases, result in the ability of cancer cells to invade and colonize new terrain. Raf can influence invasion at several levels. First, activated Raf is involved in the production of TGFβ, which promotes invasion and metastasis (Lehmann et al., 2000; Sobczak et al., 2008; Riesco-Eizaguirre et al., 2009), as well as the epithelial–mesenchymal transition that precedes invasion in response to this factor (Janda et al., 2002). Second, both B-Raf and C-Raf have essential, if opposite, roles in cell contractility and migration: B-Raf increasing Rho-dependent contractility and opposing migration in an ERK-dependent manner (Pritchard et al., 2004), and C-Raf reducing contractility and increasing migration by inhibiting the Rho effector Rok-α (Ehrenreiter et al., 2005). In addition, B-RAFV600E/MEK/ERK are responsible for upregulation of several proteins involved in migration, and support integrin signaling, inducing melanoma cell invasion and metastases (Liang et al., 2007; Klein et al., 2008; Argast et al., 2009; Old et al., 2009). The B-RafV600E/MEK/ERK axis can also increase melanoma cell contractility and invasion by repressing the gene coding for a cGMP-specific phospshodiesterase, PDE5A (Arozarena et al., 2010). The resulting increase in the cGMP pool causes a raise in intracellular Ca++ and ultimately increased contractility, which boosts the rounded, bleb-associated mode of motility adopted during invasion (Sahai and Marshall, 2003).

PDE5A expression was found to be lower in metastasis-derived patient material than in primary tumors. As ERK is likely activated in both samples, it would have been interesting to know at which level is PDE5A expression further regulated, and what are the secondary events leading to full-fledged downregulation in metastasis. One possibility here are changes in tissue architecture, which in itself can exert strong antiproliferative effects and counteract invasion. Cadherin-based cell–cell adhesion, for instance, efficiently counteracts tumor proliferation, angiogenesis and metastasis in CRAF-driven lung adenomas (Ceteci et al., 2007). Additional regulators of tissue architecture are matrix metalloproteases, which are often overexpressed in cancer (Kessenbrock et al., 2010). Increased expression of one of these enzymes, matrix metalloprotease-9, by the Raf/MEK/ERK pathway in three-dimensional breast tissue cultures leads to a remodeling of the microenvironment, which induces loss of tissue polarity and re-initiation of proliferation (Beliveau et al., 2010).

In keeping with a role for the Raf/MEK/ERK pathway in invasion, the Raf-inhibitor protein RKIP has been identified as a suppressor of metastasis in many cancers (Granovsky and Rosner, 2008). Recently, a pathway has been discovered in which RKIP, through inhibition of the Raf/MEK/ERK module, increases the processing of the let-7 miRNA. This, in turn, inhibits the chromatin-remodeling factor HMGA2, which contributes to the expression of several metastasis-promoting genes (Dangi-Garimella et al., 2009).

To colonize a new site, tumor cells must extravasate from the blood vessels. In melanoma cells, BRAFV600E promotes this process by causing the production of both tumor- and microenvironment-derived interleukin-8. This cytokine recruits polymorphonuclear leukocytes, which bind to melanoma cells ultimately facilitating their trans-endothelial passage (Liang et al., 2007).

Avoidance of immunosurveillance enables tumors to evade recognition and destruction by the immune system. Cancers escape surveillance by selecting for non-immunogenic tumor cells, such as those that have downregulated human leukocyte antigen class-I molecules and/or have become resistant to cytotoxic T-lymphocyte-induced killing (immunoselection). Alternatively, tumors can actively repress immune cells in various ways, creating an immune-privileged environment (immunosubversion) (reviewed by Zitvogel et al. (2006)). BRAFV600E, for instance, mediates immunosubversion by inducing the cytokines interleukin-10 and interleukin-6 (Sumimoto et al., 2006), and contributes to immune evasion by inducing an MEK/ERK-dependent decrease in the expression of melanoma differentiation antigens, which is recognized by antigen-specific T-lymphocytes (immunoselection). Unlike MEK inhibitors, treatment with a BRAF-specific inhibitor leaves the function of T-lymphocytes intact, raising hopes that such inhibitors might bypass immunoevasion (Boni et al., 2010).

Implementing the hallmark events described above comes at a high stress cost for tumor cells. Recently, five stress phenotypes of cancer have been defined (Luo et al., 2009): (1) DNA damage, resulting from telomere shortening, replication stress and oncogene activation, and from mutations of DNA-repair and DNA-damage checkpoint genes; (2) mitotic stress, a consequence of chromosomal instability; (3) proteotoxic stress, caused by accumulation of misfolded proteins; (4) oxidative stress mediated by the generation of reactive oxygen species within a cancer cell and (5) metabolic stress (Kroemer and Pouyssegur, 2008), a consequence of enhanced aerobic glycolysis used by cancer cells for energy production.

How does RAF contribute to overcoming stress? In the case of genotoxic stress, activated Ras and Raf can induce the expression of the mdm2 gene, leading to p53 degradation; at least in cells lacking the Mdm2 inhibitor p19ARF, this leads to reduced p53-dependent apoptosis following DNA damage (Ries et al., 2000). Downstream from p53, Ras or Raf activation by HB-EGF is responsible for the ERK-mediated induction of the cyclooxygenase-2 gene, which inhibits genotoxic stress-induced apoptosis (Han et al., 2002). Scatter Factor (hepatocyte growth factor), another growth factor that protects tumors from genotoxicity, uses Raf to signal survival through activation of nuclear factor-κB (Fan et al., 2007). In addition, an interplay between oncogenic Raf/ERK and ATM has been shown to promote homologous recombination repair in response to radiation (Golding et al., 2007), in line with previous reports showing a correlation between oncogenic Raf and radioresistance in tumors (Kasid et al., 1987, 1989, 1996). Finally, the RAF/MEK/ERK pathway protects multiple myeloma cells from DNA damage induced by treatment with a Chk1 inhibitor (Dai et al., 2008). These findings suggest that Raf and MEK inhibitors could be combined with cytostatic drugs or radiation in the therapy of cancer.

The relationships between Raf and oxidative stress are manifold: RAF/MEK/ERK activation can prevent the onset of oxidative stress in growth factor-deprived cells (Kuznetsov et al., 2008); on the other hand, generation of reactive oxygen species by derivatives of geldanamycin, a chemotherapeutic that inhibits the chaperone function of HSP90 and enforces the degradation of their client proteins, including Raf, is able to inhibit the activity of BRAFV600E (Fukuyo et al., 2008). Finally, a most interesting connection, relevant in terms of oncogene-selective therapy, has been reported recently between oncogenic activation of RAS and RAF, and the small-molecule drug erastin. Erastin causes mitochondrial dysfunction and oxidative cell death by activating voltage-dependent ion channels (voltage-dependent anion channels (VDACs)) on the mitochondria. In a panel of cancer cell lines of different origin, RAS and BRAF activation potentiated the lethality of the drug by inducing the expression of the VDACs (Yagoda et al., 2007). Thus, VDAC expression may represent a targetable weak spot in RAS- and RAF-driven tumors.

Metabolic stress, particularly lack of nutrients, imposes a number of metabolic checkpoints that cancer cells must bypass to continue proliferating under the dire conditions often found in the tumor microenvironment. BRAFV600E-expressing melanoma cells appear to solve this problem through ERK-mediated phosphorylation of the tumor suppressor and energy sensor LKB1. This phosphorylation, which occurs in the context of a physical complex including B-Raf V600E, prevents the LKB1-mediated activation of the AMP-activated protein kinase, which restricts protein synthesis (Zheng et al., 2009). The net result is normal operation despite nutrient shortage, and therefore a competitive advantage under conditions of metabolic stress.

RAF inhibitors–clinical success and challenges

From the above it is clear that Raf kinases are prime target for the design and application of molecule-target therapies of cancer, particularly melanoma. Several companies have generated Raf inhibitors currently in preclinical and clinical trials, and a drug specifically targeting BRAFV600E (PLX4032/RG7204; Plexxikon/Roche, Berkeley, CA, USA (Tsai et al., 2008; Joseph et al., 2010)) has recently produced dramatic results, with response rates of 70–80% as single agent in metastatic melanoma patients (Bollag et al., 2010; Flaherty et al., 2010). Similar results have been obtained with another ATP competitive BRAF inhibitor (GSK 2118436; GlaxoSmithKline, Brentford, UK (Kefford et al., 2010)). These drugs, which are currently being tested in clinical trials in patients affected by other solid tumors with BRAFV600E mutations, such as thyroid carcinomas or colon cancers (Arkenau et al., 2010; Puzanov et al., 2011), are reasonably well-tolerated. However, one intriguing and potentially worrying issue is the paradoxical increase in the proliferation and activation of the MEK–ERK pathway in cells not harboring the BRAFV600E mutation. The underlying mechanism is an allosteric effect of the drug, which enforces the dimerization of endogenous BRAF with CRAF or ARAF (Hatzivassiliou et al., 2010; Heidorn et al., 2010; Poulikakos et al., 2010). Within these dimers, only one active component is required for activation of the MEK–ERK pathway; therefore, at non-saturating concentrations, the inhibitors activate the pathway rather than disabling it, particularly in the presence of activated RAS, and it is possible that the rapid development of benign skin tumors in patients treated with RAF inhibitors might be fueled by such a mechanism (Degen et al., 2010; Robert et al., 2010) (Figure 4).

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

RAF inhibitors: response, resistance and drug-related tumors. In normal cells, RAF activation drives ERK activation downstream from RAS. In melanoma, BRAF mutants (V600E) with high kinase activity drive ERK activation (red arrows) independently of RAS. Cancer cells harboring these mutants are sensitive to BRAF inhibitors, which blunt kinase activity and reduce ERK activation as well as proliferation (thin arrows). Unfortunately, however, resistance arises, by mechanisms involving activation of other MEK kinases, such as COT, but also upregulation of receptor tyrosine kinases (RTKs) and of other pathways downstream from RAS (purple arrows). Finally, drug-related epidermal tumors have been observed in patients treated with RAF inhibitors. They correlate with ERK activation, which results from the ability of the drug to promote RAF dimerization. If only one subunit of the dimer is bound to the inhibitor (for instance at non-saturating inhibitor concentrations, or in the case of inhibitors with fast off-rates), the other subunit is activated and is capable of phosphorylating MEK with high efficiency, generating a tonic signal leading to increased proliferation. Thus, RAF inhibitors can paradoxically function as ERK activators, and potentially induce the development of drug-related tumors. ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.

Full figure and legend (102K)

More troublesome is the fact that melanoma cells develop chemoresistance by a number of different molecular mechanisms (reviewed by Poulikakos and Rosen, 2011), leading to relapse of drug-responsive disease. Unlike the case of imatinib resistance, often caused by mutations in the kinase domain of the target BCR-ABL (Weisberg et al., 2007), de novo mutations in B-RAF have not been observed in relapsing tumors. Rather, acquired resistance involved reactivation of the ERK pathway by switching to other MEK kinases (other RAF isoforms (Villanueva et al., 2010) or COT/Tpl2 (Johannessen et al., 2010)), or by activating mutations in NRAS (Nazarian et al., 2010), but also upregulation of receptor tyrosine kinases driving other pathways (Nazarian et al., 2010; Villanueva et al., 2010) (Figure 4). Therefore, overcoming melanoma resistance might require modulation of multiple pathways.

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Conclusions

The study of the Raf pathways has been extremely rewarding. The first serine/threonine kinase oncogene discovered has proven an excellent target in single-agent therapy of the disease it is most frequently associated with; in turn, investigation of the mode of action of RAF inhibitors has shed light on the mechanism of regulation of the cellular Raf enzyme. Animal models continue to delineate essential functions of the pathway components and the discovery of protein–protein interactions within the pathway and cross-pathways provides further potential leads for novel therapeutic strategies. Almost 30 years after its discovery, Raf is still a fascinating topic for basic and clinical researchers, and will remain so for many years to come.

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Conflict of interest

The authors declare no conflicts of interest.

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Acknowledgements

We thank all the members of the Baccarini lab for helpful discussions and apologize to all the colleagues whose work could not be cited in this review for reasons of space. The Baccarini lab is supported by the Austrian Scientific Research Fund (Grants P19530 and SFB 021) and the European Community (Grants INFLA-CARE and GROWTHSTOP).