Mutations of C-RAF Are Rare in Human Cancer Because C-RAF Has a Low Basal Kinase Activity Compared with B-RAF

Research Article

Mutations of C-RAF Are Rare in Human Cancer because C-RAF Has a Low Basal Kinase Activity Compared with B-RAF

Victoria Emuss,1 Mathew Garnett,1 Clive Mason,1 The Cancer Genome Project,2 and Richard Marais1

1The Institute of Cancer Research, Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, London, United Kingdom and 2The Wellcome Trust Sanger Institute, Hinxton, United Kingdom

Abstract binding to other proteins, binding to lipids, and phosphorylation and dephosphorylation of some residues (3, 4). The protein kinase B-RAF is mutated in f8% of human cancers. Here we show that presumptive mutants of the closely Within the kinase domain, phosphorylation of two motifs is required for activation. One of these is called the activation related kinase, C-RAF, were detected in only 4 of 545 (0.7%) segment, which must be phosphorylated on conserved threonine cancer cell lines. The activity of two of the mutated proteins is and serine residues. In B-RAF, these are T599 and S602 (originally not significantly different from that of wild-type C-RAF and designated as T598 and S601 due to a sequencing error in the these variants may represent rare human polymorphisms. The database; ref. 5) and in C-RAF, the corresponding residues are basal and B-RAF–stimulated kinase activities of a third T491 and S494. Structural studies have revealed that when the variant are unaltered but its activation by RAS is significantly activation segment is not phosphorylated, it binds to another reduced, suggesting that it may act in a dominant-negative region of the kinase domain called the glycine-rich loop, trapping manner to modulate pathway signaling. The fourth variant B-RAF in an inactive conformation. T599 phosphorylation is has elevated basal kinase activity and is hypersensitive to thought to disrupt this interaction, releasing the activation activation by RAS but does not transform mammalian cells. segment and reorientating critical residues into the correct Furthermore, when we introduce the equivalent of the most position for catalysis (6). Presumably, similar mechanisms operate common cancer mutation in B-RAF (V600E) into C-RAF, it only in the other isoforms. has a weak effect on kinase activity and does not convert The other motif that must be phosphorylated is called the C-RAF into an oncogene. This lack of activation occurs negative-charge regulatory or N-region. The N-region controls a because C-RAF lacks a constitutive charge within a motif in fundamental difference in how the RAF proteins are regulated. In the kinase domain called the N-region. This fundamental dif- C-RAF, the N-region sequence is 338SSY341Y and phosphorylation of ference in RAF isoform regulation explains why B-RAF is fre- S338 and Y341 is essential for activation by RAS and growth factors. quently mutated in cancer whereas C-RAF mutations are rare. Both sites are conserved in A-RAF (S299 and T302, respectively) but (Cancer Res 2005; 65(21): 9719-26) in B-RAF, Y340 and Y341 are replaced by aspartic acids (D448 and D449) and although S338 is conserved, it is constitutively Introduction phosphorylated (7). All RAF isoforms are only fully activated when The protein kinases of the RAF family, mitogen-activated four negative charges occupy the N-region, either from the protein/extracellular signal-regulated kinase (ERK) kinase (MEK) phosphorylated serine and tyrosine or from the phosphorylated family, and ERK family form a three-tiered cascade that is activated serine and aspartic acids. However, whereas the charges are in a RAS-dependent manner and which is an important regulator present constitutively in B-RAF, in A-RAF and C-RAF they are only of cell fate decisions (1, 2). There are three RAF proteins in mam- acquired under activating conditions. Consequently, the basal mals, A-RAF, B-RAF, and C-RAF, and they share three conserved kinase activity of B-RAF is considerably higher than that of A-RAF and C-RAF and whereas the latter two need both RAS and SRC for regions: CR1 and CR2 within the regulatory NH2 terminus and CR3 encompassing the kinase domain within the COOH terminus activation, B-RAF is fully activated by RAS alone (8). Importantly, (see Fig. 1). RAF proteins are normally cytosolic but they are for all isoforms, activation segment phosphorylation and, in the recruited to the plasma membrane by the small G-protein RAS, case of A-RAF and C-RAF, N-region phosphorylation occur at the plasma membrane, in part explaining why membrane recruitment and this is an essential step for their activation by growth factors, is essential for RAF activation by RAS and membrane-bound cytokines, and hormones. At the membrane, RAF activation occurs receptors. through a highly complex process involving conformation changes, We recently described a systematic sequencing approach that allowed us to identify somatic gain-of-function mutations in the B-RAF gene in f8% of human cancers (5, 9). Importantly, a Note: Supplementary data for this article are available at Cancer Research Online glutamic acid substitution for the valine at codon 600 (V600) (http://cancerres.aacrjournals.org/). accounts for f90% of the B-RAF mutations, demonstrating M. Garnett is currently at the Department of Oncology, Hutchison/Medical V600E Research Council Research Centre, University of Cambridge, Hills Road, Cambridge extraordinary selection for this genetic lesion. B-RAF is CB2 2XZ, United Kingdom. C. Mason is currently at the Molecular Sciences Sareum activated f500-fold; it stimulates constitutive MEK-ERK signaling Ltd., 2 Pampisford Park, London Road, Pampisford, Cambridge CB2 4EE, United in cells and transforms fibroblasts and melanocytes (6, 9–12). Kingdom. Requests for reprints: Richard Marais, Signal Transduction Team, The Institute of Over 45 other cancer-associated mutations have been described in Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom. Phone: 44-20- B-RAF, the majority clustering to the glycine-rich loop and the 7878-3856; Fax: 44-20-7352-3299; E-mail: [email protected]. I2005 American Association for Cancer Research. activation segment, the two regions of the kinase domain that are doi:10.1158/0008-5472.CAN-05-1683 responsible for trapping B-RAF in the inactive conformation (5). www.aacrjournals.org 9719 Cancer Res 2005; 65: (21). November 1, 2005

Figure 1. Amino acid changes in C-RAF. A, schematic of C-RAF. Light shaded boxes, CR1, CR2, and CR3; dark shaded boxes, N-region and the activation segment (Act. Seg.). The positions of the four mutations identified in C-RAF in the cancer cell lines are also indicated. B, alignment of the amino acid sequences for specific regions of A-RAF, B-RAF, and C-RAF from humans (Hs), mice (Mm), chickens (Gg), Xenopus (Xe), zebra fish (Dr), Drosophila melanogaster (Dm), and C. elegans (Ce). Mutations present in C-RAF in human cancer; bold, conserved in the other species. Gray box, N-region.

These mutations are thought to activate B-RAF by disrupting the described previously (7, 8, 13). Blotting for pS338 phosphorylated C-RAF inactive conformation of the kinase and allowing the active was done as described (7). Blotting for phosphorylated MEK was done using conformation to prevail (6). standard techniques and a ppMEK1/2 antibody (9121L, Cell Signaling It is clear that B-RAF is important in human cancer but the Technology, Beverly, MA). role(s) of A-RAF and C-RAF is less evident. In this study, we screen 545 cancer cell lines and over 100 tumor samples for mutations in Results A-RAF and C-RAF. We describe four coding region changes that C-RAF mutations are rare in human cancer. We previously were identified in C-RAF but do not find any such mutations in identified 43 mutations in the B-RAF gene when we sequenced the A-RAF. However, these presumed mutations in C-RAF have weak exons and intron-exon boundaries in 545 cancer cell lines but did effects on C-RAF kinase activity and none of them converts C-RAF not observe any mutations in 341 normal DNA samples (9). Here into a transforming oncogene. We have also created a V492E we have also sequenced all 16 exons of the C-RAF gene and all 15 substitution in C-RAF to mimic the common V600E mutation of exons of the A-RAF gene in these cancer cell lines and normal DNA B-RAF and find that this also has only weak kinase activity and samples, comparing the data to the sequences of A-RAF and C-RAF lacks NIH 3T3 transforming activity. The lack of C-RAF activation available on the National Center for Biotechnology Information by these mutations seems to be due to the lack of intrinsic charge database.3 Whereas 7.9% of the cancer cell lines have mutations in within the N-region, demonstrating that this motif reveals the B-RAF, only four (0.73%) have coding region variations that result in oncogenic potential of B-RAF and explaining why C-RAF is not amino acid changes in C-RAF, and there were no such mutations frequently mutated in human cancer. in A-RAF. We did not observe any such changes in either A-RAF or C-RAF in the normal DNA samples. The presumptive C-RAF mutations are a serine for proline substitution at position 207 (P207S) in Materials and Methods SW684 fibrosarcoma cells; an isoleucine for valine at 226 (V226I) in Genomic sequencing was done as described (9) using the primers shown ChaGo-K-1 lung carcinoma cells; a histidine for glutamine at 335 in Supplementary Table 1. Expression constructs for G12VRAS, C-RAF, and (Q335H) in NCI-H2087 lung adenocarcinoma cells; and a lysine for B-RAF have been described (8). Additional B-RAF and C-RAF substitutions glutamic acid at 478 (E478K) in Ls513 colorectal carcinoma cells were generated by PCR mutagenesis and verified using automated dideoxy (Table 1). sequencing. COS7 cells and NIH 3T3 cells were maintained in DMEM (Life We have also sequenced A-RAF and C-RAF from primary tumors Technologies, Paisley, Scotland) supplemented with 10% or 5% FCS, from 18 breast cancers, 26 lung cancers, 15 testis cancers, and respectively. COS7 cells were transfected with LipofectAMINE (Invitrogen, Carlsbad, CA) as described (8). NIH 3T3 transformation assays were done as described (6). Preparation of COS cell lysates, Western blotting, protein expression measurements, and RAF-coupled kinase assay have all been 3 http://www.ncbi.nlm.nih.gov/entrez/

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20 gastric cancers. In addition, we have sequenced C-RAF from 30 do not activate MEK in COS cells (Fig. 2C) and they do not colorectal cancers and its kinase domain from an additional 10 transform NIH 3T3 cells (Fig. 2D). gliomas, 9 acute lymphoid leukemias, and 20 myeloproliferative The basal kinase activity of Q335HC-RAF is also not elevated disease samples. Finally, we have sequenced the A-RAF kinase compared with that of WTC-RAF (Fig. 2A) and this variant also domain from 10 gliomas. We have not detected either A-RAF or fails to activate MEK in COS cells (Fig. 2C) or to transform NIH C-RAF mutations in any of these primary tumors, again demon- 3T3 cells (Fig. 2D). However, Q335HC-RAF activation by G12VRAS strating that C-RAF mutations are rare and A-RAF mutations are was impaired and only reached 20% to 25% of the levels seen rare or nonexistent. with WTC-RAF (Fig. 2B). Because Q335 is only three amino acids The P207S, V226I, and E478K substitutions do not occur upstream of the N-region of C-RAF (Fig. 1A), we examined if the coincident with mutations in either B-RAF or any of the RAS genes reduced activation of Q335HC-RAF is due to disrupted S338 whereas Q335H occurs coincident with activating mutations in both phosphorylation. Using an antibody that only binds to C-RAF B-RAF (L597V) and N-RAS (Q61K) in NCI-H2087 cells (Table 1). P207 when S338 is phosphorylated (7), we find that antibody binding is and V226 are between CR1 and CR2 in a region of the protein of significantly reduced in Q335HC-RAF (Fig. 3A). One interpretation which function is unknown; Q335 is between CR2 and CR3, three of this result is that the Q335H substitution disrupts antibody residues upstream of the N-region; and E478K is within the kinase binding and not S338 phosphorylation; thus, we used an alanine- domain (Fig. 1). P207, V226, and Q335 are reasonably well, but not scan mutagenic approach to define that epitope. Antibody absolutely, conserved in C-RAF from several species but they are binding is disrupted when R336, D337, and S338 are mutated to not conserved in A-RAF or B-RAF from any species or, with the alanine, but importantly not when Q335 is mutated (Fig. 3B). exception of Q335 in Caenorhabditis elegans, in the single RAF Thus, the antibody epitope seems to be ‘‘RDpS’’ and we conclude paralogues from lower organisms (Fig. 1B). In contrast, E478 is that Q335 is not part of it and that S338 phosphorylation on highly conserved, with glutamic acid being found at the Q335HC-RAF is reduced in the presence of G12VRAS. Finally, corresponding position in all RAF orthologues and paralogues because Q335HC-RAF occurs coincident with L597VB-RAF in NCI- from lower and higher organisms. Indeed, a review of the human H2087 cells (Table 1) and we recently reported the surprising genome reveals that 27.6% of all human kinases possess a finding that mutant forms of B-RAF can activate WTC-RAF (6), we glutamic or aspartic acid at the corresponding position (data not tested whether Q335HC-RAF can be activated by L597VB-RAF. Unlike shown), demonstrating a strong selection for an acidic amino acid the differences observed with G12VRAS, L597VB-RAF activates at this position in many kinases. Importantly, there are no reports Q335HC-RAF and WTC-RAF to similar levels (Fig. 3C). showing that the amino acids in B-RAF that correspond to P207, The E478K substitution activates C-RAF. In contrast to the V225, and Q335 (T312, T330, and R443, respectively) are mutated other presumptive mutants, the E478K variant does have elevated in human cancer whereas the equivalent of E478 (E586 in B-RAF) kinase activity, its basal kinase activity being 25-fold higher than is mutated and, intriguingly, also to a lysine (9). E586KB-RAF has that of WTC-RAF (Fig. 4A). The activation of C-RAF by E478K f130-fold elevated kinase activity (6). compares poorly to the enhanced activity of E586KB-RAF, which P207S, V226I, and Q335H substitutions do not activate we have shown is f130-fold more active than WTB-RAF (6). C-RAF. For characterization, we expressed myc-epitope tagged Importantly, whereas E586KB-RAF stimulates strong constitutive versions of these presumptive C-RAF mutants in COS cells and MEK-ERK signaling in COS cells (6, 14), MEK activation by E478KC- measured their activity in a kinase cascade assay using RAF is weak (Fig. 4B); furthermore, whereas E586KB-RAF transforms glutathione S-transferase (GST)-MEK, GST-ERK, and myelin basic NIH 3T3 cells, E478KC-RAF does not (Fig. 4C). Remarkably, however, protein as substrates, with ATP at a physiologically relevant whereas WTC-RAF is activated f100-fold by G12VRAS, E478KC-RAF concentration of 5 mmol/L (6). Under these conditions, C-RAF is activated over 500-fold (Fig. 4A), producing a level of activation has low basal kinase activity but is activated 80- to 120-fold by that is an impressive 13,000- to 14,000-fold higher than the basal oncogenic RAS (G12VRAS; Fig. 2A and B). The basal kinase activity of WTC-RAF. Thus, E478KC-RAF is hypersensitive to activities of P207SC-RAF and V266IC-RAF are similar to that of activation by oncogenic RAS, suggesting that this mutant can be WTC-RAF and their activation by G12VRAS is also similar to that of strongly activated by secondary signals. WTC-RAF although it was consistently lower (Fig. 2A and B). In We were intrigued by the distinct responses of C-RAF and B-RAF line with their lack of elevated basal kinase activity, these mutants to the E478K/E586K mutations and wished to investigate the

Table 1. C-RAF mutations in human cancer cell lines

Cell line Tumor type C-RAF RAS B-RAF

Amino acid Nucleotide

SW684 Fibrosarcoma P207S C619T WT WT ChaGo-K-1 Lung carcinoma V226I G676A WT WT NCI-H2087 Lung adenocarcinoma Q335H G1005T Q61KN-RAS L597V Ls513 Colorectal carcinoma E478K G1432A WT WT

NOTE: Nucleotide changes and the corresponding amino acid changes are shown for individual cell lines. The statuses of RAS and B-RAF (amino acid changes only) in these cells are also presented.

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10,000-fold, a level that is commensurate with the activity of E586KB-RAF (Fig. 4D). DDC-RAF also synergizes with E478KC-RAF to activate MEK in COS cells (Fig. 4B) and to transform NIH 3T3 cells (Fig. 4C). We also did the reverse experiment, neutralizing the charge within the N-region of E586KB-RAF by substituting S446, S447, D448, and D449 with alanines (AAAA,E586KB-RAF). Introducing these substitutions into E586KB-RAF significantly reduces its kinase activity (Fig. 4E) and to dissect the contributions made by the individual charged amino acids within the N-region, we indepen- dently substituted S446/S447 and D448/D449 with alanines. Both the S446A/S447A (AADD,E586KB-RAF) and the D448A/D449A

Figure 2. Characterization of P207SC-RAF, V226IC-RAF, and Q335HC-RAF. A, basal kinase activity; B, G12VRAS-activated kinase activity. Columns, mean from one assay done in triplicate; bars, SD. Similar results were obtained in at least three experiments. C, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or the indicated mutants. Oncogenic RAS was included where indicated. D, NIH 3T3 transformation assay. Transformation efficiency of NIH 3T3 cells by C-RAF (WT) or the indicated mutants. V600EB-RAF is included as a positive control. Columns, mean of three independent determinations; bars, SD. Right, representative results from one assay. molecular mechanism underlying this difference. As described in Introduction, a key difference in regulation of the RAF proteins is mediated by their N-regions; thus, we tested whether this region determines how C-RAF and B-RAF respond to the E478K/E586K Figure 3. RAS-dependent phosphorylation of Q335HC-RAF is impaired but its activation by mutant B-RAF is unaltered. A, Western blot for C-RAF mutations. Y340 and Y341 of C-RAF were substituted with aspartic WT Q335H DD and pS338 in COS cells expressing C-RAF or C-RAF. Where indicated, acids (creating C-RAF) to mimic the N-region of B-RAF (Fig. 1B). oncogenic RAS was coexpressed. B, Western blot for pS338 and total C-RAF in COS cells expressing C-RAF or the indicated mutants. Where indicated, As previously shown (14, 15), this double-substituted protein is G12V L597V f oncogenic RAS was coexpressed. C, RAS- and B-RAF–stimulated weakly activated ( 3-fold; not obvious in Fig. 4D) but, strikingly, C-RAF kinase activities. Columns, mean from one assay done in triplicate; DD,E478K it synergizes with E478K ( C-RAF) to activate C-RAF over bars, SD. Similar results were obtained in at least two experiments.

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Figure 4. E478KC-RAF has elevated kinase activity but is not an oncogene. A, basal and G12VRAS-stimulated C-RAF kinase activities. Columns, mean from one assay done in triplicate; bars, SD. Activities are expressed as fold activation with respect to the wild-type protein. B, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or the indicated mutants. C, NIH 3T3 transformation assay. Transformation efficiency of NIH 3T3 cells by C-RAF or B-RAF (WT ) or the indicated mutants. Columns, mean of three independent assays; bars, SD. Right, representative results from one assay. D, C-RAF kinase activity; E, B-RAF kinase activity. Columns, mean from a single assay done in triplicate; bars, SD. Similar results were obtained in at least three independent experiments.

(SSAA,E586KB-RAF) substituted proteins have significantly reduced Once again, we were struck by the different responses of C-RAF kinase activity (Fig. 4E), demonstrating that S446 phosphorylation and B-RAF to this conserved mutation; thus, we examined if this and the aspartic acids of the N-region both contribute to the is also due to the lack of N-region charge in C-RAF. When the elevated kinase activity of E586KB-RAF. Finally, we show that the V492EC-RAF mutation is combined with DDC-RAF (DD,V492EC-RAF), transforming activity of E586KB-RAF is significantly reduced when there is a synergistic augmentation in kinase activity, resulting the N-region charge is disrupted (Fig. 4C). We conclude that E478KC- in a kinase of which basal activity is elevated over 1,000-fold RAF is strongly activated by mutations that introduce a constitutive (Fig. 6A). There is also a synergistic activation of MEK in COS negative charge into its N-region whereas the activity of E586KB-RAF cells (Fig. 6B) and a synergistic augmentation of transforming is compromised by the inverse changes. activity (Fig. 6C). C-RAF activation by the V492E substitution is weak because Finally, we did the reverse experiment, neutralizing the N- its N-region lacks charge. We were intrigued to note that whereas region of V600EB-RAF by creating AAAA,V600EB-RAF. This causes an V600 of B-RAF is mutated in 6% to 7% of human cancers (5), we did f15-fold reduction in kinase activity but we note that the kinase not observe any mutations of the equivalent codon (V492) in is still f35-fold more active than WTB-RAF (Fig. 7A). Importantly, C-RAF in our 545 cancer cell lines or the 148 primary tumor AAAA,V600EB-RAF still activates MEK in COS cells (Fig. 6B) and samples. We therefore tested how a V492E substitution affected transforms NIH 3T3 cells (Fig. 7C) albeit at reduced levels. C-RAF kinase activity. V492EC-RAF is f45-fold more active than WTC-RAF (Fig. 5A), a level of activation that contrasts strongly with the f500-fold activation seen with the corresponding mutation in Discussion B-RAF (V600EB-RAF; ref. 6). Furthermore, whereas V600EB-RAF We have identified four coding region variants in C-RAF that stimulates strong constitutive MEK/ERK signaling in mammalian occur in human cancer cell lines but are not seen in normal DNA cells (9) and is transforming (Fig. 2D), V492EC-RAF only stimulates samples. These four variants convert P207 to serine, V226 to weak MEK activity in COS cells (Fig. 5B) and does not transform isoleucine, Q335 to histidine, and E478 to lysine. Dealing with NIH 3T3 cells (Fig. 5C). P207S and V226I first, we did not detect any significant changes in www.aacrjournals.org 9723 Cancer Res 2005; 65: (21). November 1, 2005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research either their basal or RAS-stimulated kinase activity when MEK was genesis, it seems that these variants do not have an obvious effect used as a substrate. It is possible that these substitutions affect on signaling or a clear effect on proliferation and with the caveat other currently unknown and MEK-independent functions of that their effects may only be evident in specific cell types, it C-RAF; however, P207 and V226 are located in a region of the seems unlikely that they play a significant role in human cancer. protein of which function is unknown and they are not even Q335 is also not conserved in all C-RAF orthologues or in the other conserved in all C-RAF orthologues or in other RAF paralogues RAF paralogues. However, it is three amino acids upstream from a variety of higher and lower organisms. Furthermore, the of the N-region and although this substitution also fails to affect corresponding residues are not mutated in B-RAF in human C-RAF basal kinase activity, it does suppress its activation by cancer. One interpretation of our data is that these variants are rare oncogenic RAS. Our alanine-scan mutagenesis data show that Q335 single-nucleotide polymorphisms that play no role in the growth of is not part of the pS338 antibody epitope, which is consistent with the cancer cell lines in which they were identified. If they are the fact that this antibody binds to B-RAF when S446 is single-nucleotide polymorphisms, they have not been previously phosphorylated (7) and B-RAF has an arginine at the position described4 and we did not observe them in our 341 normal DNA equivalent to Q335 (Fig. 1B). Our data suggest that the Q335H samples, suggesting that they are extremely rare. An alternative substitution disrupts S338 phosphorylation in the presence of explanation is that these are somatic mutations that occur due to G12VRAS, causing reduced C-RAF activation. It is not surprising that collateral DNA damage during the development of these cancers such a subtle substitution should have such a profound effect. With or that have occurred in the cell lines during in vitro culture. the exception of Q335, the amino acids surrounding the N-region of Unfortunately, in the absence of control DNA from the patients C-RAF and B-RAF are well conserved (Fig. 1B), and yet, whereas S338 from whom the cell lines were derived, it is not possible to is only phosphorylated at the plasma membrane, S446 is phosphor- determine which of these possibilities is correct. Whatever their ylated in the cytosol, demonstrating exquisite selectivity by the kinases involved. Finally, our data show that the Q335H substitution does not affect C-RAF activation by L597VB-RAF, which is consistent with our recent finding that B-RAF activates C-RAF through a distinct mechanism that is largely independent of S338 phosphorylation.5 We have been unable to study the biological function of Q335HC- RAF because we have not been able to apply RNA interference technology to NCI-H2087 cells. However, it has been shown that excessive ERK signaling can induce cell cycle arrest or senescence through induction of cell cycle inhibitory proteins such as p27 and p21 (16–21). Thus, in cancer, ERK signaling must be kept within narrow limits that are sufficient to stimulate proliferation but not so high as to induce cell cycle arrest. We speculate that in some cancers, mutations or other upstream events that stimulate excessive pathway activity are incompatible with proliferation. This may be a protective mechanism but in these circumstances, precancerous cells must develop strategies to tone down this signaling to progress. We and others recently reported the identification of mutant forms of B-RAF involving residue D594 in human cancer that are devoid of kinase activity (6, 12, 22). These account for f1% of B-RAF mutations, and thus are too common to be random, and similar mutations are not observed in either C-RAF or A-RAF. Importantly, although coincident mutations in B-RAF and RAS are very rare in human cancer (9), f30% of the inactive mutants occur in cancers that also harbor RAS mutations and we have argued that the D594 mutants could act in a dominant- negative manner to suppress excessive RAS-MEK signaling (5). Thus, it is intriguing that in addition to the Q335HC-RAF substitution, NCI-H2087 cells harbor activating mutations in B-RAF and N-RAS, which is, to our knowledge, the only example of a cancer cell line harboring amino acid changes in three components of this pathway. Perhaps if oncogenic RAS and activated B-RAF both activate C-RAF, MEK signaling is excessive and the Q335H mutation is required to reduce signaling from one of these upstream inputs. Thus, this mutant could be acting in a dominant-negative manner and we are Figure 5. C-RAF is not activated by the V492E mutation and is not transforming. currently developing genetic approaches to test this model. A, C-RAF kinase activity. Columns, mean from one assay done in triplicate; It is clear that B-RAF is important in human cancer and in the bars, SD. Activities are expressed as fold activation with respect to the wild-type protein and similar results were obtained in at least three experiments. classic sense, it is an oncogene because activated forms can B, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or V492EC-RAF. Where indicated, G12VRAS was coexpressed. C, NIH 3T3 transformation assay. Transformation efficiency of NIH 3T3 cells by C-RAF or B-RAF (WT) or the indicated mutants. Columns, mean of three independent 4 http://www.ncbi.nlm.nih.gov/ assays; bars, SD. Right, representative results from one assay. 5 M. Garnett and R. Marais, submitted for publication.

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Figure 6. V492EC-RAF becomes activated and transforming when negative charge is introduced at the N-region. A, basal and, where indicated, RAS-stimulated kinase activities. Columns, mean from one assay done in triplicate; bars, SD. Activities are expressed as fold activation with respect to the wild-type protein and similar results were obtained in at least three experiments. B, Western blot for C-RAF, ppMEK, and total MEK in COS cells expressing C-RAF or the specified mutants. Where indicated, G12VRAS was coexpressed. C, NIH 3T3 transformation assay. Transformation efficiency of NIH 3T3 cells by WTC-RAF or WTB-RAF or the indicated mutants. Columns, mean of three independent assays; bars, SD. Right, representative results from one assay. transform immortalized fibroblasts and other cell lines. Critically, and are not transforming, it is possible that they could be rare ‘‘oncogenic’’ B-RAF does not induce cancer by itself because a high polymorphisms that predispose their carriers to specific forms of proportion of common nevi harbor mutations in the B-RAF gene cancer; further studies are under way to examine this possibility. but are not cancerous (23). Notably, of the variants we identified, It may be surprising that A-RAF and C-RAF mutations in cancer only E478KC-RAF has elevated kinase activity, and yet even this are so rare because both isoforms can be converted into mutant does not seem to be a classic oncogene because it does not transforming agents in experimental systems, typically by removing transform NIH 3T3 cells. However, it is hypersensitive to activation their NH2-terminal regulatory domains (for review, see ref. 24). by RAS and synergizes with the Y340D/Y341D double mutations to Indeed, a chromosomal inversion that essentially creates just this activate C-RAF and augment transforming activity. Thus, perhaps type of agent in B-RAF has been identified in a single case of the oncogenic potential of E478KC-RAF is only apparent in some cell human thyroid cancer linked to the Chernobyl nuclear accident contexts. In common with P207SC-RAF and V226IC-RAF, we cannot (25). However, the genetic approach that we use cannot identify determine whether Q335HC-RAF and E478KC-RAF are somatic muta- large deletions, gene inversions, or translocations, and thus it tions or rare human polymorphisms. However, because Q335HC-RAF is possible that they were present in our samples but went and E478KC-RAF lack significantly elevated basal kinase activity undetected. Another surprise is that previous experimental

Figure 7. The kinase activity of V600EB-RAF is impaired by neutralization of N-region charge but it is still transforming. A, B-RAF kinase activity. Columns, mean from one assay done in triplicate; bars, SD. Activities are expressed as fold activation with respect to WTB-RAF and similar results were obtained in at least three experiments. B, Western blot for B-RAF, ppMEK, and total MEK in COS cells expressing wild-type B-RAF or the specified mutants. C, NIH 3T3 transformation assay. Transformation efficiency of NIH 3T3 cells by WTB-RAF or the indicated mutants. Columns, mean of three independent assays; bars, SD. Right, representative results from one assay. www.aacrjournals.org 9725 Cancer Res 2005; 65: (21). November 1, 2005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research approaches have been used to induce transforming point is considerably higher than that of C-RAF (8) and it is clear that mutations in full-length C-RAF, most of which are outside the E586KB-RAF and V600EB-RAF have even higher basal kinase kinase domain and a surprisingly large number are within CR2 activities. We show here that C-RAF must be activated over (26–28), and yet we did not detect any corresponding mutations 10,000-fold to achieve the same level of activity as E586KB-RAF, in our samples. Thus, although it is possible to create oncogenic and yet this is still only f25% of the activity of V600EB-RAF. versions of C-RAF, in practice mutations in human cancer seem to Furthermore, although DD,V492EC-RAF is over 1,000-fold more active be almost exclusively in B-RAF. This suggests that either the rates than WTC-RAF, this is still only 2% to 5% of the activity of V600EB- of mutation of the RAF genes differ significantly or the regulatory RAF. Presumably, this explains why AAAA,V600EB-RAF is still networks of the different isoforms differ in such a manner as to transforming: its activity is about 2,500-fold above that of WTC- select against A-RAF and C-RAF mutations. RAF. Thus, although the N-region is clearly important, it is not the Notwithstanding this reasoning, our data provide another only factor contributing to the elevated basal kinase activity of rational explanation of why C-RAF mutations are rare in human B-RAF. Further analysis of the differences between these proteins cancer. We show that the V492E substitution activates C-RAF f45- is warranted and it does not seem to be simply due to the fact that fold and over 1,000-fold when the N-region is charged. Similarly, C-RAF cannot be activated to the same level as B-RAF, as shown V600E activates B-RAF f500-fold, unless the N-region is with DD,E478KC-RAF. neutralized, then this decreases to f35-fold. Clearly, the V492/ We conclude that it is the key differences between B-RAF and V600 mutations cooperate with N-region charge to activate the C-RAF that regulate the different levels of basal kinase activities RAF proteins and, working in conjunction, they act to convert C- that account for the fact that whereas B-RAF mutations occur in RAF into an oncogene. Similar results are seen with the E478K f8% of cancers, C-RAF mutations are rare and only occur in mutation, demonstrating the importance of the N-region in specific cellular contexts. Finally, we note that our data could be revealing the transforming potential of the mutant C-RAF proteins. interpreted to suggest that approaches to target the kinase Presumably in the absence of this N-region charge, even if C-RAF responsible for phosphorylating the N-region of B-RAF would not mutations did occur in cancer, they would not enhance its activity provide effective anticancer therapies because residual trans- sufficiently to convert C-RAF into an oncogene and, hence, they do forming is activity retained. However, if agents such as these not provide an advantage and so are not selected. Alternatively, if were combined with agents that directly target oncogenic B-RAF they occurred coincident with a second event, they may even kinase activity, they could be of considerable value. stimulate excessive signaling and actually antagonize tumor progression. Presumably, the same holds true for A-RAF. Although the N-region clearly plays an important role in Acknowledgments augmenting the response of the RAF proteins to the mutants, this Received 5/16/2005; revised 7/11/2005; accepted 8/31/2005. is not the only region that seems to reveal the oncogenic potential Grant support: Cancer Research UK grant C107/A3096, The Institute of Cancer of B-RAF. We note that when the N-regions of E586KB-RAF and Research, and The Wellcome Trust. V600E The costs of publication of this article were defrayed in part by the payment of page B-RAF are neutralized, they retain some transforming activity. charges. This article must therefore be hereby marked advertisement in accordance We have previously shown that the basal kinase activity of B-RAF with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Mutations of C-RAF Are Rare in Human Cancer because C-RAF Has a Low Basal Kinase Activity Compared with B-RAF

Victoria Emuss, Mathew Garnett, Clive Mason, et al.

Cancer Res 2005;65:9719-9726.

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