B-RAF Is a Therapeutic Target in Melanoma

Maria Karasarides1, Antonio Chiloeches1,4, Robert Hayward1, Dan Niculescu-Duvaz2,Ian Scanlon2, Frank Friedlos2, Lesley Ogilvie2, Douglas Hedley2, Jan Martin2, Christopher J Marshall3, Caroline J Springer2 and Richard Marais*,1

1Signal Transduction Team, The Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK; 2Gene and Oncogene Targeting Team, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK; 3Oncogene Team, The Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

B-RAF is a serine/threonine-specific protein kinase that is MEK inhibitor U0126 blocked this constitutive ERK mutated in approximately70% of human melanomas. signalling (Figure 1b) and also blocked DNA synthesis However, the role of this signalling molecule in cancer is in these cells (Figure 1c). In contrast, ERK was not unclear. Here, we show that ERK is constitutively constitutively activated in CHL cells (Figure 1a), a activated in melanoma cells expressing oncogenic B- melanoma cell line that expresses wild-type B-RAF RAF and that this activityis required for proliferation. B- (Davies et al., 2002), and U0126 did not block their RAF depletion bysiRNA blocks ERK activity,whereas ability to synthesize DNA (Figure 1c). The low levels of A-RAF and C-RAF depletion do not affect ERK ERK activity in melanoma cells that express wild-type signalling. B-RAF depletion inhibits DNA synthesis and B-RAF compared to those that express oncogenic induces apoptosis in three melanoma cell lines and we B-RAF is consistent with previous studies (Satya- show that the RAF inhibitor BAY43-9006 also blocks moorthy et al., 2003). These results demonstrate that ERK activity, inhibits DNA synthesis and induces cell melanoma cells harbouring oncogenic B-RAF have death in these cells. BAY43-9006 targets B-RAF signal- constitutively elevated ERK activity and that MEK- ling in vivo and induces a substantial growth delayin ERK signalling is required for their proliferation. melanoma tumour xenografts. Our data demonstrate that To examine B-RAF in more detail, we used RNA oncogenic B-RAF activates ERK signalling, induces interference to deplete individual RAF isoforms. Under proliferation and protects cells from apoptosis, demon- the experimental conditions used here, each small strating that it is an important therapeutic target and thus interfering RNA (siRNA) oligonucleotide was specific provides novel strategies for clinical management of for its target protein. The A-RAF siRNA depleted A- melanoma and other cancers. RAF, but not B-RAF or C-RAF, the B-RAF siRNA Oncogene (2004) 23, 6292–6298. doi:10.1038/sj.onc.1207785 only targeted B-RAF and the C-RAF siRNA only Published online 21 June 2004 targeted C-RAF (Figure 1d). Curiously, under some conditions we observed some loss in specificity with Keywords: B-RAF; melanoma; RNA interference; pro- these reagents (data not shown) despite the fact that liferation; apoptosis; BAY43-9006 they were selected to be specific, so as an additional control, we used a nonspecific ‘scrambled’ oligonucleotide, which did not affect expression of any of the RAF isoforms (Figure 1d). B-RAF depletion suppressed the To investigate the role of oncogenic B-RAF signalling in constitutive ERK signalling in A375, Colo829 and WM- melanoma we first examined MEK-ERK signalling in 266-4 cells (Figure 1d) and as expected from the U0126 melanoma cell lines that harbour activating mutations in data, it also caused a block in DNA synthesis the B-RAF gene. Three melanoma cell lines expressing (Figure 1e). Unfortunately, we have been unable to oncogenic B-RAF (A375: V599EB-RAF; Colo829: V599EB- develop siRNA molecules that distinguish between the RAF; WM-266-4: V599DB-RAF) (Brose et al., 2002; wild type and oncogenic B-RAF and the siRNA used Tuveson et al., 2003; Davies et al., 2002) have here depletes both WTB-RAF and V599EB-RAF. Never- constitutively elevated ERK activity as shown by theless, we have shown that ERK activity is only Western blotting with an antibody that only binds to elevated in melanoma cells expressing activated B-RAF the dually phosphorylated, active forms (Figure 1a). The mutants, that B-RAF stimulates constitutive ERK signalling and that this is required for proliferation. *Correspondence: R Marais; E-mail: [email protected] Furthermore, B-RAF siRNA does not block ERK 4Current address: Departamento de Bioquimica y Biologia Molecular, signalling in RAS transformed melanoma cells (Well- Campus Universitario-Facultad de Medicina, Ctra. de Barcelona, brock et al, 2004), demonstrating that wild-type B-RAF Km.33.600, 28871 Alcala de Henares, Madrid, Spain Received 2 October 2003; revised 10 March 2004; accepted 2 April 2004; is not required for the growth of these cells. Together published online 21 June 2004 these data strongly suggest that oncogenic B-RAF B-RAF is a therapeutic target in melanoma M Karasarides et al 6293

Figure 1 Oncogenic B-RAF stimulates constitutive ERK signalling and proliferation in melanoma cells. (a) CHL, A375, Colo829 and WM-266-4 whole-cell lysates (30 mg) were prepared as previously described (Marais et al., 1997; Chiloeches et al., 2001) and Western blotted using an antiphospho-ERK (ppERK) monoclonal antibody (Sigma). (b) Western blot using anti-pp-ERK antibody of the indicated cell lines after 24 h treatment with BAY43-9006 (B; 10 mM) or UO126 (U; 10 mM). The same membrane was blotted with an anti-tubulin antibody (Sigma) as a loading control. (c)[3H]thymidine incorporation in CHL, A375, Colo829 and WM-266-4 cells in the presence of DMSO or U0126 (10 mM) as previously described (Marais et al., 1996). (d) Western blot showing expression levels of A- RAF (C-20; Santa Cruz), B-RAF (F-7; Santa Cruz), C-RAF (Transduction Labs), ppERK (Sigma) and total ERK (clone 122; (Leevers, 1993)) in A375, Colo829 and WM-266-4 (72 h after siRNA) transfected with the indicated siRNA oligonucleotides (M: Mock; S: scrambled; A: A-RAF; B: B-RAF; C: C-RAF). Transfections with siRNA were performed as we previously described (Wellbrock et al., 2004) using the following siRNA sequences: A-RAF: AAC AAC ATC TTC CTA CAT GAG; B-RAF: AAA GAA TTG GAT CTG GAT CAT (Hingorani et al., 2003); C-RAF: AAU AGU UCA GCA GUU UGG CUA; Scrambled: AAA CCG UCG AUU UCA CCC GGG. (e)[3H]thymidine incorporation into A375, Colo829 and WM-266-4 cells treated with the indicated siRNA oligonucleotides. siRNA transfections and [3H]thymidine incorporation performed as described above

Oncogene B-RAF is a therapeutic target in melanoma M Karasarides et al 6294 stimulates constitutive MEK-ERK signalling in mela- caspase activation (Erhardt et al., 1999). A role for C- noma cells and that this signalling is required for RAF in survival is also consistent with previous studies proliferation. (Mercer and Pritchard, 2003). Haematopoetic cells and Unlike B-RAF, C-RAF and A-RAF depletion did MEFs from C-raf null mice have increased rates of not suppress ERK activity in the melanoma cell lines spontaneous apoptosis and are more sensitive to (Figure 1d), but it still affected DNA synthesis apoptotic signals (Hu¨ ser et al., 2001; Mikula et al., (Figure 1e). In A375 cells, A-RAF and C-RAF siRNA 2001). Furthermore, C-RAF antisense oligonucleotides both suppressed DNA synthesis by approximately 50%. induced apoptosis in several cancer cell lines (Lau et al., In Colo829 cells, C-RAF siRNA was more efficient than 1998) that are wild type for B-RAF (Davies et al., 2002). A-RAF siRNA whereas in WM-266-4 cells, A-RAF Our data demonstrate that in melanoma cells, B-RAF siRNA was more efficient than C-RAF siRNA. In plays a major role in survival whereas C-RAF plays a Colo829 cells, C-RAF siRNA was as efficient as B-RAF lesser role. Furthermore, the C-RAF survival signal is siRNA and in WM-266-4 cells, A-RAF siRNA was as ERK independent, which is consistent with our previous efficient as B-RAF siRNA. Thus, in these cells, A-RAF data demonstrating that in MEFs the C-RAF survival and C-RAF are not required to signal to MEK and signal is independent of its kinase activity (Hu¨ ser et al., ERK under normal tissue culture conditions and they 2001). Future studies will focus on how B-RAF survival fail to compensate for B-RAF loss. However, we have signals interact with other survival pathways in mela- recently shown that in RAS transformed melanoma nocytes. For example, the proapototic protein APAF-1 cells, B-RAF depletion does not result in ERK is downregulated in approximately 40% of melanomas inactivation or suppression of DNA synthesis (Well- (Soengas et al., 2001) and it will be interesting to brock et al, 2004), suggesting that A-RAF and/or compare B-RAF survival signals in cells where APAF-1 C-RAF can compensate for B-RAF loss under some is or is not downregulated. circumstances. Nevertheless, despite the fact that Our data establish that B-RAF is required for both A-RAF and C-RAF do not appear to play a role in growth and survival of melanoma cells in vitro,sowe MEK-ERK signalling, they still contribute to the tested whether it is a therapeutic target in vivo. First we regulation of DNA synthesis in these cell lines. Possible tested whether the C-RAF inhibitor BAY43-9006 explanations for this ERK-independent role are (Lyons et al., 2001) was active against oncogenic B- that these RAF isoforms act as scaffolds or participate RAF. BAY43-9006 blocked V599EB-RAF in vitro kinase in other signalling pathways. For example, C-RAF activity with an IC50 of B43 nM (Figure 3a), so the does possess activities that are independent of its compound is somewhat less active against V599EB-RAF kinase activity (Hu¨ ser et al., 2001) and, thus, further than against activated wild-type C-RAF (IC50 B6– studies are required to elucidate the role that C-RAF 12 nM; Lyons et al., 2001). Nevertheless, BAY 43-9006 and A-RAF play in DNA synthesis in melanoma cells. blocked the constitutive ERK activity in A375, Colo829 We next examined whether B-RAF played a role in and WM-266-4 cells (Figure 1b), and since our data cell survival by examining effects on the cell-cycle demonstrate that ERK activity is dependent on B-RAF profiles of the cells. B-RAF depletion induced a but not C-RAF or A-RAF (Figure 1d), we conclude that significant increase in the sub-G1 population of all BAY 43-9006 is able to block B-RAF activity in three cell lines after 96 h (Figure 2a), induced caspase 3 melanoma cells. BAY43-9006 also blocked DNA activation (Figure 2b) and induced PARP cleavage synthesis (Figure 3b) and induced death in all three (Figure 2c). A-RAF depletion failed to induce any of lines (Figure 3c). U0126 also induced death in all three these markers of apoptosis (Figure 2) and although C- cell lines (data not shown). We do note that BAY43- RAF induced a small increase in the sub-G1 population 9006 is less active in cells than against V599EB-RAF in in Colo829 and WM-266-4 cells, it failed to stimulate vitro, inhibiting the growth of A375, Colo829 and WM- efficient caspase 3 activation or PARP cleavage 266-4 cells at concentrations of 4–6.5 mM (Table 1), (Figure 2). Taken together, these data demonstrate that possibly because the drug is not fully permeable to cells, in addition to stimulating cell proliferation, B-RAF also or because of the high ATP concentrations inside cells. provides a survival signal to these melanoma cell lines. Nevertheless, since BAY43-9006 does target ERK While C-RAF also appears to play a role in maintaining signalling in cells, blocks proliferation and induces cell cell viability, A-RAF does not seem to provide a death, we tested its activity in vivo. survival signal. WM-266-4 cells were grown as human tumour A role for B-RAF in survival is consistent with xenografts in the flanks of nude mice and after 6 days previous studies. It has been shown that inhibition of when the tumours were palpable, the mice were orally MEK sensitized one out of four melanoma cell lines to dosed with 50 mg/kg BAY43-9006 or vehicle control cisplatin, an agent that induces apoptosis (Mandic et al., once a day for 17 days. Tumour diameters were 2001) and mice that are null for B-raf die due to measured and their volumes were calculated. BAY43- catastrophic endothelial cell apoptosis in utero (Woj- 9006 elicited a significant growth retardation (55% at 17 nowski et al., 1997). Furthermore, neuronal cells that days of treatment: two-tailed Mann–Whitney test: are null for B-raf display increased apoptosis in the Po0.05) compared to the vehicle treated controls absence of nerve growth factor (Wiese et al., 2001) and (Figure 4a). The tumours were excised on the last day WTB-RAF protects fibroblasts from apoptosis in the of treatment and immuno-stained for active MEK, using absence of growth factors, apparently by blocking an antibody that only binds to phosphorylated, active

Oncogene B-RAF is a therapeutic target in melanoma M Karasarides et al 6295

Figure 2 Oncogenic B-RAF provides a survival signal to melanoma cells. Cells were transfected with the indicated siRNA oligonucleotides (M: Mock; S: scrambled; A: A-RAF; B: B-RAF; C: C-RAF). (a) Cell cycle profiles from A375, Colo829 and WM- 266-4 cells treated with the indicated siRNA oligonucleotides showing the proportion of dead cells in the sub-G1 fraction (indicated by bars) determined 96 h after siRNA transfection. Both floating and adherent cells were collected, washed in PBS and fixed in ethanol/ PBS (70/30%). Samples were treated with propidium iodide (40 mg/ml) and RNAse A (100 mg/ml), incubated at 371C for 30 min and analysed by Fluorescence-activated cell-sorting (FACS) using standard protocols. (b) Immunostaining of A375, Colo829 and WM- 266-4 cells showing caspase 3 activation 96 h after transfection with the indicated siRNA oligonucleotides (supplemented with 5% FBS). Samples were fixed in 4% formaldehyde/PBS, washed 3 Â with PBS, permeabilized (0.2% Triton X-100 : PBS) and blocked (0.1% Triton X-100 : BSA : PBS) for 30 min at room temperature. Activated caspase 3 was detected using a polyclonal antibody recognizing caspase 3 cleaved at Asp 175 (9661S; Cell Signalling) followed by an anti-rabbit FITC secondary antibody (Jackson ImmunoResearch). (c) Western blots showing PARP expression in A375, Colo829 and WM-266-4 cells at 72 h after siRNA transfection using an anti-PARP monoclonal antibody (Pharmingen). The arrow indicates full-length PARP and the asterisk the cleavage product

MEK1 and MEK2. There was a substantial reduction in consistent with a recent study demonstrating similar MEK activity in tumours from the BAY43-9006 treated results in WM793 melanoma cells (Hingorani et al., mice compared to the vehicle treated controls 2003). Our study also shows that BAY43-9006 targets B- (Figure 4b). RAF in vitro and in vivo, and was able to elicit a Our data show that oncogenic B-RAF stimulates signiﬁcant retardation in the growth of human melano- constitutive ERK activity in melanoma cells, providing ma tumour xenografts. However, we were unable to both proliferation and survival signals. These data are produce complete cures of the animals, and further

Oncogene B-RAF is a therapeutic target in melanoma M Karasarides et al 6296

Figure 3 BAY43-9006 inhibits oncogenic B-RAF signalling in cells. (a) Inhibition of V599EB-RAF in vitro kinase activity by BAY43- 9006. (b)[3H]thymidine incorporation in A375, Colo829 and WM-266-4 cells following treatment with DMSO (À) or BAY43-9006 (B; 10 mM) for 24 h. (c) Cell cycle proﬁles for A375, Colo829 and WM-266-4 cells treated with BAY43-9006 (10 mM) for the indicated times. BAY43-9006 was synthesized as previously described (Wan et al., 2004)

Table 1 IC50 values of BAY43-9006 in the indicated melanoma cell BAY43-9006 and U0126 are clearly more potent than lines siRNA at blocking B-RAF signalling in melanoma cells.

Cell line IC50 BAY43-9006 ERK was inhibited more efficiently (compare Figures 1b, d) and more rapidly (data not shown) with BAY43- A375 4.8 mM (4.4–5.2) 9006 than with B-RAF siRNA, and BAY43-9006 Colo829 5.1 mM (2.5–10.5) inhibited DNA synthesis more effectively and induced WM-266-4 6.1 mM (4.2–8.8) cell death more rapidly (Figures 1–3). This is presum- Values in parentheses indicate 95% confidence intervals. In total, 103 ably because the loss of B-RAF protein through cells were seeded in 96-well plates and cultured in 10% FBS. The next degradation occurs more slowly than its inhibition by day the cells were treated with a range of concentrations of BAY43- a small molecular inhibitor. In addition, BAY43-9006 9006 for 4–7 days and then fixed and stained with sulphorhodamine-B, may target several kinases, the combination producing a and their absorbance was determined at 590 nm. The results are expressed as percentage of control growth as a function of log (dose) more potent effect than targeting a single protein with and the IC50 determined by nonlinear regression, constraining the siRNA. Furthermore, in vivo BAY43-9006 may directly minimum to be positive (GraphPad Prism). Each experiment was target the tumour cells through B-RAF inhibition and performed in quadruplicate and repeated four times produce an antiangiogenic effect by inhibiting C-RAF, which is downstream of the VEGF receptor and provides survival signals to endothelial cells (Alavi et al., 2003). Finally, additional targets may also exist studies will aim to improve the schedulling of the drug. in the complex environment of the tumour that is not It is also possible that BAY43-9006 is simply not potent present in melanoma cells growing in tissue culture. enough to elicit a response as a monotherapy and that Our studies reveal an interesting parallel between better responses will be obtained if the drug is used in BAY43-9006 and the small molecule Imatinib (STI571, combination with more conventional anticancer agents. Glivec). Imatinib is a potent inhibitor of Abl, a tyrosine

Oncogene B-RAF is a therapeutic target in melanoma M Karasarides et al 6297

Figure 4 WM-266-4 tumour xenografts treated with BAY43- 9006. (a) The volumes of WM-266-4 tumour xenografts, during the specified time, were determined in mice treated with vehicle control or BAY43-9006. The bar indicates the treatment period. Experi- ments were conducted in accordance with the United Kingdom Home Office regulations, and United Kingdom Co-ordinating Committee on Cancer Research Guidelines (Workman et al., 1998). Xenografts were established in nude (nu/nu) female CD1 mice (29– 39 g) by s.c. injection in the right flank of a suspension of 8 Â 106 WM-266-4 cells in 200 ml PBS. After 6 days the animals were divided by stratified distribution based on tumour size into two groups of eight, and treated for 17 consecutive days with BAY43- 9006 (50 mg/kg; 10 ml/kg p.o. by gavage) or vehicle control. Tumour volumes were measured at intervals by calliper measure- ment in three dimensions, and calculated from the formula length Â breadth Â height Â 0.5236. After the final dose, the animals were killed by cervical dislocation and the tumours excised and snap frozen in liquid nitrogen. (b) Immunohistochemistry of tumour xenograft sections demonstrating levels of phosphorylated MEK in WM-266-4 tumours from mice treated with vehicle control or BAY43-9006. Cell nuclei were counterstained with TO-PRO-3. Frozen sections were fixed in 4% paraformaldehyde in PBS for 20 min. Rinsed twice in PBS and incubated for 5 min in blocking buffer (PBS, 1% BSA, 2% FBS). Active phospho-MEK was detected using a phospho-specific antibody (#9121S, Cell Signal- ling) followed by 3 Â 5 min washes in PBS and FITC-labelled conjugate (Alexa). Samples were washed in PBS and fixed again in 4% paraformaldehyde, rinsed and incubated in TO-PRO-3 (Molecular Probes) to stain the nuclei. Fluorescence micrographs were collected on a Leica confocal microscope at Â 25 magnifica- tion

where the B-RAF gene is mutated, such as 30% of low- grade ovarian cancers (Singer et al., 2003), 35–70% of papillary thyroid cancers (Cohen et al., 2003; Kimura et al., 2003) and 10–15% of colorectal cancers (Davies et al., 2002; Rajagopalan et al., 2002; Yuen et al., 2002). In conclusion, our data show that oncogenic B-RAF is an important therapeutic target in melanoma and that it offers novel strategies for the clinical management of many types of cancer. kinase that is hyper-activated in over 90% of chronic myeloid leukemia (CML) (Druker and Lydon, 2000). Like BAY43-9006, Imatinib inhibits cell proliferation, stimulates apoptosis and sensitizes cells to DNA- Acknowledgements We thank Ian Titley for technical instruction with FACS damaging chemotherapeutic drugs. Thus, agents that analysis and Hugh Paterson and David Robertson for block signalling pathways can kill cells rather than just instruction and support with the imaging facilities. We also block their proliferation and offer enormous therapeutic thank the members of the Signal Transduction Team for potential both as mono-therapies and in combination helpful discussions. This work is funded by Cancer Research with conventional cancer drugs. Importantly, B-RAF UK and the Biotechnology and Biological Sciences Research targeted therapies may ﬁnd utility in other cancers Council (Grant number 353/C14718).

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