Dual EGFR and BRAF blockade overcomes resistance to vemurafenib in BRAF mutated thyroid carcinoma cells
Notarangelo et al. Cancer Cell Int
Dual EGFR and BRAF blockade overcomes resistance to vemurafenib in BRAF mutated thyroid carcinoma cells
Tiziana Notarangelo 1
Lorenza Sisinni 1
Valentina Condelli 1
Matteo Landriscina 0 1
0 Medical Oncology Unit, Department of Medical and Surgical Sciences, University of Foggia , Viale Pinto, 1, Foggia 71100 , Italy
1 Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata , Via Padre Pio, 1, Rionero in Vulture 85028 , Italy
Background: BRAF inhibitors are effective anticancer agents in BRAF-mutated melanomas. By contrast, evidences about sensitivity of thyroid carcinomas to BRAF inhibition are conflicting and it has been proposed that BRAF V600E thyroid carcinoma cells are less sensitive to BRAF inhibitors due to activation of parallel signaling pathways. This study evaluated the hypothesis that feedback activation of EGFR signaling counteracts the cytostatic activity of vemurafenib (PLX4032) in BRAF V600E thyroid carcinoma cells. Methods: Cell proliferation, cell cycle distribution, induction of apoptosis and EGFR and AKT signaling were evaluated in thyroid carcinoma cell lines bearing the BRAF V600E mutation in response to PLX4032. Results: A partial and transient cytostatic response to PLX4032 was observed in thyroid carcinoma cell lines bearing the BRAF V600E mutation, with lack of full inhibition of ERK pathway. Interestingly, the exposure of thyroid carcinoma cells to PLX4032 resulted in a rapid feedback activation of EGFR signaling with parallel activation of AKT phosphorylation. Consistently, the dual inhibition of EGFR and BRAF, through combination therapy with PLX4032 and gefitinib, resulted in prevention of EGFR phosphorylation and sustained inhibition of ERK and AKT signaling and cell proliferation. Of note, the combined treatment with gefitinib and vemurafenib or the exposure of EGFR-silenced thyroid carcinoma cells to vemurafenib induced synthetic lethality compared to single agents. Conclusions: These data suggest that the dual EGFR and BRAF blockade represents a strategy to by-pass resistance to BRAF inhibitors in thyroid carcinoma cells.
BRAF; Thyroid carcinoma; EGFR; Vemurafenib; Gefitinib
Cancers bearing BRAF mutations represent
approximately 8% of all human malignancies, these mutations
occurring more frequently in melanomas (40–70%), and
thyroid (36–53%), colorectal (5–22%) and low grade
serous ovarian (~ 30%) carcinomas [
]. In such a
context, approximately 90% of BRAF mutations result in
the substitution of glutamic acid for valine at position
600 (BRAF V600E) . Indeed, the oncogenic activation
of BRAF leads to constitutive activation of downstream
signaling through MAPK pathway [
] and favors the
development of biologically and clinically aggressive
thyroid and colorectal malignancies, frequently resistant to
conventional anticancer therapies [
Inhibition of the BRAF V600E oncoprotein by
smallmolecule drugs, such as vemurafenib (PLX4032) or
PLX4720, results in marked antitumor activity in human
melanoma cells carrying the BRAF V600E mutation [
However, other human malignancies (i.e., thyroid and
colorectal carcinomas) are less sensitive to BRAF
inhibitors (BRAFi), regardless BRAF mutational status [
Among several mechanisms responsible for resistance,
it has been suggested that most tumors who initially
respond to BRAFi eventually develop acquired
resistance through activation of alternative pathways
leading to reactivation of cell proliferation [
the exposure of colorectal cancer cells to BRAFi results
in a feedback activation of EGFR and lack of
sensitivity to vemurafenib [
]. Prerequisite for development of
this mechanism of drug resistance is the upregulation
of EGFR, since melanoma cells devoid of EGFR
expression are sensitive to vemurafenib, lacking this feedback
activation, and the ectopic expression of EGFR induces
resistance to PLX4032 in melanoma cells [
inhibition of EGFR signaling by monoclonal antibodies (i.e.,
Cetuximab) or tyrosine kinase inhibitors (TKi; i.e.,
gefitinib or erlotinib) is synergistic with BRAF inhibition in
colon carcinoma cells [
]. Consistently with a role of HER
receptor family in resistance to BRAFi, Montero-Conde
et al. reported that BRAF-mutated thyroid carcinoma
(TC) cells exposed to PLX4032 are characterized by
transient inhibition of ERK phosphorylation with rebound
activation of HER3 signaling. Indeed, the pan-HER TKi
lapatinib prevents ERK rebound and sensitizes
BRAFmutant thyroid cancer cells to RAF or MAPK kinase
Based on this premise, this study evaluated the
hypothesis that the exposure of BRAF-mutated TC cells to
vemurafenib results in EGFR feedback activation and
that dual EGFR and BRAF blockade is superior to
single agents. This issue is extremely relevant in a clinical
perspective, since human TCs are characterized by high
expression of EGFR and poor responsiveness to EGFR
]. In addition, 25–50% of thyroid cancers
are BRAF mutated and constitutive activation of BRAF
signaling leads to aggressive malignancies, lacking typical
traits of thyroid differentiation [
] and, thus, poorly
responsive to radioiodine therapy .
Cell cultures, siRNAs and chemicals
Papillary BCPAP, poorly differentiated WRO,
anaplastic BHT101 and FRO TC cell lines were purchased from
DSMZ (Braunschweig, Germany). BHT101, FRO and
BCPAP cell lines are characterized by the BRAF V600E
mutation, being WRO cells wild type for BRAF gene
]. Cell line authentication was verified before starting
this study by STR profiling, according to ATCC
product description, and by BRAF mutational status. All cell
lines were cultured in DMEM containing 10% (v/v) fetal
bovine serum (FBS), 2 mM glutamine, and 100 U/mL
penicillin and streptomycin. BHT101 cells were cultured
in the same medium supplemented with 20% (v/v) FBS.
Unless otherwise specified, reagents were purchased
from Sigma-Aldrich (Milan, Italy). BRAF inhibitor
PLX4032 (vemurafenib) was purchased from Selleck
Chemicals (Huston, USA). Gefitinib was kindly
provided by AstraZeneca, pertuzumab was kindly provided
by Roche (Basel, Switzerland). Drugs were dissolved in
dimethylsulfoxide (DMSO) and the same DMSO volume
was added to untreated control.
SiRNA of EGFR was purchased from Qiagen (siRNA
Cat. No. GS1956). For control experiments, cells were
transfected with a similar amount of negative siRNA
(Qiagen, Cat. No. SI03650318). For knock-down
experiments, siRNAs were diluted to a final concentration of
40 nM and transiently transfected by using the HiPerFect
Transfection Reagent (Qiagen), according to
Growth curves and MTT assay
Growth rates were assessed upon seeding of cells in
sixwell plates at the concentration of 4 × 104 cells/well. Cell
lines were incubated in the presence and the absence of
specified drug concentrations, harvested after 24, 48
and 72 h and counted in a Burker chamber (three
countings per sample). Incubation with drugs was carried out
continuously, and drug containing fresh medium was
changed at 48 h intervals.
Cell viability was evaluated using the dimethylthiazol
diphenyltetrazolium bromide (MTT) (Sigma-Aldrich,
Italy) dye assay as previously described [
cells were seeded into 24-well plates (1 × 104 cells/well)
and treated as described in Figure Legends. After drug
removal, cells were incubated in a drug-free medium
for 48 h, and, subsequently, in presence of 600 μM MTT
solution for additional 3 h at 37 °C to allow MTT
metabolism into formazan crystals. The formazan crystals were
finally solubilized by adding 200 µL of 0.04 N HCl in
isopropanol to each microplate well. Adsorbance at 540 nm
was measured using a Bio-Tek microplate reader (model
EL-340; BioMetallics, Priceston, NJ). Wells containing
only DMEM, FBS and MTT were used as controls. Each
experiment was performed three times using four
replicates for each drug concentration.
Cell cycle analysis
Cells were incubated in a culture medium supplemented
with 20 mmol/L 5-bromo-20-deoxyuridine (BrdUrd)
for 20 min and harvested. Subsequent to incubation in
a solution containing 3 N HCl for 30 min at room
temperature to obtain DNA denaturation, cell pellets were
further incubated in the presence of anti-BrdUrd FITC
(Becton–Dickinson) for 1 h at room temperature in the
dark. After washing with PBS, cells were further
incubated with 6 mg/mL propidium iodate (PI) for 20 min
and then evaluated using FACSCalibur™
Total cell lysates were obtained by the homogenization
of cell pellets in a cold lysis buffer (20 mmol/L Tris, pH
7.5 containing 300 mmol/L sucrose, 60 mmol/L KCl,
15 mmol/L NaCl, 5% (v/v) glycerol, 2 mmol/L EDTA,
1% (v/v) Triton X-100, 1 mmol/L PMSF, 2 mg/mL
aprotinin, 2 mg/mL leupetin, and 0.2% (w/v)
deoxycholate) for 2 min at 4 °C and further sonication for 30 s
on ice. Immunoblot analysis was performed as
previously reported [
]. The following antibodies from Santa
Cruz Biotechnology were used: mouse monoclonal
antiGAPDH (sc-47724), rabbit polyclonal anti-phosphoEGFR
(Tyr1173, sc-12351). The following antibodies from Cell
Signaling Technology were also used: mouse monoclonal
anti-phospho44/42 MAPK (pErk1/2, #9106), rabbit
polyclonal anti-phosphoAKT (Ser473, #9271), rabbit
polyclonal anti-AKT (#9272), rabbit polyclonal anti-EGFR
(#4267). Rabbit polyclonal anti-MAPK (Erk1/2, #ABS44)
antibody was purchased from Millipore Merck.
Apoptosis was evaluated by citofluorimetric analysis
of Annexin-V and 7-amino-actinomycin-D
(7-AAD)positive cells using the fluorescein isothiocyanate
(FITC)-Annexin-V/7-AAD kit (Beckman Coulter, Milan,
Italy). Stained cells were analyzed using the
FACSCalibur™ (Becton–Dickinson). Positive staining for
AnnexinV as well as double staining for Annexin-V and 7-AAD
were interpreted as signs of early and late phases of
apoptosis respectively [
The paired Student’s t test was used to establish the
statistical significance between different levels of growth
rate, cell cycle distribution and apoptosis compared to
the respective controls. Statistically significant values
(P < 0.05) are reported in Figure Legends. All
experiments were independently performed at least three
Vemurafenib partially inhibits cell growth and ERK signaling in thyroid carcinoma cells
To establish the sensitivity of thyroid cancer cell lines
to BRAFi, growth rate (Fig. 1a), cell viability (Fig. 1b)
and cell cycle distribution (Fig. 1c) were evaluated in
response to PLX4032 in TC cell lines harboring the
BRAF V600E mutation (i.e., BHT101, FRO and BCPAP
cells) or BRAF-wild type WRO cells. Indeed, PLX4032
was ineffective in BRAF wild-type WRO cells (Fig. 1a,
b) and induced a dose dependent inhibition of cell
growth in BRAF V600E BHT101, FRO and BCPAP
cell lines (Fig. 1a, b). Noteworthy, PLX4032
inhibition of BRAF-mutated TC cell lines proliferation was
incomplete, reaching a maximum of 60%
downregulation of cell growth (Fig. 1a, b). Consistently, the
exposure of BRAF V600E BHT101, FRO and BCPAP cells
to PLX4032 resulted in the accumulation of cells in
G0-G1 phase with a parallel attenuation of S phase
(Fig. 1c) and this correlated with inhibition of ERK
phosphorylation (Fig. 1d). As expected, the exposure
of BRAF-wild type WRO cells to PLX4032 was
ineffective in delaying cell cycle progression (Fig. 1c) and
induced a paradoxical activation of ERK
phosphorylation (Fig. 1d).
BRAF inhibition results in feedback activation of EGFR phosphorylation in BRAF V600E thyroid carcinoma cells
Since these data suggest that BRAF V600E TC cells lines
are not fully responsive to vemurafenib, the hypothesis
that BRAF pharmacological inhibition results in
feedback activation of EGFR signaling was further evaluated
in BRAF-mutated BHT101, BCPAP and FRO cell lines.
Thus, TC cells were exposed to 10 μM PLX4032 for short
(4–8 h depending on the cell line) or longer (15 and 24 h)
periods and evaluated for EGFR phosphorylation (Fig. 2).
Interestingly, vemurafenib treatment induced a rapid
feedback activation of EGFR phosphorylation between
4 and 8 h (Fig. 2a–c), this suggesting that EGFR
signaling is induced by BRAF inhibition in BRAF-mutated TC
cell lines. In addition, the kinetic of ERK signaling
inhibition in response to PLX4032 showed a rapid and
sustained reactivation of ERK signaling after 15–24 h of
treatment (Fig. 2a–c). Consistently, vemurafenib induced
the rebound activation of AKT phosphorylation, which
occurred early in FRO and BHT101 cells (Fig. 2a, c) and
at later time points in BCPAP cells (Fig. 2b). These data
suggest that vemurafenib induces rebound activation of
EGFR signaling in BRAFV600E TC cells.
Dual EGFR and BRAF blockade induces inhibition of cell proliferation, suppression of ERK signaling and synthetic lethality
In further experiments, the hypothesis that dual
blockade of EGFR and BRAF signaling results in potentiation
of BRAFi single agent activity was further evaluated.
Thus, the cytostatic activity of combined therapy with
1–10 μM PLX4032 and the EGFR inhibitor, gefitinib
(1–10 μM) was evaluated in comparison with PLX4032
or gefitinib single agents in BRAF-mutated BHT101,
FRO and BCPAP TC cell lines (Fig. 3). Indeed, the
combined blockade of EGFR and BRAF resulted in a
more significant inhibition of cell proliferation (Fig. 3a)
and cell cycle progression, with increased
accumulation of cells in G0–G1 phase and attenuation of S phase
(Fig. 3b). Noteworthy, the combined inhibition of EGFR
and BRAF prevented the feedback activation of EGFR
phosphorylation (Fig. 4a) and the parallel activation of
AKT phosphorylation (Fig. 4a) and induced a prolonged
suppression of ERK signaling (Fig. 4b). Since the
feedback activation of HER3 signaling is involved in acquired
resistance to vemurafenib [
] and HER3 pathway is
activated upon heterodimerization with HER2 receptor, this
activation being blocked by pertuzumab [
inhibition of HER2/HER3 heterodimerization by pertuzumab
was tested in combination with BRAFi and compared to
the dual blockade of BRAF and EGFR in TC cells.
Interestingly, the dual treatment with pertuzumab and
vemurafenib resulted in a cytostatic activity comparable to the
dual blockade of EGFR and BRAF (Additional file 1:
Finally, the hypothesis that the combined blockade of
EGFR and BRAF results in synthetic lethality was
evaluated. Interestingly, while gefitinib and PLX4032 single
agents exhibited no or minimal cytotoxic activity, the
exposure of FRO, BCPAP and BHT101 TC cells to
combination therapy with gefitinib and PLX4032 resulted in
10–40% induction of apoptotic cell death (Fig. 5a). The
specificity of cytostatic and cytotoxic activity of
combination therapy with gefitinib and vemurafenib was
confirmed upon EGFR silencing and subsequent exposure to
vemurafenib in FRO, BCPAP and BHT101 (Fig. 5b). Of
note, vemurafenib induced a more significant cell cycle
arrest (Fig. 5c) and higher levels of apoptosis (Fig. 5d)
in a low EGFR background. These data support the
concept that the dual blockade of EGFR and BRAF results in
increased cytostatic activity and induction of synthetic
lethality compared to BRAFi single agent.
Early responses involved in adaptive resistance to
BRAFi have been described in human cancer cells [
Among several proposed mechanisms, the occurrence
of genomic events, as secondary NRAS mutations and
BRAF alternative splicing or other alterations, both
upstream and downstream to BRAF, leading to
reactivation of ERK pathway, provided the rational for
combination treatment with BRAF inhibitors and other agents
to circumvent or delay resistance [
]. In addition, the
activation of alternative pathways are also responsible
for resistance to BRAFi, leading mostly to selection of
resistant clones that cause tumor regrowth and
progressive disease [
]. This issue is extremely relevant in
BRAFmutated TCs that are characterized by loss of thyroid
specific characters [
] and poor responsiveness to
radioiodine therapy [
] and, thus, require new therapeutic
options. In such a context, evidences about sensitivity of
BRAF V600E TCs to vemurafenib single agent are
conflicting with initial studies showing sustained responses
] and subsequent reports showing transient
This study was designed to evaluate the role of
feedback activation of EGFR signaling in counteracting the
cytostatic activity of vemurafenib in BRAF-mutated
TC cell lines. Our data suggest that (1) BRAF V600E
TC cell lines are transiently responsive to
vemurafenib, (2) vemurafenib treatment elicits a feedback
activation of EGFR pathway and (3) simultaneous
blockade of BRAF and EGFR results in potentiation of
vemurafenib single agent activity, prolonged
suppression of ERK and AKT signaling and induction of
This evidence is consistent with previous studies, in
colon carcinoma and melanoma cells, suggesting that
EGFR expression dictates the activity of BRAF
inhibitors. Indeed, melanoma cells with poor EGFR
background are highly responsive to BRAF inhibitors,
whereas colon carcinoma cells exhibit a rapid feedback
activation of EGFR phosphorylation, being
characterized by high EGFR expression [
]. Consistently, the
upregulation of EGFR in melanoma cells results in loss
of activity of BRAF inhibitors and dual inhibition of
EGFR and BRAF re-establishes vemurafenib activity
in colon carcinoma cells [
]. In such a context, TCs are
characterized by high expression of EGFR and sustained
activation of its downstream signaling [
representing a mechanism of escape from EGFR inhibitors
]. Consistently, our data suggest that transient
exposure of BRAF V600E TC cells to vemurafenib results in a
rapid phosphorylation of EGFR and this correlates with
reactivation of ERK and AKT signaling. Intriguingly,
the simultaneous blockade of EGFR and BRAF results
in a more significant and prolonged suppression of ERK
and AKT signaling and induction of synthetic lethality
compared to single agents. The specificity of this
pharmacological activity is supported by data obtained with
transient silencing of EGFR expression and subsequent
treatment with vemurafenib, which results in arrest of
cell cycle progression and synthetic lethality.
An issue that needs to be discussed is whether there is a
relationship between EGFR rebound activation and AKT
and ERK phosphorylation in response to vemurafenib.
Indeed, our data show that AKT phosphorylation occurs
at earlier time points compared to ERK re-activation,
which is a later event. Interestingly, combined exposure
of TC cells to gefitinib and vemurafenib results in
sustained suppression of both AKT and ERK signaling in
parallel with inhibition of EGFR phosphorylation, this
suggesting that these events are likely to be part of a
common adaptive response. While this issue deserves further
investigation, much evidence supports the relevance of
AKT activation in driving poor response to inhibitors of
the RAF/RAS/MEK axis in TC cells lines. Indeed, AKT
pathway is highly active in BRAF-mutated TC cell lines
] and its phosphorylation is enhanced by inhibition
of MEK/ERK signaling, regardless the BRAF mutational
]. In addition, the simultaneous suppression of
MEK/ERK and PI3K/AKT pathways abrogates
compensatory mechanisms of tumor survival and causes
synergistic cytotoxicity in TC cell lines [
It is noteworthy that the combined blockade of HER2/
HER3 heterodimerization and BRAF signaling is
comparable to the combination of gefitinib and vemurafenib
in terms of cytostatic activity. Indeed, Montero-Conde
et al. reported that exposure of BRAF-mutated TC cells
to PLX4032 results in rebound activation of HER3
signaling and inhibition of HER family receptor with lapatinib
prevents ERK rebound activation and sensitizes
BRAFmutant TC cells to RAF or MAPK kinase inhibitors [
Thus, it is likely that feedback activation of multiple
signaling pathways, most of them involving the HER family
receptor, is responsible for resistance to BRAFi in TC
Clinically relevant is the observation that combined
inhibition of EGFR and BRAF signaling is more
effective than vemurafenib or gefitinib single agents and
results in induction of synthetic lethality. Indeed.
BRAFmutated TCs are aggressive malignancies frequently
poor responsive to radioiodine therapy [
]. In such a
perspective, BRAF/MEK/ERK signaling is emerging as a
potential target is these malignancies. The BRAF
inhibitor, dabrafenib and the MEK inhibitor, solumetinib were
evaluated for their capacity to re-induce iodine uptake
in iodine-refractory BRAF-mutated human TCs with
potentially interesting results [
]. In addition, the
multi-targeted TK inhibitors sorafenib and lenvantinib
obtained the approval by FDA as effective treatments in
these malignancies . In such a complex scenario, our
data provide a rationale for evaluating dual EGFR and
BRAF blockade as potential therapeutic option in
BRAFmutated radioiodine-refractory TCs. Notably, this
strategy already provided interesting results in other human
malignancies at either preclinical or clinical level. Indeed,
pharmacological agents blocking EGFR signaling
combined with BRAFi inhibited orthotopic glioma xenografts
and increased apoptosis, with resultant significant
extension of animal survival [
]. In addition, vemurafenib in
combination with cetuximab and irinotecan showed
valuable clinical activity and a reasonable toxicity profile in
pretreated metastatic colorectal carcinomas [
This study suggests that vemurafenib single agent activity
is significantly impaired in BRAF V600E TC cells by
feedback activation of EGFR signaling pathway and that dual
inhibition of EGFR and BRAF may represent a strategy to
potentiate BRAFi single agents.
Additional file 1: Figure S1. Cell cycle distribution in BRAF V600E FRO
and BCPAP thyroid carcinoma cell lines exposed to 10 μM PLX4032, 10
μM gefitinib, 1 μM pertuzumab or the combination of PLX4032 with both
agents for 24 h. Statistical significance respect to vemurafenib single
agent: **p < 0.001.
Study concept and Design, ML; Acquisition of data, TN, LS; Analysis and
interpretation of data, TN, LS, VC, ML; Drafting of the manuscript, TN, ML;
Critical revision of the manuscript, TN, LS, VC, ML; Obtained funding, ML; Study
supervision, ML. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or
Consent for publication
Ethics approval and consent to participate
This work was supported by AIRC (Grant IG2015-16738) and Italian Ministry of
Health (Grant GR-2010-2310057) to ML.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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