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[CANCER RESEARCH 63, 5198–5202, September 1, 2003]
Advances in Brief
Suppression of BRAF
V599E
in Human Melanoma Abrogates Transformation
1
Sunil R. Hingorani, Michael A. Jacobetz, Gavin P. Robertson, Meenhard Herlyn, and David A. Tuveson
2
Abramson Family Cancer Research Institute and Abramson Cancer Center at the University of Pennsylvania [S. R. H., M. A. J., D. A. T.] and Department of Medicine [S. R. H.,
D. A. T.], University of Pennsylvania and Wistar Institute [M. H.], Philadelphia, Pennsylvania 19104, and Departments of Pharmacology, Dermatology, and Pathology,
Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [G. P. R.]
Abstract
Activating mutations in the BRAF serine/threonine kinase are found in
>70% of human melanomas, of which >90% are BRAF
V599E
. We sought
to investigate the role of the BRAF
V599E
allele in malignant melanoma. We
here report that suppression of BRAF
V599E
expression by RNA interfer
-
ence in cultured human melanoma cells inhibits the mitogen-activated
protein kinase cascade, causes growth arrest, and promotes apoptosis.
Furthermore, knockdown of BRAF
V599E
expression completely abrogates
the transformed phenotype as assessed by colony formation in soft agar.
Similar targeting of BRAF
V599E
or wild-type BRAF in human fibrosar
-
coma cells that lack the BRAF
V599E
mutation does not recapitulate these
effects. Moreover, these results are specific for BRAF, as targeted inter-
ference of CRAF in melanoma cells does not significantly alter their
biological properties. Thus, when present, BRAF
V599E
appears to be es
-
sential for melanoma cell viability and transformation and, therefore,
represents an attractive therapeutic target in the majority of melanomas
that harbor the mutation.
Introduction
Malignant melanoma will afflict ⬎50,000 people in the United
States this year and result in ⬎7,000 deaths (1). The incidence of
melanoma is rising among the most rapidly of all malignancies (2).
When diagnosed early, melanoma is highly curable by wide surgical
excision. However, in patients with deep local invasion, or with
spread to lymph nodes or distant sites, the disease is highly resistant
to all forms of therapy. The median survival for patients with meta-
static melanoma is 6–9 months (3).
Recently, activating mutations in the BRAF gene were described in
a majority of melanomas and benign nevi, suggesting an important
role for this oncogene in melanocyte biology and disease (4–6). More
than 60% of malignant melanomas were found to contain a specific
mutation, BRAF
V599E
, the product of which possesses constitutive
kinase activity. BRAF is a member of the Raf family of serine/
threonine kinases, along with CRAF and ARAF, which serve as
immediate effectors of the ras GTPases (7). Activation of the Raf/
MEK
3
/ERK, or MAPK, signaling cascade promotes cellular prolifer-
ation and survival. The highly homologous Raf family members have
overlapping but distinct biochemical activities and biological func-
tions. We therefore sought to determine whether Raf family members,
and specifically BRAF
V599E
, are required in melanoma cells for main
-
tenance of the transformed state. Accordingly, the biochemical sig-
naling properties and cellular phenotypes of melanoma cells were
assessed after depletion of B-Raf, B-Raf
V599E
, and C-Raf proteins
by RNAi.
Materials and Methods
RNAi Sequences and Preparation. Small inhibitory duplex RNAs (PRO-
LIGO, Boulder, CO) were prepared and reconstituted in annealing buffer as
described (8, 9). The sense strands of the siRNA duplexes were as follows:
Lamin A/C: CUggACUUCCAgAAgAACATT; Com-4: AgAAUUggAUCUg-
gAUCAUTT; Mu-A: gCUACAgAgAAAUCUCgAUTT; C1: UgUgC-
gAAAUggAAUgAgCTT. Duplex shRNA oligos were cloned into the HindIII
and BglII sites in pSUPER.retro (Oligoengine, Seattle, WA), and insert fidelity
was confirmed by sequencing both strands with the following primers: forward
– ttatccagccctcactcc; reverse – gtgttctgggaaatcacc. The sense strands of the
shRNA pSUPER.retro inserts were as follows:
Com-1: gatccccTGGATACCGTTACATCTTCttcaagagaGAAGATGTAA
CGGTATCCAtttttggaaa.
Com-2: gatccccTCCCAGAGTGCTGTGCTGTttcaagagaACAGCACAG-
CACTCTGGGAtttttggaaa.
Com-3: gatccccTTGGTTGGGACACTGATATttcaagagaATATCAGTG-
TCCCAACCAAtttttggaaa.
Com-4: gatccccAGAATTGGATCTGGATCATttcaagagaATGATCCAG-
ATCCAATTCTtttttggaaa.
Mu-A: gatccccGCTACAGAGAAATCTCGATttcaagagaATCGAGATT-
TCTCTGTAGCtttttggaaa.
Mu-B: gatccccGAGAAATCTCGATGGAGTGttcaagagaCACTCCATC-
GAGATTTCTCtttttggaaa.
C1: gatccccTGTGCGAAATGGAATGAGCttcaagagaGCTCATTCCATT-
TCGCACAtttttggaaa.
BRAF cDNA. Human wild-type BRAF and BRAF
V599E
were cloned from
mRNA and sequenced to confirm fidelity. 5⬘ HA epitope tags were cloned into
both cDNAs by PCR. Full-length BRAF cDNAs were subsequently cloned into
pBABE.puro.
Cell Culture and Transfection. WM793 melanoma cells were derived
from a vertical growth phase tumor as described previously (10), and HT1080
and HEK cells were obtained from American Type Culture Collection. Cells
were cultured under standard conditions (37°C in humidified atmosphere
containing 5%CO
2
) and grown in DMEM supplemented with 25 mM HEPES
(pH 7.4), 10% FCS, penicillin (100 units/ml), and streptomycin (100
g/ml).
To achieve transient suppression of gene expression, cells were plated in
six-well dishes at 50–60% confluency and transfected with 5
g of duplex
RNA plus 6
l of OLIGOFECTAMINE (Life Technologies, Inc., Carlsbad,
CA) per the manufacturer’s recommendations and as described (8, 9).
The specificity of the targeting sequences was determined by transient
cotransfection of HEK cells with pBABE.puro.HA-tagged BRAF or
pBABE.puro.HA-tagged BRAF
V599E
and shRNA vectors (11). For stable trans
-
fection experiments, cells were plated at 50–80% confluency in 100-mm
dishes and transfected with 4
g of plasmid DNA and 12
l of Fugene 6
(Roche, Indianapolis, IN) per the manufacturer’s instructions. Twenty-four h
after transfection, cells were selected in media containing 2
g/ml Puromycin
for 60–72 h and then collected for biochemical and cellular assays.
Immunoblotting. Adherent cells were washed with ice-cold PBS and lysed
and scraped in boiling SDS lysis buffer (10 m
M Tris, 1% SDS, 50 mM NaF, and
1m
M VO4). Lysates were boiled for 5 min, the DNA was sheared, and
insoluble debris was removed by microcentrifugation (14,000 rpm for 10 min).
Received 6/2/03; accepted 7/9/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported in part by NIH Grant R25-CA87812 (S. R. H.), the McCabe Foundation
(D. A. T.), the Abramson Cancer Center of the University of Pennsylvania Pilot Projects
Program and Grant IRG-78-002-26 from the American Cancer Society (D. A. T.), The
Mary L. Smith Charitable Lead Trust (D. A. T.), and NIH Grants CA-25874, CA-47159,
CA-76674, and CA-10815 (M. H.).
2
To whom requests for reprints should be addressed, at University of Pennsylvania,
Department of Medicine, Philadelphia, PA 19104. E-mail: tuvesond@mail.med.
upenn.edu.
3
The abbreviations used are: MEK, mitogen-activated protein kinase kinase; ERK,
extracellular signal-regulated kinase; siRNA, small interfering RNA; HEK, human
embryonic kidney; HA, hemagglutinin; shRNA, short hairpin RNA; MAPK, mitogen-
activated protein kinase; RNAi, RNA interference; TBS, Tris-buffered saline.
5198
Protein concentrations were determined with bicinchoninic acid (Pierce, Rock-
ford, IL). Samples (15
g of total protein per lane) were resolved by reducing
SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride mem-
branes (Millipore, Bedford, MA). Membranes were blocked and incubated
with primary antibodies in TBS [150 mM Tris-HCL (pH 8.0) and 150 mM
NaCl] ⫹ 3% BSA for antiphospho antibodies or 5% nonfat dry milk/TBS for
other antibodies. The membranes were subsequently washed (TBS/0.1%
Tween 20), incubated with horseradish peroxidase-conjugated secondary an-
tibodies, and washed again before being processed with enhanced chemilumi-
nescence plus (Amersham Biosciences, Little Chalfont, United Kingdom).
Membranes were probed sequentially for the indicated proteins after washing
in stripping buffer [50 mM Glycine (pH 2.5) and 0.05% Tween 20] for 15 min
at 55°C. Primary antibodies were procured from the following sources: an-
tiphosphorylated and total MEK (Cell Signaling, Beverly, MA), anti-HA
(Sigma, St. Louis, MO), and anti-Lamin A/C (Vector Laboratories, Burl-
ingame, CA) and antibodies against actin, B-Raf, and C-Raf were all obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-
conjugated secondary antibodies were from Jackson Immunoresearch (West
Grove, PA).
Proliferation, Apoptosis, and Transformation Assays. After selection,
shRNA-transfected cells were plated onto glass coverslips in media. The next
day, cells were incubated with 1 mM BrdUrd (Sigma) for 4 h, and positive
nuclei detected with anti-BrdUrd FITC per manufacturer’s instructions (Roche,
Indianapolis, IN). Apoptosis was detected with the In Situ Cell Death Detec-
tion kit per the manufacturer’s instructions (Roche). Three high-powered fields
were counted manually to determine the percentage of cells in S phase and the
degree of apoptosis, respectively. Nuclear staining was detected with 4⬘,6-
diamidino-2-phenylindole (Sigma). Soft agar assays were performed by plating
50,000 cells/60-mm dish in 0.34% agar/media suspension over a solidified
0.5% agar layer. Dishes were replenished every 5–7 days.
Results
The recent development of facile methods for both transient and
stable suppression of gene expression by RNAi has provided powerful
tools for the study of mammalian cell genetics (8, 11, 12). These
methods exploit a conserved biological response to short duplex RNA
that results in post-transcriptional gene silencing (13). To evaluate the
role of BRAF expression in human melanoma cells, we generated a
series of stably expressing vectors against discrete regions of the
human BRAF coding sequence (Fig. 1A). We then tested the speci-
ficity of these reagents in HEK cells that had been transiently cotrans-
fected with HA epitope-tagged wild-type and mutant BRAF vectors
(Fig. 1B). Targeting sequences common to both wild-type and mutant
alleles (Com-1, 2, 3, and 4) caused various degrees of knockdown of
BRAF expression in HEK cells as demonstrated by immunoblots
against the expressed protein, with Com-4 being the most effective. A
mutant-specific vector (Mu-A) was also created that essentially com-
pletely abolished BRAF
V599E
expression while keeping wild-type
BRAF expression intact.
After establishing the efficacy and specificity of various RNAi
constructs described above, we designed corresponding duplex siRNA
species to study the effects of endogenous BRAF knockdown in
melanoma cells because of the higher transfection efficiencies achiev-
able by this method and the ability to obviate the need for antibiotic
selection. WM793 human melanoma cells were derived from a ver-
tical growth phase tumor (10) and have been shown to harbor the
BRAF
V599E
allele (14). Com-4 and Mu-A siRNA significantly inhib
-
ited BRAF expression and its attendant downstream signaling as
measured by phosphorylated MEK levels, although these effects were
relatively short lived and reversed by 72–96 h after transfection (Fig.
2). Transfection of siRNA against Lamin A/C as a control revealed
efficient and more durable suppression of target expression, reflecting
either greater efficacy or stability of this transfected RNA duplex or
perhaps lower endogenous levels of lamin mRNA synthesis.
Despite the marked biochemical sequelae to BRAF knockdown by
siRNA, the effects were transient and rapidly reversible, and there were
no overt phenotypic consequences. Thus, to study the potential biological
consequences of more durable perturbations of these pathways, we used
our panel of shRNA vectors to stably knock down BRAF in melanoma
cells. Transfection of Com-4 and Mu-A shRNA vectors into WM793
cells effectively and stably suppressed BRAF expression and MEK phos-
phorylation (Fig. 3A). Cells transfected with control vector had no dis-
cernible effect on these parameters. On the other hand, although it was
possible to stably suppress BRAF expression by Com-4 in HT1080
human fibrosarcoma cells, no corresponding inhibition of MEK phos-
phorylation was observed, indicating that these cells activate the MAPK
pathway by another mechanism. As expected, transfection with Mu-A
had no discernible effect on B-Raf levels because these cells do not
contain the BRAF
V599E
mutation.
Several cellular consequences to stable BRAF suppression were
readily apparent. First, the morphology of WM793 cells changed
dramatically, becoming much larger and flatter and also less refractile
(Fig. 3B). In addition, far fewer WM793 cells were recovered after
Fig. 1. Design and characterization of vector-based RNAi of human BRAF. A, sche-
matic representation of human BRAF cDNA demonstrating sites selected for targeting. B,
efficacy of shRNA vectors in suppressing BRAF and BRAF
V599E
expression. Subconfluent
adherent HEK cells were either not transfected (⫺) or cotransfected with pBABE.pu-
ro.HA-BRAF (left panels) or pBABE.puro.HA-BRAF
V599E
(right panels), and the series of
shRNA plasmid constructs were directed against BRAF. After transfection (48–72 h),
whole cell extracts were prepared and analyzed for HA expression; actin was used as a
loading control. This experiment was performed three times with similar results.
Fig. 2. Transient suppression of wild-type and mutant BRAF by siRNA in WM793
cells. Subconfluent cell cultures were transfected with siRNA directed against Lamin A/C
(L), BRAF (4), and BRAF
V599E
(A). Whole cell lysates were then prepared at 24-, 48-, 72-,
and 96-h post-transfection and analyzed for specific protein expression by immunoblot-
ting with the indicated antibodies. The position of the M
r
45,000 phospho-MEK 1/2
proteins is indicated; the identity of the faster migrating band is unknown. This experiment
was performed three times with similar results.
5199
BRAF
V599E
SUPPRESSION IN HUMAN MELANOMA
transfection with Com-4, as compared with control vector or Mu-A,
suggesting that wild-type B-Raf function may also be important for
the viability of these cells. No appreciable differences in morphology
or cell number were noted in parallel transfections of HT1080 cells
(Fig. 3B). In addition, Com-4 and Mu-A-transfected WM793 cells
exhibited markedly lower proliferative rates; the percentage of cells in
S phase was ⬃4 ⫹/⫺ 2% and 0.8 ⫹/⫺ 1%, respectively, as compared
with 19 ⫹/⫺ 2% for cells transfected with empty vector (Fig. 3C).
The proliferation of HT1080 cells was not affected by transfection
with any of these vectors, again despite achieving significant knock-
down of B-Raf levels (Fig. 3C). WM793 cells also exhibited increased
levels of apoptosis after stable suppression of BRAF, whereas human
fibrosarcoma cells again remained unaffected by similar manipula-
tions (Fig. 3D).
These results demonstrated that BRAF-dependent signaling was
necessary for the optimal proliferation and survival of human mela-
noma WM793 cells and dispensable for human fibrosarcoma cells.
We wondered whether these effects were specific for the BRAF family
member of the Raf kinases or extended to the heretofore more exten-
sively studied homologue, CRAF. We therefore generated CRAF-
specific duplex siRNA species and stably expressing shRNA vectors
and tested their abilities to suppress CRAF expression and inhibit
downstream phosphorylation of MEK. As shown in Fig. 4A, knock-
down of C-Raf protein levels by siRNA followed a slower time course
than that of B-Raf and remained more durably suppressed. Notably,
however, there appeared to be no effect on MEK phosphorylation
despite nearly complete suppression of CRAF. Thus, at least in
WM793 melanoma cells, CRAF appears not to be required for MEK
activation. A stably expressing shRNA vector directed against the
same sequence similarly knocked down CRAF expression with no
attendant affect on MEK phosphorylation (Fig. 4B).
Finally, the ability to manifest anchorage-independent growth, an
established feature of cellular transformation, was assessed in mela-
noma cells after the knockdown of BRAF, BRAF
V599E
, or CRAF.
WM793 cells transfected with empty vector readily formed colonies
in soft agar (Fig. 4C). Cells in which C-Raf levels had been stably
knocked down formed colonies almost as readily. Transfection with
either Com-4 or Mu-A, however, essentially completely abrogated the
ability of these cells to manifest anchorage-independent growth.
Therefore, BRAF is uniquely required for cellular transformation in
WM793 cells.
Discussion
Oncogenesis is generally viewed as a multistep process character-
ized by the progressive acquisition of genetic mutations and func-
tional capabilities (15). The hope for curative therapies lies in the
proposition that key genetic events exist which represent unique
points of vulnerability for cancer cells. Indeed, despite the complexity
of genetic and epigenetic alterations in cancer cells and their micro-
environment, recent evidence demonstrates that the specific inhibition
of one, or perhaps a few, critical pathways in tumor cells may be
sufficient to kill them and provide significant clinical benefit. As
examples, treatment of patients with stable phase chronic myeloge-
Fig. 3. Effects of stable suppression of endogenous BRAF and BRAF
V599E
in WM793 and HT1080 cells. Subconfluent cultures of WM793 or HT1080 cells were transfected with
pSUPER.retro (vector), shRNA directed against BRAF (Com-4), or shRNA directed against BRAF
V599E
(Mu-A). After selection in puromycin, the biochemical and biological properties
of these cells were assessed. A, immunoblot analysis of B-Raf, activated MEK (p-MEK), total MEK (t-MEK), and actin levels. The positions of the M
r
45,000 phospho- and total-MEK
1/2 proteins are indicated. B, phase contrast microscopy of cells in culture (magnification: ⫻100). C, proliferative index as measured by BrdUrd incorporation. D, degree of apoptosis
as assessed by TUNEL reactivity. BrdUrd and TUNEL data are expressed as the means ⫹/⫺ SD of direct counting of three high-powered fields. These experiments were performed
twice with similar results.
5200
BRAF
V599E
SUPPRESSION IN HUMAN MELANOMA
nous leukemia or advanced gastrointestinal stromal tumors with ima-
tinib mesylate, a small molecule inhibitor of the ABL and KIT tyrosine
kinases, respectively, induces dramatic remissions with minimal tox-
icity (16, 17). Importantly, the responses of chronic myelogenous
leukemia and gastrointestinal stromal tumor patients to imatinib cor-
relates with the drug’s ability to inhibit the kinase activities of Bcr-
Abl and mutant c-kit (18, 19), respectively. These results suggest that
essential pathways may exist in other malignancies and that biochem-
ical confirmation of the effectiveness of molecularly targeted thera-
pies may be predictive of clinical efficacy.
Our findings suggest that the BRAF
V599E
mutation commonly
found in malignant melanomas may represent a therapeutic target
analogous to BCR-ABL and KIT. We have demonstrated here that
knockdown of BRAF expression and inhibition of downstream sig-
naling in WM793 human melanoma cells causes growth arrest and
promotes apoptosis under adherent conditions, and prevents colony
formation in suspension. These observations have been preliminarily
extended to a second melanoma cell line known to contain the
BRAF
V599E
mutation (data not shown). These effects were specific to
BRAF, as suppression of CRAF failed to inhibit downstream phos-
phorylation of MEK and did not appreciably alter the biological
properties of these cells. Moreover, these effects were specific to
melanoma cells, because human fibrosarcoma cells were impervious
to suppression of BRAF expression.
Fig. 4. Effects of transient and stable suppression of BRAF or CRAF expression in WM793 cells. In A, WM793 cells were transfected with siRNA directed against Lamin A/C (L),
BRAF (4), BRAF
V599E
(A), and CRAF (C) and analyzed for specific protein expression at 24, 48, and 96 h post-transfection. In B, WM793 cells were stably transfected with
pSUPER.retro (vector) or shRNA directed against CRAF (C-1), subjected to antibiotic selection, and assessed for knockdown of C-Raf, activated MEK (p-MEK), total MEK (t-MEK),
and actin levels. The positions of the M
r
45,000 phospho- and total-MEK 1/2 proteins are indicated. In C, WM793 cells were stably transfected with pSUPER.retro, Com-4 shRNA
against BRAF, C-1 shRNA against C-Raf, or Mu-A shRNA against BRAF
V599E
and plated onto semisolid media. Colonies were counted and photographed after 30 days of growth
(magnification: ⫻40). These results are representative of two independent experiments.
5201
BRAF
V599E
SUPPRESSION IN HUMAN MELANOMA
Currently, a Raf kinase inhibitor, BAY 43–9006 (20), is undergoing
worldwide clinical evaluation in Phase I and II trials in patients with
a variety of malignancies, including melanoma. However, BAY 43–
9006 inhibits both B-Raf and C-Raf kinase activities,
4
and any ben-
eficial or adverse effects of treatment may therefore result from
simultaneous inhibition of both kinases. Our results suggest that
targeted inhibition of B-Raf specifically in such tumors may be
equally efficacious and perhaps associated with less toxicity.
That CRAF expression was dispensable for the transformed pheno-
type in human melanoma cells was somewhat surprising. As the first
of the three Raf family isoforms identified, a large body of evidence
exists exploring the transforming properties of CRAF in mammalian
cell systems. These properties, however, are exquisitely dependent on
cellular context (7); for example, although a constitutively active form
of CRAF can readily transform NIH 3T3 cells, it is unable to do so in
RIE-1 cells (21). More recent experiments involving targeted disrup-
tion and mutation of Raf isoforms in mice implicate B-Raf as the more
potent activator of MEK in many cell and tissue types (22). The
minimal effects on transformation of CRAF suppression in the mel-
anoma cells studied here suggest that this isoform may not always be
the predominant effector of MAPK signaling in human cells either.
In summary, we find that suppression of BRAF
V599E
in WM793
human melanoma cells abrogates their transformed phenotype and
conclude, therefore, that agents that specifically inhibit activated
BRAF, and not CRAF, might be particularly efficacious in melanomas,
and perhaps other tumor types, that harbor activating mutations in this
proto-oncogene.
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