- Email: [email protected]
New insight into BRAF mutations in cancer
New insight into BRAF mutations in cancer Nathalie Dhomen and Richard Marais There has been much recent progress in our understanding of the role played by the RAS–RAF–MEK–ERK cascade in human cancer. RAS is an oncogene and this pathway is known to promote proliferation and malignant transformation. More recently, however, RAF has become the focus of attention, particularly in melanoma, where approximately 70% of cases carry mutations in the BRAF gene. The majority of the mutations in BRAF in cancer are activating, but rare mutants that cannot activate MEK have provided new insight into RAF signalling networks that exist in cancer and normal cells. Surprisingly, germline mutations in BRAF that occur in rare genetic syndromes have also recently been described. The induction of BRAF mutations in melanoma depends on the type of UV exposure that the skin receives, and some studies have suggested the existence of susceptibility loci that make it more likely that some individuals will acquire these mutations. Importantly, genetic profiling and microarray studies have provided insight into the spectrum of melanomas in which BRAF plays a role and also revealed intriguing new data that could be important for the diagnosis and treatment of human cancers. Addresses Institute of Cancer Research, Cancer Research UK Centre for Cell and Molecular Biology, 237 Fulham Road, London, SW3 6JB, UK Corresponding author: Marais, Richard ([email protected])
Current Opinion in Genetics & Development 2007, 17:31–39 This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Sara A Courtneidge and Benjamin G Neel
0959-437X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2006.12.005
ARAF mutations have not been found [7,8,9,10,11]. The lack of ARAF and CRAF mutations appears to be because there is a fundamental difference in how BRAF is regulated compared with ARAF and CRAF. Whereas BRAF can be activated by single amino acid substitutions, CRAF and ARAF require two mutations for oncogenic activation [1,9], and given that this is likely to be a very rare event, it is not seen. The most common BRAF mutation, which accounts for more than 90% of cases of cancer involving this gene, is a glutamic acid for valine substitution at position 600 (V600E) [7]. BRAFV600E is activated approximately 500-fold and it induces constitutive ERK signaling through hyperactivation of the RAS–MEK–ERK pathway, and constitutive Nuclear factor kappa-B (NF-kB) signalling in response to this hyperactivation (Figure 1, cellular responses), stimulating proliferation, survival and transformation. Although MEK is activated following its direct phosphorylation by oncogenic BRQAF, it is currently unclear how NF-kB signaling is activated. Cells that express BRAFV600E can grow as tumours in nude mice [12–18], and continued expression of BRAFV600E is necessary for tumour growth and progression [19]. These data show that BRAF is an oncogene that stimulates many of the hallmarks of cancer and yet, surprisingly, BRAF is mutated in up to 80% of the benign skin lesions called naevi [20,21,22] and in pre-malignant colon polyps [23,24]. Indeed, BRAFV600E induces senescence in pigmented skin cells called melanocytes, through transcriptional upregulation of the cell cycle inhibitor p16INK4a [25,26]. Thus, BRAF plays an important role in cancer induction, maintenance and progression, and given that it is mutated early in the initiation process, this might be a founder event. However, by itself oncogenic BRAF is not sufficient for cancer and must cooperate with other processes to induce the fully cancerous state.
Lessons from melanoma Introduction RAF is a serine–threonine-specific protein kinase that is activated downstream of the small G-protein RAS (Figure 1). RAF activates the MAP kinase extracellular signal-regulated kinase (MEK), which in turn activates the extracellular signal-regulated kinase (ERK). In mammals, there are three highly conserved RAF genes, ARAF (otherwise known as A-Raf), BRAF (B-Raf) and CRAF (c-Raf or Raf-1), and knockout studies in mice suggest that their protein products play distinct roles [1–6]. Somatic mutations in BRAF occur in approximately 7% of human cancers, whereas CRAF mutations are rare and www.sciencedirect.com
Importantly, up to 70% of human melanomas, a form of skin cancer that arises from the melanocyte lineage, harbour mutations in BRAF [7]. Melanocytes reside predominantly in the skin, where they contribute to skin and hair pigmentation, but they are also present in other tissues. Upon exposure to UV light, skin cells called keratinocytes stimulate melanocytes to produce the pigment melanin, which is used to protect the skin from further UV damage — the tanning response [27]. Melanoma has a genetic component, but it is widely accepted (although some uncertainty still remains) that the only known environmental risk is UV irradiation. Melanoma can be segregated into four groups according to the site of Current Opinion in Genetics & Development 2007, 17:31–39
32 Genetic and cellular mechanisms of oncogenesis
Figure 1
The RAS–RAF–MEK–ERK pathway. Growth factors bind to receptor tyrosine kinases (RTKs), resulting in RAS activation. RAF proteins are one of a family of effector proteins activated by RAS, and they in turn stimulate the activation of MEK, which subsequently stimulates ERK activity. ERK phosphorylates both cytosolic and nuclear proteins, thereby mediating the cellular responses cells make when this pathway when this pathway is activated.
the primary lesion and, therefore, to the amount of UV exposure that is perceived to have induced the melanoma [28]. Two of these groups arise on sites of high UV exposure, either accumulated owing to chronic exposure throughout life (i.e. chronic sun damage [CSD]), such as occurs on the face, or as a consequence of episodes of acute, high-intensity exposure (non-CSD), such as occurs on the back or trunk. The other two groups are from sites of low or no UV exposure, such as the palms of the hands or the soles of the feet (acral melanomas), or on internal sites involving mucosal membranes or the uveal tract of the eye (mucosal and uveal melanoma, respectively). 81% of non-CSD melanomas have BRAF or NRAS mutations, but mutations in these genes are rare in Current Opinion in Genetics & Development 2007, 17:31–39
CSD, acral and mucosal melanomas [28]. Thus, in melanoma, mutant BRAF is associated with episodes of sunburn, but not with melanomas from sun-protected sites, or indeed from sites exposed to chronic UV damage. Notably, BRAF mutations are also absent in basal and squamous cell carcinomas, skin cancers that are associated with chronic UV exposure and which are about 10 times more common than melanoma [29]. Thus, although UV light appears to induce BRAF mutations in melanoma, the link is not simple, a point that is emphasized by the observation that the V600E mutation requires a GTG to GAG change, a mutation that does not conform to a typical UV-damaged DNA signature [7]. Indeed, the majority of mutations in BRAF in melanoma do not conform to a common UV signature [30]. It should be noted that the V600E mutation can occur in a UV-independent manner. BRAFV600E is found in a high proportion of ovarian (30%), thyroid (30%) and colorectal (15%) cancers [7], in which UV exposure simply cannot be the cause of the mutation, and the V600E mutation arises in mouse liver tumours induced by Nnitrosodiethylamine [31]. How then does UV induce BRAF mutations in melanoma? The nature of the UV exposure might be important, because cycles of acute, high-intensity exposure — particularly at a young age — are more efficient at inducing the mutation than is chronic exposure. One possibility is that the mutations are not induced directly by UV irradiation but are a secondary consequence of exposure. Note that melanin production results in the accumulation of highly toxic oxidizing agents, and so its increased synthesis following acute UV exposure could cause increased DNA damage in the melanocytes [32,33]. Furthermore, the inflammation and erythema that accompany sunburn could contribute. Melanocytes are notoriously resistant to apoptosis [34], and recently we demonstrated that TNFa (Tumour necrosis factor a), normally secreted by cells of the immune system to induce death, can in fact stimulate the survival of melanoma in cells expressing BRAFV600E [35]. This survival pathway may cause failure of anti-BRAF therapies in the clinic because it will provide a survival advantage to melanoma cells. However it may also play a role in the induction of melanoma, because the monocytes and macrophages that infiltrate sunburnt skin might provide survival signals to melanocytes harbouring otherwise lethal levels of DNA damage. Taken together, these data suggest that V600 is a hyper-mutable site that is particularly sensitive to some forms of DNA damaging agents. An alternative explanation is that that BRAFV600E is just an extremely efficient oncogene and so cells that acquire this mutation have a high chance of progressing to cancer, whereas other BRAF mutants are less efficient. If this is the case, then the biological basis of the selection is not at all clear and it is difficult to explain why BRAF mutations are only associated with some types of cancer. This therefore suggests that there are important biological selection pressures that www.sciencedirect.com
New insight into BRAF mutations in cancer Dhomen and Marais 33
also contribute to the selection of BRAFV600E-containing cancers.
Melanocortin-1 receptor: a susceptibility locus for BRAF mutations? Another possibility is that inherited susceptibility factors predispose some individuals to the acquisition of BRAF mutations in their melanocytes. One such locus might be the G-protein-coupled Melanocortin-1 receptor (MC1R), which encodes a receptor for the a-melanocyte-stimulating hormone (a-MSH). In response to UV light, keratinocytes secrete a-MSH, which activates MC1R on melanocytes, stimulating cAMP synthesis and thereby melanin production [36]. MC1R is an attractive candidate as a melanoma susceptibility gene. It is highly polymorphic, with variants that differ in signalling strength. The fully active variant is considered to be wild type, but partially (r) or completely (R) impaired signalling variants exist [37,38]. In the absence of functional MC1R, keratinocyte-derived aMSH fails to stimulate efficient melanin production. This fault is rescued by topical application of the cAMP agonist forskolin [39], stimulating a false tanning response and emphasizing the important role played by aMSH signalling in UV protection. The r and R variants are associated with phenotypic traits such as fair skin, freckling and red hair — hence the r and R idioms — and individuals carrying these alleles have increased sensitivity to UV light [40]. In the past year alone, several studies have confirmed that MC1R is a lowpenetrance susceptibility locus, although the strength of this association is debated [41–46], possibly because MC1R susceptibility has pigmentation-dependent and -independent components. That MC1R variation predisposes some individuals to BRAF mutations comes from a study of non-CSD melanomas. In these cancers, BRAF mutations are associated with the r or R variants and are less prevalent in the presence of wild type MC1R [38]. Although this study used a small cohort of patients, the results suggest that BRAF is mutated more readily when MC1R signalling is impaired. Unfortunately, the prevalence of RAS mutations was not reported in this study, but statistical arguments predict that RAS mutations should be more common in cancers with intact MC1R signalling. However, this seems counterintuitive in light of other observations. When cAMP levels in melanocytes are elevated, PKA (Protein kinase A) phosphorylates CRAF, locking it in an inactive conformation and preventing it from signalling downstream of RAS (Figure 2) [47]. Consequently, because cAMP levels appear to be constantly elevated in melanocytes, CRAF does not signal and only BRAF can couple RAS to MEK. Surprisingly, however, when RAS is mutated in melanoma cells, it is CRAF that couples RAS to MEK, and BRAF no longer signals [47]. Thus, melanoma cells seem to activate MEK through one of two pathways, either directly from mutant BRAF or from www.sciencedirect.com
mutant RAS but through CRAF (Figure 2). It is unclear why mutant RAS does not signal to BRAF, but RAS activates BRAF more strongly than it does CRAF [48], raising the possibility that, if BRAF is used, ERK will be hyperactivated and stimulate cell cycle arrest or senescence rather than proliferation, which is clearly incompatible with tumour progression [30]. To counteract this, the cells have to switch to CRAF, but to allow CRAF to signal, PKA must be inactivated (Figure 2), and in agreement with this, melanoma cells in which RAS is mutated do not respond to aMSH, whereas half of the melanomas in which BRAF is mutated still respond to aMSH, demonstrating that MCR1 signaling is still tolerated in the presence of oncogenic BRAF [47].
Melanoma genetics: the devil is in the detail It appears that melanoma arises through a process of ‘Darwinian evolution’, the course of which is determined by whether RAS or BRAF is mutated. These events could occur in committed melanocytes that have already selected BRAF as the major signalling isoform, or they might occur in an earlier, stem cell fraction that signals through either RAF isoform. Depending on the precise starting point, the molecular mechanisms will be different, but in both cases MC1R signalling must be inactivated when RAS is mutated. Given that cells that acquire mutation in BRAF do not need the extra step of MC1R inactivation, BRAF offers a shorter path to melanocyte transformation, and this could also contribute to the higher rate of BRAF than RAS mutations in melanoma. However, as stated, it could simply be that BRAF is more mutable, or that the other pathways that RAS can activate play a negative role in melanoma progression and thus BRAF mutant melanomas are more successful. It will be interesting to determine if similar signalling complexity exists in colorectal, ovarian and thyroid cancer, the other cancers in which BRAF and RAS are mutated. Clearly, these findings could have important prognostic and therapeutic implications. Several studies have attempted to address the therapeutic implications of BRAF in melanoma. It has been suggested that BRAF mutant melanomas respond to treatment more rapidly than melanomas without mutant BRAF and that patients with BRAF mutant tumours enjoy longer diseasefree survival [49]. However, the response is likely to be more complex. When several genetic aberrations that are associated with melanoma are considered, 12 distinct genetic profiles can be defined [50]. The most common (36%) consists of tumours with aberrations in CDKN2A (Cyclin-dependent kinase inhibitor 2A) — this encodes the tumour suppressors p16INK4a and p14ARF (p19ARF in mouse) — together with mutations in BRAF. These patients enjoyed enhanced survival, again suggesting that BRAF mutations provide a favourable prognosis. However, when BRAF mutations are accompanied by aberrations in multiple tumour suppressor genes, survival is much reduced. Current Opinion in Genetics & Development 2007, 17:31–39
34 Genetic and cellular mechanisms of oncogenesis
Figure 2
Clearly, more comprehensive analysis is required if we are to understand the contribution made by BRAF and NRAS mutations to disease progression, prognosis and response to therapy. To this end, gene expression profiling has been used to examine the plethora of molecular changes that occur in melanoma. A comparison of naevi, primary melanomas and metastatic melanoma samples [51] revealed, as expected, that these lesions can be distinguished from each other. It was even possible to distinguish the radial and vertical growth phase primary lesions. This is an important distinction, because vertical growth phase lesions are thought to be more advanced lesions that have metastatic potential [52]. Intriguingly, metastatic melanomas could even be separated into two distinct groups, although the clinical significance of this division is as yet unclear. A molecular signature of invasion in cutaneous melanoma has been identified [53], as has a signature that can differentiate poorly and highly invasive uveal melanoma cells [54]. Finally, melanoma cell lines with differing capacity to metastasize to the lungs of nude mice can also be differentiated by these approaches [55]. Studies have also focused on defining gene profiles that are regulated by oncogenic BRAF or NRAS. In one study [56] of 10 melanoma cell lines, 65 genes were shown to distinguish wild type BRAF from BRAFV600E lines, whereas expression of 109 genes changed when wild type NRAS and NRASQ61R lines were compared. Fifty-six genes were common to both the BRAF and NRAS sets, and many of these genes encode members or regulators of the RAS–RAF–MEK–ERK pathway, whereas others are involved in metastasis or invasion. In a second study [57], 83 genes were shown to discriminate melanomas with mutant BRAF from melanomas with wild type BRAF. Despite the fact that it was possible to distinguish oncogenic NRAS melanomas from wild type RAS melanomas, it was not possible to distinguish BRAF and NRAS melanomas from each other. These studies suggest that it is possible to define a ‘BRAF signature’. However, these conclusions have been challenged. Re-analysis of the data using multiple testing Mutations in RAS and RAF alter signalling through the MC1R and RAS– RAF–MEK–ERK pathways. (a) In melanocytes wild type for RAS and BRAF, RAS signals to MEK through BRAF as opposed to CRAF (solid versus dashed arrows). CRAF is inhibited by signalling from amelanocyte-stimulating hormone (a-MSH) through the Melanocortin 1 receptor (MC1R). This stimulates the production of cAMP, catalysed by adenylyl cyclase (AC), which in turn activates protein kinase A (PKA). PKA phosphorylates CRAF, thereby inhibiting its activity. (b) When RAS is mutated (RASMUT), it switches its signalling from BRAF to CRAF (solid versus dashed arrows), and the RAS–RAF–MEK–ERK is hyper-activated (thick arrows). This is accompanied by a reduction in signalling through MC1R (dashed arrows), thus enabling CRAF to be activated. (c) When BRAF is mutated (BRAFMUT), the RAS–RAF–MEK–ERK is again hyperactivated, but this occurs by direct activation of MEK by BRAF.
Current Opinion in Genetics & Development 2007, 17:31–39
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New insight into BRAF mutations in cancer Dhomen and Marais 35
controls reduced the 83 gene set to a single gene whose expression changes could be linked to BRAF mutation status [58]. Indeed, when the data from three independent DNA microarray cohorts were analysed, it was possible to define three distinct ‘types’ of melanoma that differ in their proliferative and metastatic potential [58], but mutant BRAF was not restricted to any of these groups, leading the authors to conclude that it is not possible to identify a BRAF (or NRAS) signature profile. Furthermore, the genes highlighted in each of the studies supporting the putative existence of a ‘BRAF signature’ are different, again suggesting that such a signature profile cannot be easily defined. One problem with these studies is that where cells were grown in 10% foetal calf serum, a condition in which the RAF–RAF–MEK–ERK pathway is activated, the contribution from oncogenic BRAF or oncogenic NRAS could have been masked. Microarrays can clearly be used to distinguish different forms of melanoma, but to date this approach has not convincingly enabled us to identify a BRAF or NRAS signature. More studies are therefore required to allow us to harness the power of this approach, but care must be taken in sample preparation and analysis. An alternative and perhaps more promising approach is to simply use the BRAF and NRAS mutation status in combination with comparative genomic hybridization. Using this approach, it is possible to classify melanomas into CSD, non-CSD, acral and mucosal melanomas with 70% confidence [28]. While this level of accuracy is no yet powerful enough for routine clinical use, these studies suggest that if the accuracy can be improved, this approach could be extremely useful for diagnosis and for determining prognosis and making therapeutic potential. Furthermore, it can reveal intriguing new insight into the genetics of melanoma. For example, it has been shown that in melanomas in which BRAF and NRAS are wild type, the genes encoding cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1) are often amplified. Notably, CDK4 and CCND1 transcription is activated downstream of BRAF signalling, implying that their upregulation is crucial in all melanomas, that this can occur through alternative molecular mechanisms and, therefore, that these proteins could be important therapeutic targets for all forms of melanoma.
New RAF networks and germline mutations Perhaps one of the most unexpected findings recently made is that BRAF can activate CRAF (Figure 3). The mechanism appears to involve direct binding of BRAF to CRAF, followed by trans-phosphorylation of CRAF by BRAF [59,60]. CRAF is activated by both wild type BRAF and the mutant forms found in cancer. Wild type BRAF binds to CRAF in a RAS-dependent manner, whereas mutant BRAF binds in a RAS-independent manner [60–63]. Furthermore, this appears to be a oneway relationship, because CRAF does not activate BRAF www.sciencedirect.com
Figure 3
RAF signalling networks in normal and cancer cells. (a) Wild type BRAF activates CRAF in a RAS-dependent manner, and both BRAF and CRAF can activate MEK. In addition, CRAF also activates other effector pathways such as ASK1, MST2, Bcl-2 and NFkB. (b) High activity BRAF mutants activate CRAF independently of RAS and although CRAF can signal to MEK (dashed arrow), BRAF activity is the dominant route leading to MEK activation (bold arrow). However, CRAF can still signal to the other effector pathways. (c) Low activity BRAF mutants are unable to signal to MEK (dashed arrow) but are able to activate CRAF independently of RAS, thereby signalling to MEK and also to the other signalling pathways.
[60]. Thus, BRAF can activate CRAF, and both proteins can then signal to MEK (Figure 3). This signalling network appears to be very important in some cancer contexts. Although our discussion so far has been restricted to BRAFV600E, because this is the most common and therefore clinically the most important mutant, close to 70 other mutations have been identified in BRAF in cancer (www.sanger.ac.uk/genetics/CGP/cosmic/). Crucially, the activities of mutant forms of BRAF range from those with extremely high activity such as BRAFV600E (500-fold activated) to some mutants whose activity is actually Current Opinion in Genetics & Development 2007, 17:31–39
36 Genetic and cellular mechanisms of oncogenesis
impaired and which are therefore unable to activate MEK directly [59]. Importantly however, although the impaired activity mutants are unable to activate MEK, they can still activate CRAF, which is then responsible for stimulating MEK signalling in these cells. The importance of this RAF cross-talking network in normal cell signalling is unclear. However, in the cells that express the impaired activity mutants, it is essential for MEK activation. Notably, CRAF has been implicated in other signalling pathways, especially survival, where CRAF has been shown to bind to apoptosis-stimulating kinases such as mammalian sterile 20-like kinase 2 (MST2) and apoptosis-signal-regulating kinase 1 (ASK1) [64,65], and to kinases such as Roka [66], which controls cytoskeletal rearrangements (Figure 3). CRAF is also implicated in survival mediated by Bcl-2 and NFkB activation [67,68], although the mechanisms of regulation are unclear. The regulation of these additional effectors by CRAF is independent of its kinase activity in some cases, but access to these pathways may be important for regulation of survival and processes such as migration. In the context of cancers in which BRAF is mutated, the activation of CRAF by BRAF may provide access to these novel functions of CRAF, which would normally be activated downstream of RAS and this may be very important to tumour progression. Another surprising recent discovery is the existence of germline mutations in RAS, BRAF and MEK in individuals affected by Noonan, Costello and cardio-facio-cutaneous (CFC) syndromes [69–72], rare genetic disorders that result in a variety of developmental defects such as cardiac anomalies together with short stature and distinct facial abnormalities. The mutations in these individuals are gain-of-function because they stimulate constitutive MEK signalling and in agreement with the role of this pathway in cancer, individuals with Noonan and Costello syndromes have increased risk of specific types of tumours such as rhabdomyosarcoma, certain types of leukaemia and neuroblastoma [73]. Notably, as in cancer, both activating and inactivating mutations have been found in these genetic syndromes [72], and we presume that the inactivating mutants stimulate MEK indirectly through CRAF, as occurs in cancer.
activity of the mutants, BRAFV600E was only two fold more active than wild type BRAF [72] rather than the 500-fold previously reported. The residues that are involved generally only produce more modestly active mutations, and we suspect that the most active mutants would not be tolerated. In this respect, it is worth noting that the KRAS mutations in Noonan syndrome are the lower activity versions. It is presumably only the weaker activating mutants that are tolerated during development. Nevertheless, these are effectively gain-of-function mutations that stimulate elevated, but modest, signalling through the pathway. It is currently unclear how the spectrum of deformities associated with these syndromes are induced by this elevated signalling, or if this knowledge can be used to alleviate some of the non-developmental symptoms suffered by these individuals, but clearly further studies are warranted, particularly using animal models, to investigate the role of this pathway in these syndromes.
Conclusions In the years since BRAF was identified as a potent oncogene, we have learned much about the biology of this pathway in cancer. Although most studies have focussed on melanoma, these studies will have implications for other cancers. It is worth noting that although BRAF is mutated in a higher proportion of melanomas than in colorectal cancers, more people die of colorectal cancer with mutant BRAF than die of BRAF melanomas, because colorectal cancer is a significantly more common disease. Genetic approaches have taught us a great deal about the genetics of cancers in which BRAF is mutated, and these studies are bound to have an enormous impact on the clinical management of these cancers. The existence of rare mutations in BRAF that suppress, rather than stimulate, BRAF kinase activity leads to the discovery that BRAF can activate CRAF in both normal and cancer cells. Furthermore, the surprising existence of RAS, BRAF and MEK mutations in Noonan, Costello and CFC syndromes suggests that weak activating mutations can be tolerated during development. Clearly future genetic approaches will continue to provide important insight into BRAF signalling in cancer, particularly as new tools, such as mouse models [74], become more widely available.
Acknowledgements The specific mutations that are found in cancer are rare in these genetic syndromes, although about half of the syndrome mutations involve codons that are also mutated in cancer. Curiously, despite its prevalence in cancer, BRAFV600E has not been discovered in these genetic syndromes, although it has been reported that some of the mutants in these syndromes are at least as activating as the V600E substitution. However, this seems unlikely. BRAFV600E causes embryonic lethality in mouse models [74], showing that BRAFV600E is not tolerated during development, and in the assay used to measure the Current Opinion in Genetics & Development 2007, 17:31–39
This work is funded Cancer Research UK (Ref C107/A3096) and the Institute of Cancer Research.
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Current Opinion in Genetics & Development 2007, 17:31–39
Current Opinion in Genetics & Development 2007, 17:31–39 This review comes from a themed issue on Genetic and cellular mechanisms of oncogenesis Edited by Sara A Courtneidge and Benjamin G Neel
0959-437X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2006.12.005
ARAF mutations have not been found [7,8,9,10,11]. The lack of ARAF and CRAF mutations appears to be because there is a fundamental difference in how BRAF is regulated compared with ARAF and CRAF. Whereas BRAF can be activated by single amino acid substitutions, CRAF and ARAF require two mutations for oncogenic activation [1,9], and given that this is likely to be a very rare event, it is not seen. The most common BRAF mutation, which accounts for more than 90% of cases of cancer involving this gene, is a glutamic acid for valine substitution at position 600 (V600E) [7]. BRAFV600E is activated approximately 500-fold and it induces constitutive ERK signaling through hyperactivation of the RAS–MEK–ERK pathway, and constitutive Nuclear factor kappa-B (NF-kB) signalling in response to this hyperactivation (Figure 1, cellular responses), stimulating proliferation, survival and transformation. Although MEK is activated following its direct phosphorylation by oncogenic BRQAF, it is currently unclear how NF-kB signaling is activated. Cells that express BRAFV600E can grow as tumours in nude mice [12–18], and continued expression of BRAFV600E is necessary for tumour growth and progression [19]. These data show that BRAF is an oncogene that stimulates many of the hallmarks of cancer and yet, surprisingly, BRAF is mutated in up to 80% of the benign skin lesions called naevi [20,21,22] and in pre-malignant colon polyps [23,24]. Indeed, BRAFV600E induces senescence in pigmented skin cells called melanocytes, through transcriptional upregulation of the cell cycle inhibitor p16INK4a [25,26]. Thus, BRAF plays an important role in cancer induction, maintenance and progression, and given that it is mutated early in the initiation process, this might be a founder event. However, by itself oncogenic BRAF is not sufficient for cancer and must cooperate with other processes to induce the fully cancerous state.
Lessons from melanoma Introduction RAF is a serine–threonine-specific protein kinase that is activated downstream of the small G-protein RAS (Figure 1). RAF activates the MAP kinase extracellular signal-regulated kinase (MEK), which in turn activates the extracellular signal-regulated kinase (ERK). In mammals, there are three highly conserved RAF genes, ARAF (otherwise known as A-Raf), BRAF (B-Raf) and CRAF (c-Raf or Raf-1), and knockout studies in mice suggest that their protein products play distinct roles [1–6]. Somatic mutations in BRAF occur in approximately 7% of human cancers, whereas CRAF mutations are rare and www.sciencedirect.com
Importantly, up to 70% of human melanomas, a form of skin cancer that arises from the melanocyte lineage, harbour mutations in BRAF [7]. Melanocytes reside predominantly in the skin, where they contribute to skin and hair pigmentation, but they are also present in other tissues. Upon exposure to UV light, skin cells called keratinocytes stimulate melanocytes to produce the pigment melanin, which is used to protect the skin from further UV damage — the tanning response [27]. Melanoma has a genetic component, but it is widely accepted (although some uncertainty still remains) that the only known environmental risk is UV irradiation. Melanoma can be segregated into four groups according to the site of Current Opinion in Genetics & Development 2007, 17:31–39
32 Genetic and cellular mechanisms of oncogenesis
Figure 1
The RAS–RAF–MEK–ERK pathway. Growth factors bind to receptor tyrosine kinases (RTKs), resulting in RAS activation. RAF proteins are one of a family of effector proteins activated by RAS, and they in turn stimulate the activation of MEK, which subsequently stimulates ERK activity. ERK phosphorylates both cytosolic and nuclear proteins, thereby mediating the cellular responses cells make when this pathway when this pathway is activated.
the primary lesion and, therefore, to the amount of UV exposure that is perceived to have induced the melanoma [28]. Two of these groups arise on sites of high UV exposure, either accumulated owing to chronic exposure throughout life (i.e. chronic sun damage [CSD]), such as occurs on the face, or as a consequence of episodes of acute, high-intensity exposure (non-CSD), such as occurs on the back or trunk. The other two groups are from sites of low or no UV exposure, such as the palms of the hands or the soles of the feet (acral melanomas), or on internal sites involving mucosal membranes or the uveal tract of the eye (mucosal and uveal melanoma, respectively). 81% of non-CSD melanomas have BRAF or NRAS mutations, but mutations in these genes are rare in Current Opinion in Genetics & Development 2007, 17:31–39
CSD, acral and mucosal melanomas [28]. Thus, in melanoma, mutant BRAF is associated with episodes of sunburn, but not with melanomas from sun-protected sites, or indeed from sites exposed to chronic UV damage. Notably, BRAF mutations are also absent in basal and squamous cell carcinomas, skin cancers that are associated with chronic UV exposure and which are about 10 times more common than melanoma [29]. Thus, although UV light appears to induce BRAF mutations in melanoma, the link is not simple, a point that is emphasized by the observation that the V600E mutation requires a GTG to GAG change, a mutation that does not conform to a typical UV-damaged DNA signature [7]. Indeed, the majority of mutations in BRAF in melanoma do not conform to a common UV signature [30]. It should be noted that the V600E mutation can occur in a UV-independent manner. BRAFV600E is found in a high proportion of ovarian (30%), thyroid (30%) and colorectal (15%) cancers [7], in which UV exposure simply cannot be the cause of the mutation, and the V600E mutation arises in mouse liver tumours induced by Nnitrosodiethylamine [31]. How then does UV induce BRAF mutations in melanoma? The nature of the UV exposure might be important, because cycles of acute, high-intensity exposure — particularly at a young age — are more efficient at inducing the mutation than is chronic exposure. One possibility is that the mutations are not induced directly by UV irradiation but are a secondary consequence of exposure. Note that melanin production results in the accumulation of highly toxic oxidizing agents, and so its increased synthesis following acute UV exposure could cause increased DNA damage in the melanocytes [32,33]. Furthermore, the inflammation and erythema that accompany sunburn could contribute. Melanocytes are notoriously resistant to apoptosis [34], and recently we demonstrated that TNFa (Tumour necrosis factor a), normally secreted by cells of the immune system to induce death, can in fact stimulate the survival of melanoma in cells expressing BRAFV600E [35]. This survival pathway may cause failure of anti-BRAF therapies in the clinic because it will provide a survival advantage to melanoma cells. However it may also play a role in the induction of melanoma, because the monocytes and macrophages that infiltrate sunburnt skin might provide survival signals to melanocytes harbouring otherwise lethal levels of DNA damage. Taken together, these data suggest that V600 is a hyper-mutable site that is particularly sensitive to some forms of DNA damaging agents. An alternative explanation is that that BRAFV600E is just an extremely efficient oncogene and so cells that acquire this mutation have a high chance of progressing to cancer, whereas other BRAF mutants are less efficient. If this is the case, then the biological basis of the selection is not at all clear and it is difficult to explain why BRAF mutations are only associated with some types of cancer. This therefore suggests that there are important biological selection pressures that www.sciencedirect.com
New insight into BRAF mutations in cancer Dhomen and Marais 33
also contribute to the selection of BRAFV600E-containing cancers.
Melanocortin-1 receptor: a susceptibility locus for BRAF mutations? Another possibility is that inherited susceptibility factors predispose some individuals to the acquisition of BRAF mutations in their melanocytes. One such locus might be the G-protein-coupled Melanocortin-1 receptor (MC1R), which encodes a receptor for the a-melanocyte-stimulating hormone (a-MSH). In response to UV light, keratinocytes secrete a-MSH, which activates MC1R on melanocytes, stimulating cAMP synthesis and thereby melanin production [36]. MC1R is an attractive candidate as a melanoma susceptibility gene. It is highly polymorphic, with variants that differ in signalling strength. The fully active variant is considered to be wild type, but partially (r) or completely (R) impaired signalling variants exist [37,38]. In the absence of functional MC1R, keratinocyte-derived aMSH fails to stimulate efficient melanin production. This fault is rescued by topical application of the cAMP agonist forskolin [39], stimulating a false tanning response and emphasizing the important role played by aMSH signalling in UV protection. The r and R variants are associated with phenotypic traits such as fair skin, freckling and red hair — hence the r and R idioms — and individuals carrying these alleles have increased sensitivity to UV light [40]. In the past year alone, several studies have confirmed that MC1R is a lowpenetrance susceptibility locus, although the strength of this association is debated [41–46], possibly because MC1R susceptibility has pigmentation-dependent and -independent components. That MC1R variation predisposes some individuals to BRAF mutations comes from a study of non-CSD melanomas. In these cancers, BRAF mutations are associated with the r or R variants and are less prevalent in the presence of wild type MC1R [38]. Although this study used a small cohort of patients, the results suggest that BRAF is mutated more readily when MC1R signalling is impaired. Unfortunately, the prevalence of RAS mutations was not reported in this study, but statistical arguments predict that RAS mutations should be more common in cancers with intact MC1R signalling. However, this seems counterintuitive in light of other observations. When cAMP levels in melanocytes are elevated, PKA (Protein kinase A) phosphorylates CRAF, locking it in an inactive conformation and preventing it from signalling downstream of RAS (Figure 2) [47]. Consequently, because cAMP levels appear to be constantly elevated in melanocytes, CRAF does not signal and only BRAF can couple RAS to MEK. Surprisingly, however, when RAS is mutated in melanoma cells, it is CRAF that couples RAS to MEK, and BRAF no longer signals [47]. Thus, melanoma cells seem to activate MEK through one of two pathways, either directly from mutant BRAF or from www.sciencedirect.com
mutant RAS but through CRAF (Figure 2). It is unclear why mutant RAS does not signal to BRAF, but RAS activates BRAF more strongly than it does CRAF [48], raising the possibility that, if BRAF is used, ERK will be hyperactivated and stimulate cell cycle arrest or senescence rather than proliferation, which is clearly incompatible with tumour progression [30]. To counteract this, the cells have to switch to CRAF, but to allow CRAF to signal, PKA must be inactivated (Figure 2), and in agreement with this, melanoma cells in which RAS is mutated do not respond to aMSH, whereas half of the melanomas in which BRAF is mutated still respond to aMSH, demonstrating that MCR1 signaling is still tolerated in the presence of oncogenic BRAF [47].
Melanoma genetics: the devil is in the detail It appears that melanoma arises through a process of ‘Darwinian evolution’, the course of which is determined by whether RAS or BRAF is mutated. These events could occur in committed melanocytes that have already selected BRAF as the major signalling isoform, or they might occur in an earlier, stem cell fraction that signals through either RAF isoform. Depending on the precise starting point, the molecular mechanisms will be different, but in both cases MC1R signalling must be inactivated when RAS is mutated. Given that cells that acquire mutation in BRAF do not need the extra step of MC1R inactivation, BRAF offers a shorter path to melanocyte transformation, and this could also contribute to the higher rate of BRAF than RAS mutations in melanoma. However, as stated, it could simply be that BRAF is more mutable, or that the other pathways that RAS can activate play a negative role in melanoma progression and thus BRAF mutant melanomas are more successful. It will be interesting to determine if similar signalling complexity exists in colorectal, ovarian and thyroid cancer, the other cancers in which BRAF and RAS are mutated. Clearly, these findings could have important prognostic and therapeutic implications. Several studies have attempted to address the therapeutic implications of BRAF in melanoma. It has been suggested that BRAF mutant melanomas respond to treatment more rapidly than melanomas without mutant BRAF and that patients with BRAF mutant tumours enjoy longer diseasefree survival [49]. However, the response is likely to be more complex. When several genetic aberrations that are associated with melanoma are considered, 12 distinct genetic profiles can be defined [50]. The most common (36%) consists of tumours with aberrations in CDKN2A (Cyclin-dependent kinase inhibitor 2A) — this encodes the tumour suppressors p16INK4a and p14ARF (p19ARF in mouse) — together with mutations in BRAF. These patients enjoyed enhanced survival, again suggesting that BRAF mutations provide a favourable prognosis. However, when BRAF mutations are accompanied by aberrations in multiple tumour suppressor genes, survival is much reduced. Current Opinion in Genetics & Development 2007, 17:31–39
34 Genetic and cellular mechanisms of oncogenesis
Figure 2
Clearly, more comprehensive analysis is required if we are to understand the contribution made by BRAF and NRAS mutations to disease progression, prognosis and response to therapy. To this end, gene expression profiling has been used to examine the plethora of molecular changes that occur in melanoma. A comparison of naevi, primary melanomas and metastatic melanoma samples [51] revealed, as expected, that these lesions can be distinguished from each other. It was even possible to distinguish the radial and vertical growth phase primary lesions. This is an important distinction, because vertical growth phase lesions are thought to be more advanced lesions that have metastatic potential [52]. Intriguingly, metastatic melanomas could even be separated into two distinct groups, although the clinical significance of this division is as yet unclear. A molecular signature of invasion in cutaneous melanoma has been identified [53], as has a signature that can differentiate poorly and highly invasive uveal melanoma cells [54]. Finally, melanoma cell lines with differing capacity to metastasize to the lungs of nude mice can also be differentiated by these approaches [55]. Studies have also focused on defining gene profiles that are regulated by oncogenic BRAF or NRAS. In one study [56] of 10 melanoma cell lines, 65 genes were shown to distinguish wild type BRAF from BRAFV600E lines, whereas expression of 109 genes changed when wild type NRAS and NRASQ61R lines were compared. Fifty-six genes were common to both the BRAF and NRAS sets, and many of these genes encode members or regulators of the RAS–RAF–MEK–ERK pathway, whereas others are involved in metastasis or invasion. In a second study [57], 83 genes were shown to discriminate melanomas with mutant BRAF from melanomas with wild type BRAF. Despite the fact that it was possible to distinguish oncogenic NRAS melanomas from wild type RAS melanomas, it was not possible to distinguish BRAF and NRAS melanomas from each other. These studies suggest that it is possible to define a ‘BRAF signature’. However, these conclusions have been challenged. Re-analysis of the data using multiple testing Mutations in RAS and RAF alter signalling through the MC1R and RAS– RAF–MEK–ERK pathways. (a) In melanocytes wild type for RAS and BRAF, RAS signals to MEK through BRAF as opposed to CRAF (solid versus dashed arrows). CRAF is inhibited by signalling from amelanocyte-stimulating hormone (a-MSH) through the Melanocortin 1 receptor (MC1R). This stimulates the production of cAMP, catalysed by adenylyl cyclase (AC), which in turn activates protein kinase A (PKA). PKA phosphorylates CRAF, thereby inhibiting its activity. (b) When RAS is mutated (RASMUT), it switches its signalling from BRAF to CRAF (solid versus dashed arrows), and the RAS–RAF–MEK–ERK is hyper-activated (thick arrows). This is accompanied by a reduction in signalling through MC1R (dashed arrows), thus enabling CRAF to be activated. (c) When BRAF is mutated (BRAFMUT), the RAS–RAF–MEK–ERK is again hyperactivated, but this occurs by direct activation of MEK by BRAF.
Current Opinion in Genetics & Development 2007, 17:31–39
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New insight into BRAF mutations in cancer Dhomen and Marais 35
controls reduced the 83 gene set to a single gene whose expression changes could be linked to BRAF mutation status [58]. Indeed, when the data from three independent DNA microarray cohorts were analysed, it was possible to define three distinct ‘types’ of melanoma that differ in their proliferative and metastatic potential [58], but mutant BRAF was not restricted to any of these groups, leading the authors to conclude that it is not possible to identify a BRAF (or NRAS) signature profile. Furthermore, the genes highlighted in each of the studies supporting the putative existence of a ‘BRAF signature’ are different, again suggesting that such a signature profile cannot be easily defined. One problem with these studies is that where cells were grown in 10% foetal calf serum, a condition in which the RAF–RAF–MEK–ERK pathway is activated, the contribution from oncogenic BRAF or oncogenic NRAS could have been masked. Microarrays can clearly be used to distinguish different forms of melanoma, but to date this approach has not convincingly enabled us to identify a BRAF or NRAS signature. More studies are therefore required to allow us to harness the power of this approach, but care must be taken in sample preparation and analysis. An alternative and perhaps more promising approach is to simply use the BRAF and NRAS mutation status in combination with comparative genomic hybridization. Using this approach, it is possible to classify melanomas into CSD, non-CSD, acral and mucosal melanomas with 70% confidence [28]. While this level of accuracy is no yet powerful enough for routine clinical use, these studies suggest that if the accuracy can be improved, this approach could be extremely useful for diagnosis and for determining prognosis and making therapeutic potential. Furthermore, it can reveal intriguing new insight into the genetics of melanoma. For example, it has been shown that in melanomas in which BRAF and NRAS are wild type, the genes encoding cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1) are often amplified. Notably, CDK4 and CCND1 transcription is activated downstream of BRAF signalling, implying that their upregulation is crucial in all melanomas, that this can occur through alternative molecular mechanisms and, therefore, that these proteins could be important therapeutic targets for all forms of melanoma.
New RAF networks and germline mutations Perhaps one of the most unexpected findings recently made is that BRAF can activate CRAF (Figure 3). The mechanism appears to involve direct binding of BRAF to CRAF, followed by trans-phosphorylation of CRAF by BRAF [59,60]. CRAF is activated by both wild type BRAF and the mutant forms found in cancer. Wild type BRAF binds to CRAF in a RAS-dependent manner, whereas mutant BRAF binds in a RAS-independent manner [60–63]. Furthermore, this appears to be a oneway relationship, because CRAF does not activate BRAF www.sciencedirect.com
Figure 3
RAF signalling networks in normal and cancer cells. (a) Wild type BRAF activates CRAF in a RAS-dependent manner, and both BRAF and CRAF can activate MEK. In addition, CRAF also activates other effector pathways such as ASK1, MST2, Bcl-2 and NFkB. (b) High activity BRAF mutants activate CRAF independently of RAS and although CRAF can signal to MEK (dashed arrow), BRAF activity is the dominant route leading to MEK activation (bold arrow). However, CRAF can still signal to the other effector pathways. (c) Low activity BRAF mutants are unable to signal to MEK (dashed arrow) but are able to activate CRAF independently of RAS, thereby signalling to MEK and also to the other signalling pathways.
[60]. Thus, BRAF can activate CRAF, and both proteins can then signal to MEK (Figure 3). This signalling network appears to be very important in some cancer contexts. Although our discussion so far has been restricted to BRAFV600E, because this is the most common and therefore clinically the most important mutant, close to 70 other mutations have been identified in BRAF in cancer (www.sanger.ac.uk/genetics/CGP/cosmic/). Crucially, the activities of mutant forms of BRAF range from those with extremely high activity such as BRAFV600E (500-fold activated) to some mutants whose activity is actually Current Opinion in Genetics & Development 2007, 17:31–39
36 Genetic and cellular mechanisms of oncogenesis
impaired and which are therefore unable to activate MEK directly [59]. Importantly however, although the impaired activity mutants are unable to activate MEK, they can still activate CRAF, which is then responsible for stimulating MEK signalling in these cells. The importance of this RAF cross-talking network in normal cell signalling is unclear. However, in the cells that express the impaired activity mutants, it is essential for MEK activation. Notably, CRAF has been implicated in other signalling pathways, especially survival, where CRAF has been shown to bind to apoptosis-stimulating kinases such as mammalian sterile 20-like kinase 2 (MST2) and apoptosis-signal-regulating kinase 1 (ASK1) [64,65], and to kinases such as Roka [66], which controls cytoskeletal rearrangements (Figure 3). CRAF is also implicated in survival mediated by Bcl-2 and NFkB activation [67,68], although the mechanisms of regulation are unclear. The regulation of these additional effectors by CRAF is independent of its kinase activity in some cases, but access to these pathways may be important for regulation of survival and processes such as migration. In the context of cancers in which BRAF is mutated, the activation of CRAF by BRAF may provide access to these novel functions of CRAF, which would normally be activated downstream of RAS and this may be very important to tumour progression. Another surprising recent discovery is the existence of germline mutations in RAS, BRAF and MEK in individuals affected by Noonan, Costello and cardio-facio-cutaneous (CFC) syndromes [69–72], rare genetic disorders that result in a variety of developmental defects such as cardiac anomalies together with short stature and distinct facial abnormalities. The mutations in these individuals are gain-of-function because they stimulate constitutive MEK signalling and in agreement with the role of this pathway in cancer, individuals with Noonan and Costello syndromes have increased risk of specific types of tumours such as rhabdomyosarcoma, certain types of leukaemia and neuroblastoma [73]. Notably, as in cancer, both activating and inactivating mutations have been found in these genetic syndromes [72], and we presume that the inactivating mutants stimulate MEK indirectly through CRAF, as occurs in cancer.
activity of the mutants, BRAFV600E was only two fold more active than wild type BRAF [72] rather than the 500-fold previously reported. The residues that are involved generally only produce more modestly active mutations, and we suspect that the most active mutants would not be tolerated. In this respect, it is worth noting that the KRAS mutations in Noonan syndrome are the lower activity versions. It is presumably only the weaker activating mutants that are tolerated during development. Nevertheless, these are effectively gain-of-function mutations that stimulate elevated, but modest, signalling through the pathway. It is currently unclear how the spectrum of deformities associated with these syndromes are induced by this elevated signalling, or if this knowledge can be used to alleviate some of the non-developmental symptoms suffered by these individuals, but clearly further studies are warranted, particularly using animal models, to investigate the role of this pathway in these syndromes.
Conclusions In the years since BRAF was identified as a potent oncogene, we have learned much about the biology of this pathway in cancer. Although most studies have focussed on melanoma, these studies will have implications for other cancers. It is worth noting that although BRAF is mutated in a higher proportion of melanomas than in colorectal cancers, more people die of colorectal cancer with mutant BRAF than die of BRAF melanomas, because colorectal cancer is a significantly more common disease. Genetic approaches have taught us a great deal about the genetics of cancers in which BRAF is mutated, and these studies are bound to have an enormous impact on the clinical management of these cancers. The existence of rare mutations in BRAF that suppress, rather than stimulate, BRAF kinase activity leads to the discovery that BRAF can activate CRAF in both normal and cancer cells. Furthermore, the surprising existence of RAS, BRAF and MEK mutations in Noonan, Costello and CFC syndromes suggests that weak activating mutations can be tolerated during development. Clearly future genetic approaches will continue to provide important insight into BRAF signalling in cancer, particularly as new tools, such as mouse models [74], become more widely available.
Acknowledgements The specific mutations that are found in cancer are rare in these genetic syndromes, although about half of the syndrome mutations involve codons that are also mutated in cancer. Curiously, despite its prevalence in cancer, BRAFV600E has not been discovered in these genetic syndromes, although it has been reported that some of the mutants in these syndromes are at least as activating as the V600E substitution. However, this seems unlikely. BRAFV600E causes embryonic lethality in mouse models [74], showing that BRAFV600E is not tolerated during development, and in the assay used to measure the Current Opinion in Genetics & Development 2007, 17:31–39
This work is funded Cancer Research UK (Ref C107/A3096) and the Institute of Cancer Research.
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