© 2006 Nature Publishing Group
BRAF mutation predicts sensitivity to MEK
David B. Solit1,3, Levi A. Garraway4,6, Christine A. Pratilas2,3, Ayana Sawai3, Gad Getz6, Andrea Basso3†,
Qing Ye3, Jose M. Lobo3, Yuhong She3, Iman Osman7, Todd R. Golub5,6, Judith Sebolt-Leopold8,
William R. Sellers4,6& Neal Rosen1,3
The kinase pathway comprising RAS, RAF, mitogen-activated
protein kinase kinase (MEK) and extracellular signal regulated
kinase (ERK) is activated in most human tumours, often through
gain-of-function mutations of RAS and RAF family members1.
and pharmacologic analysis, we find that mutation of BRAF is
associated with enhanced and selective sensitivity to MEK inhi-
bition when compared to either ‘wild-type’ cells or cells harbour-
ing aRAS mutation. This MEK dependency wasobservedinBRAF
mutant cells regardless of tissue lineage, and correlated with both
downregulation ofcyclinD1protein expression andtheinduction
of G1 arrest. Pharmacological MEK inhibition completely abro-
gated tumour growth in BRAF mutant xenografts, whereas RAS
mutant tumours were only partially inhibited. These data suggest
an exquisite dependency on MEK activity in BRAF mutant
tumours, and offer a rational therapeutic strategy for this geneti-
cally defined tumour subtype.
Activating RAS and BRAF mutations typically demonstrate
mutual exclusivity in tumours1–3. This suggests an epistatic relation-
ship whereby either mutation is sufficient to deregulate a common
effector pathway such as the MEK–ERK kinase cascade. If so,
tumours arising as a result of mutation to either RAS or BRAF
should harbour similar downstream dependencies that might repre-
sent useful therapeutic targets. To test this hypothesis, we examined
the consequences of MEK–ERK pathway inhibition in a collection of
cancer cell lines that exhibited differing mechanisms of MAP kinase
pathway deregulation. Cell lines containing the NRAS(Q61R) or
BRAF(V600E) mutations (present in ,15% and ,50% of melano-
mas, respectively) were analysed alongside a panel of cancer cell lines
Several of these RAS/BRAF-WT cell lines exhibit levels of ERK
phosphorylation comparable to those observed in the setting of
RAS or RAF mutation.
MEK1/2 are dual-specificity kinases that phosphorylate and acti-
vate ERK, the classical MAP kinase4. To inhibit MEK–ERK, we used
the potent and selective MEK inhibitor CI-1040 (ref. 5). CI-1040 is a
non-competitive inhibitor of MEK1/2 with a Kiof 300nM in vitro5,6.
The only other known CI-1040 target is the MEK5 kinase; however,
its inhibition occurs at a 100-fold greater concentration than that
required for inhibition of MEK1/2 (ref. 7). Because ERK is the only
known MEK substrate, we reasoned that selective MEK inhibition
might clarify the role of the MAP kinase pathway in differing genetic
CI-1040 inhibited MEK (as measured by phosphorylated ERK
(p-ERK) levels) with a half-maximal inhibitory concentration (IC50)
of 100–500nM in all cell lines tested (Fig. 1 and data not shown). In
in a manner that correlated with the mechanism of ERK activation
(Fig. 1a). Whereas RAS/BRAF-WT cells exhibited resistance to
Figure 1 | The BRAF(V600E) mutation confers sensitivity to the MEK
inhibitor CI-1040. a, CI-1040 IC50values as a function of BRAFand NRAS
mutational status. b, Immunoblot of p-ERK and total ERK, demonstrating
that CI-1040 inhibits MAPK activity with IC50values ranging from 100 to
500nM. Cells were treated for 24h. MEK inhibition caused profound
downregulation of cyclin D1 expression in BRAF mutant tumour cells. In
contrast, cyclin D1 declined only modestly in SKMEL103 melanoma cells
with the NRAS(Q61R) mutation and in SKMEL31 RAS/BRAF-WT cells.
wild-type RAS and BRAF.
1Department of Medicine,2Department of Pediatrics, and3Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,
New York, New York 10021, USA.4Department of Medical Oncology and5Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney
Street, Boston, Massachusetts 02115, USA.6Broad Institute of Harvard and MIT, 320 Charles Street, Cambridge, Massachusetts 02141, USA.7Departments of Medicine and
Urology, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA.8Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor,
Michigan 48105, USA. †Present address: Schering-Plough, 2015 Galloping Hill Rd, Kenilworth, New Jersey 07033, USA.
Vol 439|19 January 2006|doi:10.1038/nature04304
© 2006 Nature Publishing Group
CI-1040 even at concentrations in vast excess of those required for
ERK inhibition, cells harbouring a BRAF mutation were exquisitely
sensitive, with IC50values of 0.024–0.111mM (Fig. 1a). Surprisingly,
RAS mutant cells did not demonstrate the same sensitivity despite
effective inhibition of p-ERK (Fig. 1b and data not shown). These
data raised the possibility that RAS and BRAF mutant cancer cells
might be differentially dependent on signalling mechanisms that
involve MEK, despite their known epistatic relationship in human
To explorethis hypothesisin anunbiased manner, weinterrogated
the large-scale chemical sensitivity data available for the NCI60
cancer cell lines8using supervised learning methods previously
applied to the analysis of gene-expression data. NCI60 cell lines
were partitioned into two classes according to the presence or
absence of the BRAF(V600E) mutation. We then performed a
supervised analysis9where the mean 2log10(GI50) values for each
compound in the BRAF(V600E) and non-mutant classes were
compared using a variance fixed t-test metric and ranked according
to T-score (the GI50 is the concentration that inhibits cell growth by
50%). Thirty-six compounds exhibited significantly increased
potency against the BRAF(V600E) class distinction (Fig. 2a and
Supplementary Table S1; false discovery rate (FDR) ¼ 0.25, nominal
P value ,3 £ 1024). The top-scoring compound against the
BRAF(V600E) class was hypothemycin (a resorcylic acid lactone,
the homologues of which possess potent and selective MEK inhibi-
tory activity), which was found to inhibit p-ERK at a potency
comparable to CI-1040 (Supplementary Fig. S1)10,11. Additional
top-scoring compounds included protein LF (anthrax lethal factor),
a zinc metalloproteinase known to inactivate MEK through enzy-
matic cleavage12, and PD98059 (ref. 13), a well-characterized MEK
inhibitor. Thus, at least three of the most potent compounds against
the BRAF(V600E) class distinction appeared to exert their effects
through MEK inhibition. These results were consistent with the
CI-1040 analysis and suggest that BRAF mutation might confer a
preferential sensitivity to MEK inhibition in human cancer cells.
NCI60 cell lines that harbour RAS mutations are non-overlapping
with respect to the BRAF(V600E) mutation, supporting the notion
analysis of the NCI60 data was repeated, using the class distinction
RAS mutant versus wild-type RAS. Surprisingly, and in contrast to
the results observed for the BRAF(V600E) class distinction, no
compound surpassed the Bonferroni significance threshold in the
RAS mutant class (Fig. 2b). Conceivably, RAS and BRAF mutations
genetic heterogeneity. Thus, we performed an additional supervised
distinctions. a–c, Colourgrams show BRAF mutant (a) or RAS mutant (b)
versus the remaining NCI60 cells, or for mutant RAS versus mutant BRAF
lines (c). Columns denote NCI60 cell lines; rows denote compounds; colour
denotes the number of standard deviations above (red) or below (blue) the
mean for all cell lines (top 100 compounds for each class distinction shown;
see Methods). NSC numbers, names, variance-fixed T-scores (absolute
values; see Methods) and asymptotic P values are shown for top-scoring
compounds. Blue font indicates known MEK inhibitors. d, Relative GI50
values for the MEK inhibitor hypothemycin in non-haematological NCI60
cell lines. Black bars indicate BRAF wild-type cells; blue bars indicate
BRAF(V600E) cells; asterisks indicate non-melanoma cell lines with the
NATURE|Vol 439|19 January 2006
© 2006 Nature Publishing Group
If these classes do indeed manifest common genetic dependencies,
compounds that target the relevant mechanisms (for example, the
MEK–ERK pathway) should fail to score by this class distinction.
However, hypothemycin again distinguished BRAF and RAS mutant
cells (Fig. 2c); protein LF also retained a high rank.
Because most BRAF(V600E) cell lines analysed were melanoma-
derived, the enhanced sensitivity to MEK inhibition may have
reflected a melanocytic lineage effect independent of the
BRAF(V600E) mutation; however, several lines of evidence rendered
this possibility unlikely. First, in the NCI60 analyses all
BRAF(V600E) cell lines exhibited markedly reduced hypothemycin
GI50 values relative to the mean across the sample set, regardless of
tissue type (Fig. 2d). Colo205, an NCI60 colon cancer line with
the BRAF(V600E) mutation, was also found to exhibit sensitivity to
CI-1040 at an equivalent level to the melanoma cells (Fig. 1a). In
addition, the two melanoma lines lacking a BRAF mutation were
clearly indifferent to the effects of hypothemycin. Finally, only one of
the breast/prostate cell lines demonstrated similar sensitivity to
the drug: the breast cancer line DU-4475 (IC5024nM). Notably,
sequencing of BRAF in this cell line showed that it also contained a
V600E mutation. Thus, the sensitivity of cancer cell lines to MEK
inhibition correlated most closely with BRAF mutation status.
In many cell types, RAS–RAF–MEK–ERK signalling is required
for both D-cyclin expression and assembly of the cyclin D–cdk4
complex14. The marked sensitivity of BRAF mutant cells to MEK
inhibitors allowed us to examine the functional consequences of
MAPkinaseblockade inthiscontext. TreatmentofBRAF mutantcell
lines withCI-1040 causedamarkeddecline inD-cyclinproteinlevels
(Figs 1b and 3a, d). In the SKMEL28 cell line, this decline was
followed by loss of RB phosphorylation and a profound G1 cell cycle
arrest (Fig. 3). G1 arrest was accompanied by apoptosis in several
a BRAF-mutant context exerts both cytocidal and cytostatic effects.
had no effect on cyclin D1 protein expression in the vast majority of
RAS/BRAF-WT cells, as shown in Fig. 1b for the MCF7, BT-474 and
A431 cell lines. BT-474 and A431 exhibited robust MAPK activity
driven by HER2/neu and EGFR, respectively, suggesting that cyclin
D1 expression and G1 progression are driven by MEK/ERK-
independent mechanisms in certain RAS/BRAF-WT cells.
To determine whether the differential sensitivity to MEK inhi-
bition observed for BRAF mutant cancer cells was re-capitulated
in vivo, mice harbouring xenograft tumours were treated with the
MEK inhibitor PD0325901. PD0325901 is a derivative of CI-1040
that has improved oral bioavailability and induces a longer duration
of target suppression15. The effects of PD0325901 on tumour cells
in vitro are qualitatively identical to those of CI-1040, including the
marked selectivity for BRAF mutant cell lines, but occur at 100-fold
lower concentrations (Supplementary Fig. S2).
Daily treatment with PD0325901 at doses of 5 and 25mgkg21
completely suppressed the growth of SKMEL28 and Colo205
BRAF(V600E) mutant xenografts (Fig. 4a and Supplementary
Fig. S3; P , 0.01 for both 5 and 25mg kg21versus control,
P ¼ 0.16 for 5 versus 25mgkg21). Growth suppression was associ-
ated with loss of D-cyclin expression, induction of p27 and
hypophosphorylation of RB (Fig. 4e and Supplementary Fig. S4).
In contrast, PD0325901 treatment of SKMEL103 (NRAS(Q61R)),
SKMEL30 (NRAS(Q61R)) and SKMEL31 (RAS/BRAF-WT) xeno-
grafts at a dose of 5mgkg21only delayed tumour growth, with
complete growth suppression requiring 25mgkg21(Fig. 4b, c and
SupplementaryFig.S3b;P , 0.01for5versus25mgkg21,and5and
25mgkg21versus control). BT-474 xenografts (BRAF/RAS-WT)
were completely insensitive to PD0325901 (Fig. 4d). PD0325901
treatment at the doses studied was non-toxic and resulted in
RB phosphorylation, cyclin D expression and proliferation as
measured by Ki67 were unaffected by MEK inhibition in
Figure 3 | MEK inhibition causes loss of D-cyclin expression, RB
hypophosphorylation and G1 arrest in BRAF mutant cancer cells.
a, Immunoblot showing the kinetics of change in p-ERK, D-cyclin
expression and RB in SKMEL28 cells treated with 1mM CI-1040. b, CI-1040
induced a G1 growth arrest in BRAF mutant tumour cells but not in
RAS/BRAF-WT breast cancer cells (BT-474 shown). c, d, CI-1040 induces
apoptosis in some but not all cancer cell lines with the BRAF(V600E)
mutation as measured by FACS analysis (c) and PARP cleavage (d).
NATURE|Vol 439|19 January 2006
© 2006 Nature Publishing Group
correlation between basal p-ERK levels and PD0325901 sensitivity
(Supplementary Fig. S4). Thus, the MEK dependency characteristic
of BRAF mutant tumour cells in vitro was also apparent in vivo.
Excess MAP kinase pathway activation occurs commonly in
human tumours. In melanoma and other solid tumours, mutation
of BRAF and RAS occurs frequently and tends to exhibit mutual
exclusivity, suggesting that each mutation confers a similar selective
advantage1. However,our findings suggest that tumourcellscarrying
BRAF mutations are much more reliant on MEK–ERK signalling
than are RAS mutant cells, or cells that activate MAP kinase byother
means. Thus, BRAF mutant cancer cells may harbour a critical
dependency on MEK–ERK that renders them highly sensitive to
pharmacological MEK inhibition.
BRAF mutations occur at a high frequency in melanomas, but are
also observed in colon, lung and several other tumour types1,2.
Expression of BRAF(V600E) in non-transformed melanocytes
leads to constitutive ERK activation and tumorigenicity in mice,
and depletion of BRAF but not A-RAF or C-RAF in BRAF(V600E)
mutant melanoma cells reduces ERK activity16,17. Our data suggest
that D-cyclin expression is also deregulated and ERK-dependent in
BRAF-mutant tumours. Cyclin D downregulation may therefore
mediate at least some of the anti-proliferative effects observed after
MEK inhibition. On the other hand, MEK inhibition had little effect
on D-cyclin expression in most BRAF/RAS-WT tumour cells. In
these cells, mutations in the PTEN or phosphatidylinositol-3-OH
kinase (PI(3)K) genes, or activation of other pathways, may drive
D-cyclin expression in an ERK-independent fashion18,19. Our results
are also consistent with a model in which ERK regulates G1
progression only in certain lineages (for example, melanocytes);
presumably, such lineage differences in cell growth control contrib-
ute to the imbalanced frequency of RAS and BRAF mutations
observed across tumour types.
RAS-dependent transformation has been found previously to
require activation of cyclin D1 (refs 20–23). As both oncogenic
RAF and activated ERK also induce cyclin D1 expression24,25, it has
been presumed that in human tumours with RAS mutation, cyclin
D1 expression was controlled by RAS-mediated MEK–ERK acti-
vation. However, our results suggest that in certain genetic contexts,
including some tumours with RAS mutation, ERK signalling may be
dispensable for cyclin D1 expression and cell proliferation. RAS
family members have multiple other targets, such as PI(3)K and
RalGDS; these may exert more prominent oncogenic effects in
certain tumour subtypes, thereby reducing the requirement for
MEK–ERKactivation26,27. Our findings therefore raise the possibility
mutant tumours. Instead, direct RAS inhibitors or combinatorial
strategies may be required.
Thus far, the use of BRAF inhibitors in clinical trials has met with
mixed results. On the other hand, the favourable therapeutic index
and selectivity of MEK inhibitors may provide an appealing thera-
peutic strategy for BRAF mutant cancers. We therefore propose
clinical trials of MEK inhibitors in which patients are stratified
based on BRAF mutational status.
Cell culture. CI-1040 and PD0325901 were obtained from Pfizer Global
Research and Development. Drugs were dissolved in DMSO to yield 10mM
stock solutions and stored at 2208C. All SKMEL lines were obtained from
A. Houghton and P. Chapman with the remainder obtained from the ATCC.
Cells were maintained in either RPMI or a 1:1 mixture of DMEM:F12 medium
supplemented with 2mM glutamine, 50Uml21each of penicillin and strepto-
mycin, and 10% heat-inactivated fetal bovine serum, and incubated at 378C in
Alamar blue cell proliferation assay. Cells were plated in 96-well plates at a
density of 2,000–5,000 cellsper well. After24h, cells were treated with a range of
drug concentrations prepared by serial dilution. The cells were exposed to
Alamar blue (AccuMed International, OH) 3–5days after drug treatment, and
plates were read using a fluorescence spectrophotometer.
Western blot analysis. Treated cells were harvested, washed with PBS and lysed
1mM Na3VO4, 1mM phenylmethylsulphonylfluoride, and 10mgml21each of
leupeptin, aprotinin and soybean trypsin inhibitor) for 30min on ice. Lysates
were centrifuged at 13,200 r.p.m. for 10min and the protein concentration of
the supernatant was determined by BCA assay (Pierce). Equal amounts of
total protein were resolved by SDS–PAGE and transferred onto nitrocellulose
membranes. Blots were probed overnight at 48C with antibody raised against
the protein of interest. Anti-MAP kinase, phospho-MAP kinase, RB and cleaved
PARP antibodies were obtained from Cell Signaling Technology. Anti-cyclin
D1, anti-cyclin D2 and anti-cyclin D3, and p27 antibodies, were obtained from
Santa Cruz Biotechnology. After incubation with horseradish peroxidase-
and stained with ethidium bromide. Detection and quantification of apoptotic
cells (sub-G1) were performed by flow cytometric analysis.
Animal studies. Four- to six-week-old nu/nu athymic female mice were
obtained from the National Cancer Institute, Frederick Cancer Center and
maintained inventilated caging. Experiments were carried out under an IACUC
mutant xenografts. a, PD0325901 suppressed the growth of SKMEL28
(BRAF(V600E)) xenografts at both the 5 and 25mgkg21dose levels. b, c, In
contrast, 5mgkg21PD0325901 only delayed the growth of SKMEL103
(RAS(Q61R)) and SKMEL31 (RAS/RAF-WT) xenografts, with complete
growth suppression requiring the higher dose. d, BT-474 xenografts
(RAS/RAF-WT) were refractory to MEK inhibition (n ¼ 10 mice per
xenograft tumours but caused downregulation of D-cyclins, induction of
only in the SKMEL28 xenografts. Tumour lysates were derived from mice
euthanized 8h after the final treatment. All error bars indicate standard
NATURE|Vol 439|19 January 2006
© 2006 Nature Publishing Group Download full-text
animals in research were followed. Tumours were generated by injecting
0.5–1.0 £ 107tumour cells together with reconstituted basement membrane
(Matrigel, Collaborative Research). For the BT-474 model, before tumour cell
inoculation, 0.72mgpellet2117b-estradiol pellets (Innovative Research of
America) were inserted subcutaneously. Before initiation of treatment, mice
were randomized to receive PD0325901 at a dose of 5 and 25mgkg21or vehicle
only as control. PD0325901 was formulated in 0.5% hydroxypropyl methyl-
cellulose plus 0.2% Tween 80, and administered byoral gavage. Mice were killed
tumour were measured) was measured in control and treated groups using a
calliper. The data are expressed as the increase or decrease in tumour volume in
mm3(mm3¼ p/6 £ (larger diameter £ (smaller diameter)2). Treatment arms
were compared using the Wilcoxon rank sums test. To prepare lysates, tumour
tissue was homogenized in 2% SDS lysis buffer and then processed as described
above. For immunohistochemical studies, xenograft tumours were fixed
overnight in paraformaldahyde followed by dehydration in graded ethanols.
Statistical methods. Pharmacological data (2log10(GI50)) for 42,796 com-
pounds were downloaded from the NCI website (http://dtp.nci.nih.gov/docs/
cancer/cancer_data.html). The GI50 data were used to populate a matrix with
existed, the entry with the largest number of replicates was included; in cases
where multiple entries had the same number of replicates, the largest mean
(2log10(GI50)) value for the NCI60 data set was selected. Incomplete data were
class distinctions described. BRAF mutant status was determined based on
GI50 data are non-gaussian with many (2log10(GI50)) values at or near 4, a
variance-fixed t-test was used to calculate significance. Here, the mean and
median standard deviation was calculated for compounds for which the mean
2log10(GI50) values across the NCI60 set were between 6 and 7. For both
calculations, the standard deviation was near 0.4; thus, this value was used as a
with the top variance-fixed T-scores for the relevant class distinctions were
selected for additional analysis; in Fig. 2, the absolute values of these scores are
indicated. GI50 values and distributions for selected compounds were analysed
through the NCI Developmental Therapeutic website.
Received 10 May; accepted 4 October 2005.
Published online 6 November 2005.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements The authors thank H. Ju, W. L. Wong and H. Tseng for
technical assistance. This work was supported by grants from the National
Institutes of Health (L.A.G., C.A.P., G.G., T.R.G., W.R.S. and N.R.), the William
H. Goodwin and Alice Goodwin Foundation for Cancer Research, the MSKCC
Experimental Therapeutics Program (D.B.S. and N.R.), the Waxman Foundation
(D.B.S. and N.R.), the Howard Hughes Medical Institute (G.G. and T.R.G.),
Golfers Against Cancer (D.B.S. and N.R.) and the American Society of Clinical
Oncology (D.B.S. and C.A.P.).
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare competing
financial interests: details accompany the paper at www.nature.com.
Correspondence and requests for materials should be addressed to N.R.
NATURE|Vol 439|19 January 2006