Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB

Abstract

Extracellular lysyl oxidases (LOX and LOXL1–LOXL4) are critical for collagen biosynthesis. LOXL2 is a marker of poor survival in oral squamous cell cancer. We investigated mechanisms by which tumor cell secreted LOXL2 targets proximal mesenchymal cells to enhance tumor growth and metastasis. This study identified the first molecular mechanism for LOXL2 in the promotion of cancer via its enzymatic modification of a non-collagenous substrate in the context of paracrine signaling between tumor cells and resident fibroblasts. The role and mechanism of active LOXL2 in promoting oral cancer was evaluated and employed a novel LOXL2 small molecule inhibitor, PSX-S1C, administered to immunodeficient, and syngeneic immunocompetent orthotopic oral cancer mouse models. Tumor growth, histopathology, and metastases were monitored. In vitro mechanistic studies with conditioned tumor cell medium treatment of normal human oral fibroblasts were carried out in the presence and absence of the LOXL2 inhibitor to identify signaling mechanisms promoted by LOXL2 activity. Inhibition of LOXL2 attenuated cancer growth and lymph node metastases in the orthotopic tongue mouse models. Immunohistochemistry data indicated that LOXL2 expression in and around tumors was decreased in mice treated with the inhibitor. Inhibition of LOXL2 activity by administration of PXS-S1C to mice reduced tumor cell proliferation, accompanied by changes in morphology and in the expression of epithelial to mesenchymal transition markers. In vitro studies identified PDGFRβ as a direct substrate for LOXL2, and indicated that LOXL2 and PDGF-AB together secreted by tumor cells optimally activated PDGFRβ in fibroblasts to promote proliferation and the tendency toward fibrosis via ERK activation, but not AKT. Optimal fibroblast proliferation in vitro required LOXL2 activity, while tumor cell proliferation did not. Thus, tumor cell-derived LOXL2 in the microenvironment directly targets neighboring resident cells to promote a permissive local niche, in addition to its known role in collagen maturation.

Full text

Mahjour et al. Oncogenesis (2019) 8:34
https://doi.org/10.1038/s41389-019-0144-0 Oncogenesis
ARTICLE Open Access
Mechanism for oral tumor cell lysyl oxidase
like-2 in cancer development: synergy with
PDGF-AB
Faranak Mahjour
1
,VrindaDambal
1
, Neha Shrestha
1
,VarunSingh
1
, Vikki Noonan
2
, Alpdogan Kantarci
3
and
Philip C. Trackman
1
Abstract
Extracellular lysyl oxidases (LOX and LOXL1–LOXL4) are critical for collagen biosynthesis. LOXL2 is a marker of poor
survival in oral squamous cell cancer. We investigated mechanisms by which tumor cell secreted LOXL2 targets
proximal mesenchymal cells to enhance tumor growth and metastasis. This study identified the first molecular
mechanism for LOXL2 in the promotion of cancer via its enzymatic modification of a non-collagenous substrate in the
context of paracrine signaling between tumor cells and resident fibroblasts. The role and mechanism of active LOXL2
in promoting oral cancer was evaluated and employed a novel LOXL2 small molecule inhibitor, PSX-S1C, administered
to immunodeficient, and syngeneic immunocompetent orthotopic oral cancer mouse models. Tumor growth,
histopathology, and metastases were monitored. In vitro mechanistic studies with conditioned tumor cell medium
treatment of normal human oral fibroblasts were carried out in the presence and absence of the LOXL2 inhibitor to
identify signaling mechanisms promoted by LOXL2 activity. Inhibition of LOXL2 attenuated cancer growth and lymph
node metastases in the orthotopic tongue mouse models. Immunohistochemistry data indicated that LOXL2
expression in and around tumors was decreased in mice treated with the inhibitor. Inhibition of LOXL2 activity by
administration of PXS-S1C to mice reduced tumor cell proliferation, accompanied by changes in morphology and in
the expression of epithelial to mesenchymal transition markers. In vitro studies identified PDGFRβas a direct substrate
for LOXL2, and indicated that LOXL2 and PDGF-AB together secreted by tumor cells optimally activated PDGFRβin
fibroblasts to promote proliferation and the tendency toward fibrosis via ERK activation, but not AKT. Optimal
fibroblast proliferation in vitro required LOXL2 activity, while tumor cell proliferation did not. Thus, tumor cell-derived
LOXL2 in the microenvironment directly targets neighboring resident cells to promote a permissive local niche, in
addition to its known role in collagen maturation.
Introduction
Oral squamous cell carcinoma (OSCC) accounts for
more than 90% of oral cavity cancers and is the sixth most
common cancer in the world
1,2
. Tobacco smoking and
excessive alcohol are major risk factors with synergistic
effects
3
. Metastasis to cervical lymph nodes in patients
with OSCC occurs in almost half of patients
4
and recur-
rent metastasis occurs in 20–30% of patients after treat-
ment
5,6
. These cancers have a poor 5-year survival rate,
and cause significant morbidity due to limiting speech,
food intake, and other aspects of oral and systemic health.
Lysyl oxidases (LOXs) catalyze the oxidative deamination
of the ɛ-amino group of lysine and hydroxylysine residues in
the telopeptide regions of procollagens to form peptidyl
aldehydes, which results in biosynthetic cross-linking of
collagen
7
. Overexpressed LOXs promote cancer
© The Author(s) 2019
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, whi ch permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated other wise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Correspondence: Philip C. Trackman (trackman@bu.edu)
1
Boston University Henry M. Goldman School of Dental Medicine, Department
of Molecular and Cell Biology, Boston, MA 02118, USA
2
Boston University Henry M. Goldman School of Dental Medicine, Division of
Oral & Maxillofacial Pathology, Boston, MA 02118, USA
Full list of author information is available at the end of the article.
Oncogenesis
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progression in part due to excess modification of the
extracellular matrix that can stimulate invasion and
metastasis
8
. The LOX family consists of five members: LOX
andthefourrelatedgenesLOX-like-1–4(LOXL1–LOXL4).
Cancer progression and metastasis depends on the inter-
actions between cancer cells and the tumor microenviron-
ment, modifying tumor and stromal cell proliferation,
extracellular matrix production and turnover, drug resis-
tance, cell migration, and metastasis. Collagen accumula-
tion, fibrosis, and dense or stiff microenvironments
promote metastasis in solid tumors including breast cancer
through focal adhesion formation
8,9
. LOXs may additionally
interact with and/or oxidize other non-collagen proteins,
which in turn regulate cell signaling pathways
10–13
,which
can modulate cancer progression.
LOX-like-2 (LOXL2) has emerged as a biomarker for
OSCC and its overexpression is associated with poor
prognosis in patients
14
. The underlying mechanisms of the
effect of LOXL2 on invasiveness of cancer are not well
understood. LOXL2 nuclear interaction with Snail that
regulates E-cadherin and leads to epithelial–mesenchymal
transition (EMT) and invasiveness has been suggested
13
.
Other studies suggested that interaction of LOXL2 with
histones and nuclear proteins leads to EMT and invasive-
ness. However, there is no evidence of direct oxidation of
these nuclear proteins by LOXL2 and the mechanism of
action of LOXL2 remains unclear
7,13,15
. With a poor sur-
vival rate of OSCC, high occurrence of metastasis, and
difficulties faced by patients with conventional treatments,
there is a clear need to develop new targeted therapeutic
approaches to address oral cancer and metastasis. The
current study determined the effects and mechanism of
secreted LOXL2 as a mediator of the progression and
invasiveness of OSCC in two orthotopic in vivo mouse
models, employing a novel LOXL2 pharmacologic small
molecule inhibitor. Reduced tumor growth and metastasis
and apparent inhibition of EMT was found in response to
the inhibitor. Studies of mechanisms in vitro support that
tumor cell-derived LOXL2 directly stimulated stromal
fibroblast proliferation and activity by enhancing PDGF-
AB-mediated signaling after modification of PDGFRβ,while
having no proliferative effect on tumor cells themselves.
These interactions define a novel interaction that occurs in
the microenvironment emanating from tumor cells and that
targets stromal mesenchymal cells. These findings provide a
mechanistic basis for potential novel therapeutic approa-
ches to address tumor growth, fibrosis, and metastasis.
Methods
Histopathology of LOXL2 and LOX expression in human
oral cancer biopsies
Tissue blocks from three to five biopsies from different
donors corresponding to dysplasia, differentiated OSCC,
and poorly differentiated oral cancer, respectively, from the
Boston University School of Dental Medicine Pathology
diagnostic services laboratory were obtained under an
approved IRB protocol (#31869). Sections (6 µm) were
stained with hematoxylin and eosin, or immunostained for
LOX or LOXL2 with counterstaining with hematoxylin
16,17
.
Orthotopic tongue immunodeficient mouse model
All animal experiments were approved by the Boston
University Institutional Animal Care and Use Committee,
protocol #15354. HSC3 cells were a generous gift from Dr.
Roberto Weigert (NIDCR, Bethesda, MD) and were vali-
dated by STR profiling by Genetica DNA Laboratories at
the time of initiation of the studies. Cells were
mycoplasma-free prior to use. HSC3 cells transduced with
a lentivirus expressing the red fluorescent protein (RFP)
DsRed were cultured according to the methods described
previously
18
. Cell suspension (480,000 cells in 40 µl) was
injected to the tongue of each nude mouse (NCRNU mice
6-week-old female, Taconic) under anesthesia (3% iso-
flurane). Diet gels containing electrolytes and nutrients
were made available to the mice. Tumor growth was
measured by caliper measurements and distant metastasis
was detected by IVIS in mice injected with HSC3 ecto-
pically expressing DsRed. There were four experimental
groups (n=8 per group) in this in vivo experiment: 1.
Mice injected with HSC3 cells transduced with DsRed; 2.
Mice injected with HSC3 cells transduced with DsRed and
the LOXL2 inhibitor PXS-S1C (30 mg/kg in DMSO i.p.;
three times per week); 3. Mice injected with HSC3 cells
transduced with DsRed, PXS-S1C (10 mg/kg in DMSO i.
p.; three times per week); 4. Control, group mice with no
HSC3 injected cells, and no PXS-1C applied to serve as
IVIS negative controls. Three weeks later, the mice were
euthanized using isoflurane overdose followed by cervical
dislocation.
Orthotopic tongue immunocompetent mouse model
LY2 cells were cultured and processed as above. Ali-
quots of 40 µl of the cell suspension (480,000 cells) were
injected to the tongue of each immunocompetent mouse
(6-week-old female BALB/c) under anesthesia (3% iso-
flurane), and diet gels containing electrolytes and nutri-
ents (76A, ClearH20) were made available to the mice.
There were four experimental groups in this in vivo
experiment (n=12 per group): 1. Mice injected with LY2
cells; 2. Mice injected with LY2 cells, PXS-S1C (30 mg/kg
in DMSO i.p., three times per week); 3. Mice injected with
LY2 cells, PXS-S1C (10 mg/kg in DMSO i.p., three times
per week); 4. Control, group mice (n=8, without injec-
tion). Mice were euthanized after 6 weeks.
LOXL2 inhibitor treatment of mice
PXS-S1C was first dissolved in sterile DMSO at 2.2 or
6.6 mg/ml for a dosing of 10 or 30 mg/kg, respectively.
Mahjour et al. Oncogenesis (2019) 8:34 Page 2 of 17
Oncogenesis
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PSX-S1C was administered by intraperitoneal injection
12–24 h before the injection of cancer cells. The PXS-S1C
injection was continued at a frequency of three times
per week.
The tongues and lymph nodes were fixed in 4% paraf-
ormaldehyde, paraffin-embedded and were sectioned
(6 µm). Tongues were fixed for histology, hematoxylin and
eosin staining, Sirius red staining (Sirius red in picric
acid), and immunohistochemistry (IHC) staining with
anti-Ki67 antibody (ab15580, Abcam) or anti-proliferating
cell nuclear antigen antibody (PCNA, ab92552, Abcam),
anti-LOXL2 antibody (GTX105085, GeneTex), anti-E-
cadherin antibody (610181, BD Transduction Labora-
tories), and anti-vimentin antibody (ab92547, Abcam).
The images of the stained slides were taken with a Zeiss
microscope with lens objectives of ×4, ×10, and ×20.
Analyses of IHC slides was performed in a blinded fashion
by a lab member, who was not informed regarding the
identity of each experimental group.
Cell culture
Human gingival fibroblasts (HGF) and cancer cells were
grown separately at 37 °C and 5% CO
2
in a humidified
incubator. The HGF were taken from three healthy
donors aged 25–35 years undergoing crown lengthening
procedures. The study protocol was approved by Boston
University Medical Center Institutional Review Board
committee. HSC3 cells are highly aggressive SCC cancer
cells with metastatic characteristics derived from human
tongue and were generated by multiple successive pas-
sages of a tumor in mice
19
. LY2 cells are aggressive SCC
cancer cells with metastatic characteristics and were
derived from a BALB/c mouse keratinocyte tumor, and
were kindly provided by Dr. Nadarajah Vigneswaran and
Dr. Wolfgang Zacharias
20
. UMSCC2 cells are human
OSCC cells obtained from Dr. Thomas Carey at the
University of Michigan
21
and SCC71 human oral cancer
keratinocytes were obtained from Dr. Rheinwald at Har-
vard University
22
. SCC25 and CAL27 cells are human
tongue SCC cells and were obtained from ATCC.
The growth medium used for gingival fibroblasts was
Dulbecco’s modified Eagle’s medium (DMEM, high glu-
cose 11965-092, Thermo Fisher Scientific), 10% fetal
bovine serum (FBS, F0926, Sigma), 1% non-essential
amino acid (NEAA, 11140050, Gibco), and 1% penicillin/
streptomycin (15140122, Gibco)
12
. For serum depletion,
HGF or cancer cells were washed two times with PBS and
treated with serum-free DMEM containing 1% Penicillin/
Streptomycin for 24 h. To produce conditioned media
(CM), cancer cells were grown to 90% visual confluence.
After washing twice with PBS, they were treated with
serum-free DMEM containing 1% Penicillin/Streptomy-
cin for 24 h and the media were collected. Additional
reagents were recombinant human PDGF-BB (#100-14B)
and PDGF-AB (100-00AB) were purchased from Pepro-
tech. PDGF-CC (1687-CC-025) was purchased from R&D
Systems. β-aminopropionitrile (BAPN) was purchased
from Sigma/Aldrich.
Cell proliferation assay
CyQUANT Cell Proliferation Assay kit (C7026, Life
Technologies) measures the DNA content in cells in
culture. Gingival fibroblasts were seeded at the density of
10,000 cells per well in a 24-well culture plate. After 6 h
they were washed with PBS twice and treated with either
HSC3 CM only, CM with PXS-S1C (1 µM), or CM with
5μM PDGFR inhibitor Tyrphostin (also known as
AG1296) for 24 h under a serum-free condition. The CM
was aspirated and the cells were washed in PBS. Cells
were then stained according to the CyQuant instructions.
Cells were suspended and transferred to a well of a 96-
well black microplate (Corning
®
96-Well Solid Poly-
styrene Microplate, CLS3915), fluorescence was measured
with excitation at 480 nm and emission at 520 nm using
Berthold Technologies TriStar LB 941 plate reeader.
Real-time qPCR
HSC3 cells were washed with PBS and then treated with
serum-free medium with or without PXS-S1C (1 µM) for
24 h. RNA was isolated using RNeasy Mini Kit according to
the manufacturer’s instructions. cDNA was made by using
High-Capacity cDNA Reverse Transcription Kit (4368814,
Thermo Fisher Scientific). The cDNA concentration was
measured by NanoDrop spectrophotometer and then sub-
jected to qPCR using TaqMan Universal PCR Master Mix
(4304437, Thermo Fisher Scientific) and TaqMan probes
for LOX (Hs00942480_m1 Gene LOX), LOXL1
(Hs00935937_m1 Gene LOXL1), LOXL2 (Hs00158757_m1
Gene LOXL2), LOXL3 (Mm01184865_m1 Gene Loxl3),
LOXL4 (Hs00260059_m1 Gene LOXL4), and 18S
(Hs99999901_s1 18S Human probe) as a control.
Measurement of protein levels and western blots
To measure protein concentration in CM, gingival
fibroblasts, LY2, or HSC3 cells were washed with PBS two
times and then serum-free medium (5 ml) was added to
the cells and after 24 h were collected. The CM were
concentrated from 25 to 1 ml using Amicon Ultra-15
Centrifugal filter devices (Z717185, Millipore Sigma) and
protein concentrations measured with Nano Orange
Protein Quantification kit (N6666, Invitrogen), and sub-
jected to western blotting for LOXL2. Samples (20 µg)
were analyzed.
To measure protein expression in cell layers, cells were
washed with PBS and lysed with SDS–PAGE sample
buffer. Protein level of the cell lysates was measured with
the Nano Orange Protein Quantification kit. Then they
were subjected to western blotting. Primary antibodies
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employed were anti-LOXL2 antibody (#GTX105085,
GeneTex), rabbit mAb against phospho-PDGF Receptor β
(Tyr751, #4549, Cell Signaling), rabbit mAb against
phospho-PDGF Receptor β(Tyr771, #3173, Cell Signal-
ing), rabbit mAb against phospho-PDGF Receptor α
(Tyr849)/PDGF Receptor β(Tyr857, #3170, Cell Signal-
ing), rabbit mAb against PDGF Receptor β(#3169, Cell
Signaling), rabbit mAb against phospho-p44/42 MAPK
(ERK 1/2, Thr202/Tyr204, #4377, Cell Signaling), anti-
p44/42 MAPK antibody (ERK 1/2, #9102, Cell Signaling),
anti-phospho-AKT antibody (Ser473, #9271, Cell Signal-
ing), and rabbit anti-AKT mAb (#4691, Cell Signaling)
Signals were visualized with HyGlo Quick spray (1001354,
Denville Scientific) or SuperSignal™West Femto Max-
imum Sensitivity Substrate (34095, Thermo Fisher Sci-
entific) recorded with a G:BOX, Syngene, optimized so
that the intensity of bands was not saturated. The mem-
branes were stripped using Western Blot stripping buffer
(21059, Thermo Fisher Scientific) and re-probed with
antibodies against either β-tubulin (HRP Conjugate,
#5346, Cell Signaling) or β-actin (#4970, Cell Signaling) as
a loading control. Using ImageJ software, the band
intensities were analyzed and normalized to β-actin or
β-tubulin signals.
Knockdown of PDGF A and PDGF B
A set of three PDGF A and two PDGF B MISSION
small hairpin RNA (shRNA) lentiviral transduction par-
ticles (lentiviral-based shRNA vectors) were used to knock
down PDGF A and PDGF B in HSC3 tumor cells. Hex-
adimethrine bromide (8 µg/ml) and PDGFB MISSION
shRNA Lentiviral Transduction Particles Human
(TRCN0000010411 and TRCN0000010412), PDGFA
MISSION shRNA Lentiviral Transduction Particle
Human (TRCN0000158353, TRCN0000157461, and
TRCN0000156591) and MISSION
®
pLKO.1-puro non-
target shRNA control transduction particles were added
to the cells at 2 MOI and incubated for 20 h at 37 °C in a
humidified incubator with 5% CO
2
. The HSC3 cells were
transduced at 70–80% visual confluence overnight. The
medium was replaced with fresh medium and puromycin
(final concentration of 2 µg/ml) was added the following
day. Drug-resistant cells were expanded to become
confluent.
Sandwich ELISA for PDGF-AB
CM samples and standards were added to the 96-well
ELISA plate (ThermoFisher, #EHPDGF AB) followed by
incubation at 4 °C with gentle shaking overnight. The
wells were washed and processed as described by the
manufacturer. Absorbances were determined at wave-
lengths of 450 nm and at 550 nm and the concentration of
PDGF-AB dimer in each sample determined using a
standard curve generated at the same time.
Pulldown assay
Gingival fibroblasts were serum starved and then trea-
ted with CM with or without PXS-S1C (1 µM) for 24 h.
Fibroblast proteins were extracted and the carbonyl-
containing proteins were bioyinylated with biotin hydra-
zide because the hydrazide (−NH−NH
2
) as a reactive
group reacts with carbonyls to form hydrazine bond
(R1R2C =NNH
2
) followed by sodium cyanoborohydride
(NaBH
3
CN) reduction. Proteins were then subjected to
pulldown assays using an avidin-coupled affinity resin
(Neutravidin, Pierce Scientific). Blots of samples before
and after purification were subjected to western blot and
visualized with streptavidin-coupled HRP and anti-
PDGFRβantibody to assess for aldehydes and
PDGFRβ-aldehydes in response to the CM treatment.
Results
LOX family expression in human OSCC
High levels of LOXL2 were previously associated with a
poor prognosis of head and neck squamous cell carci-
noma
23
. To independently determine if the other LOX
paralogues were expressed in oral cancer, we investigated
The Cancer Genome Atlas (TCGA) data set for OSCC of
the tongue compared to non-affected tongue tissue from
the same 334 subjects, focusing on the relative gene
expression of all five LOX paralogues, LOX and
LOXL1–LOXL4. Data in Table S1 demonstrate that in
oral cancer tissues LOXL2 was by far the most sig-
nificantly upregulated LOX paralogue (9.8-fold), while
other paralogues were modestly upregulated (1.6–2.3-
fold). To further characterize expression of LOXL2 and
LOX in human tongue oral cancer, several biopsies
obtained from the Boston University School of Dental
Medicine Pathology diagnostic services laboratory were
obtained under an approved IRB protocol. Figure 1shows
representative histopathology and IHC indicating that
both LOX and LOXL2 were expressed in dysplasia, dif-
ferentiated oral cancer and poorly differentiated oral
cancer. In dysplasia, LOXL2 and LOX were highly
expressed in the outer keratinized oral epithelium, with
some staining observed extending into the stratum spi-
nosum. LOXL2 was expressed by more cells than LOX
consistent with TCGA data in Table S1. In differentiated
oral cancer, LOX and LOXL2 were clearly expressed in
tumor nests, and in associated elongated cells with a
mesenchymal morphology. In poorly differentiated oral
cancer, LOX staining appeared to be mostly restricted to
the epithelium that exhibited light counterstaining con-
sistent with keratinocyte edema. By contrast,
LOXL2 staining occurred throughout the specimen in
both tumor cells and in surrounding stroma. Taken
together, TCGA and IHC studies of human squamous cell
tongue cancer support that LOXL2 is expressed at high
levels in poorly differentiated and well-differentiated oral
Mahjour et al. Oncogenesis (2019) 8:34 Page 4 of 17
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cancer by tumor cells and mesenchymal cells, and further
support the notion that LOXL2 likely contributes to the
etiology of metastatic disease. High abnormal expression
of LOX and LOXL2 was also observed in the epithelium
of dysplastic tissue, suggesting a possible role in dysplasia
that may ultimately be a factor in cancer development in
combination with mutations and environmental influ-
ences. Due to the highest expression of LOXL2 in tongue
oral cancer in the TCGA data, and the human histo-
pathology and IHC in Fig. 1, we next focused on the study
of expression and mechanisms by which secreted LOXL2
could promote oral cancer.
In vivo mouse studies
To define the effect of LOXL2 on oral cancer growth
and metastasis, effects of the small molecule LOXL2
inhibitor PXS-S1C were investigated in both immunode-
ficient and immunocompetent mouse models. PXS-S1C is
a novel small molecule active site-directed irreversible
inhibitor whose structure is shown in Fig. S1A, and was
provided to us by Pharmaxis Corporation, LLC, Australia.
The specificity of the inhibitor is shown in detail in Fig.
S1B from which Ic50s reported earlier were determined
and compared to other LOX family enzymes, and other
copper-dependent amine oxidases
12
. Irreversibility of
LOXL2 inhibition by PXS-S1C was established in Fig. S1C
in which the the Kitz–Wilson plot shows first-order
inactivation kinetics consistent with PXS-S1C acting as an
enzyme-activated irrevesible inhibitor
24
, with kinetic
parameters shown in Fig. S1D.
LOXL2 promotes tumor growth and oral cancer metastasis
in immunodeficient mice
HSC3 cells are human aggressive tongue-derived
OSCCs
19
, which were transduced to express RFP and
were injected into the tongues of nude mice followed by
injection of PXS-S1C (10 and 30 mg/kg) with a frequency
of three times a week for 3 weeks by intraperitoneal
injection. LOXL2 inhibition was found to attenuate tumor
growth in tongues as determined by caliper measure-
ments (Fig. 2a). Tracking cancer cells by IVIS after
3 weeks of injections of vehicle or PXS-S1C (30 mg/kg)
demonstrated that inhibition of LOXL2 by PXS-S1C sig-
nificantly decreased the distribution of cancer cells to
other tissues (Fig. 2b, c).
Histopathologic analysis
H&E-stained sections of the primary HSC3 tumors
showed features of differentiated infiltrating keratinizing
squamous cell carcinoma. The tumors contained sheets
and islands of atypical squamous epithelial cells infiltrat-
ing the connective tissue stroma and insinuating deep
between skeletal muscle fibers. HSC3 tumor cells were
infiltrative but demarcated given the lobular growth pat-
tern and cellular cohesion. Keratin pearl formation and
patchy dyskeratosis were appreciated. A mild pre-
dominantly acute inflammatory cell infiltrate occurred at
Fig. 1 Histology and immunohistochemistry of human dysplasia and oral cancer biopsies for LOX and LOXL2. Biopsy samples were selected
by the pathology service at Boston University Henry M. Goldman School of Dental Medicine and tissue sections were prepared and stained. Slides
made from one selected subject from 3 to 5 subjects sampled in each category of dysplasia, differentiated oral cancer, and poorly differentiated oral
cancer, respectively, are shown. Stained slides were imaged using an automated slide imager, and images were processed using Case Viewer
software version 2.2 (Budapest, Hungary). Data indicate that LOXL2 was highly expressed in a variety of cancer cells and associated mesenchymal
cells in human oral cancer, while LOX expression was more restricted
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Fig. 2 LOXL2 promotes human tongue orthotopic cancer growth and metastasis in mice. a PXS-S1C attenuates human tongue tumor growth
in mice, and band cPSX-S1C significantly decreases tumor cell spreading. Tumor volume of mouse tongues were measured every 3 days. Data are
means ± SD. ANOVA, p: 0.0001, Tukey’s multiple comparisons test, **p< 0.001, ***p< 0.0001 indicate difference among the groups (n=8 per group).
bIVIS imaging for red fluorescent protein-labeled HSC3 cells 21 days after commencing injections of vehicle or PXS-S1C (30 mg/kg). The fluorescence
signals were optimized for DsRed protein at excitation 570 nm and emission 620 nm. cQuantification of fluorescence signal area shows a significant
difference between PXS-S1C-treated and non-treated groups. Data are mean ± SD. Student’st-test, *p< 0.05 indicates difference between the groups.
dHistology of HSC3 cell orthotopic tongue tumors. Hematoxylin and eosin staining a mouse tongue is shown 18 days after implantation. The photo
is representative of histological features of HSC3 orthotopic tumors. The images were taken at ×4 and ×20 magnifications. Scale bar =100 µm. PXS-
S1C attenuates expression of (e) Ki67 and (f) LOXL2 in LY2 orthotopic tumors in immunodeficient mice. eImmunohistochemistry staining of tongue
sections with anti-Ki-67 antibody shows that PXS-S1C reduced Ki-67 staining in orthotopic HSC3 tumors in mice. Scale bar =100 µm. Data are mean
± SD. ANOVA, p< 0.01, Tukey’s multiple comparisons test, *p< 0.05 indicate difference among the groups (n=8 per group). fStaining of tongue
tissues with anti-LOXL2 antibody shows that PXS-S1C reduced LOXL2 staining in orthotopic HSC3 tumors in mice. Scale bar =100 µm. Data are
mean ± SD. ANOVA, p< 0.01, Tukey’s multiple comparisons test, *p< 0.05 indicate difference among the groups (n=8 per group)
Mahjour et al. Oncogenesis (2019) 8:34 Page 6 of 17
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the periphery of the tumor characterized by scattered
polymorphonuclear leukocytes. Focal perivascular and
perineural invasion were noted within tumors (Fig. 2d).
IHC staining of PXS-S1C-treated mice demonstrated
reduced Ki-67 staining (proliferation marker) compared
to no inhibitor-treated control mice (Fig. 2e) and lower
expression of LOXL2 in tongue tumor samples in treated
mice (Fig. 2f). The latter finding was unexpected, because
PXS-S1C is a mechanism-based inhibitor of LOXL2
activity, and was not designed to directly target LOXL2
transcription.
PXS-S1C inhibits oral cancer growth and metastasis
in immunocompetent mice
The effect of PXS-S1C on cancer growth and metastasis
in an immunocompetent mouse model was investigated
by injection of LY2 to the tongue of syngeneic immuno-
competent mice (Balb/c mice)
20
followed by intraper-
itoneal injections of the LOXL2 inhibitor (10 and
30 mg/kg) three times a week as above, and mice were
sacrificed after 6 weeks. Tongues exhibited lesions from
all mice that had been injected with LY2 cells as expected
(Fig. 3a). Interestingly, LOXL2 inhibitor treatment
reduced the frequency of lymph node enlargement com-
pared to the non-treatment group (Fig. 3a, b). The data
suggest that LOXL2 enzyme activity significantly pro-
motes oral cancer to cervical lymph node metastasis.
PXS-S1C lowers tumor LOXL2 levels and proliferating cancer
cells
H&E-stained sections of the primary LY2 tongue tumors
showed histopathologic features of poorly to moderately
differentiated infiltrating squamous cell carcinoma (Fig.
3c). The tumors revealed multifocal infiltrating islands of
pleomorphic squamous epithelial cells. Atypical mitotic
figures were readily identified. The tumor margins were
poorly demarcated. The tumor cells were discohesive and
haphazardly arranged and notable for single-cell invasion.
LY2 tumors demonstrated significant perineural and
perivascular invasion. The surrounding connective tissue
stroma exhibited a patchy acute and chronic inflammatory
infiltrate throughout (Fig. 3c). IHC staining of the tongue
tissues indicate that treatment of mice with LOXL2 inhi-
bitor PXS-S1C decreased levels of PCNA (Fig. 4a) and
LOXL2 (Fig. 4b) in LY2 tongue tumors.
Inhibition of LOXL2 activity dramatically changed the
morphological appearance of LY2 cancer cells in the
tongue and lymph nodes. Treatment with PXS-S1C
induced tumor cell changes consistent with transforma-
tion from a poorly differentiated to a well-differentiated
morphology characterized by a more cohesive tumor cell
arrangement at the border of the tumors in the tongues
(Fig. 4c). In lymph nodes, tumor cells were arranged as
sheets and islands of atypical squamous epithelial cells in
the inhibitor-treated mice compared to a haphazard
arrangement of tumor cells in untreated mice (Fig. 4c).
These findings suggest that active LOXL2 promotes epi-
thelial to mesenchymal transtition (EMT). To further
assess for EMT, IHC staining of tongue tissues with E-
cadherin and vimentin was carried out. IHC staining of
tongue tissues shows that PXS-S1C treatment increased
E-cadherin expression at the border of the LY2-derived
tongue tumors and lymph node metastases (Fig. 4d). In
contrast, PXS-S1C treatment reduced vimentin expres-
sion in the same tumors (Fig. 4e).
Increased collagen accumulation is a hallmark of tumors
pronetometastasize.InlightofthefunctionofLOXL2in
EMT and collagen synthesis, we investigated collagen
accumulation in LY2 tumors. Measurement of Sirius red
staining intensity in LY2 tumor and non-tumor areas
showed that collagen accumulation increased in LY2 tumors
in comparison with adjacent non-tumor regions (Fig. 5a).
PXS-S1C treatment appeared to reduce collagen accumu-
lation within and around LY2 tongue tumors (Fig. 5a).
In vitro studies in human cells
Cancer cell-derived LOXL2 stimulates oral fibroblast
proliferation and signaling
Fibroblasts are a major component of the cancer
microenvironment that contributes to cancer progression
and metastasis
25
. Due to high expression of LOXL2 in
tumor cells observed above, and the fact that LOXL2 is
principally a secreted enzyme, we next evaluated the
hypothesis that LOXL2 secreted from tumor cells in some
way directly targets surrounding fibroblasts. To assess for
effects of tumor cell-derived LOXL2 on oral fibroblasts,
the effect of human tumor cell-conditioned medium (CM)
on the proliferative response of HGF in the presence or
absence of the LOXL2 inhibitor PXS-S1C was next
investigated.
Fibroblasts are targets of LOXL2
Cultured primary human oral fibroblasts were treated
with non-CM, or cancer cell CM from the following oral
cancer cell lines: HSC3, CAL27, SCC71, SCC25, and
UMSCC2. PXS-S1C or vehicle was then added to CM to a
final concentration of 1 µM. After 24 h of treatment,
fibroblasts were subjected to CyQUANT DNA accumu-
lation assays. Data indicate that PXS-S1C-attenuated
human gingival fibroblast proliferative responses to all
oral cancer cell CM tested. Proliferation of the HSC3 cell
line was inhibited to the greatest degree by PXS-S1C (Fig.
5b). Mechanistic studies were next carried out with the
HSC3 cell line due to its known aggressive character
19
and
the strong inhibition of proliferation observed.
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LOXL2 mRNA and protein expression in oral cancer tumor
cell lines
LOXL2 was found to be the highest LOX expressed by
the HSC3 cell line (Fig. 5c). To assess the expression of
LOXL2 protein in HSC3 and LY2 invasive oral cancer cell
lines, and primary gingival fibroblasts, the cells were
serum depleted for 24 h. Then the CM of cancer cells and
gingival fibroblasts were collected and concentrated by
ultrafiltration and subjected to western blotting for
LOXL2. The data show that LOXL2 protein is secreted by
HSC3 cells, and that HSC3 cells appear to express higher
levels of LOXL2 than LY2 cells and HGF (Fig. 5d). LOXL2
was detected as multiple bands with different molecular
weight because it undergoes posttranslational modifica-
tions including N-glycosylation, and proteolytic proces-
sing of its SRCR domains
26–28
.
Tumor cell secreted LOXL2 promotes fibroblast proliferation
via PDGFR
To investigate the mechanism by which LOXL2 stimu-
lates fibroblast proliferation in response to HSC3 CM,
primary fibroblasts were serum depleted for 24 h and then
treated with HSC3 CM for 5 or 10 min, and whole cell
layer lysates were prepared. The effect on activation of
signaling kinases was determined employing the receptor
tyrosine kinase signaling array (RTK signaling array) indi-
cated in the section “Methods”. Data show the activation of
PDGFR, AKT, and ERK in human oral fibroblasts after
treatment with CM, while other RTKs assayed were not
activated under the conditions used (Fig. S2).
To investigate whether LOXL2 contributes to oral
fibroblast stimulation by tumor cells via PDGFRs, oral
fibroblasts were serum depleted for 24 h and then treated
Fig. 3 LOXL2 promotes syngeneic orthotopic tongue tumor growth and metastasis to cervical lymph nodes in immunocompetent mice. a
Gross features of tongues and lymph nodes of the mice in all groups. Circles mark grossly oversized lymph nodes. bNumber of the mice with
abnormal size of lymph nodes (left panel) was reduced by the treatment with PXS-S1C. Number of the mice with normal versus abnormal size of
lymph nodes. Normal sized LN in control mice =0.053 ± 0.01 cm
2
,*p< 0.05. Chi-Square test (4 × 2 analysis) p=0.008, Chi-Square test, p=0.008,
indicates difference in number of normal and abnormal sized LN among the groups. Average size of the lymph nodes in each group (right panel).
ANOVA, p< 0.05, Tukey’s multiple comparison test *p< 0.05 indicates difference between the groups. Fisher’s exact test (2 × 2 analysis). Control vs.
LY2, p=0.001, mice injected with LY2 have larger LNs than controls. Fisher’s exact test (2 × 2 analysis) LY2 vs. both LY2 +PXS-S1C 10 and 30 mg/kg
group ogether, p=0.03, LOX inhibitor reduces the frequency of mice having enlarged LNs. Fisher’s exact test (2 × 2 analysis), LY2 +PXS-S1C
10 mg/kg vs. LOXL2 +30 mg/kg, p> 0.05 There is no statistical difference between the two different doses of LOX inhibitor. cHistology of LY2
orthotopic tongue tumors. Hematoxylin and eosin staining of LY2 orthotopic tongue tumor. The images are representative of histological features of
LY2 tumor. The images were taken at ×4 and ×20 objectives. Scale bar =100 µm
Mahjour et al. Oncogenesis (2019) 8:34 Page 8 of 17
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for 24 h with non-CM, CM, CM with PXS-S1C (1 µM) or
CM with PDGFR inhibitor AG 1296 (5 µM). Following the
treatment, the fibroblast proliferative response (DNA
accumulation) was determined by CyQUANT assay. The
data show that both PDGFR inhibitor and LOXL2
inhibitor treatments decreased the CM-stimulated pro-
liferative response of oral fibroblasts (Fig. 6a). These
data suggested that LOXL2 participates in tumor cell
CM-induced stimulation of the proliferation of human oral
fibroblasts by interacting with fibroblast PDGF signaling.
Fig. 4 LOXL2 promotes proliferation and EMT of primary tongue and cervical lymph node metastases. PXS-S1C attenuates expression of (a)
PCNA and (b) LOXL2 in LY2 orthotopic tongue tumors in immunocompetent mice after 6 weeks of treatment, scale bar =100 µm. Data are mean ±
SD. ANOVA, p< 0.05, Tukey’s multiple comparisons test, *p< 0.05 indicate difference among the groups (n=12 per group). PSX-S1C treatment of LY2
tongue orthotopic tumors in mice alters tongue and cervical lymph node (c) tumor cell morphology, (d) E-cadherin expression and (e) vimentin
expression. The images were taken at ×10 and ×20 magnifications. Scale bar =100 µm. Images are from mice at the 6-week time point
Mahjour et al. Oncogenesis (2019) 8:34 Page 9 of 17
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Fig. 5 LOXL2 promotes collagen accumulation in syngeneic tongue oral cancer in mice, while human oral tumor cells-derived
LOXL2 stimulates oral fibroblast proliferation in vitro. a Collagen accumulation in orthotopic tongue LY2 tumors by Sirius red staining of LY2
tumors in the tongue. A representative image is shown from one of 12 mice at the 6-week time point. Scale bar =100 µm. Treatment with PXS-S1C
appeared to reduce the amount of collagen, particularly at the apparent interfaces of tumor with surrounding non-tumor tissue. The images were
taken at ×10 and ×20 magnifications. bStimulation of human gingival fibroblast proliferation induced by CM of different oral cancer cell lines was
inhibited by PXS-S1C treatment. Gingival fibroblasts were serum depleted for 24 h and then treated with cancer cell CM with and without PXS-S1C
(1 µM) in serum-free conditions for 24 h, and DNA accumulation measured by CyQUANT assays: [(HSC3 CM)–(HSC3 +PXS-S1C)]/HSC3 CM × 100].
Data are means ± SD. Experiments were done with six replicate samples for each cell line. ANOVA, p< 0.0001, Tukey’s multiple comparison test. **p<
0.001 indicate differences among different groups. cLOXL2 is the most abundantly expressed paralogue by HSC3 cells. RNAs isolated from serum-
depleted HSC3 cells was subjected to qPCR for all five lysyl oxidase paralogues using Taqman probes. Data are means ± SEM. This experiment was
performed three times independently with triplicate samples. The RNA levels were normalized to 18S rRNA. ANOVA One way, P< 0.01 among all LOX
family members. dLOXL2 protein is secreted at high levels by HSC3 cells. CM from human HSC3 cells, mouse LY2 cells and normal human gingival
fibroblasts were collected under serum-free conditions, concentrated by 25-fold, and subjected to western blotting and visualized with anti-LOXL2
antibody. β-tubulin was used as a loading control. Data are means ± SEM. This experiment was done three times independently. ANOVA, p< 0.002,
Tukey’s multiple comparison test *p< 0.05 indicate difference among the groups
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To assess whether inhibition of LOXL2 affects the
PDGFR activation in response to CM, gingival fibroblasts
were serum depleted and then treated for 5 or 10 min with
no-CM, CM, or CM with LOXL2 inhibitor (1 µM PXS-
S1C). Equal amounts of proteins were subjected to
western blotting for phosphorylated (p-) PDGFRβY771,
p-PDGFRβY857, p-PDGFRβY751, PDGFRβ, and βactin
(loading control). PXS-S1C attenuated phosphorylation of
PDGFRβY771 and βY857 in treated fibroblasts. However,
PDGFRβY751 phosphorylation was unexpectedly not
Fig. 6 (See legend on next page.)
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inhibited by PXS-S1C in response to HSC3 CM treatment
of oral fibroblasts (Fig. 6b).
PXS-S1C attenuates fibroblast proliferation stimulated
by cancer cell CM via ERK, but not AKT signaling
To further investigate the signaling pathway that med-
iates the decreased proliferation of HSC3 CM-treated oral
fibroblasts treated with PXS-S1C, downstream signaling
was assessed. Human serum-depleted oral fibroblasts
were treated for 15 or 30 min with non-CM, CM, or CM
with PXS-S1C (1 µM). The proteins were extracted and
subjected to western blotting for activated AKT and
ERK1/2. The blots showed that PXS-S1C surprisingly did
not attenuate the activation AKT but did inhibit ERK1/
2 signaling in fibroblasts treated with HSC3 CM (Fig. 6c).
In summary, data indicate that LOXL2 activity present in
HSC3-CM helps to promote activation of oral fibroblast
PDGFR at Y771 and Y857 phosphorylation sites but not
Y751 in PDGFR in response to CM treatment. Moreover,
the LOXL2 activity helps to promote CM-stimulated
fibroblast ERK1/2 phosphorylation, but not AKT
phosphorylation.
PDGF-AB but not PDGF-BB mimics the effect of HSC3 cancer
cell CM on oral fibroblasts
PDGF signaling is best known to activate AKT and not
ERK1/2 in the context of cancer. We next considered the
hypothesis that a novel PDGF ligand secreted by HSC3
cells may be responsible for the observed ERK1/2 acti-
vation in oral fibroblasts.
To evaluate the HSC3-secreted PDGF ligands that drive
oral fibroblast PDGFR activation and with which LOXL2
may collaborate, the phosphorylation of PDGFRβ
stimulated by different PDGF ligands with and without
the LOXL2 inhibitor PXS-S1C was assessed. Serum-
depleted human oral fibroblasts were treated with no
PDGF, PDGF-BB, PDGF-CC, and PDGF-AB with vehicle
or PXS-S1C (1 µM) and evaluated for activation of
PDGFR, ERK 1/2, and AKT as above. Data indicate that
PXS-S1C attenuated phosphorylation of all PDGFRβ
phosphorylation sites including PDGFRβY751, and AKT
in activating phosphorylations assayed in response to
PDGF-BB (Fig. 6d), while PXS-S1C did not attenuate
phosphorylation of PDGFRβin response to PDGF-CC
(Fig. S3). Interestingly, in response to HSC3 CM treat-
ment of oral fibroblasts, PXS-S1C attenuated phosphor-
ylation of PDGFRβY771 and βY857 but not PDGFRβ
Y751 (Fig. 6e). PXS-S1C attenuated ERK1/2-related
PDGF signaling, while AKT was not affected by PXS-S1C
(Fig. 7a). Thus, data show that PDGF-AB, but not PDGF-
BB or PDGF-CC, precisely mimics the effects of HSC3
CM on gingival fibroblasts.
PDGF-A or PDGF-B knockdown in HSC3 cells inhibits
proliferation of oral fibroblasts induced by HSC3 CM
To confirm independently that PDGF-AB is the ligand
secreted by HSC3 cells that stimulates oral fibroblast
proliferation in collaboration with LOXL2 activity, shRNA
lentiviral particles were used to knock down PDGF-A or
PDGF-B in HSC3 cells. CM from knock-down cells were
then assayed for PDGF-AB levels by ELISA, and the same
media samples were assayed for the ability to stimulate
proliferation of oral fibroblasts. The concentration of
PDGF-AB ligand in knockdown and control HSC3 CM
was next measured using a PDGF-AB ELISA which spe-
cifically recognizes the PDGF-AB dimer and not PDGF-
(see figure on previous page)
Fig. 6 HSC3 oral tumor cell-derived LOXL2 stimulates oral fibroblast proliferation and cell signaling in collaboration with PDGF-AB. LOXL2
inhibitor PXS-S1C attenuates HSC3 CM-stimulated human oral fibroblast (a) proliferation, (b) phosphorylation of PDGFRβat the Y771 and Y857 but not
Y751 residues, and (c) ERK activation, but not AKT. aHuman gingival fibroblast proliferation was reduced after 24-h treatment with PXS-S1C (1 µM) or
AG 1296 (5 µM) in the HSC3 CM as determined by the CyQUANT assay. Data are means SEM. This experiment was done three times independently
with six replicate samples. ANOVA, p< 0.0001, Dunnett’s multiple comparisons test, ***p< 0.0001 indicates significant difference between treated
groups; while
##
p< 0.001,
###
p< 0.0001 indicate significant differences from non-CM group. band cGingival fibroblasts were serum depleted and then
treated with non-CM, and CM with and without PXS- S1C (1 µM) and cell layer protein samples were subjected to western blot. Data are means ± SEM.
The experiment was performed with three times independently with primary human gingival fibroblasts isolated from three different donors.
Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, *p< 0.05
indicates a significant difference from PXS-S1C treated group. Dunnett’s multiple comparison test, #p< 0.05,
##
p< 0.001 indicate significant differences
from Non-CM group. dPXS-S1C attenuates PDGF-BB stimulated phosphorylation of all three PDGFRβphosphorylation sites Y771, Y857 and Y751, and
AKT activation in oral fibroblasts. Gingival fibroblasts were serum depleted and then treated with no PDGF-BB, and PDGF-BB (10 ng/ml) with and
without PXS-S1C (1µM). The protein samples were subjected to western blot. Data are means ± SEM. The experiment was done with three times
independently with primary human gingival fibroblasts isolated from three different donors. Representative blots are shown. Data from all three
experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, *p< 0.05, **p< 0.001, and ***p< 0.0001 indicate difference from
PXS-S1C-treated group. Dunnett’s multiple comparison test,
#
p< 0.05,
##
p< 0.001,
###
p< 0.0001 indicate difference from No PDGF group. ePDGF-AB
mimics the effects of HSC3 cell CM on oral fibroblasts in phosphorylation of PDGFRβ. Gingival fibroblasts were serum starved and then treated with no
PDGF-AB, and PDGF-AB (10 ng/ml) with and without PXS-S1C (1 µM). The protein samples were subjected to western blot. Data are means ± SEM. The
experiment was done with three times independently with primary human gingival fibroblasts isolated from three different donors. Representative
blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidak’s multiple comparison test, *p< 0.05 indicates
difference from PXS-S1C-treated group. Dunnett’s multiple comparison test,
#
p< 0.05 indicates difference from No PDGF group
Mahjour et al. Oncogenesis (2019) 8:34 Page 12 of 17
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AA or PDGF-BB. Data indicated that the concentration of
PDGF-AB ligand was decreased significantly in the
knocked-down HSC3 medium (Fig. 7b). Serum-depleted
primary human oral fibroblasts were then treated with
aliquots of the same CM of knock-down or control cells
for 24 h and finally subjected to CyQUANT assay to
assess proliferative responses to CM from knocked-down
tumor cells. The result shows that fibroblast proliferation
was significantly lower after PDGF ligand knockdowns in
HSC3 cells in comparison with CM from HSC3 cells
transduced with non-target shRNA control particles
(HSC3 control) (Fig. 7c). To investigate whether the level
of PDGF-A or PDGF-B knock down in HSC3 cells cor-
relates with the proliferative response in fibroblasts
Fig. 7 (See legend on next page.)
Mahjour et al. Oncogenesis (2019) 8:34 Page 13 of 17
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treated with HSC3 CM, the relationship between the
gingival fibroblast proliferation inhibition derived from
Fig. 7b and the relative level of PDGF-AB concentration
found in in Fig. 7c was analyzed by linear regression. The
data indicate that the stronger knockdowns correlate well
with lower proliferative responses to CMs (Fig. 7d). Taken
together, data indicate that PDGF-AB is the major factor
derived from HSC3 cells that drives oral fibroblast pro-
liferation in collaboration with LOXL2. Interestingly, the
DNA synthesis of knock-down HSC3 cells themselves
showed no significant difference in cell proliferation
compared to the no knock-down HSC3 control group
(Fig. S4). Thus, PDGF-AB signaling did not affect pro-
liferation of HSC3 cells, and PDGF-AB targets only
fibroblasts in the microenvironment.
LOXL2 in CM oxidizes PDGFRβin oral fibroblasts
We next considered the hypothesis that LOXL2 could
optimize PDGF receptor signaling in response to PDGF-
AB by oxidizing exposed lysine residues of the PDGFRβ
receptor protein, possibly similar to that found for the
LOX paralogue (not LOXL2) on smooth muscle cells and
megakaryocytes
10,11
. We therefore developed an assay to
identify LOXL2-dependent generation of aldehydes on
PDGFRβin oral fibroblasts treated with HSC3-CM. Oral
fibroblasts were serum depleted and then treated with
HSC3 CM with or without PXS-S1C (1 µM) for 24 h. Cell
layer proteins were extracted and the aldehyde-containing
proteins were affinity labeled with biotin hydrazide.
Proteins were then subjected to a pulldown assay with an
avidin-coupled affinity resin (Neutravidin), followed by
SDS–PAGE and western blotting for PDGFRβ. Blots of
samples before and after purification were subjected to
western blot and visualized with anti-PDGFRβantibody to
assess for PDGFRβ-aldehydes in response to CM treat-
ment. Western blots of the samples probed with anti-
PDGFRβantibody showed that LOXL2 inhibitor PXS-
S1C decreased the oxidation of PDGFR compared to
control samples lacking the LOXL2 inhibitor (Fig. 7e). We
conclude that PDGFRβis a substrate for LOXL2.
PXS-S1C effects on HSC3 cell growth and LOXL2 expression
We considered the notion that secreted LOXL2 could
stimulate the proliferation of tumor cells in addition to
neighboring stromal cells. To determine the effect of
inhibition of LOXs and specifically LOXL2 on tumor cells,
the serum-stimulated proliferative response of HSC3 cells
in the presence or absence of PXS-S1C or BAPN, which
inhibits all LOX paralogues including LOXL2, was
investigated. HSC3 cells were serum-depleted overnight
and treated with PXS-S1C (1 µM) or BAPN (0.5 mM) in
medium containing 2.5% serum for serum stimulation of a
proliferative response. After 24 h of treatment, the change
in DNA accumulation was determined as a measure of the
proliferative response by CyQUANT assay. The data
indicate that neither PXS-S1C nor BAPN attenuated
HSC3 cell growth in vitro (Fig. 7f). HSC3 cells grown in
the presence of 2.5% serum in the presence or absence of
(see figure on previous page)
Fig. 7 PDGF-AB, but not PDGF-BB, is secreted by HSC3 tumor cells, PDGFRβis a substrate for LOXL2, and LOXL2 promotes its own
synthesis in HSC3 cells. a PDGF-AB mimics the effects of HSC3 cancer cell CM on oral fibroblasts in phosphorylation of ERK1/2, and not AKT. Oral
fibroblasts were serum depleted and then treated with no PDGF-AB, and PDGF-AB (10 ng/ml) with and without PXS-S1C (1µM). The protein samples
were subjected to western blot. Data are means ± SEM. The experiment was done with three times independently with primary human gingival
fibroblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative
analyses. Sidak’s multiple comparison test, *p< 0.05 indicates difference from PXS-S1C-treated group. Dunnett’s multiple comparison test,
#
p< 0.05 and
###
p< 0.0001 indicate difference from No PDGF group. bPDGF-A and PDGF-B knockdown in HSC3 cells blocks HSC3 CM stimulation of oral fibroblast
proliferation. The concentration of PDGF-AB ligand was decreased significantly in CM of knockdown HSC3 cells as compared with non-target control
cell CM. PDGF-A and PDGF-B were, respectively, knocked down with independent shRNAs (A1, A2, or A3 for PDGF-A; and B1 and B2 for PDGF’B). The
concentration of PDGF-AB ligand in knocked down HSC3 CM was measured using a PDGF-AB-specific ELISA kit. Data are mean± SD. This experiment
was done with triplicate samples. ANOVA, p< 0.0001, Dunnett’s multiple comparisons test
#
p< 0.05,
##
p< 0.001,
###
p< 0.0001 indicate difference from
Control HSC3 group. cThe proliferation of oral fibroblasts treated with knocked down HSC3 medium for 24 h was assessed by CyQUANT assay. Data are
mean ± SEM. This experiment was performed three times independently with primary human gingival fibroblasts isolated from three different donors.
ANOVA, p: 0.0001, Dunnett’s multiple comparisons test:
#
p< 0.05, indicate difference from HSC3 Control group. dThe degree of PDGF-A or PDGF-B
knockdown correlate linearly with decreased proliferative responses to HSC3 CM. The relationship between fibroblast proliferation inhibition and the
relative level of PDGF-AB concentration was analyzed using linear regression. Correlation coefficient r:−0.93, Rsquared: 0.87, p-value: 0.006. Data
indicate that PDGF-AB specifically is the ligand in HSC3 CM that stimulates oral fibroblast proliferation. eCarbonyl pull down assay for PDGFRβin oral
fibroblasts treated with HSC3 CM in the absence or presence of PXS-S1C. Human oral fibroblasts were treated with HSC3 CM in the absence or
presence of 1 µM PXS-S1C followed by biotin hydrazide derivitization and affinity pulldown with a streptavidin affinity resin (Neutravidin). Input samples
and proteins eluted by boiling in SDS–PAGE were subjected to Western blotting for PDGFRβ. Data are representative of two experiments with the same
outcome from two different gingival fibroblast donors. fPXS-S1C and BAPN did not inhibit serum-stimulated proliferative response of HSC3 tumor cells.
HSC3 cells were serum-depleted overnight and treated with PXS-S1C (1 µM) or BAPN (0.5 mM) in medium containing 2.5% serum for serum stimulation
of a proliferative response. Data are means± SEM. ANOVA, p< 0.0001, Tukey’s multiple comparisons *p< 0.05 indicates difference among the groups. g
PXS-S1C decreased the expression of LOXL2 in HSC3 cells in vitro. Relative LOXL2 mRNA levels in HSC3 cell line with and without PXS-S1C after 24h
treatment was measured. Data are means ± SEM. This experiment was done three times independently with triplicate samples. ANOVA, p: 0.04, Sidak’s
multiple comparisons test *p< 0.05 indicates difference from non-treated HSC3 group. The RNA levels were normalized to 18S rRNA
Mahjour et al. Oncogenesis (2019) 8:34 Page 14 of 17
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PXS-S1C were next assessed for relative RNA levels of all
five LOX paralogues. Interestingly, data indicate that PXS-
S1C decreased the expression specifically of LOXL2 and
no other paralogue in HSC3 cells in vitro (Fig. 7g). Data
suggest that LOXL2 activity regulates its own expression
in HSC3 tumors cells by a feed-forward autocrine
pathway.
Discussion
OSCC is one of the most common cancers in the world,
with poor survival and a high recurrence rate
1,29
. Devel-
opment of OSCC is typically evolves at a low frequency
from hyperplasia and dysplasia to carcinoma in situ and
finally to invasive metastatic OSCC. Oral cancer can also
develop independent of this typical progression
30,31
.In
either case, the development of OSCC is accompanied by
changes in intercellular signaling between tumor cells and
non-tumor cells, such as fibroblasts in the tumor micro-
environment leading to dysregulation of gene expression
and protein products facilitating cancer progression and
metastasis
8,32,33
. Understanding of the molecular
mechanisms underlying OSCC progression is essential to
afford the opportunity to develop novel strategies to
suppress tumor progression. The findings of previous
studies have shown that LOXL2 is highly expressed in a
variety of cancers in humans including head and neck
squamous cell carcinoma, breast, colon, skin, and gastric
cancer, and its high expression correlates with metastasis
and poor prognosis
23,34–37
. Studies on overexpression of
LOXL2 have not precisely indicated underlying mechan-
isms by which LOXL2 contributes to OSCC progression
and metastasis. The direct targets of LOXL2 in cancer
remain unclear. Therefore, we sought to investigate
mechanisms by which LOXL2 induces progression and
invasiveness in OSCC, and propose that LOXL2 is a
potential target for OSCC therapy.
Immunocompromised and immunocompetent mouse
models of oral cancer employed here both demonstrated
significantly elevated LOXL2 levels, consistent with data
in humans noted above and from Fig. 1and the TCGA
resource (Table S1). The application of a novel and highly
selective small molecule inhibitor of LOXL2-attenuated
tumor growth and metastatic spread to a significant
degree in both mouse models. Although complete inhi-
bition of cancer growth was not accomplished, the pos-
sibility is raised that LOXL2 inhibitors in combination
with other therapeutic approaches including immu-
notherapy could result in potentially effective strategies to
address oral cancer. The LY2 cells in the immuno-
competent model appeared morphologically to feature
characteristics of aggressive poorly differentiated cells,
and appear to undergo what resembles a reversal of EMT
in response to the LOXL2 inhibitor. This notion is sup-
ported by the increased levels of E-cadherin and lower
levels of vimentin staining in both tongue tumors and
lymph nodes observed in PXS-S1C-treated mice, and
further supports the idea that LOXL2 significantly con-
tributes to oral cancer development and that LOXL2
inhibitors may be of benefit in addressing oral cancer.
LOXL2 promotion of EMT has been reported in several
cancer models previously
14,35,38–43
, and the use of the
novel selective LOXL2 inhibitor employed here resulting
in apparent MET further supports the role of LOXL2 in
cancer progression.
There is now a considerable body of literature indicating
that elevated LOXL2 in particular is associated with a
variety of cancers, including oral cancer. As reviewed
previously by us and others
44–50
, proposed mechanisms
range from enzymatic and non-enzymatic nuclear activities
that promote EMT, to extracellular enzyme-mediated
indirect or direct activation of FAK and proliferative
responses in tumor cells. Molecular details of these rela-
tionships are still largely under investigation by a variety of
research teams. Here, we entertained the notion that tumor
cell-secreted LOXL2 may target non-tumor mesenchymal
cells to stimulate proliferation. The resulting abundant
fibrogenic cells, which produce collagens and LOXs and
MMPs, would then ultimately modify the surrounding
microenvironment by contributing to high collagen
synthesis and cross-linking to promote an environment
conducive to either cancer cell growth and/or metastasis.
Our studies, led us step by step, to the finding that oral
tumor cells secreted LOXL2 in combination with PDGF-
AB, enhance ERK signaling and oral fibroblast prolifera-
tion. The mechanism of action of LOXL2 that enhances
PDGF receptor sensitivity to PBGF-AB appears to consist
of direct oxidation of lysine residues on PDGFRβ, similar to
what occurs in normal vascular smooth muscle cells in
response to the LOX paralogue
51
. The identity of the lysine
residues in PDGFRβthat are oxidized remain to be
determined and is under investigation.
PDGF signaling as a driver of oral cancer
PDGF signaling has been reported to be a driver of EMT
in normal development and in a variety of cancers
52–58
.In
oral cancer in particular, elevated levels of PDGFRβhave
been identified in associated stromal cells
59
, consistent
with our hypothesis that PDGF ligands emanating from
tumor cells have biological activity in the tumor micro-
environment. Moreover, PDGF ligands drive metastatic
oral cancer cell line migration mediated by PDGFRβin
tumor cells
58
. It was previously reported that optimization
of PDGF signaling in vascular smooth muscle cells by
LOX-dependent oxidation of PDGFRβaccompanied
enhanced smooth muscle cell chemotaxis and migration.
Note that LOX is not LOXL2. Thus, we show here for the
first time that LOX and LOXL2 appear to share the ability
to oxidize and optimize the function of PDGFRβ.
Mahjour et al. Oncogenesis (2019) 8:34 Page 15 of 17
Oncogenesis
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Our studies suggest that the outcome of PDGF signaling
differs between oral tumor cells and oral fibroblasts. Sti-
mulation of proliferation of fibroblasts, and stimulation of
tumor cell EMT appear to be the respective outcomes.
This conclusion is based on our extensive data shown
above that LOXL2-stimulated proliferation of oral fibro-
blasts depends on LOXL2 activity and PDGF-AB activa-
tion of ERK. By contrast, in vivo modulation of tumor cell
morphology, E-cadherin and vimentin levels, and the lack
of in vitro inhibition of tumor cell proliferation by the
LOXL2 inhibitor, point to a possible EMT function rather
than proliferative stimulation of tumor cell growth. As
noted, PDGFRβin oral tumor cells has been shown to
mediate migration, which can be a consequence of the
EMT process
58
. An additional complexity is the apparent
feed forward requirement of tumor cells for LOXL2
activity to maintain LOXL2 expression (Fig. 7g) that may
be independent of PDGF signaling.
Figure 8provides a summary of the new understanding
of an extracellular role for LOXL2 in promoting oral
cancer. In summary, we show that tumor cell LOXL2
targets proximal mesenchymal fibrogenic cells by a novel
microenvironment tumor-promoting mechanism, while
small molecule LOXL2 inhibition can improve oral cancer
outcomes. Moreover, this study provides the first mole-
cular mechanism for enzymatically active LOXL2 in the
promotion of cancer via its modification of a non-
collagenous substrate in the context of paracrine signal-
ing between tumor cells and resident fibroblasts. The
successful use of a selective LOXL2 pharmacologic inhi-
bitor to address oral cancer in two in vivo mouse models
provides pre-clinical evidence that supports the notion
that similar inhibitors may have therapeutic potential
against oral cancer.
Acknowledgements
We thank Dr. Stefano Monti, Boston University, for help analyzing the TCGA
data set. We thank Wolfgang Jarolimek of Pharmaxis Corp., LLC, Frenchs Forest,
NSW Australia for the generous gift of the LOXL2 inhibitor PXS-S1C, and for
respective supporting data. LY2 cells were kindly provided by Dr. Nadarajah
Vigneswaran and Dr. Wolfgang Zacharias, University of Texas Health Science
Center, Dental Branch, Houston, TX. This research was supported by funding
from Pharmaxis Ltd., Frenchs Forest, NSW, Australia, and partially supported by
the Evans Center for Interdisciplinary Biomedical Research ARC on the
“Etiology and Pathogenesis of Oral Cancer”at Boston University to P.C.T., by a
Fellowship from Boston University School of Dental Medicine to F.M., and by
NIH/NIDCR R21 DE023973 to P.C.T. The Boston University Medical Campus IVIS
Imaging Core was funded by NIH-NCRR, S10RR024523.
Authors’contributions
F.M. carried out experiments, analyzed data, and wrote the draft of the
manuscript. V.D. carried out immunohistochemistry experiments and analyzed
data. N.S. carried out experiments and analyzed data. V.S. carried out some
experiments. V.N. reviewed human histopathology slides and analyzed data. A.
K. analyzed data and edited the manuscript. P.C.T. conceived of the project,
supervised all aspects, and edited the manuscript. All authors read and
approved the manuscript.
Author details
1
Boston University Henry M. Goldman School of Dental Medicine, Department
of Molecular and Cell Biology, Boston, MA 02118, USA.
2
Boston University
Henry M. Goldman School of Dental Medicine, Division of Oral & Maxillofacial
Pathology, Boston, MA 02118, USA.
3
Forsyth Institute, Cambridge, MA 02142,
USA
Conflict of interest
P.C.T. has served as a consultant for Pharmaxis Corporation, and Pharmaxis
partially funded this work. The remaining authors declare that they have no
conflict of interest.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41389-019-0144-0).
Received: 6 March 2019 Revised: 4 April 2019 Accepted: 23 April 2019
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