Eur J Oral Sci 2017; 1–11 Ó 2017 Eur J Oral Sci

DOI: 10.1111/eos.12356 European Journal of

Printed in Singapore. All rights reserved
Oral Sciences

E. E. Hinsley1, C. E. de Oliveira2,
Angiotensin 1-7 inhibits angiotensin S. Hunt1, R. D. Coletta2,
D. W. Lambert1
II-stimulated head and neck cancer 1
Academic Unit of Oral and Maxillofacial
Pathology, School of Clinical Dentistry,

progression University of Sheffield, Sheffield, UK;

2
Department of Oral Diagnosis, School of
Dentistry, University of Campinas, Piracicaba,
Brazil

Hinsley EE, de Oliveira CE, Hunt S, Coletta RD, Lambert DW. Angiotensin 1-7
inhibits angiotensin II-stimulated head and neck cancer progression.
Eur J Oral Sci 2017; 00: 1–11. © 2017 Eur J Oral Sci
Angiotensin II (Ang II) is the product of the proteolytic action of angiotensin-con-
verting enzyme (ACE) on the precursor peptide, angiotensin I (Ang I). In addition
to its vasoactive properties, Ang II is able to stimulate angiogenesis and act as a
mitogen, promoting cellular proliferation. Recently, evidence has emerged that Ang
II is also able to promote tumour invasion, a key step in the metastatic cascade,
although the mechanisms by which it does so remain largely obscure. Here we show Daniel W. Lambert, School of Clinical
Dentistry, University of Sheffield, Sheffield
that Ang II is able to promote the invasion and migration of head and neck squa-
S10 2TA, UK
mous cell carcinoma (HNSCC) cells both in an autocrine manner and by triggering
stromal tumour–paracrine interactions. The effects of Ang II on autocrine and para- E-mail: d.w.lambert@sheffield.ac.uk
crine signalling pathways are mediated by angiotensin receptor 1 (AT1R) and inhib-
ited by angiotensin 1-7 (Ang 1-7), a peptide produced from Ang II by the action of Key words: a disintegrin and
angiotensin-converting enzyme 2 (ACE2). These data are the first to demonstrate a metalloproteinase; epidermal growth factor
role for the renin–angiotensin system in oral carcinogenesis and raise the possibility receptor; fibroblasts; head and neck
of utilizing AT1R receptor antagonists and/or Ang 1-7 as novel therapeutic agents squamous cell carcinoma; mas receptor
for HNSCC. Accepted for publication April 2017

Head and neck cancer, predominantly comprised of head
and neck squamous cell carcinoma (HNSCC), is the
sixth most common cancer worldwide. Although com-
monly associated with risk factors such as advanced age,
smoking, and alcohol consumption, the incidence of this
cancer is increasing among younger and low-risk groups.
Head and neck squamous cell carcinoma has a poor
prognosis, with surgery remaining the front-line thera-
peutic approach (1). Compared with other common can-
cers, the molecular landscape of HNSCC is relatively
poorly understood, hampering the development of novel
therapeutic approaches. Recent studies have indicated
that the tumour stroma plays a critical role in the pro-
gression of HNSCC (2–4), suggesting that factors within
the stroma may represent viable targets for a new genera-
tion of therapeutic agents.
The tumour stroma is a complex milieu of extracellu-
lar matrix components and a variety of cell types, of
which fibroblasts are the most numerous (5). We have
recently demonstrated that fibroblast-derived factors
are able to invoke phenotypic changes in adjacent
epithelial cells, promoting tumour progression (6). In
turn, factors in the stromal environment, including
those generated by cancer cells and by fibroblasts them-
selves, promote phenotypic changes in the fibroblasts
(4). These include the acquisition of a more migratory,
contractile phenotype and increased expression of
myofibroblast markers, such as a-smooth muscle actin
(a-SMA) and matrix metalloproteinase-2 (MMP-2) (7).
Although fibroblasts are known to promote migration
and invasion of HNSCC cells, little is known of the
factors that promote these interactions and direct
remodelling of stromal fibroblasts. We have recently
shown that a small peptide, endothelin-1 (ET-1), is able
to promote paracrine signalling between oral fibroblasts
and HNSCC cells via epidermal growth factor receptor
(EGFR) transactivation (2).
Angiotensin II (Ang II), like ET-1, is a peptide
produced by the action of a metalloproteinase, in
this case angiotensin-converting enzyme (ACE).
Angiotensin II is a key component of the renin–an-
giotensin system (RAS) and exerts its actions on
target cells by binding to one of two G protein–cou-
pled receptors, angiotensin receptor 1 (AT1R) and
the less well-characterized angiotensin receptor 2
(AT2R) (8). It is becoming increasingly evident that
in addition to its systemic effects on blood pressure
and fluid homeostasis, Ang II is able to exert effects
at the local tissue level (9). A functional RAS has
been detected in a number of tissues, including the
oral mucosa (10).
Binding of Ang II to AT1R triggers receptor phospho-
rylation, activating a variety of intracellular and extracel-
lular events, including arrestin recruitment (11) and
EGFR transactivation (12). Angiotensin II-mediated
EGFR transactivation, a result of AT1R-induced
2 Hinsley et al.
activation of a disintegrin and metalloproteinase
(ADAM) proteinase-mediated EGFR ligand release,
leading to autocrine or paracrine activation of EGFR
signalling, has been demonstrated to promote migration
of dermal keratinocytes and fibroblasts (13). Increased
AT1R expression has been identified in a number of
tumours, including ovarian (14), breast (15), and bladder
(16). Furthermore, Ang II is known to promote tumour
growth (17) and angiogenesis (18), and AT1R blockers
and ACE inhibitors have been demonstrated to reduce
growth and vascularization of a wide range of tumours,
including those of the lung, prostate, and oral cavity,
suggesting a role for Ang II in cancer pathogenesis [re-
viewed in (19)].
The discovery in 2000 of a homologue of ACE, termed
ACE2, revealed a new paradigm in angiotensin peptide
biology (20). Angiotensin-converting enzyme 2 prote-
olytically cleaves Ang II to produce angiotensin 1-7 (Ang
1-7), a peptide that also binds to a G protein–coupled
receptor, MasR (9). The cellular consequences of MasR
activation remain unclear but it is apparent that Ang 1-7
appears able to counteract the effects of Ang II (8).
Recently, it was reported that administration of Ang 1-7
reduces lung cancer growth (21) and invasion (22) and
breast cancer-associated fibrosis by inhibiting extracellu-
lar signal-regulated kinase (ERK) activation (23).
Previous reports have reported a functional, tissue-
level (as opposed to systemic) RAS in the oral mucosa
(10), and the ACE inhibitor perindopril has been
shown to reduce the growth of head and neck carci-
noma in vivo (24), suggesting a role for Ang II in dis-
ease aetiology. Here we examine, for the first time, the
contribution of the local RAS to HNSCC pathogenesis.
We identify aberrant expression of AT1R in HNSCC
cells and demonstrate that its ligand, Ang II, is able to
stimulate both HNSCC cells and oral fibroblasts to
promote tumour cell chemotaxis and invasion. Further-
more, we demonstrate the ability of the Ang II metabo-
lite, Ang 1-7, to block the protumorigenic effects of
Ang II. Taken together, these findings suggest that
AT1R blockade and/or Ang 1-7 administration, may be
a novel therapeutic opportunity in HNSCC.
Material and methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM), Roswell
Park Memorial Institute medium (RPMI-1460), Ham’s
F12 nutrient mix, fetal bovine serum (FBS), Dulbecco’s
PBS, L-glutamine, adenine, transferrin, 3,30 ,5-triiodo-L-
thyronine, hydrocortisone, epidermal growth factor and
trypsin/ethylenediaminetetraacetic acid were all purchased
from BioWhittaker (Wokingham, UK). Reagents were
purchased from Sigma-Aldrich (Poole, UK), unless other-
wise stated, and were of the highest analytical grade.
Routine cell culture and transfection
The HNSCC-derived cell lines SCC4, H357, and Cal27,
and primary oral fibroblasts [collected with informed
consent and South Sheffield Ethics Committee approval,
as described previously (25) and used within passages 3–
10] were routinely cultured in DMEM supplemented with
2 mM L-glutamine and 10% (v/v) FBS. Normal oral ker-
atinocytes (NOKs) [collected with informed consent and
South Sheffield Ethics Committee approval, as described
previously (26), and used up to passage 3] and D19 and
D20 [derived from oral dysplasia lesions, described previ-
ously (27)], B16 [derived from an HNSCC primary tumour
and a cervical lymph node metastasis, respectively (28)],
and B22 cell lines were all routinely cultured in Green’s
medium consisting of a 3:1 ratio of DMEM and Ham’s
F12 supplemented with 2 mM L-glutamine, 180 lM ade-
nine, 5 lg ml-1 of transferrin, 0.2 lM 3,30 ,5-triiodo-L-thyro-
nine, 4 lg ml-1 of hydrocortisone, 10 ng ml-1 of epidermal
growth factor, and 10% (v/v) FBS. All cells were grown in
antibiotic-free medium at 37°C and 5% (v/v) CO2. Oral
fibroblasts were transfected at approximately 60% conflu-
ence using Oligofectamine reagent (Fisher Scientific,
Loughborough, UK) with small interfering RNA (siRNA)
to ADAM17 or a negative-control non-targeting oligonu-
cleotide (final concentration 50 nM; Fisher Scientific) in
Opti-MEM media (Fisher Scientific). Cells were incubated
for 24 h, after which media and cells were collected for
further experimentation and/or analysis.
Cell treatments
Oral fibroblasts and HNSCC cells were serum starved for
24 h before experimentation. For the preparation of con-
ditioned media (CM), oral fibroblasts were treated with
Ang II (100 nM; Sigma-Aldrich) and/or Ang 1-7 (100 nM;
Sigma-Aldrich) for 4 h at 37°C and 5% (v/v) CO2. Where
indicated, fibroblasts were pretreated with telmisartan, an
AT1R antagonist (1 lM; Sigma-Aldrich), A-779, a MasR
antagonist (1 lM; Sigma-Aldrich), or GM6001, an MMP
inhibitor (10 lM; Merck Millipore, Feltham, UK) for
30 min before addition of and treatment with mitogenic
peptides for 4 h. Media was collected from fibroblasts
after incubation, filtered, and added to the lower chamber
of invasion or migration assay plates, before cells were
seeded, as described, into the upper chamber. For prepara-
tion of oral fibroblasts for immunoblotting, cells were trea-
ted with either Ang II (100 nM) or transforming growth
factor beta (TGF-b; 20 ng ml-1) for 48 h at 37°C and 5%
(v/v) CO2.
RNA isolation and quantitative PCR
The RNeasy mini kit (Qiagen, Manchester, UK) was used
to extract total RNA from all cell lines, and from primary
NOKs and oral fibroblasts, according to the manufac-
turer’s instructions. RNA from each sample was quantified
using a NanoDrop spectrophotometer (Fisher Scientific).
A High Capacity cDNA Reverse Transcription kit (Fisher
Scientific) was used for synthesis of cDNA according to
the manufacturer’s instructions. cDNA was subsequently
analysed by SYBR Green or TaqMan quantitative PCR
(qPCR). Primers used for SYBR Green quantification
were as follows: U6 forward, 50 -CTCGCTTCGGCAGCA
CA-30 and U6 reverse, 50 -AACGTTCACGAATTTGCGT-
30 ; AT1R forward, 50 -ACCTGGCTATTGTTCACCCAA-30
and AT1R reverse, 50 -ACAAGCATTGTGCGTCGAAG-30 ;
AT2R forward, 50 -GTTCCCCTTGTTTGGTGTAT-30 and
AT2R reverse, 50 -CATCTTCAGGACTTGGTCAC-30 ;
MasR forward, 50 -TTCATCCCGTTCAGAAGACA-30

and MasR reverse, 50 -CCAATGGCAAGCAGAAATA
GA-30 ; a-SMA forward, 50 -GAAGAAGAGGACAGCAC
TG-30 and a-SMA reverse, 50 -TCCCATTCCCACCATC
AC-30 . TaqMan probes for ACE, ACE2, and b2mi-
croglobulin were obtained from Fisher Scientific. All val-
ues were normalized to U6 (SYBR) or b2microglobulin
(TaqMan) expression levels. Where indicated, amplicons
were also analysed by agarose gel electrophoresis and
visualized with ethidium bromide under ultraviolet (UV)
transillumination.
Cell migration and invasion assays
SCC4 or H357 cells at 90% confluence were serum starved
for 24 h before experimentation. Then the cells were tryp-
sinized and resuspended in DMEM, containing 0.1% (w/
v) BSA, at 5 9 105 cells ml-1. Supplements including Ang
II (100 nM) and/or Ang 1-7 (100 nM) were added and
200 ll of cell suspension was added to the top of the tran-
swell chamber. Then, 500 ll of DMEM containing 10%
(v/v) FCS or CM from fibroblasts was added to the lower
chamber (as indicated in individual figure legends). The
EGFR antagonist, AG 1478 (125 nM; Sigma-Aldrich),
was incubated with the SCC4 cell suspension for 30 min
at 37°C and 5% (v/v) CO2 before the addition of the
SCC4 cell suspension to the chamber. After 24 h (migra-
tion) or 48 h (invasion), cells were swabbed away from the
inside of the transwell chamber and cells adhering to the
underside of the chamber were fixed for 10 min in 100%
(v/v) methanol. Migrated cells were stained with 0.1% (w/
v) crystal violet and counted by light microscopy at 40x
magnification. Three fields of view from each insert were
counted. For the scratch assay, oral fibroblasts were
seeded in a six-well plate (100,000 cells/well) and allowed
to adhere overnight. Cells were serum starved for 24 h
and a scratch was made in each well using a 200-ll pipette
tip. Media was removed, and cells were washed in PBS
before the addition of peptides or inhibitors. Mitomycin C
(Sigma-Aldrich), at 1 lg ml-1, was added to each well to
prevent proliferation. Each well was photographed at two
points along the scratch at 0 and 24 h. The distance
between each edge of the scratch was measured.
Invasion assays were prepared using Matrigel (growth
factor depleted; Sigma-Aldrich) diluted 1/45 (v/v) with
DMEM. In these assays, 100 ll of diluted Matrigel was
added to the top of each transwell chamber. The inserts
were left overnight at 37°C and used the following day.
Gel contraction assay
Oral fibroblasts (250,000 cells/well) were resuspended in
DMEM containing 10% (v/v) FBS and were mixed with
rat tail collagen (7.5 mg ml-1 in 10x DMEM). The pH of
the cell + collagen mixture was adjusted to pH 7 using
0.1 M sodium hydroxide (NaOH). Then, 300 ll of the
cell + collagen mixture was added to 24-well plates. The
cell + collagen mixture was left to incubate for 45 min
before the addition of 500 ll of DMEM containing 10%
(v/v) FBS. This medium was removed after 4 h and
replaced with 500 ll of serum-free medium and matrices
were incubated overnight at 37°C and 5% (v/v) CO2.
Using a scalpel, the gels were carefully loosened from the
edges of the well and treated with serum-free medium con-
taining thrombin (0.5 U ml-1) or Ang II (100 nM). Colla-
gen lattices were photographed and the distance
contracted by the gels was measured.
Angiotensin peptides in head and neck cancer 3
Immunoblotting
Cells were trypsinized, washed with PBS, and pelleted.
Protein was extracted using radioimmunoprecipitation
assay (RIPA) lysis buffer (Merck Millipore, Feltham, UK)
containing Complete Mini Protease Inhibitor Cocktail
(Roche, Welwyn Garden City, UK; used according to the
manufacturer’s instructions) and Benzonase (Sigma-
Aldrich; used according to the manufacturer’s instruc-
tions). Protein concentration was measured using the BCA
Protein Assay Kit (Fisher Scientific) with BSA as a
standard. Total protein extracts (50 lg) were separated by
3-8% (v/v) acrylamide SDS-PAGE and transferred to
nitrocellulose membrane. Following blocking of non-specific
protein binding in 5% (w/v) dried milk and 3% (w/v) BSA in
Tris-buffered saline containing 0.5% (v/v) Tween 20, mem-
branes were incubated with antibodies directed to a-SMA
(1:1,000; Sigma-Aldrich) or b-actin (1:4,000; Sigma-Aldrich).
Horseradish peroxidase-conjugated secondary antibodies
(Sigma) were diluted 1:2,000. All antibodies were diluted in
5% (w/v) dried milk and 3% (w/v) BSA in Tris-buffered
saline containing 0.5% (v/v) Tween 20. Immunoreactive
bands were visualized using enhanced chemiluminescence
(ECL) (Fisher Scientific). Densitometry was performed using
Adobe Photoshop (San Jose, CA, USA).
Statistical analyses
Data are expressed as mean  standard error of the mean. Sta-
tistical analyses, where appropriate, were made between two
groups using the Student’s t-test or Mann–Whitney U-test. A
value of P < 0.05 was considered statistically significant.
Results
Ang II stimulates migration and invasion of HNSCC
cells via AT1R
We first examined the expression of AT1R and AT2R in
a panel of primary human NOKs and fibroblasts, and in
cell lines derived from oral dysplasia lesions, primary
HNSCC, and a local metastasis. Expression of AT1R
was found to be markedly elevated in a number of pri-
mary carcinoma cell lines (Fig. 1A) compared with nor-
mal, dysplastic, and metastatic cells. High levels of AT1R
transcript were also detected in normal oral fibroblasts
(NOFs; Fig 1A). Angiotensin receptor 2 was detected in
all cell lines and NOKs examined, with no significant dif-
ferences in AT2R expression observed between cells from
normal and diseased tissue (Fig. 1B). Angiotensin recep-
tor 2 was undetectable in NOFs using this methodology.
Having established the expression of AT1R and AT2R in
HNSCC cells, we next examined the effect of exogenous
Ang II on HNSCC cell motility. Two HNSCC cell lines,
H357 and SCC4 (both derived from squamous cell carci-
noma of the tongue), were selected on the basis of their
high levels of AT1R expression. The ability of both cell
lines to migrate and invade was significantly increased
following treatment with Ang II (Fig. 1C–F). The stimu-
lation of migration was completely abrogated by the
prior application of a specific AT1R antagonist,
telmisartan, but not by the AT2R antagonist, P123319
(Fig. 1G,H).
4 Hinsley et al.

Fig. 1. Angiotensin receptor 1 (AT1R) mediates angiotensin II (Ang II)-stimulated migration and invasion of head and neck squa-
mous cell carcinoma (HNSCC). Total RNA was prepared from normal primary oral keratinocytes (NOKs), normal primary oral
fibroblasts (NOFs), and from a range of cell lines derived from oral dysplasias, squamous cell carcinomas, and a local lymph
node metastasis. Expression of AT1R (A) and AT2R (B) was assessed by quantitative PCR (qPCR). Migration (C) and invasion
(E) of SCC4 and migration (D) and invasion (F) of H357, in response to treatment with Ang II, were analysed as described in
the Material and methods. The effect of telmisartan, an AT1R antagonist, and PD123319, an AT2R antagonist, on migration of
SCC4 cells in response to Ang II was assessed (G and H, respectively). The results are plotted as mean values relative to
untreated control wells  standard error of the mean, of three separate fields of view from three independent experiments.
*P < 0.05 relative to control. ns, not significant.

Fibroblasts potentiate the effect of Ang II on HNSCC

motility
Given the high levels of AT1R detected in NOFs, we
next sought to determine whether interaction of Ang II
with NOFs could trigger promigratory paracrine
signalling between fibroblasts and cancer cells. Treat-
ment of SCC4 and H357 cells with CM collected from
NOFs incubated with Ang II significantly increased
migration and invasion (Fig. 2A–D) to a degree
greater than that observed in the absence of such CM
 Angiotensin peptides in head and neck cancer 5

Fig. 2. Fibroblasts potentiate the effects of angiotensin II (Ang II) on head and neck squamous cell carcinoma (HNSCC) migra-
tion and invasion. Conditioned media (CM) was collected and filtered from fibroblasts treated with Ang II or PBS for 4 h, before
being added to the lower chamber of a transwell plate, as described in the Material and methods. Migration (A) and invasion (C)
of SCC4 cells, and migration (B) and invasion (D) of H357 cells, were then assessed. The ability of telmisartan to block the
observed migration of SCC4 cells was also analysed (E). The results are plotted as mean values relative to untreated control wells
 standard error of the mean, of three separate fields of view from three independent experiments. *P < 0.05 relative to control,
**P < 0.05 relative to Ang II treated.

Ang II invokes myofibroblast transdifferentiation of

(~three-fold increase in the presence of CM vs. 1.5-fold
increase in the absence of CM, for SCC4). Pretreatment
of fibroblasts with telmisartan blocked the stimulation
of migration and invasion (Fig. 2E), as expected given
the absence of AT2R in these cells. Pretreatment of
fibroblasts with GM6001 blocked the Ang II-mediated
stimulation of migration, implicating the ADAM-
mediated release of a soluble factor (Fig. 3A,B). The
ability of AG 1478 treatment of SCC4 cells to block
the stimulatory effect of CM collected from Ang II-
treated fibroblasts implicates the involvement of EGFR
signalling (Fig. 3C). We have previously shown a role
for ADAM17, a membrane-anchored protease responsi-
ble for the proteolytic activation of a number of physi-
ologically significant substrates, in stromal-epithelial
signalling in HNSCC (2). We therefore examined the
effect of abrogating ADAM17 expression on the non-
cell autonomous actions of Ang II; siRNA-mediated
knockdown of ADAM17 in fibroblasts blocked the
Ang II-mediated increase in SCC4 migration on indi-
rect co-culture (Fig. 3D). A significant reduction in
ADAM17 transcript confirmed the efficacy of the
siRNA-mediated knockdown (Fig. 3E).
NOFs
As myofibroblast transdifferentiation has previously
been shown to promote HNSCC cell motility, we next
analysed whether Ang II treatment of NOFs resulted in
the acquisition of an activated, myofibroblast-like phe-
notype. Treatment with TGF-b1 was used in these
experiments as a positive control as TGF-b1 is widely
reported to induce myofibroblast differentiation in vitro
(29). qRT-PCR analysis of RNA extracted from NOFs
treated with Ang II revealed a significant increase in
the expression of a-SMA, a widely utilized marker of
myofibroblasts (Fig. 4A). A significant increase was
also observed in the expression of MMP-2, previously
reported to be up-regulated in myofibroblasts (Fig. 4B).
Immunoblotting also indicated a small increase in
a-SMA protein (Fig. 4C). The stimulation of a-SMA
expression by Ang II was completely blocked by prior
application of telmisartan, suggesting the involvement
of AT1R (Fig. 4D). Collagen contraction assays indi-
cated the acquisition of a more contractile phenotype
upon Ang II treatment, of a similar magnitude to that
induced by thrombin, a known stimulator of fibroblast
6 Hinsley et al.

Fig. 3. Angiotensin II (Ang II) stimulates head and neck squamous cell carcinoma (HNSCC) migration via epidermal growth factor
receptor (EGFR) transactivation. Angiotensin II is known to induce phenotypic changes by transactivating EGFR signalling by a mech-
anism involving a disintegrin and metalloproteinase (ADAM)-mediated proteolysis of EGFR ligands (A). The effect of inhibiting both
ADAM proteolysis, by treating normal oral fibroblasts (NOFs) with the broad-spectrum ADAM/matrix metalloproteinase (MMP) inhi-
bitor, GM6001 (A, B), and EGFR signalling in SCC4 cells, using the EGFR antagonist, AG1478 (A, C), was assessed using migration
assays, as described in the Material and methods. Small interfering RNA (siRNA) targeted to siRNA (siA17) or a non-targeting negative
control oligonucleotide (-ve) were transiently transfected into NOFs treated with Ang II or left untreated, and the conditioned medium
was used in a migration assay to assess chemotaxis of SCC4 cells, as described in the Materials and methods (D). Results are plotted rela-
tive to untreated control wells  standard error of the mean of three separate fields of view from three independent experiments. Total
RNA was extracted from transfected NOFs and subjected to quantitative RT-PCR using probes for ADAM17 and B2M (as a reference
gene) to assess siRNA-mediated knockdown (E; n = 3). *P < 0.05 relative to control, **P < 0.05 relative to Ang II treated.

Ang 1-7 inhibits Ang II-induced cancer cell motility

contraction (Fig. 4E). Angiotensin II-treated NOFs
were also observed to be more migratory than their Angiotensin 1-7, the product of the proteolytic action
vehicle-treated counterparts (Fig. 4F). of ACE2 on Ang II, is reported to oppose the
 Angiotensin peptides in head and neck cancer 7

Fig. 4. Angiotensin II (Ang II) provokes myofibroblast transdifferentiation of normal oral fibroblasts (NOFs). NOFs were treated
with Ang II or transforming growth factor beta (TGF-b) for 48 h before cells were harvested and RNA extracted or lysates pre-
pared as described in the Material and methods. Alpha smooth muscle actin (a-SMA) (A) and matrix metalloproteinase 2 (MMP-
2) (B) transcript levels were assessed by quantitative PCR (qPCR); values are shown normalized to U6 expression levels in the
same samples. Cell lysates were separated by SDS-PAGE and immunoblotted for a-SMA and b-actin (as a loading control). A
representative blot is shown (C) and the intensity of the band corresponding to a-SMA, determined by densitometry and normal-
ized to b-actin levels in the same sample, is indicated under each lane. qPCR for a-SMA was also carried out on RNA extracted
from NOFs pretreated with telmisartan before addition of Ang II for 48 h (D). Results are plotted relative to untreated control
wells, as the mean value  standard error of the mean of three independent experiments, carried out in triplicate. *P < 0.05. Oral
fibroblasts were seeded into collagen as described in the Material and methods. Following overnight incubation in serum-free
medium, NOFs were treated with Ang II or thrombin for 30 min. The gels were photographed and the distance contracted was
measured (E). A scratch was made in oral fibroblasts cultured as a monolayer. Cells were treated with Ang II or TGF-b, and the
diameter of the scratch was measured before treatment and after 24 h. Photographs were taken (F) and percentage migration rela-
tive to control was calculated. The data represent the mean value of at least three experiments  standard error of the mean.
*P < 0.05.

profibrotic actions of Ang II in animal models of renal
and cardiovascular disease, acting through its receptor,
MasR. Therefore, we next analysed the expression of
ACE2 and MasR in our panel of cells to determine
whether the oral mucosa may be capable of generating
and/or responding to Ang 1-7. The highest ACE2 levels
were observed in NOKs, with lower levels observed in
a number of HNSCC cell lines, and ACE2 was unde-
tectable in NOFs (Fig. 5A). MasR was readily detect-
able in HNSCC-derived cells, with lower levels present
in NOFs and NOKs (Fig. 5B). Having established that
HNSCC cells express both ACE2 and MasR, we next
examined the effect of Ang 1-7 on their motility and
invasion, both in the presence and the absence of Ang
II. Co-application of Ang 1-7 with Ang II significantly
abrogated the ability of Ang II to promote cell motility
and invasion directly (Fig. 5C,D).
Given that detectable levels of MasR transcript were
observed in NOFs, albeit low compared with HNSCC
cells (Fig. 5B, inset), we next analysed the effect of
exogenous Ang 1-7 on Ang II-stimulated paracrine sig-
nalling between NOFs and HNSCC cells. As observed
for direct application, treatment of SCC4 cells with
CM from fibroblasts incubated with Ang 1-7 and Ang
II significantly inhibited the stimulation of cell migra-
tion and invasion observed in response to Ang II alone
8 Hinsley et al.

Fig. 5. Angiotensin 1-7 (Ang 1-7) abrogates angiotensin II (Ang II)-stimulated migration and invasion of head and neck squa-
mous cell carcinoma (HNSCC) cells. Total RNA was prepared from normal primary oral keratinocytes (NOKs), normal primary
oral fibroblasts (NOFs), and from a range of cell lines derived from oral dysplasias, squamous cell carcinomas, and a local lymph
node metastasis. Expression of angiotensin-converting enzyme 2 (ACE2) (A) and a G protein–coupled receptor, MasR (B), was
assessed by quantitative PCR (qPCR). Migration (C) and invasion (D) of SCC4 in response to treatment with Ang II and Ang 1-
7 was analysed as described in the Material and methods. The results are plotted as mean values relative to untreated control
wells  standard error of the mean, of three separate fields of view from three independent experiments. *P < 0.05 relative to
control; **P < 0.05 relative to Ang II treated. ns, not significant.

(Fig. 6A,B). Angiotensin 1-7 was also able to block the
Ang II-stimulated increase in a-SMA expression in
NOFs, suggesting that Ang 1-7 is able inhibit the
fibroblast-specific actions of Ang II (Fig. 6C) and that
MasR is functional in these cells.
Discussion
Evidence supporting a role for the RAS in carcinogene-
sis and tumour progression has been mounting in
recent years and is currently the focus of considerable
interest (19). Changes in the levels of components of
the RAS have been observed in a number of tumour
types, including lung and prostate cancer, and inhibi-
tion of various elements of the pathway has been
shown to reduce tumour growth and metastasis [re-
viewed previously (30)]. Here we have examined, for
the first time, the role of the RAS in head and neck
cancer progression with the aim of identifying novel
therapeutic targets for this frequently fatal disease.
A recent report identified the presence of all the com-
ponents required for a functional local tissue RAS in
the human oral mucosa (10), in keeping with previous
reports in other species (31, 32). In light of these find-
ings, we examined the levels of the two receptors for
the major bioactive peptide generated by the RAS, Ang
II, in a panel of NOKs, NOFs, and cell lines derived
from oral dysplasias and oral squamous cell carcinomas
(the most common form of HNSCC) and observed a
marked up-regulation of AT1R expression in all the
HNSCC cell lines examined compared with NOKs,
cells propagated from dysplastic lesions, and those
from a metastatic lesion. Alterations in AT1R expres-
sion have been detected in other malignancies,
including prostate and breast (15, 33); in the latter,
meta-analysis of a large number of gene-expression pro-
filing data sets recently identified AT1R as one of the
most prominently overexpressed genes. This is the first
report, however, of aberrant AT1R expression in
HNSCC and suggests that malignant cells may become
sensitive to the mitogenic effects of local Ang II. This is
given credence by the finding that treatment of two
HNSCC-derived cell lines with Ang II resulted in a
modest, but significant, increase in motility and inva-
sive capacity; the ability of telmisartan, but not the
AT2R antagonist, PD123319, to inhibit this provides
further evidence that Ang II mediates these effects via
AT1R.
In the current study, high levels of AT1R were also
detected in NOFs; CM collected from NOFs treated
with Ang II was able to stimulate the motility of oral
cancer cells, suggesting that AT1R in oral fibroblasts is
functional and promotes stromal-tumour cell interac-
tions. The ability of an ADAM/MMP inhibitor and an
EGFR antagonist to block the promigratory effects of
CM from Ang II-treated oral fibroblasts indicates, as
we recently reported for another mitogenic peptide,
 Angiotensin peptides in head and neck cancer 9

Fig. 6. Angiotensin 1-7 (Ang 1-7) blocks stimulation of normal oral fibroblasts (NOFs) by angiotensin II (Ang II). NOFs were
pretreated for 30 min with Ang 1-7 before addition of Ang II or vehicle control for 4 h. Conditioned media (CM) was harvested
and applied to the lower chamber of migration (A, C) or matrigel invasion (B) assay plates, before addition of SCC4 cells to the
upper chamber. SCC4 migration and invasion was quantified as described in ‘Materials and methods’. Results are mean values
plotted relative to untreated control wells  standard error of the mean, of three separate fields of view from three independent
experiments. *P < 0.05 relative to control, **P < 0.05 relative to Ang II treated. Quantitative reverse transcription PCR (RT-
qPCR) for a-SMA was carried out on RNA extracted from NOFs pretreated with Ang 1-7 before addition of Ang II for 48 h;
values are expressed relative to U6 in the same samples (C).

ET-1 (2), that EGFR transactivation may play a role in
the effects of Ang II on HNSCC motility. Epidermal
growth factor receptor transactivation mediates patho-
physiological signalling triggered by Ang II in other tis-
sues, such as aortic smooth muscle cells (34), and
aberrant EGFR signalling is a frequently observed fea-
ture of head and neck cancers (35). Further work is
required to elucidate Ang II-induced signalling path-
ways in HNSCC.
Invasion and local metastatic spread of head and
neck cancer is increasingly recognized to involve com-
plex interactions between tumour cells and the sur-
rounding stroma. The most numerous cells of the
stroma, fibroblasts, frequently adopt an activated,
myofibroblastic phenotype in response to contextual
signals such as TGF-b in the tumour microenvironment
(5). These cancer-associated fibroblasts (CAFs) have
altered morphology resulting from the expression of
a-SMA and secrete elevated levels of ECM compo-
nents, such as MMP-2, which promote cancer cell inva-
sion. Indeed, our unpublished data and a recent study
demonstrate that TGF-b treatment of oral fibroblasts
provokes a myofibroblast transdifferentiation, which
increases pro-invasive paracrine signalling (3). Here we
show that Ang II provokes the acquisition of a myofi-
broblast phenotype in oral fibroblasts and increases
their ability to stimulate cancer cell migration and inva-
sion in a paracrine manner. Although the ability of
Ang II to stimulate myofibroblast transdifferentiation
in pathologies, such as lung fibrosis, is known (36), this
is, to our knowledge, the first report of Ang II-
mediated fibroblast remodelling influencing cancer cell
behaviour. The ability of an AT1R antagonist to block
the fibroblast phenotypic changes induced by Ang II,
at least in vitro, raises the possibility of utilizing such
antagonists in the clinic to inhibit metastasis and to
reduce fibrosis to improve penetration of existing
chemotherapeutic agents.
Evidence is mounting in the literature for the possi-
ble clinical applications of Ang 1-7, the product of
ACE2 action on Ang II, with beneficial effects of Ang
1-7 administration observed in a number of pathologies
10 Hinsley et al.
[reviewed previously (8)]. Angiotensin 1-7 has been
shown to reduce lung cancer proliferation, angiogene-
sis, and invasion (21, 22); it is also reported to reduce
fibrosis associated with breast cancers (23). In HNSCC
cells, we observed beneficial effects only in the presence
of Ang II; no significant changes in cancer cell motility
or fibroblast phenotype were observed in response to
Ang 1-7 treatment alone. This is unsurprising given the
well-documented ability of Ang 1-7 to oppose the
actions of Ang II, but is in apparent contradiction to
previous findings in lung cancer cells (22), which
reported inhibition of invasion by Ang 1-7 in the
absence of Ang II, suggesting that the effects of Ang 1-
7 may be tissue specific. Our finding, that the response
to Ang 1-7 is dependent on the actions of Ang II, raises
the possibility that application of Ang 1-7 may be tar-
geted to those tumours (and associated CAFs) express-
ing AT1R and Mas. A recent phase I clinical trial in
patients with solid tumours, including one with head
and neck cancer, indicated that Ang 1-7 (delivered sub-
cutaneously) is well tolerated and with some clinical
benefit (37). Although further studies are required to
determine its efficacy, these data, together with the
in vitro evidence presented here, suggest that Ang 1-7
and/or AT1R receptor antagonists are promising new
therapeutic agents for HNSCC.
Acknowledgements – We thank Dr Craig Murdoch, Dr Helen Col-
ley and Dr Keith Hunter (University of Sheffield, UK) for kindly
providing cells and Prof. Paula Farthing, Dr Simon Whawell
(University of Sheffield, UK) for helpful suggestions. Funding was
obtained from British Society of Oral and Maxillofacial Patholo-
gists (to D.W.L.). E.E.H. was supported by a University of Shef-
field PhD Scholarship.
Conflicts of interest – None declared.
References
1. ARGIRIS A, KARAMOUZIS MV, RABEN D, FERRIS RL. Head
and neck cancer. Lancet 2008; 371: 1695–1709.
2. HINSLEY EE, HUNT S, HUNTER KD, WHAWELL SA, LAMBERT
DW. Endothelin-1 stimulates motility of head and neck squa-
mous carcinoma cells by promoting stromal-epithelial interac-
tions. Int J Cancer 2012; 130: 40–47.
3. MARSH D, SUCHAK K, MOUTASIM KA, VALLATH S, HOPPER C,
JERJES W, UPILE T, KALAVREZOS N, VIOLETTE SM, WEINREB
PH, CHESTER KA, CHANA JS, MARSHALL JF, HART IR, HACK-
SHAW AK, PIPER K, THOMAS GJ. Stromal features are predic-
tive of disease mortality in oral cancer patients. J Pathol
2011; 223: 470–481.
4. KELLERMANN MG, SOBRAL LM, DA SILVA SD, ZECCHIN KG,
GRANER E, LOPES MA, KOWALSKI LP, COLETTA RD. Mutual
paracrine effects of oral squamous cell carcinoma cells and
normal oral fibroblasts: induction of fibroblast to myofibrob-
last transdifferentiation and modulation of tumor cell prolifer-
ation. Oral Oncol 2008; 44: 509–517.
5. KALLURI R, ZEISBERG M. Fibroblasts in cancer. Nat Rev Can-
cer 2006; 6: 392–401.
6. KABIR TD, LEIGH RJ, TASENA H, MELLONE M, COLETTA RD,
PARKINSON EK, PRIME SS, THOMAS GJ, PATERSON IC, ZHOU
D, MCCALL J, SPEIGHT PM, LAMBERT DW. A miR-335/COX-
2/PTEN axis regulates the secretory phenotype of senescent
cancer-associated fibroblasts. Aging 2016; 8: 1608–1635.
7. SOBRAL LM, ZECCHIN KG, NASCIMENTO DE, AQUINO S, LOPES
MA, GRANER E, COLETTA RD. Isolation and characterization
of myofibroblast cell lines from oral squamous cell carcinoma.
Oncol Rep 2011; 25: 1013–1020.
8. LAMBERT DW, HOOPER NM, TURNER AJ. Angiotensin-con-
verting enzyme 2 and new insights into the renin-angiotensin
system. Biochem Pharmacol 2008; 75: 781–786.
9. HAMMING I, COOPER ME, HAAGMANS BL, HOOPER NM, KOR-
STANJE R, OSTERHAUS AD, TIMENS W, TURNER AJ, NAVIS G,
VAN GOOR H. The emerging role of ACE2 in physiology and
disease. J Pathol 2007; 212: 1–11.
10. NAKAMURA T, HASEGAWA-NAKAMURA K, SAKODA K, MAT-
SUYAMA T, NOGUCHI K. Involvement of angiotensin II type 1
receptors in interleukin-1beta-induced interleukin-6 produc-
tion in human gingival fibroblasts. Eur J Oral Sci 2011; 119:
345–351.
11. LEE MH, EL-SHEWY HM, LUTTRELL DK, LUTTRELL LM. Role
of beta-arrestin-mediated desensitization and signaling in the
control of angiotensin AT1a receptor-stimulated transcrip-
tion. J Biol Chem 2008; 283: 2088–2097.
12. BHOLA NE, GRANDIS JR. Crosstalk between G-protein-
coupled receptors and epidermal growth factor receptor in
cancer. Front Biosci 2008; 13: 1857–1865.
13. YAHATA Y, SHIRAKATA Y, TOKUMARU S, YANG L, DAI X,
TOHYAMA M, TSUDA T, SAYAMA K, IWAI M, HORIUCHI M,
HASHIMOTO K. A novel function of angiotensin II in skin
wound healing. Induction of fibroblast and keratinocyte
migration by angiotensin II via heparin-binding epidermal
growth factor (EGF)-like growth factor-mediated EGF
receptor transactivation. J Biol Chem 2006; 281: 13209–
13216.
14. SUGANUMA T, INO K, SHIBATA K, KAJIYAMA H, NAGASAKA T,
MIZUTANI S, KIKKAWA F. Functional expression of the angio-
tensin II type 1 receptor in human ovarian carcinoma cells
and its blockade therapy resulting in suppression of tumor
invasion, angiogenesis, and peritoneal dissemination. Clin
Cancer Res 2005; 11: 2686–2694.
15. RHODES DR, ATEEQ B, CAO Q, TOMLINS SA, MEHRA R, LAX-
MAN B, KALYANA-SUNDARAM S, LONIGRO RJ, HLEGESON BE,
BHOJANI MS, REHEMTULLA A, KLEER CG, HAYES DF, LUCAS
PC, VARAMBALLY S, CHINNAIYAN AM. AGTR1 overexpression
defines a subset of breast cancer and confers sensitivity to
losartan, an AGTR1 antagonist. Proc Natl Acad Sci U S A
2009; 106: 10284–10289.
16. TANAKA N, MIYAJIMA A, KOSAKA T, MIYAZAKI Y, SHIROTAKE
S, SHIRAKAWA H, KIKUCHI E, OYA M. Acquired platinum
resistance enhances tumour angiogenesis through angiotensin
II type 1 receptor in bladder cancer. Br J Cancer 2011; 105:
1331–1337.
17. AMAYA K, OHTA T, KITAGAWA H, KAYAHARA M, TAKAMURA
H, FUJIMURA T, NISHIMURA G, SHIMIZU K, MIWA K. Angio-
tensin II activates MAP kinase and NF-kappaB through
angiotensin II type I receptor in human pancreatic cancer
cells. Int J Oncol 2004; 25: 849–856.
18. FUJITA M, HAYASHI I, YAMASHINA S, FUKAMIZU A, ITOMAN
M, MAJIMA M. Angiotensin type 1a receptor signaling-depen-
dent induction of vascular endothelial growth factor in
stroma is relevant to tumor-associated angiogenesis and
tumor growth. Carcinogenesis 2005; 26: 271–279.
19. GEORGE AJ, THOMAS WG, HANNAN RD. The renin-angioten-
sin system and cancer: old dog, new tricks. Nat Rev Cancer
2010; 10: 745–759.
20. LAMBERT DW, CLARKE NE, TURNER AJ. Not just angiotensi-
nases: new roles for the angiotensin-converting enzymes. Cell
Mol Life Sci 2010; 67: 89–98.
21. GALLAGHER PE, TALLANT EA. Inhibition of human lung can-
cer cell growth by angiotensin-(1–7). Carcinogenesis 2004; 25:
2045–2052.
22. NI L, FENG Y, WAN H, MA Q, FAN L, QIAN Y, LI Q, XIANG
Y, GAO B. Angiotensin-(1-7) inhibits the migration and inva-
sion of A549 human lung adenocarcinoma cells through inac-
tivation of the PI3K/Akt and MAPK signaling pathways.
Oncol Rep 2012; 27: 783–790.
23. COOK KL, METHENY-BARLOW LJ, TALLANT EA, GALLAGHER
PE. Angiotensin-(1–7) reduces fibrosis in orthotopic breast
tumors. Cancer Res 2010; 70: 8319–8328.

24. YASUMATSU R, NAKASHIMA T, MASUDA M, ITO A, KURATOMI
Y, NAKAGAWA T, KOMUNE S. Effects of the angiotensin-I con-
verting enzyme inhibitor perindopril on tumor growth and
angiogenesis in head and neck squamous cell carcinoma cells.
J Cancer Res Clin Oncol 2004; 130: 567–573.
25. HEARNDEN V, LOMAS H, MACNEIL S, THORNHILL M, MURDOCH
C, LEWIS A, MADSEN J, BLANAZS A, ARMES S, BATTAGLIA G.
Diffusion studies of nanometer polymersomes across tissue
engineered human oral mucosa. Pharm Res 2009; 26: 1718–
1728.
26. COLLEY HE, HEARNDEN V, JONES AV, WEINREB PH, VIOLETTE
SM, MACNEIL S, THORNHILL MH, MURDOCH C. Development
of tissue-engineered models of oral dysplasia and early inva-
sive oral squamous cell carcinoma. Br J Cancer 2011; 105:
1582–1592.
27. WASEEM A, ALI M, ODELL EW, FORTUNE F, TEH MT. Down-
stream targets of FOXM1: CEP55 and HELLS are cancer
progression markers of head and neck squamous cell carci-
noma. Oral Oncol 2010; 46: 536–542.
28. MCGREGOR F, MUNTONI A, FLEMING J, BROWN J, FELIX DH,
MACDONALD DG, PARKINSON EK, HARRISON PR. Molecular
changes associated with oral dysplasia progression and acqui-
sition of immortality: potential for its reversal by 5-azacyti-
dine. Cancer Res 2002; 62: 4757–4766.
29. HINSLEY EE, KUMAR S, HUNTER KD, WHAWELL SA, LAMBERT
DW. Endothelin-1 stimulates oral fibroblasts to promote oral
cancer invasion. Life Sci 2012; 91: 557–561.
30. AGER EI, NEO J, CHRISTOPHI C. The renin-angiotensin system
and malignancy. Carcinogenesis 2008; 29: 1675–1684.
31. OHSAWA M, OHUCHI N, TANIGUCHI Y, KIZAWA Y, KOIKE K,
IWAMOTO K, HAYASHI K, MURAKAMI H. Inhibition of
Angiotensin peptides in head and neck cancer 11
angiotensin II- and endothelin-1-stimulated proliferation by
selective MEK inhibitor in cultured rabbit gingival fibrob-
lasts. Fundam Clin Pharmacol 2005; 19: 677–685.
32. OHUCHI N, KOIKE K, SANO M, KUSAMA T, KIZAWA Y, HAYA-
SHI K, TANIGUCHI Y, OHSAWA M, IWAMOTO K, MURAKAMI H.
Proliferative effects of angiotensin II and endothelin-1 on gui-
nea pig gingival fibroblast cells in culture. Comp Biochem
Physiol C Toxicol Pharmacol 2002; 132: 451–460.
33. UEMURA H, ISHIGURO H, NAKAIGAWA N, NAGASHIMA Y,
MIYOSHI Y, FJINAMI K, SAKAGUCHI A, KUBOTA Y. Angioten-
sin II receptor blocker shows antiproliferative activity in pros-
tate cancer cells: a possibility of tyrosine kinase inhibitor of
growth factor. Mol Cancer Ther 2003; 2: 1139–1147.
34. YANG X, ZHU MJ, SREEJAYAN N, REN J, DU M. Angiotensin
II promotes smooth muscle cell proliferation and migration
through release of heparin-binding epidermal growth factor
and activation of EGF-receptor pathway. Mol Cells 2005; 20:
263–270.
35. POMERANTZ RG, GRANDIS JR. The role of epidermal growth
factor receptor in head and neck squamous cell carcinoma.
Curr Oncol Rep 2003; 5: 140–146.
36. MARSHALL RP, GOHLKE P, CHAMBERS RC, HOWELL DC, BOT-
TOMS SE, UNGER T, MCANULTY RJ, LAURENT GJ. Angioten-
sin II and the fibroproliferative response to acute lung injury.
Am J Physiol Lung Cell Mol Physiol 2004; 286: L156–L164.
37. PETTY WJ, MILLER AA, MCCOY TP, GALLAGHER PE, TALLANT
EA, TORTI FM. Phase I and pharmacokinetic study of angio-
tensin-(1-7), an endogenous antiangiogenic hormone. Clin
Cancer Res 2009; 15: 7398–7404.