Mutant BRAF Induces DNA Strand Breaks, Activates DNA Damage Response Pathway, and Up-Regulates Glucose Transporter-1 in Nontransformed Epithelial Cells

Article (PDF Available)inAmerican Journal Of Pathology 180(3):1179-88 · March 2012with41 Reads
DOI: 10.1016/j.ajpath.2011.11.026 · Source: PubMed
Abstract
Although the oncogenic functions of activating BRAF mutations have been clearly demonstrated in human cancer, their roles in nontransformed epithelial cells remain largely unclear. Investigating the cellular response to the expression of mutant BRAF in nontransformed epithelial cells is fundamental to the understanding of the roles of BRAF in cancer pathogenesis. In this study, we used two nontransformed cyst108 and RK3E epithelial cell lines as models in which to compare the phenotypes of cells expressing BRAF(WT) and BRAF(V600E). We found that transfection of the BRAF(V600E), but not the BRAF(WT), expression vector suppressed cellular proliferation and induced apoptosis in both cell types. BRAF(V600E) generated reactive oxygen species, induced DNA double-strand breaks, and caused subsequent DNA damage response as evidenced by an increased number of pCHK2 and γH2AX nuclear foci as well as the up-regulation of pCHK2, p53, and p21. Because BRAF and KRAS (alias Ki-ras) mutations have been correlated with GLUT1 up-regulation, which encodes glucose transporter-1, we demonstrated here that expression of BRAF(V600E), but not BRAF(WT), was sufficient to up-regulate GLUT1. Taken together, our findings provide new insights into mutant BRAF-induced oncogenic stress that is manifested by DNA damage and growth arrest by activating the pCHK2-p53-p21 pathway in nontransformed cells, while it also confers tumor-promoting phenotypes such as the up-regulation of GLUT1 that contributes to enhanced glucose metabolism that characterizes tumor cells.
Tumorigenesis and Neoplastic Progression
Mutant BRAF Induces DNA Strand Breaks, Activates
DNA Damage Response Pathway, and Up-Regulates
Glucose Transporter-1 in Nontransformed Epithelial Cells
Jim Jinn-Chyuan Sheu,*
Bin Guan,
Fuu-Jen Tsai,* Erin Yi-Ting Hsiao,*
Chih-Mei Chen,* Raquel Seruca,
§
Tian-Li Wang,
and Ie-Ming Shih
From the Human Genetic Center,*China Medical University
Hospital, Taichung, Taiwan; the School of Chinese Medicine,
China Medical University, Taichung, Taiwan; the Departments of
Pathology, Oncology, Gynecology and Obstetrics,
Johns Hopkins
Medical Institutions, Baltimore, Maryland; and the Institute of
Molecular Pathology and Immunology,
§
The University of Porto,
Porto, Portugal
Although the oncogenic functions of activating BRAF mu-
tations have been clearly demonstrated in human cancer,
their roles in nontransformed epithelial cells remain
largely unclear. Investigating the cellular response to the
expression of mutant BRAF in nontransformed epithelial
cells is fundamental to the understanding of the roles of
BRAF in cancer pathogenesis. In this study, we used two
nontransformed cyst108 and RK3E epithelial cell lines as
models in which to compare the phenotypes of cells ex-
pressing BRAF
WT
and BRAF
V600E
. We found that transfec-
tion of the BRAF
V600E
, but not the BRAF
WT
, expression
vector suppressed cellular proliferation and induced
apoptosis in both cell types. BRAF
V600E
generated reactive
oxygen species, induced DNA double-strand breaks, and
caused subsequent DNA damage response as evidenced by
an increased number of pCHK2 and
H2AX nuclear foci as
well as the up-regulation of pCHK2, p53, and p21. Because
BRAF and KRAS (alias Ki-ras) mutations have been corre-
lated with GLUT1 up-regulation, which encodes glucose
transporter-1, we demonstrated here that expression of
BRAF
V600E
, but not BRAF
WT
, was sufficient to up-regulate
GLUT1. Taken together, our findings provide new in-
sights into mutant BRAF-induced oncogenic stress
that is manifested by DNA damage and growth arrest
by activating the pCHK2-p53-p21 pathway in non-
transformed cells, while it also confers tumor-pro-
moting phenotypes such as the up-regulation of
GLUT1 that contributes to enhanced glucose metabo-
lism that characterizes tumor cells. (Am J Pathol 2012,
180:1179 –1188; DOI: 10.1016/j.ajpath.2011.11.026)
BRAF probably represents the most frequently mutated
oncogene within the kinase family and activating point
mutation at the hot spot V600E of BRAF has been found
in several types of human neoplasms, most frequently in
melanoma,
1
papillary thyroid carcinoma,
2,3
high-grade
malignant astrocytoma
4
and ovarian low-grade serous
neoplasms.
5
BRAF protein is a downstream effector of
KRAS and participates in the signal transduction of the
mitogen activated protein kinase (MAPK) pathway that
controls cellular growth, differentiation, and survival.
6,7
Dimerization of the BRAF kinase domain with KSR or with
other RAF molecules has been recently shown to be
central to its activation mechanism.
8
Activating mutations
in BRAF and KRAS appear to exert equivalent tumor-
promoting effects as based on the mutual exclusive mu-
tation in both genes.
5,9
Constitutive activation of BRAF
due to V600E mutation activates the MAPK pathway and
results in up-regulation of several genes with tumor-pro-
moting functions including cyclin D1,
10,11
and targeting
BRAF and its downstream effectors has emerged as a
new therapeutic strategy for those tumors harboring the
BRAF mutation.
12–16
Ovarian low-grade serous tumor represents a unique
type of ovarian epithelial neoplasm and is distinct from
ovarian high-grade serous carcinoma, the conventional
type of ovarian cancer, based on their clinical, patholog-
ical, and molecular features.
17,18
Ovarian low-grade se-
Supported by China Medical University (CMU97-001), the National Sci-
ence Council (NSC98-2320-B-039-033-MY3), Taiwan, and by NIH/NCI
RO1CA103937, RO1CA129080, RO1CA116184, RO1CA148826 and
U24CA160036.
Accepted for publication November 14, 2011.
Address reprint requests to Ie-Ming Shih, M.D., Ph.D., Cancer Re-
search Building-2, Room 305, 1550 Orleans Street, Johns Hopkins Med-
ical Institutions, Baltimore, MD 21231, or Jim Jinn-Chyuan Sheu, Ph.D.,
the Human Genetic Center, 2 Yuh-Der Road, China Medical University
Hospital,Taichung, 40447, Taiwan. E-mail:ishih@jhmi.edu or jimsheu@mail.
cmu.edu.tw.
The American Journal of Pathology, Vol. 180, No. 3, March 2012
Copyright © 2012 American Society for Investigative Pathology.
Published by Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ajpath.2011.11.026
1179
rous tumors include a benign form, serous borderline
tumor, and the malignant counterpart, low-grade serous
carcinoma. Low-grade serous carcinoma develops from
serous borderline tumor, which in turn may arise from an
ovarian serous cystadenoma. Both ovarian low-grade se-
rous carcinoma and serous borderline tumor harbor
BRAF,KRAS,orERBB2 sequence mutation in more than
50% of cases.
5,19–21
Expression of active MAPK was
more frequently observed in low-grade serous tumors
than in high-grade ovarian serous carcinomas that have
rare mutations in either BRAF or KRAS.
22
Moreover, BRAF
and KRAS mutation status is a useful predictor of sensi-
tivity to MEK inhibition in ovarian cancer.
11,23
Interest-
ingly, BRAF or KRAS mutations can be detected in mor-
phologically normal-appearing cyst epithelium that is
adjacent to a serous borderline tumor but not in the cys-
tadenomas without concurrent borderline tumors, suggest-
ing the mutations may occur early during tumor progression
of ovarian low-grade serous tumors.
24
Although the onco-
genic roles of BRAF mutations have been established in
mouse models,
25
it remains largely unclear what are the
biological effects of BRAF mutations in the very beginning of
tumor formation such as in nontransformed epithelial cells.
Thus, in this study, we ectopically expressed either
BRAF
V600E
or BRAF
WT
in nontransformed epithelial cells
isolated from ovarian cystadenoma and RK3E cells, an ep-
ithelial cell model frequently used to test the oncogenic
effects, to determine the phenotypes in both cell lines. Fur-
thermore, a recent study has demonstrated that BRAF ex-
pression is required for the expression of GLUT1, which
encodes glucose transporter-1, and glucose deprivation is
associated with the development of KRAS pathway muta-
tions in tumor cells.
26
Thus, in this study, we also tried to
determine whether mutant BRAF plays a causal role in
up-regulating GLUT1 expression in our cellular model.
Materials and Methods
Cell Growth Assay
Expression vectors including the empty vector, wild-type
BRAF (BRAF
WT
), and mutant BRAF (BRAF
V600E
) were
kind gifts from Dr. Raquel Seruca (Institute of Molecular
Pathology and Immunology of the University of Porto,
Portugal). To determine the effects of BRAF
V600E
on non-
transformed epithelial cells, we established the cyst108
cell line, which was derived from a benign ovarian serous
cystadenoma. The reason to use the epithelial cells from
a cystadenoma was because cystadenoma represents
the immediate precursor lesion of serous borderline tu-
mor. To establish cyst108, we scraped the epithelial cells
directly from a benign serous cystadenoma after incubat-
ing a fragment of cystadenoma with 0.5% trypsin and
EDTA at 37°C for 15 minutes. The epithelial cells were
then rigorously suspended to obtain a single cell popu-
lation. After overnight culture, the cells were immortalized
with SV40 large T antigen, and the epithelial cells were
enriched using cold trypsin treatments to eliminate stro-
mal cells afterward. Cyst108 cells were maintained in
RPMI1640 medium supplemented with 10% fetal bovine
serum and have been passed for at least 30 passages;
these cells exhibited epithelioid morphology under
phase-contrast microscopy and expressed epithelial cell
markers, including cytokeratin 18 and Epi-CAM in 98%
of cells. They showed contact inhibition in vitro and were not
tumorigenic in nu/nu mice for more than 3 months. In this
study, we also included the RK3E cell line because it has
been widely used to assess transformation ability of poten-
tial oncogenes.
27–33
Cells transfected with BRAF
V600E
were
grown in 96-well plates at a density of 3000 cells per well. As
controls, the empty vector and BRAF
WT
vector were also
transfected into the cells. Cell number was measured daily
for 4 consecutive days using the SYBR green I staining
method (Molecular Probes, Eugene, OR). The data were
expressed as mean SD from five replicates.
Detection of Reactive Oxygen Species
Cyst108 and RK3E cells were seeded on chamber slides
(Nunc, Roskilde, Denmark), and subsequently trans-
fected with empty vector, BRAF
WT
, or BRAF
V600E
vectors
on the second day. Seventy-two hours after transfection,
cells were stained with 5
mol/L CellROX Deep Red
reagent (Invitrogen, Carlsbad, CA) in complete medium
for 30 minutes at 37°C, followed by three washes with
PBS and fixation with 4% formaldehyde. Cell nuclei were
counterstained with DAPI (Sigma, St. Louis, MO). Reac-
tive oxygen species (ROS)-positive cells were detected
and counted under a fluorescent microscope (excitation
wave: 644 nm and emission wave: 665 nm). The data
were expressed as mean SD from triplicates.
DNA Strand Break Assay
DNA strand breaks were quantified using a Comet assay
kit (Trevigen, Gaithersburg, MD) as previously de-
scribed.
33,34
Briefly, transfected cells were harvested in
ice-cold PBS, and the cell number was adjusted at a
density of 1 10
5
cells/mL. Cells were mixed with
LMAgarose at 1:10 ratio (v/v) and spread onto the Com-
etSlide immediately. After gel solidification, cells on
slides were lysed, and DNA in the cells was denatured by
using the buffers provided by the kit. Fragmented DNA
strands were separated from nuclei by electrophoresis
and detected by SYBR Green staining. The percentage of
comet-like nuclei (with DNA strand breaks) was counted
under a fluorescent microscope from five randomly se-
lected high-power fields (40) with each approximately
containing 100 nuclei. UVC-treated cells at sublethal dose
were used as the positive control in this assay.
Immunofluorescence Staining
To determine whether BRAF
V600E
expression resulted in
a DNA damage response, transfected cells were seeded
in chamber slides at a density of 5000 cells per well. At
different time points, cells were fixed with para-formalde-
hyde and incubated with anti–phospho-CHK2 (pCHK2)
antibody (clone ab38461; Abcam, Cambridge, MA) or
anti-
H2AX antibody (clone ab11174; Abcam) for 2 hours
followed by rhodamine-conjugated anti-rabbit antibody
1180 Sheu et al
AJP March 2012, Vol. 180, No. 3
(Jackson ImmunoResearch Laboratories, West Grove,
PA) and nuclei were counterstained with DAPI (Sigma, St.
Louis, MO). Cells transfected with an empty vector or
BRAF
WT
vector were used as controls.
Western Blot Analysis
Protein lysates from different groups were collected at
different time points after gene transfection. Proteins
were then separated by SDS-PAGE and transferred onto
polyvinylidene fluoride (PVDF) membranes. To determine
whether BRAF
V600E
caused activation of the DNA dam-
age response pathway, we performed Western blot anal-
ysis by hybridizing membranes with antibodies against
H2AX (clone ab11174; Abcam), phosphor-CHK2 (clone
ab38461; Abcam), p53 (clone sc-6243; Santa Cruz Bio-
technology, Santa Cruz, CA), and p21 (clone sc-6246;
Santa Cruz Biotechnology), all of which are involved in
the DNA damage response pathway
35,36
for 2 hours at
room temperature. Antibodies against glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) were used as the
loading control. To detect GLUT1, the membranes were
hybridized with an affinity-purified rabbit anti-GLUT1
polyclonal antibody (Millipore, Bedford, MA). After three
washes with 0.01% Tween 20 in Tris-buffered saline, the
membranes were blotted with horseradish peroxidase-
conjugated anti-mouse (Pierce, Rockford, IL) or anti-rab-
bit (Cell Signaling Technology, Danvers, MA) antibodies
for 1 hour at room temperature. Protein bands were re-
vealed by chemiluminescence (Amersham Biosciences,
Arlington Heights, IL).
GLUT1 Immunohistochemistry
Paraffin-embedded tissues from 33 cases of ovarian low-
grade serous tumors (serous borderline tumors and low-grade
serous carcinomas) were obtained from the Department of
Pathology at the Johns Hopkins Hospital, Baltimore, MD. Ac-
quisition of tissue specimens and clinical information were
approved under the regulations of the institutional review
board. There were 10 cases of low-grade serous carcinomas
that metastasized or disseminated to intraperitoneal soft tis-
sues. For immunohistochemistry, the unstained slides were
subjected to antigen retrieval by boiling the slides in citrate
buffer (pH 6.0) (Zymed, South San Francisco, CA) for 20 min-
utes. After blocking, samples were then stained with an affinity-
purified rabbit anti-GLUT1 polyclonal antibody (Millipore)
at a 1:600 dilution at room temperature for 1 hour. An
EnVisionSystem peroxidase kit (DAKO, Carpentaria, CA)
was used for chromogen development. Immunointensity was
independently scored by two investigators based on mem-
brane immunoreactivity and labeled as negative (0), weakly
positive (1), moderately positive (2), and strongly positive
(3) groups. For discordant cases, a third investigator scored,
and the final intensity score was determined by the majority
scores.
Results
To determine the effect of mutant BRAF on cyst108 and
RK3E cells, we transfected both cell lines with constructs
that expressed mutant (V600E) and wild-type BRAF and
compared the proliferative activity of cells in vitro (Figure 1,A
Figure 1. Expression of mutant BRAF (V600E) suppresses cellular proliferation and induces pCHK2 and
H2AX nuclear foci. Forty-eight hours after transfecting
epithelial cells, robust expression levels of wild-type and mutant BRAF can be detected in cyst108 cells (A) and in RK3E cells (B). A reduced cellular proliferation
was recorded in cyst108 cells (C) and in RK3E cells (D) that expressed mutant BRAF. Forty-eight hours after transfection, cells were stained for pCHK2 and
H2AX.
Nuclear foci for pCHK2 and
H2AX immunofluoresence were observed in mutant BRAF-expressing cyst108 cells (E) and RK3E cells (F). UV light-treated cells serve
as the positive control for immunofluoresence staining. MT, mutant; WT, wild type.
BRAF Mutation and DNA Damage 1181
AJP March 2012, Vol. 180, No. 3
and B). As shown in Figure 1, C and D, both cyst108 and
RK3E cells expressing wild-type BRAF continued grow-
ing as in the vector control groups. By contrast, the pro-
liferative activity significantly decreased in cyst108 and
RK3E cells when they expressed BRAF
V600E
. One of the
explanations for the growth-inhibitory effects by mutant
BRAF is oncogenic stress that describes growth arrest
and cellular senescence as a result of expression of
oncogenes in otherwise normal cells.
37,38
Because on-
cogenic stress is a poorly defined process, we sought to
determine whether it was directly related to DNA damage
response. First, we performed immunofluoresence stain-
ing for two representative markers of DNA damage, in-
cluding phosphorylated checkpoint kinase 2 (pCHK2)
and phosphorylated histone 2AX (
H2AX) in cyst108 and
RK3E cells. We found that BRAF
V600E
-expressing cells
demonstrated an increased number of nuclear foci of
pCHK2 and
H2AX as compared to the cells in the con-
trol groups, including BRAF
WT
-expressing, vector con-
trol, and parental cells (Figure 1, E and F). UV-irradiated
cells served as the positive control which induced numer-
ous pCHK2 and
H2AX foci in the nuclei. Western blot
analysis further demonstrated a time-dependent increase
in protein levels of pCHK2,
H2AX, p53, and p21 in
BRAF
V600E
, but not in BRAF
WT
, transfected cyst108 and
RK3E cells (Figure 2, A and B).
The above findings indicated that expression of mutant
BRAF activated the DNA damage pathway and subse-
quently induced growth arrest because of up-regulation
of p53 and p21 proteins. This observation also suggests
that DNA strand breaks occur due to mutant BRAF, but
not wild-type BRAF, expression. To determine whether
this was the case, we directly visualized the individual
cells with DNA strand breaks in cyst108 and RK3E cells.
On electrophoresis, DNA with double-strand breaks mi-
grated out of the nuclei, forming a comet tail–like struc-
ture, whereas the undamaged DNA remained within the
nuclei. Figure 2, C and D, showed a higher percentage of
comet-like cells in the BRAF
V600E
-expressing group than
in control group transfected with BRAF
WT
or vector only
as early as 48 hours after transfection. These findings
suggest that ectopic expression of mutant BRAF proteins
caused DNA strand breaks, initiated DNA damage re-
sponse and subsequently up-regulated p53 and p21,
leading to growth arrest in nontransformed epithelial
cells. Although DNA double-strand breaks have several
causes, we asked in this study whether expression of
mutant BRAF
V600E
was associated with generation of re-
active oxygen species, which were detected by CellROX
Deep Red reagent. Both cyst108 and RK3E cells were
analyzed, and their percentage of positive cells was de-
termined under a fluorescent microscope. As shown in
Figure 3, the percentage of positive cells was signifi-
cantly higher in cells expressing BRAF
V600E
than cells
expressing BRAF
WT
or in vector control cells.
Several studies have shown that oncogene-induced
DNA damage response serves as a molecular pressure
to select tumorigenic clones during cancer develop-
ment.
39–41
To study whether BRAF
V600E
can also pro-
mote tumor progression, we selected two cyst108 cell
clones, MT-1 and MT-2, that were refractory to
BRAF
V600E
-induced cell death at a low cell density (1000
cells/25 cm
2
). Two BRAF
WT
cell clones, WT-1 and WT-2,
were also analyzed as controls. Anchorage-independent
assay showed that cell clones that overexpress either
BRAF
WT
or BRAF
V600E
formed colonies in soft-agar (Figure
4A). Constitutive expression of BRAF
V600E
confirmed MT-1
and MT-2 clones to be highly transformed as evidenced by
more colonies (Figure 4A). Consistent with our previous
study,
42
Western blot analysis (Figure 4B) and quantitative
PCR (Figure 4C) confirmed lower expression levels of Arf in
BRAF
V600E
-treated, but not in BRAF
WT
-treated, clones that
accounted for the failure of p53 up-regulation.
Previous study demonstrated that BRAF mutation was
associated with up-regulation of GLUT1, which was respon-
sible for an increased glucose uptake and promotion of
Figure 2. Mutant BRAF activates DNA damage
response pathway and induces DNA strand
breaks. Western blot analysis demonstrates a
time-dependent increase in protein expression
of p53, pCHK2,
H2AX, and p21 in cyst108 cells
(A) and in RK3E cells (B). GAPDH serves as the
protein loading control. Cells with DNA strand
breaks were analyzed by the Comet assay. For
both cyst108 cells (C) and RK3E cells (D), a
significantly higher percentage of comet-like nu-
clei are found in the BRAF
V600E
expressing
group than in control group transfected with
BRAF
WT
or vector only 48 hours after transfec-
tion. MT, mutant; WT, wild type.
1182 Sheu et al
AJP March 2012, Vol. 180, No. 3
cellular survival and growth in cancer cells.
26
However, it is
not known whether mutant BRAF is sufficient to up-regulate
GLUT1 expression. Therefore, we applied Western blot
analysis to demonstrate that expression of GLUT1 signifi-
cantly increased in cyst108 and RK3E cells expressing
BRAF
V600E
as compared to those cells expressing
BRAF
WT
36 hours and 48 hours after transfection (Figure
5, A and C). Similarly, real-time quantitative PCR also
demonstrated a significant increase at the mRNA levels
of GLUT1 in cells expressing BRAF
V600E
(Figure 5, B and
D). To extrapolate the in vitro finding to human speci-
mens, we performed immunohistochemistry of GLUT1 on
a panel of 33 cases of ovarian low-grade serous tumors,
including 23 serous borderline tumors and 10 low-grade
serous carcinomas, and correlated their GLUT1 immuno-
reactivity and the mutation status of BRAF and KRAS.
Consistent with our previous study,
5
we found that muta-
tions in BRAF and KRAS were mutually exclusive, and
Figure 3. Measurement of reactive oxygen species in cyst108 (A) and RK3E cells (B). CellROX Deep Red reagent was used to detect the reactive oxygen species
and percentage of positive cells was recorded under a fluorescent microscope. Cell nuclei were counter stained with DAPI (blue fluorescence). The percentage
of positive cells (red fluorescence in cytoplasm) is significantly higher in cells expressing BRAF
V600E
than cells expressing BRAF
WT
or vector control cells.
Figure 4. Long-term expression of BRAF
V600E
promotes a more transforming phenotype in cyst108
cells. A soft-agar assay was performed to detect the tumorigenic activity of cell clones that overexpress
BRAF
V600E
or BRAF
WT
under G418 selection (A). Western blot analysis (B) and quantitative PCR (C)
were performed to detect expression levels of BRAF, Arf, and p53 in selected cell clones. GAPDH was
used as the protein loading control. Vector-treated cells serve as controls in both assays. MT, mutant;
WT, wild type.
BRAF Mutation and DNA Damage 1183
AJP March 2012, Vol. 180, No. 3
mutations in either one of the genes were detected in 18
(55%) of 33 specimens. Specifically, mutations of BRAF
or KRAS were found in 6 (60%) of 10 metastatic/dissem-
inated low-grade serous carcinomas, whereas the muta-
tions were recorded in 12 (52%) of 23 serous borderline
tumors.
Immunohistochemically, we found that all 33 tumor
samples were positive for GLUT1 staining except one
serous borderline tumor (case 19) (Figure 6A). GLUT1
immunoreactivity was only detected in the cell membrane
and cytoplasm of tumor cells, but not in stromal cells. All
of the tumor specimens harboring either BRAF or KRAS
mutations showed GLUT1 immunostaining; however,
there was no significance in staining intensity among
groups with BRAF mutation, KRAS mutation, and wild-
type (P0.1, Mann-Whitney test). All 10 cases of low-
grade serous carcinomas expressed GLUT1 protein, of
which the immunostaining intensity score ranged from 1
to 3. Representative photomicrographs of GLUT1 stain-
ing in three advanced-stage low-grade serous carcino-
mas with different mutation status of BRAF and KRAS are
illustrated in Figure 6B.
Discussion
Using both ovarian cystadenoma epithelial cells and
RK3E cells as the models, we were able to demonstrate
that mutant BRAF induced growth arrest in both cell types
and such “oncogenic stress” is attributed, at least in part,
by the DNA damage response pathway that subse-
quently activates p53 and p21. We provide cogent evi-
dence in this report that expression of mutant, but not
wild-type, BRAF directly causes DNA strand breaks and
accounts for the activation of the DNA damage response.
Transcription-induced DNA double-strand breaks have
been proposed to occur when novel transcription is in-
duced during tumor development.
43
It is likely that ex-
pression of mutant BRAF, like deregulated expression of
Myc,
44
induces oxidative stress that is responsible for
topoisomerase TOP2B-dependent DNA double-strand
breaks in epithelial cells. In fact, we have also observed
that expression of mutant BRAF was associated with gen-
eration of reactive oxygen species in epithelial cells. As
occurs in oncogenic stress, increased p53 levels due to
the ATM-pCHK2-p53-p21 pathway activation lead to cell
growth arrest at G1 or G2/M and/or in apoptosis.
45
Con-
sistent with this view, it has also been reported that
ovarian serous borderline tumors have a much lower
proliferative activity
46,47
and a significantly lower TP53
mutation frequency than ovarian high-grade serous
carcinoma, the conventional type of ovarian cancer,
that harbor neither BRAF nor KRAS mutations.
48
Al-
though this is our preferred view, other mechanisms for
mutant BRAF-induced p53 activation should be also
pointed out. For example, a recent study demonstrates
that the Jnk pathway signaling is involved in the acti-
vation of p53 in response to both KRAS and Neu on-
cogene expression.
49
The results from this study may help further understand-
ing of the molecular pathogenesis of ovarian low-grade
serous carcinoma. It can be speculated that epithelial cells
from a serous borderline tumor may evolve a mechanism to
restrain tumor progression.
39–41
In response to BRAF mu-
tations, activation of ATM/pCHK2/p53/p21 is thus important
to suppress tumor cell proliferation. Although not frequently
occurring, serous borderline tumors may progress to low-
grade serous carcinomas, which are frankly malignant neo-
plasm and are often associated with high morbidity and
mortality. How do tumor cells in low-grade serous carci-
noma overcome the growth inhibitory effect due to activat-
ing mutations of BRAF? We propose that additional mo-
lecular genetic alterations occur during progression from
Figure 5. Increased GLUT1 protein expression
in cells that express mutant BRAF. Western blot
analysis was performed 36 hours and 48 hours
after transfection. Expression of GLUT1 is signif-
icantly increased in cyst108 (A) and RK3E cells
(C) expressing BRAF
V600E
as compared to those
cells expressing BRAF
WT
at both time points.
GAPDH serves as the protein loading control.
Quantitative PCR was performed to detect
mRNA levels of GLUT1 in cyst108 (B) and RK3E
cells (D) 48 hours after gene transfection. MT,
mutant.
1184 Sheu et al
AJP March 2012, Vol. 180, No. 3
a serous borderline tumor to a low-grade serous carci-
noma, and such molecular alterations abolish the check-
point controlled by the ATM-p53 pathway, allowing cells
to proliferate despite the presence of mutant BRAF-in-
duced DNA damage and up-regulation of p53 and p21.
To this end, a recent study based on whole exome se-
quencing reveals rare somatic mutations in ovarian low-
grade serous carcinomas except activating mutations in
KRAS and BRAF, suggesting that molecular genetic
changes other than sequence mutations may be respon-
sible for tumor progression.
21
In fact, a previous study
that analyzed the genome-wide copy number altera-
tions in ovarian serous neoplasms has reported that
hemizygous ch1p36 deletion and ch9p21 homozygous
or hemizygous deletions were much more common in
ovarian low-grade serous carcinomas than in serous
borderline tumor.
42
The ch1p36 region contains sev-
eral candidate tumor suppressors, including miR-34a,
which is required for DNA damage response and is the
direct p53 target that mediates its tumor suppressor
functions.
50,51
Similarly, the ch9p21 region corre-
sponding to the CDKN2A/B locus encodes three well-
known tumor suppressor proteins, p14 (Arf), p16, and
p15. CDKN2A and CDKN2B share similar function in
inhibiting cyclin-dependent kinase. Arf is a potent tu-
mor suppressor that blocks cell cycle progression by
interfering with the p53-negative regulator, MDM2,
thereby stabilizing p53 protein expression. Besides,
the expression level of CDKN2A was enhanced in re-
sponse to oncogene-induced stress such as by the
activation of the RAS/RAF/MEK signaling pathway.
Thus, deletions or silencing of miR-34a and CDKN2A/B
loci may uplift the p53 checkpoint on BRAF mutations
and permit tumor cells to escape from cell-cycle arrest
and become more aggressive, as shown in Figure 4.
The above view is supported by the fact that expres-
sion of BRAF
V600E
in the lung epithelium or in melano-
cytes fails to result in frankly malignancy unless tumor
suppressor genes such as Pten are inactivated.
52,53
Figure 6. Expression of GLUT1 in low-grade ovarian serous tumors and the correlation of GLUT1 immunoreactivity with mutation status of BRAF and KRAS.
Immunohistochemistry was performed in 33 cases of low-grade ovarian serous tumors including 23 serous borderline tumors and 10 advanced stage low-grade
serous carcinomas. A: The GLUT1 immunostaining intensity is shown for all cases, which are grouped into BRAF mutation, KRAS mutation, and wild-type groups.
Mutations of KRAS and BRAF are mutually exclusive. Low-grade serous carcinomas are labeled with asterisks, otherwise they are serous borderline tumors. B:
Representative photomicrographs of low-grade serous carcinomas from each group are illustrated, and their mutation status in KRAS and BRAF is indicated below.
The case numbers correspond to those shown above.
BRAF Mutation and DNA Damage 1185
AJP March 2012, Vol. 180, No. 3
If BRAF mutations result in growth arrest in serous
borderline tumor, why is this genotype clonally selected
and can be detected in low-grade serous carcinomas
even when they are at advanced stages (Figure 6A)? We
reasoned that once tumor cells bypass the oncogene-
induced growth arrest, they may benefit from tumor-pro-
moting phenotypes conferred by BRAF mutations, such
as metabolic switches among several tumor-promoting
functions. To explore this possibility, we focused on
GLUT1, a gene that encodes glucose transporter-1,
which has been implicated to play a critical role in regu-
lating glucose metabolism and energy consumption in
cancer cells.
54
The reason to focus on GLUT1 stems from
a recent report showing that either BRAF or KRAS muta-
tion is required for GLUT1 overexpression and glucose
deprivation contributes to the development of mutations
in BRAF and KRAS in colorectal cancer cells.
26
In that
report, the glycolysis inhibitor, 3-bromopyruvate, prefer-
entially inhibited the growth of cells with either BRAF or
KRAS mutations, suggesting that cancer cells may de-
velop dependency on increased glucose metabolism
due to GLUT1 overexpression. The observation in the
current study demonstrating that BRAF mutation induced
up-regulation of GLUT1 expression, thus, provides direct
evidence that mutant BRAF is not only required, but also
sufficient, to up-regulate GLUT1 expression.
We also report in this study that the great majority of
low-grade ovarian serous tumors express GLUT1. More
specifically, although all borderline tumors harboring ei-
ther BRAF or KRAS mutations express GLUT1, those
cases with wild-type BRAF and KRAS also show GLUT1
immunoreactivity except in one case. This observation
indicates that some low-grade ovarian serous tumors with
wild-type BRAF and KRAS up-regulate GLUT1 using dif-
ferent mechanism not directly related to mutations of
BRAF or KRAS, such as those mediated by hypoxia.
55
Our data demonstrate that mutation of BRAF and KRAS
represents one of the mechanisms to up-regulate GLUT1.
It can be speculated that for those tumors with either
BRAF or KRAS mutation, blocking the pathway by MEK or
BRAF inhibitors may be responsible for tumor suppres-
sion due to down-regulation of GLUT1 expression. For
those tumors with wild-type BRAF and KRAS, MEK or
BRAF inhibitor may not work well because alternative
mechanisms are used by those tumor cells to up-regulate
GLUT1. Because expression of GLUT1 has been shown
as a reliable marker to predict positive fluorodeoxyglu-
cose uptake by positron emission tomography in ovarian
cancer,
56
our data suggest the potential to apply fluoro-
deoxyglucose uptake imaging to detect low-grade ovar-
ian serous carcinomas.
In conclusion, our findings provide new insights into
further defining oncogenic stress induced by mutant
BRAF in nontransformed epithelial cells. We demonstrate
for the first time that expression of mutant, but not
wild-type, BRAF leads to DNA double-strand breaks,
followed by activation of pCHK2-p53 DNA damage
response pathway that is responsible for growth inhi-
bition and tumor suppression. On the other hand, sim-
ilar to other oncogenes that regulate cellular metabo-
lism in favor of tumor growth,
57
mutant BRAF also
confers oncogenic phenotypes by up-regulating
GLUT1 of which abundant GLUT1 proteins contribute
to enhanced glucose uptake and metabolism that
characterize cancer cells.
Acknowledgments
We are grateful for the technical support from I-Wen Chiu
and Carmen Chan (China Medical University Hospital)
and for the kind gift of BRAF expression vectors from Dr.
Raquel Seruca (University of Porto, Portugal).
References
1. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague
J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R,
Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A,
Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake
H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones
K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri
G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST,
Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ,
Wooster R, Stratton MR, Futreal PA: Mutations of the BRAF gene in
human cancer. Nature 2002, 417:949 –954
2. Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, Westra
WH, Ladenson PW, Sidransky D: BRAF mutation in papillary thyroid
carcinoma. J Natl Cancer Inst 2003, 95:625– 627
3. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA:
High prevalence of BRAF mutations in thyroid cancer: genetic evi-
dence for constitutive activation of the RET/PTCRAS- BRAF signaling
pathway in papillary thyroid carcinoma. Cancer Res 2003, 63:1454
1457
4. Schiffman JD, Hodgson JG, VandenBerg SR, Flaherty P, Polley MY,
Yu M, Fisher PG, Rowitch DH, Ford JM, Berger MS, Ji H, Gutmann
DH, James CD: Oncogenic BRAF mutation with CDKN2A inactivation
is characteristic of a subset of pediatric malignant astrocytomas.
Cancer Res 2010, 70:512–519
5. Singer G, Oldt R 3rd, Cohen Y, Wang BG, Sidransky D, Kurman RJ,
Shih Ie M: Mutations in BRAF and KRAS characterize the develop-
ment of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003,
95:484 – 486
6. Ji H, Wang Z, Perera SA, Li D, Liang MC, Zaghlul S, McNamara K,
Chen L, Albert M, Sun Y, Al-Hashem R, Chirieac LR, Padera R,
Bronson RT, Thomas RK, Garraway LA, Janne PA, Johnson BE, Chin
L, Wong KK: Mutations in BRAF and KRAS converge on activation of
the mitogen-activated protein kinase pathway in lung cancer mouse
models. Cancer Res 2007, 67:4933– 4939
7. Downward J: Targeting RAS signalling pathways in cancer therapy.
Nat Rev Cancer 2003, 3:11–22
8. Rajakulendran T, Sahmi M, Lefrancois M, Sicheri F, Therrien M: A
dimerizationdependent mechanism drives RAF catalytic activation.
Nature 2009, 461:542–545
9. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B,
Velculescu VE: Tumorigenesis: rAF/RAS oncogenes and mismatch-
repair status. Nature 2002, 418:934
10. Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, Ye Q,
Lobo JM, She Y, Osman I, Golub TR, Sebolt-Leopold J, Sellers WR,
Rosen N: BRAF mutation predicts sensitivity to MEK inhibition. Nature
2005, 439:358 –362
11. Pohl G, Ho CL, Kurman RJ, Bristow R, Wang TL, Shih Ie M: Inactiva-
tion of the mitogenactivated protein kinase pathway as a potential
target-based therapy in ovarian serous tumors with KRAS or BRAF
mutations. Cancer Res 2005, 65:1994 –2000
12. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin
J, Dummer R, Garbe C, Testori A, Maio M, Hogg D, Lorigan P, Lebbe
C, Jouary T, Schadendorf D, Ribas A, O’Day SJ, Sosman JA, Kirk-
wood JM, Eggermont AM, Dreno B, Nolop K, Li J, Nelson B, Hou J,
Lee RJ, Flaherty KT, McArthur AG: Improved survival with vemu-
rafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011,
364:2507–2516
1186 Sheu et al
AJP March 2012, Vol. 180, No. 3
13. Vultur A, Villanueva J, Herlyn M: BRAF inhibitor unveils its potential
against advanced melanoma. Cancer Cell 2010, 18:301–302
14. Vultur A, Villanueva J, Herlyn M: Targeting BRAF in advanced
melanoma: a first step toward manageable disease. Clin Cancer Res
2011, 17:1658 –1663
15. Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis
M, Cipolla AK, Wubbenhorst B, Xu X, Gimotty PA, Kee D, Santiago-
Walker AE, Letrero R, D’Andrea K, Pushparajan A, Hayden JE, Brown
KD, Laquerre S, McArthur GA, Sosman JA, Nathanson KL, Herlyn M:
Acquired resistance to BRAF inhibitors mediated by a RAF kinase
switch in melanoma can be overcome by cotargeting MEK and
IGF-1R/PI3K. Cancer Cell 2010, 18:683– 695
16. Tuveson DA, Weber BL, Herlyn M: BRAF as a potential therapeutic
target in melanoma and other malignancies. Cancer Cell 2003,
4:95–98
17. Shih Ie M, Kurman RJ: Ovarian tumorigenesis: a proposed model
based on morphological and molecular genetic analysis. Am J Pathol
2004, 164:1511–1518
18. Cho KR, Shih Ie M: Ovarian cancer. Annu Rev Pathol 2009, 4:287–313
19. Nakayama K, Nakayama N, Kurman RJ, Cope L, Pohl G, Samuels Y,
Velculescu VE, Wang TL, Shih Ie M: Sequence mutations and ampli-
fication of PIK3CA and AKT2 genes in purified ovarian serous neo-
plasms. Cancer Biol Ther 2006, 5:779 –785
20. Sieben NL, Macropoulos P, Roemen GM, Kolkman-Uljee SM, Jan
Fleuren G, Houmadi R, Diss T, Warren B, Al Adnani M, De Goeij AP,
Krausz T, Flanagan AM: In ovarian neoplasms, BRAF, but not KRAS,
mutations are restricted to low-grade serous tumours. J Pathol 2004,
202:336 –340
21. Jones S, Wang TL, Kurman RJ, Nakayama K, Velculescu VE, Vogel-
stein B, Kinzler KW, Papadopoulos N, Shih IM. Low-grade serous
carcinomas of the ovary contain very few point mutations. J Pathol
2012, 226:413– 420
22. Hsu C-Y, Bristow R, Cha M, Wang BG, Ho C-L, Kurman RJ, Wang T-L,
Shih I-M: Characterization of active mitogen-activated protein kinase
in ovarian serous carcinomas. Clin Cancer Res 2004, 10:6432– 6436
23. Nakayama N, Nakayama K, Yeasmin S, Ishibashi M, Katagiri A, Iida
K, Fukumoto M, Miyazaki K: KRAS or BRAF mutation status is a useful
predictor of sensitivity to MEK inhibition in ovarian cancer. Br J
Cancer 2008, 99:2020 –2028
24. Ho C-L, Kurman RJ, Dehari R, Wang T-L, Shih I-M: Mutations of BRAF
and KRAS precede the development of ovarian serous borderline
tumors. Cancer Res 2004, 64:6915– 6918
25. Charles RP, Lezza G, Amendola E, Dankort D, McMahon M: Muta-
tionally activated BRAFV600E elicits papillary thyroid cancer in the
adult mouse. Cancer Res 2011, 71:3863–3871
26. Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H,
Schmidt K, Willson JK, Markowitz S, Zhou S, Diaz LA Jr, Velculescu
VE, Lengauer C, Kinzler KW, Vogelstein B, Papadopoulos N: Glucose
deprivation contributes to the development of KRAS pathway muta-
tions in tumor cells. Science 2009, 325:1555–1559
27. Kolligs FT, Hu G, Dang CV, Fearon ER: Neoplastic transformation of
RK3E by mutant beta-catenin requires deregulation of Tcf/Lef tran-
scription but not activation of c-myc expression. Mol Cell Biol 1999,
19:5696 –5706
28. Kolligs FT, Nieman MT, Winer I, Hu G, Van Mater D, Feng Y, Smith IM,
Wu R, Zhai Y, Cho KR, Fearon ER: ITF-2, a downstream target of the
Wnt/TCF pathway, is activated in human cancers with beta-catenin
defects and promotes neoplastic transformation. Cancer Cell 2002,
1:145–155
29. Komiya T, Park Y, Modi S, Coxon AB, Oh H, Kaye FJ: Sustained
expression of Mect1-Maml2 is essential for tumor cell growth in
salivary gland cancers carrying the t(11;19) translocation. Oncogene
2006, 25:6128 – 6132
30. Bommer GT, Jager C, Durr EM, Baehs S, Eichhorst ST, Brabletz T, Hu
G, Frohlich T, Arnold G, Kress DC, Goke B, Fearon ER, Kolligs FT:
DRO1, a gene down-regulated by oncogenes, mediates growth inhi-
bition in colon and pancreatic cancer cells. J Biol Chem 2005, 280:
7962–7975
31. Foster KW, Ren S, Louro ID, Lobo-Ruppert SM, McKie-Bell P, Grizzle
W, Hayes MR, Broker TR, Chow LT, Ruppert JM: Oncogene expres-
sion cloning by retroviral transduction of adenovirus E1A-immortal-
ized rat kidney RK3E cells: transformation of a host with epithelial
features by c-MYC and the zinc finger protein GKLF. Cell Growth
Differ 1999, 10:423– 434
32. Hendrix ND, Wu R, Kuick R, Schwartz DR, Fearon ER, Cho KR:
Fibroblast growth factor 9 has oncogenic activity and is a down-
stream target of Wnt signaling in ovarian endometrioid adenocarci-
nomas. Cancer Res 2006, 66:1354 –1362
33. Sheu JJ, Guan B, Choi JH, Lin A, Lee CH, Hsiao YT, Wang TL, Tsai FJ,
Shih IM: Rsf-1, a chromatin remodeling protein, induces DNA dam-
age and promotes genomic instability. J Biol Chem 2010, 285:38260 –
38269
34. Singh NP, McCoy MT, Tice RR, Schneider EL: A simple technique for
quantitation of low levels of DNA damage in individual cells. Exp Cell
Res 1988, 175:184 –191
35. Kastan MB, Bartek J: Cell-cycle checkpoints and cancer. Nature
2004, 432:316 –323
36. Shiloh Y: ATM and related protein kinases: safeguarding genome
integrity. Nat Rev Cancer 2003, 3:155–168
37. Haigis KM, Wistuba II, Kurie JM: Lung premalignancy induced by
mutant B-Raf, what is thy fate? To senesce or not to senesce, that is
the question. Genes Dev 2007, 21:361–366
38. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW: Oncogenic
ras provokes premature cell senescence associated with accumula-
tion of p53 and p16INK4a. Cell 1997, 88:593– 602
39. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg
P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas J, Bartek J:
DNA damage response as a candidate anticancer barrier in early
human tumorigenesis. Nature 2005, 434:864 –870
40. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N,
Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC, Takaoka M,
Nakagawa H, Tort F, Fugger K, Johansson F, Sehested M, Andersen
CL, Dyrskjot L, Orntoft T, Lukas J, Kittas C, Helleday T, Halazonetis
TD, Bartek J, Gorgoulis VG: Oncogene-induced senescence is part
of the tumorigenesis barrier imposed by DNA damage checkpoints.
Nature 2006, 444:633– 637
41. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A,
Liloglou T, Venere M, Ditullio RA Jr, Kastrinakis NG, Levy B, Kletsas
D, Yoneta A, Herlyn M, Kittas C, Halazonetis TD: Activation of the
DNA damage checkpoint and genomic instability in human precan-
cerous lesions. Nature 2005, 434:907–913
42. Kuo KT, Guan B, Feng Y, Mao TL, Chen X, Jinawath N, Wang Y,
Kurman RJ, Shih IM, Wang TL: Analysis of DNA copy number alter-
ations in ovarian serous tumors identifies new molecular genetic
changes in low-grade and high-grade carcinomas. Cancer Res 2009,
69:4036 – 4042
43. Haffner MC, De Marzo AM, Meeker AK, Nelson WG, Yegnasubrama-
nian S: Transcription-induced DNA double strand breaks: both onco-
genic force and potential therapeutic target? Clin Cancer Res 2011,
17:3858 –3864
44. Sagun KC, Carcamo JM, Golde DW: Antioxidants prevent oxidative
DNA damage and cellular transformation elicited by the over-expres-
sion of c-MYC. Mutat Res 2006, 593:64 –79
45. Vogelstein B, Lane D, Levine AJ: Surfing the p53 network. Nature
2000, 408:307–310
46. Garzetti GG, Ciavattini A, Goteri G, De Nictolis M, Stramazzotti D,
Lucarini G, Biagini G: Ki67 antigen immunostaining (MIB 1 monoclo-
nal antibody) in serous ovarian tumors: index of proliferative activity
with prognostic significance. Gynecol Oncol 1995, 56:169 –174
47. Huettner PC, Weinberg DS, Lage JM: Assessment of proliferative
activity in ovarian neoplasms by flow and static cytometry. Correlation
with prognostic features. Am J Pathol 1992, 141:699 –706
48. Singer G, Stöhr R, Cope L, Dehari R, Hartmann A, Cao DF, Wang TL,
Kurman RJ, Shih IeM: Patterns of p53 mutations separate ovarian
serous borderline tumors and low- and high-grade carcinomas and
provide support for a new model of ovarian carcinogenesis: a muta-
tional analysis with immunohistochemical correlation. Am J Surg
Pathol 2005, 29:218 –224
49. Haigis KM, Sweet-Cordero A: New insights into oncogenic stress. Nat
Genet 2011, 43:177–178
50. Kato M, Paranjape T, Muller RU, Nallur S, Gillespie E, Keane K,
Esquela-Kerscher A, Weidhaas JB, Slack FJ: The mir-34 microRNA is
required for the DNA damage response in vivo in C. elegans and in
vitro in human breast cancer cells. Oncogene 2009, 28:2419 –2424
51. He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, Xue W, Zender
L, Magnus J, Ridzon D, Jackson AL, Linsley PS, Chen C, Lowe SW,
Cleary MA, Hannon GJ: A microRNA component of the p53 tumour
suppressor network. Nature 2007, 447:1130 –1134
BRAF Mutation and DNA Damage 1187
AJP March 2012, Vol. 180, No. 3
52. Dankort D, Filenova E, Collado M, Serrano M, Jones K, McMahon M:
A new mouse model to explore the initiation, progression, and ther-
apy of BRAFV600E-induced lung tumors. Genes Dev 2007, 21:379
384
53. Dankort D, Curley DP, Cartlidge RA, Nelson B, Karnezis AN, Damsky
WE Jr, You MJ, DePinho RA, McMahon M, Bosenberg M: Braf(V600E)
cooperates with Pten loss to induce metastatic melanoma. Nat Genet
2009, 41:544 –552
54. Amann T, Hellerbrand C: GLUT1 as a therapeutic target in hepatocel-
lular carcinoma. Expert Opin Ther Targets 2009, 13:1411–1427
55. Marotta D, Karar J, Jenkins WT, Kumanova M, Jenkins KW, Tobias
JW, Baldwin D, Hatzigeorgiou A, Alexiou P, Evans SM, Alarcon R,
Maity A, Koch C, Koumenis C: In vivo profiling of hypoxic gene
expression in gliomas using the hypoxia marker EF5 and
laser-capture microdissection. Cancer Res 2011, 71:779 –789
56. Kurokawa T, Yoshida Y, Kawahara K, Tsuchida T, Okazawa H,
Fujibayashi Y, Yonekura Y, Kotsuji F: Expression of GLUT-1 glu-
cose transfer, cellular proliferation activity and grade of tumor
correlate with [F-18]-fluorodeoxyglucose uptake by positron emis-
sion tomography in epithelial tumors of the ovary. Int J Cancer
2004, 109:926 –932
57. Levine AJ, Puzio-Kuter AM: The control of the metabolic switch in
cancers by oncogenes and tumor suppressor genes. Science 2010,
330:1340 –1344
1188 Sheu et al
AJP March 2012, Vol. 180, No. 3
    • "Phosphorylation of p53 is classically regarded as a crucial step of p53 stabilisation, particularly at Ser15 that has been reported to stabilise p53 by inhibiting the interaction between p53 and MDM2 [36]. Of note, Ser15 is phosphorylated after DNA damage and other types of stress such as mutant BRAF inducing DNA strand breaks and activating DNA damage response pathway [38] . Thus, in BRAF mutated cells, Ser15 phosphorylation may be viewed as a marker of functional reactivation of p53. "
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    Full-text · Article · Mar 2016
    • "S1H,I). Similar to HRAS-induced OIS, previous data suggest that BRAF-induced OIS leads to cellular hyperproliferation in vitro and in vivo (Zhu et al. 1998; Dankort et al. 2007) and that in vivo human nevi display features of an activated DDR (Gorgoulis et al. 2005; d'Adda di Fagagna 2008; Sheu et al. 2012). Thus, we performed MLL1 knockdown in diBRAF melanocytes, and, similar to the fibroblast system, this led to a reduction of both MLL1 and SASP gene expression (Supplemen- talFig. "
    [Show abstract] [Hide abstract] ABSTRACT: Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces “SASP-like” inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.
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    • "Previous reports have suggested that V5-BRAF(V600E) expression may result in chromosomal breaks and a DNA damage signal in cells as measured by comet assay, micronuclei formation, or induction of phosphorylated γH2aX [10,11]. We extended these observations by directly scoring metaphase preparations for damaged chromosomes. "
    [Show abstract] [Hide abstract] ABSTRACT: The oncogenic BRAF(V600E) mutation is common in melanomas as well as moles. The roles that this mutation plays in the early events in the development of melanoma are poorly understood. This study demonstrates that expression of BRAF(V600E) is not only clastogenic, but synergizes for clastogenesis caused by exposure to ultraviolet radiation in the 300 to 320 nM (UVB) range. Expression of BRAF(V600E) was associated with induction of Chk1 pS280 and a reduction in chromatin remodeling factors BRG1 and BAF180. These alterations in the Chk1 signaling pathway and SWI/SNF chromatin remodeling pathway may contribute to the clastogenesis and UVB sensitivity. These results emphasize the importance of preventing sunburns in children with developing moles.
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