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

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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.
LOXL2 promotes human tongue orthotopic cancer growth and metastasis in mice. a PXS-S1C attenuates human tongue tumor growth in mice, and b and c PSX-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). b IVIS 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. c Quantification of fluorescence signal area shows a significant difference between PXS-S1C-treated and non-treated groups. Data are mean ± SD. Student's t-test, *p < 0.05 indicates difference between the groups. d Histology 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. e Immunohistochemistry 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). f Staining 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)
… 
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. b Number 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. c Histology 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
… 
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. b Stimulation 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. c LOXL2 is the most abundantly expressed paralogue by HSC3 cells. RNAs isolated from serumdepleted 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. d LOXL2 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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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 LOXL1LOXL4) 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 identied the rst molecular
mechanism for LOXL2 in the promotion of cancer via its enzymatic modication of a non-collagenous substrate in the
context of paracrine signaling between tumor cells and resident broblasts. 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 immunodecient, 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 broblasts 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 identied PDGFRβas a direct substrate
for LOXL2, and indicated that LOXL2 and PDGF-AB together secreted by tumor cells optimally activated PDGFRβin
broblasts to promote proliferation and the tendency toward brosis via ERK activation, but not AKT. Optimal
broblast 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 2030% of patients after treat-
ment
5,6
. These cancers have a poor 5-year survival rate,
and cause signicant 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 articles Creative Commons license, unless indicated other wise in a credit line to the material. If
material is not included in the articles 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 modication of the
extracellular matrix that can stimulate invasion and
metastasis
8
. The LOX family consists of ve members: LOX
andthefourrelatedgenesLOX-like-14(LOXL1LOXL4).
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, brosis, 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
1013
,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 epithelialmesenchymal
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
difculties 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
broblast proliferation and activity by enhancing PDGF-
AB-mediated signaling after modication of PDGFRβ,while
having no proliferative effect on tumor cells themselves.
These interactions dene a novel interaction that occurs in
the microenvironment emanating from tumor cells and that
targets stromal mesenchymal cells. These ndings provide a
mechanistic basis for potential novel therapeutic approa-
ches to address tumor growth, brosis, and metastasis.
Methods
Histopathology of LOXL2 and LOX expression in human
oral cancer biopsies
Tissue blocks from three to ve 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 immunodecient 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 proling 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 uorescent 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-
urane). 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 isourane 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-
urane), 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 rst 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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
PSX-S1C was administered by intraperitoneal injection
1224 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 xed in 4% paraf-
ormaldehyde, parafn-embedded and were sectioned
(6 µm). Tongues were xed 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 broblasts (HGF) and cancer cells were
grown separately at 37 °C and 5% CO
2
in a humidied
incubator. The HGF were taken from three healthy
donors aged 2535 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 broblasts was
Dulbeccos modied Eagles medium (DMEM, high glu-
cose 11965-092, Thermo Fisher Scientic), 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 conuence.
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 broblasts 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), uorescence 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 manufacturers instructions. cDNA was made by using
High-Capacity cDNA Reverse Transcription Kit (4368814,
Thermo Fisher Scientic). The cDNA concentration was
measured by NanoDrop spectrophotometer and then sub-
jected to qPCR using TaqMan Universal PCR Master Mix
(4304437, Thermo Fisher Scientic) 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
broblasts, 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 lter devices (Z717185, Millipore Sigma) and
protein concentrations measured with Nano Orange
Protein Quantication 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 SDSPAGE sample
buffer. Protein level of the cell lysates was measured with
the Nano Orange Protein Quantication kit. Then they
were subjected to western blotting. Primary antibodies
Mahjour et al. Oncogenesis (2019) 8:34 Page 3 of 17
Oncogenesis
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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 Scientic) or SuperSignalWest Femto Max-
imum Sensitivity Substrate (34095, Thermo Fisher Sci-
entic) 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 Scientic) 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
humidied incubator with 5% CO
2
. The HSC3 cells were
transduced at 7080% visual conuence overnight. The
medium was replaced with fresh medium and puromycin
(nal concentration of 2 µg/ml) was added the following
day. Drug-resistant cells were expanded to become
conuent.
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 broblasts 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 (NHNH
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 afnity resin
(Neutravidin, Pierce Scientic). Blots of samples before
and after purication 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 ve LOX paralogues, LOX and
LOXL1LOXL4. Data in Table S1 demonstrate that in
oral cancer tissues LOXL2 was by far the most sig-
nicantly upregulated LOX paralogue (9.8-fold), while
other paralogues were modestly upregulated (1.62.3-
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
Oncogenesis
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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 inu-
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 dene the effect of LOXL2 on oral cancer growth
and metastasis, effects of the small molecule LOXL2
inhibitor PXS-S1C were investigated in both immunode-
cient 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 specicity 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 KitzWilson plot shows rst-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 immunodecient 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-
nicantly 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 inltrating keratinizing
squamous cell carcinoma. The tumors contained sheets
and islands of atypical squamous epithelial cells inltrat-
ing the connective tissue stroma and insinuating deep
between skeletal muscle bers. HSC3 tumor cells were
inltrative but demarcated given the lobular growth pat-
tern and cellular cohesion. Keratin pearl formation and
patchy dyskeratosis were appreciated. A mild pre-
dominantly acute inammatory cell inltrate 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 signicantly decreases tumor cell spreading. Tumor volume of mouse tongues were measured every 3 days. Data are
means ± SD. ANOVA, p: 0.0001, Tukeys multiple comparisons test, **p< 0.001, ***p< 0.0001 indicate difference among the groups (n=8 per group).
bIVIS imaging for red uorescent protein-labeled HSC3 cells 21 days after commencing injections of vehicle or PXS-S1C (30 mg/kg). The uorescence
signals were optimized for DsRed protein at excitation 570 nm and emission 620 nm. cQuantication of uorescence signal area shows a signicant
difference between PXS-S1C-treated and non-treated groups. Data are mean ± SD. Studentst-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 magnications. Scale bar =100 µm. PXS-
S1C attenuates expression of (e) Ki67 and (f) LOXL2 in LY2 orthotopic tumors in immunodecient 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, Tukeys 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, Tukeys 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 nding 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
sacriced 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 signicantly 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 inltrating squamous cell carcinoma (Fig.
3c). The tumors revealed multifocal inltrating islands of
pleomorphic squamous epithelial cells. Atypical mitotic
gures were readily identied. The tumor margins were
poorly demarcated. The tumor cells were discohesive and
haphazardly arranged and notable for single-cell invasion.
LY2 tumors demonstrated signicant perineural and
perivascular invasion. The surrounding connective tissue
stroma exhibited a patchy acute and chronic inammatory
inltrate 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 ndings 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 broblast
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 broblasts. To assess for
effects of tumor cell-derived LOXL2 on oral broblasts,
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 broblasts 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
nal concentration of 1 µM. After 24 h of treatment,
broblasts were subjected to CyQUANT DNA accumu-
lation assays. Data indicate that PXS-S1C-attenuated
human gingival broblast 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 broblasts, the cells were
serum depleted for 24 h. Then the CM of cancer cells and
gingival broblasts were collected and concentrated by
ultraltration 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 modica-
tions including N-glycosylation, and proteolytic proces-
sing of its SRCR domains
2628
.
Tumor cell secreted LOXL2 promotes broblast proliferation
via PDGFR
To investigate the mechanism by which LOXL2 stimu-
lates broblast proliferation in response to HSC3 CM,
primary broblasts 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 broblasts after
treatment with CM, while other RTKs assayed were not
activated under the conditions used (Fig. S2).
To investigate whether LOXL2 contributes to oral
broblast stimulation by tumor cells via PDGFRs, oral
broblasts 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, Tukeys multiple comparison test *p< 0.05 indicates difference between the groups. Fishers exact test (2 × 2 analysis). Control vs.
LY2, p=0.001, mice injected with LY2 have larger LNs than controls. Fishers 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. Fishers 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
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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 broblast 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 broblasts (Fig. 6a). These
data suggested that LOXL2 participates in tumor cell
CM-induced stimulation of the proliferation of human oral
broblasts by interacting with broblast 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, Tukeys 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 magnications. 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 broblast 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 magnications. bStimulation of human gingival broblast proliferation induced by CM of different oral cancer cell lines was
inhibited by PXS-S1C treatment. Gingival broblasts 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, Tukeys 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 ve 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
broblasts 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,
Tukeys 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 broblasts
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 broblasts. 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 broblasts (Fig. 6b).
PXS-S1C attenuates broblast 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
broblasts treated with PXS-S1C, downstream signaling
was assessed. Human serum-depleted oral broblasts
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 broblasts treated with HSC3 CM (Fig. 6c).
In summary, data indicate that LOXL2 activity present in
HSC3-CM helps to promote activation of oral broblast
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
broblast ERK1/2 phosphorylation, but not AKT
phosphorylation.
PDGF-AB but not PDGF-BB mimics the effect of HSC3 cancer
cell CM on oral broblasts
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 broblasts.
To evaluate the HSC3-secreted PDGF ligands that drive
oral broblast 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 broblasts 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 broblasts, 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 broblasts.
PDGF-A or PDGF-B knockdown in HSC3 cells inhibits
proliferation of oral broblasts induced by HSC3 CM
To conrm independently that PDGF-AB is the ligand
secreted by HSC3 cells that stimulates oral broblast
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 broblasts. The concentration of
PDGF-AB ligand in knockdown and control HSC3 CM
was next measured using a PDGF-AB ELISA which spe-
cically recognizes the PDGF-AB dimer and not PDGF-
(see gure on previous page)
Fig. 6 HSC3 oral tumor cell-derived LOXL2 stimulates oral broblast proliferation and cell signaling in collaboration with PDGF-AB. LOXL2
inhibitor PXS-S1C attenuates HSC3 CM-stimulated human oral broblast (a) proliferation, (b) phosphorylation of PDGFRβat the Y771 and Y857 but not
Y751 residues, and (c) ERK activation, but not AKT. aHuman gingival broblast 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, Dunnetts multiple comparisons test, ***p< 0.0001 indicates signicant difference between treated
groups; while
##
p< 0.001,
###
p< 0.0001 indicate signicant differences from non-CM group. band cGingival broblasts 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 broblasts isolated from three different donors.
Representative blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidaks multiple comparison test, *p< 0.05
indicates a signicant difference from PXS-S1C treated group. Dunnetts multiple comparison test, #p< 0.05,
##
p< 0.001 indicate signicant 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 broblasts. Gingival broblasts 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 broblasts isolated from three different donors. Representative blots are shown. Data from all three
experiments were subjected to quantitative analyses. Sidaks multiple comparison test, *p< 0.05, **p< 0.001, and ***p< 0.0001 indicate difference from
PXS-S1C-treated group. Dunnetts 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 broblasts in phosphorylation of PDGFRβ. Gingival broblasts 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 broblasts isolated from three different donors. Representative
blots are shown. Data from all three experiments were subjected to quantitative analyses. Sidaks multiple comparison test, *p< 0.05 indicates
difference from PXS-S1C-treated group. Dunnetts multiple comparison test,
#
p< 0.05 indicates difference from No PDGF group
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AA or PDGF-BB. Data indicated that the concentration of
PDGF-AB ligand was decreased signicantly in the
knocked-down HSC3 medium (Fig. 7b). Serum-depleted
primary human oral broblasts were then treated with
aliquots of the same CM of knock-down or control cells
for 24 h and nally subjected to CyQUANT assay to
assess proliferative responses to CM from knocked-down
tumor cells. The result shows that broblast proliferation
was signicantly 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 broblasts
Fig. 7 (See legend on next page.)
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treated with HSC3 CM, the relationship between the
gingival broblast 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 broblast pro-
liferation in collaboration with LOXL2. Interestingly, the
DNA synthesis of knock-down HSC3 cells themselves
showed no signicant 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
broblasts in the microenvironment.
LOXL2 in CM oxidizes PDGFRβin oral broblasts
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 broblasts treated with HSC3-CM. Oral
broblasts 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 afnity labeled with biotin hydrazide.
Proteins were then subjected to a pulldown assay with an
avidin-coupled afnity resin (Neutravidin), followed by
SDSPAGE and western blotting for PDGFRβ. Blots of
samples before and after purication 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 specically 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 gure 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 broblasts in phosphorylation of ERK1/2, and not AKT. Oral
broblasts 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
broblasts isolated from three different donors. Representative blots are shown. Data from all three experiments were subjected to quantitative
analyses. Sidaks multiple comparison test, *p< 0.05 indicates difference from PXS-S1C-treated group. Dunnetts 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 broblast
proliferation. The concentration of PDGF-AB ligand was decreased signicantly 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 PDGFB). The
concentration of PDGF-AB ligand in knocked down HSC3 CM was measured using a PDGF-AB-specic ELISA kit. Data are mean± SD. This experiment
was done with triplicate samples. ANOVA, p< 0.0001, Dunnetts multiple comparisons test
#
p< 0.05,
##
p< 0.001,
###
p< 0.0001 indicate difference from
Control HSC3 group. cThe proliferation of oral broblasts 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 broblasts isolated from three different donors.
ANOVA, p: 0.0001, Dunnetts 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 broblast proliferation inhibition and the
relative level of PDGF-AB concentration was analyzed using linear regression. Correlation coefcient r:0.93, Rsquared: 0.87, p-value: 0.006. Data
indicate that PDGF-AB specically is the ligand in HSC3 CM that stimulates oral broblast proliferation. eCarbonyl pull down assay for PDGFRβin oral
broblasts treated with HSC3 CM in the absence or presence of PXS-S1C. Human oral broblasts were treated with HSC3 CM in the absence or
presence of 1 µM PXS-S1C followed by biotin hydrazide derivitization and afnity pulldown with a streptavidin afnity resin (Neutravidin). Input samples
and proteins eluted by boiling in SDSPAGE were subjected to Western blotting for PDGFRβ. Data are representative of two experiments with the same
outcome from two different gingival broblast 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, Tukeys 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, Sidaks
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
Oncogenesis
Content courtesy of Springer Nature, terms of use apply. Rights reserved
PXS-S1C were next assessed for relative RNA levels of all
ve LOX paralogues. Interestingly, data indicate that PXS-
S1C decreased the expression specically 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
nally 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 broblasts 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 ndings 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
23,3437
. 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
signicantly 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 signicant
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 signicantly con-
tributes to oral cancer development and that LOXL2
inhibitors may be of benet in addressing oral cancer.
LOXL2 promotion of EMT has been reported in several
cancer models previously
14,35,3843
, 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
4450
, 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
brogenic 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 nding that oral
tumor cells secreted LOXL2 in combination with PDGF-
AB, enhance ERK signaling and oral broblast 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
5258
.In
oral cancer in particular, elevated levels of PDGFRβhave
been identied 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
rst 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 broblasts. Sti-
mulation of proliferation of broblasts, 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 bro-
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 brogenic cells by a novel
microenvironment tumor-promoting mechanism, while
small molecule LOXL2 inhibition can improve oral cancer
outcomes. Moreover, this study provides the rst mole-
cular mechanism for enzymatically active LOXL2 in the
promotion of cancer via its modication of a non-
collagenous substrate in the context of paracrine signal-
ing between tumor cells and resident broblasts. 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 Cancerat 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.
Authorscontributions
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
Conict 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
conict of interest.
Publishers note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional afliations.
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
References
1. Cooper, J. S. et al. National Cancer Database report on cancer of the head and
neck: 10-year update. Head Neck 31,748758 (2009).
2. Musharraf, S. G. et al. Metabolite proling of preneoplastic and neoplastic
lesions of oral cavity tissue samples revealed a biomarker pattern. Sci. Rep. 6,
38985 (2016).
3. da Silva, S. D. et al. Advances and applications of oral cancer basic research.
Oral Oncol. 47,783791 (2011).
4. Wreesmann, V. B. et al. Inuence of extracapsular nodal spread extent on
prognosis of oral squamous cell carcinoma. Head Neck 38(Suppl. 1),
E1192E1199 (2016).
5. Moriwaki, K. et al. TRKB tyrosine kinase receptor is a potential therapeutic
target for poorly differentiated oral squamous cell carcinoma. Oncotarget.9,
2522525243 (2018).
Fig. 8 Summary of interactions between oral tumor cells and
broblasts that contribute to oral cancer development.
LOXL2 secreted by tumor cells (a) oxidizes lysine residues on PDGFRβ
in proximal broblasts (b), in addition, to its classical role in collagen
maturation (not shown). PDGF-AB secreted by tumor cells is
consequently able to more efciently stimulate PDGF signaling (c),
resulting in increased ERK1/2 activation and cell proliferation (d).
LOXL2 production by tumor cells is required for maintaining
LOXL2 synthesis is a feed-forward pathway (e), whose mechanism
remains to be determined
Mahjour et al. Oncogenesis (2019) 8:34 Page 16 of 17
Oncogenesis
Content courtesy of Springer Nature, terms of use apply. Rights reserved
6. Wang,B.,Zhang,S.,Yue,K.&Wang,X.D.Therecurrenceandsurvivaloforal
squamous cell carcinoma: a report of 275 cases. Chin. J. Cancer 32,614618
(2013).
7. Matsuura, S. et al. Lysyl oxidase is associated with increased thrombosis and
platelet reactivity. Blood 127, 14931501 (2016).
8. Xiao, Q. & Ge, G. Lysyl oxidase, extracellular matrix remodeling and cancer
metastasis. Cancer Microenviron. 5,261273 (2012).
9. Velez, D. O. et al. 3D collagen architecture induces a conserved migratory and
transcriptional response linked to vasculogenic mimicry. Nat. Commun. 8,
1651 (2017).
10. Eliades, A. et al. Control of megakaryocyte expansion and bone marrow
brosis by lysyl oxidase. J. Biol. Chem. 286, 2763027638 (2011).
11. Lucero, H. A. & Kagan, H. M. Lysyl oxidase: an oxidative enzyme and effector of
cell function. Cell.Mol.LifeSci.63, 23042316 (2006).
12. Saxena, D. et al. Multiple functions of lysyl oxidase like-2 in oral broproli-
ferative processes. J. Dent. Res. 97, 12771284 (2018).
13. Peinado, H. et al. A molecular role for lysyl oxidase-like 2 enzyme in snail
regulation and tumor progression. Embo J. 24,34463458 (2005).
14. Moreno-Bueno, G. et al. Lysyl oxidase-like 2 (LOXL2), a new regulator of cell
polarity required for metastatic dissemination of basal-like breast carcinomas.
EMBO Mol. Med. 3,528544 (2011).
15. Iturbide, A. et al. LOXL2 oxidizes methylated TAF10 and controls TFIID-
dependent genes during neural progenitor differentiation. Mol. Cell 58,
755766 (2015).
16. Assaggaf, M. A., Kantarci, A., Sume, S. S. & Trackman, P. C. Prevention of
phenytoin-induced gingival overgrowth by lovastatin in mice. Am.J.Pathol.
185, 15881599 (2015).
17. Beerlage, C. et al. Hypoxia-inducible factor 1-regulated lysyl oxidase is involved
in Staphylococcus aureus abscess formation. Infect. Immun. 81, 25622573
(2013).
18. Bais, M. V., Kukuruzinska, M. & Trackman, P. C. Orthotopic non-metastatic and
metastatic oral cancer mouse models. Oral Oncol. 51, 476482 (2015).
19. Momose, F. et al. Variant sublines with different metastatic potentials selected
in nude mice from human oral squamous cell carcinomas. J. Oral Pathol. Med.
18,391395 (1989).
20. Vigneswaran,N.,Wu,J.,Song,A.,Annapragada,A.&Zacharias,W.Hypoxia-
induced autophagic response is associated with aggressive phenotype and
elevated incidence of metastasis in orthotopic immunocompetent murine
models of head and neck squamous cell carcinomas (HNSCC). Exp. Mol.
Pathol. 90,215225 (2011).
21. Zhao, M. et al. Assembly and initial characterization of a panel of 85 geno-
mically validated cell lines from diverse head and neck tumor sites. Clin. Cancer
Res. 17, 72487264 (2011).
22. Rheinwald, J. G. & Beckett, M. A. Tumorigenic keratinocyte lines requiring
anchorage and broblast support cultured from human squamous cell car-
cinomas. Cancer Res. 41, 16571663 (1981).
23. Peinado, H. et al. Lysyl oxidase-like 2 as a new poor prognosis marker of
squamous cell carcinomas. Cancer Res. 68, 45414550 (2008).
24. Abeles, R. H. Suicide enzyme inactivators. Basic Life Sci. 25,287305 (1983).
25. Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6,392401
(2006).
26. Resnick, D., Pearson, A. & Krieger, M. The SRCR superfamily: a family reminis-
cent of the Ig superfamily. Trends Biochem. Sci. 19,58(1994).
27. Xu, L. et al. Post-translational modications of recombinant human lysyl
oxidase-like 2 (rhLOXL2) secreted from Drosophila S2 cells. J. Biol. Chem. 288,
53575363 (2013).
28. Lopez-Jimenez, A. J., Basak, T. & Vanacore, R. M. Proteolytic processing of lysyl
oxidase-like-2 in the extracellular matrix is required for crosslinking of base-
ment membrane collagen IV. J. Biol. Chem. 292,1697016982 (2017).
29. Thomson, P. J. Perspectives on oral squamous cell carcinoma prevention-
proliferation, position, progression and prediction. J. Oral Pathol. Med. 47,
803807 (2018).
30. Goodson, M. L., Sloan, P., Robinson, C. M., Cocks, K. & Thomson, P. J. Oral
precursor lesions and malignant transformationwho, where, what, and
when? Br. J. oral. Maxillofac. Surg. 53,831835 (2015).
31. Speight,P.M.,Khurram,S.A.&Kujan,O. Oral potentially malignant disorders:
risk of progression to malignancy. Oral Surg. Oral Med. Oral Pathol. Oral Radiol.
125,612627 (2018).
32. Kanojia, D. & Vaidya, M. M. 4-nitroquinoline-1-oxide induced experimental oral
carcinogenesis. Oral Oncol. 42,655667 (2006).
33. Sweeny, L., Zimmermann, T. M., Liu, Z. & Rosenthal, E. L. Evaluation of tyrosine
receptor kinases in the interactions of head and neck squamous cell carci-
noma cells and broblasts. Oral Oncol. 48,12421249 (2012).
34. Torres, S. et al. LOXL2 is highly expressed in cancer-associated broblasts and
associates to poor colon cancer survival. Clin. Cancer Res. 21,48924902 (2015).
35. Salvador, F. et al. Lysyl oxidase-like protein LOXL2 promotes lung metastasis of
breast cancer. Cancer Res. 77,58465859 (2017).
36. Martin, A. et al. Lysyl oxidase-like 2 represses Notch1 expression in the skin to
promote squamous cell carcinoma progression. Embo J. 34,10901109 (2015).
37. Peng, L. et al. Secreted LOXL2 is a novel therapeutic target that promotes
gastric cancer metastasis via the Src/FAK pathway. Carcinogenesis 30,
16601669 (2009).
38. Ninomiya, G. et al. Signicance of lysyl oxidaselike 2 gene expression on the
epithelialmesenchymal status of hepatocellular carcinoma. Oncol. Rep. 39,
26642672 (2018).
39. Park, P. G. et al. Role of LOXL2 in the epithelialmesenchymal transition and
colorectal cancer metastasis. Oncotarget 8, 8032580335 (2017).
40. Cuevas, E. P. et al. LOXL2 drives epithelialmesenchymal transition via acti-
vation of IRE1-XBP1 signalling pathway. Sci. Rep. 7, 44988 (2017).
41. Wang, Y. et al. Escin Ia suppresses the metastasis of triple-negative breast
cancer by inhibiting epithelial-mesenchymal transition via down-regulating
LOXL2 expression. Oncotarget 7,2368423699 (2016).
42. Canesin, G. et al. Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel
partners in E-cadherin repression and early metastasis colonization. Oncogene
34,951964 (2015).
43. Cuevas, E. P. et al. LOXL2 catalytically inactive mutants mediate epithelial-to-
mesenchymal transition. Biol. Open 3,129137 (2014).
44. Cano, A., Santamaria, P. G. & Moreno-Bueno, G. LOXL2 in epithelial cell plas-
ticity and tumor progression. Future Oncol. 8,10951108 (2012).
45. Iturbide, A., Garcia de Herreros, A. & Peiro, S. A new role for LOX and LOXL2
proteins in transcription regulation. FEBS J. 282,17681773 (2015).
46. Li, T. et al. Lysyl oxidase family members in urological tumorigenesis and
brosis. Oncotarget 9,2015620164 (2018).
47. Nishioka,T.,Eustace,A.&West,C.Lysyloxidase:frombasicsciencetofuture
cancer treatment. Cell Struct. Funct. 37,7580 (2012).
48. Semenza, G. L. Molecular mechanisms mediating metastasis of hypoxic breast
cancer cells. Trends Mol. Med. 18,534543 (2012).
49. Wu, L. & Zhu, Y. The function and mechanisms of action of LOXL2 in cancer
(Review). Int. J. Mol. Med. 36,12001204 (2015).
50. Trackman, P. C. Lysyl oxidase isoforms and potential therapeutic opportunities
for brosis and cancer. Expert Opin. Ther. Targets 20,935945 (2016).
51. Lucero, H. A. et al. Lysyl oxidase oxidizes cell membrane proteins and
enhances the chemotactic response of vascular smooth muscle cells. J. Biol.
Chem. 283,2410324117 (2008).
52. Hiram-Bab, S. et al. Platelet-derived growth factor BB mimics serum-induced
dispersal of pancreatic epithelial cell clusters. J. Cell Physiol. 229,743751
(2014).
53. Wang,Y.,Qiu,H.,Hu,W.,Li,S.&Yu,J.Over-expressionofplatelet-derived
growth factor-D promotes tumor growth and invasion in endometrial cancer.
Int. J. Mol. Sci. 15,47804794 (2014).
54. Schlegel,N.C.,vonPlanta,A.,Widmer,D.S.,Dummer,R.&Christofori,G.PI3K
signalling is required for a TGFbeta-induced epithelial-mesenchymal-like
transition (EMT-like) in human melanoma cells. Exp. Dermatol. 24,2228
(2015).
55. Wang, R. et al. The PDGF-D/miR-106a/Twist1 pathway orchestrates epithelial-
mesenchymal transition in gemcitabine resistance hepatoma cells. Oncotarget
6, 70007010 (2015).
56. Neri, S. et al. Fibroblast-led cancer cell invasion is activated by epithelial-
mesenchymal transition through platelet-derived growth factor BB secretion
of lung adenocarcinoma. Cancer Lett. 395,2030 (2017).
57. Chen, J. et al. PDGF-D promotes cell growth, aggressiveness, angiogenesis and
EMT transformation of colorectal cancer by activation of Notch1/Twist1
pathway. Oncotarget 8,99619973 (2017).
58. Zhang,H.,Sun,J.D.,Yan,L.J.&Zhao,X.P.PDGF-D/PDGFRbetapromotes
tongue squamous carcinoma cell (TSCC) progression via activating p38/AKT/
ERK/EMT signal pathway. Biochem. Biophys. Res. Commun. 478,845851
(2016).
59. Kartha, V. K. et al. PDGFRbeta is a novel marker of stromal activation in oral
squamous cell carcinomas. PLoS ONE 11, e0154645 (2016).
Mahjour et al. Oncogenesis (2019) 8:34 Page 17 of 17
Oncogenesis
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... In addition to the traditional ECM substrates, platelet growth factor receptor β (PDGFRβ) was recently identified as a cell-surface substrate of matrix (ECM) proteins such as tropoelastin and collagen type IV to initiate their crosslinking and promote ECM remodeling [3][4][5]. In addition to the traditional ECM substrates, platelet growth factor receptor β (PDGFRβ) was recently identified as a cell-surface substrate of LOXL2 [6]. Elevated expression of LOXL2 has been associated with poor prognosis in metastatic/invasive cancers and fibrotic disorders [7][8][9]. ...
... Elevated expression of LOXL2 has been associated with poor prognosis in metastatic/invasive cancers and fibrotic disorders [7][8][9]. Small molecule inhibitors and inhibitory RNAs have shown to effectively retard disease progression in both in vitro and in vivo studies [6,[9][10][11][12][13][14][15][16]. Despite its prominence as a therapeutic target, little progress has been made on structure-based drug design for LOXL2 specific inhibitors because of the lack of 3D structural information of the catalytically competent (mature) form of LOXL2. ...
... The higher affinity of fl-LOXL2 to the ECM that we previously observed [4] could partly be attributed to the basicity of the SRCR2 domain. LOXL2 has been shown to oxidatively deaminate Lys residue(s) of the extracellular domain of PDGFRβ to an aldehyde [6]. The pI value of the extracellular domain of PDGFRβ is predicted to be 4.83 and 4.71 by Isoelectric Compute pI/MW [35] and Point Calculator 2.0 [36], respectively. ...
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Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.
... The formation of aldehydes initiates crosslinking of the extracellular matrix (ECM) proteins, such as elastin and collagen leading to ECM remodeling. Significant upregulation of LOXL2 has been observed in metastatic/invasive phenotypes of tumor cells and tumors as well as fibrotic disorders [3][4][5][6][7]. Small molecule-based inhibitors of LOXL2 have been under development and some exhibited promising results from in vitro and in vivo studies [8][9][10][11][12][13]. ...
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Lysyl oxidase-like 2 (LOXL2) catalyzes the oxidative deamination of peptidyl lysines and hydroxylysines to promote extracellular matrix remodeling. Aberrant activity of LOXL2 has been associated with organ fibrosis and tumor metastasis. The lysine tyrosylquinone (LTQ) cofactor is derived from Lys653 and Tyr689 in the amine oxidase domain via post-translational modification. Based on the similarity in hydrodynamic radius and radius of gyration, we recently proposed that the overall structures of the mature LOXL2 (containing LTQ) and the precursor LOXL2 (no LTQ) are very similar. In this study, we conducted a mass spectrometry-based disulfide mapping analysis of recombinant LOXL2 in three forms: a full-length LOXL2 (fl-LOXL2) containing a nearly stoichiometric amount of LTQ, Δ1-2SRCR-LOXL2 (SRCR1 and SRCR2 are truncated) in the precursor form, and Δ1-3SRCR-LOXL2 (SRCR1, SRCR2, SRCR3 are truncated) in a mixture of the precursor and the mature forms. We detected a set of five disulfide bonds that is conserved in both the precursor and the mature recombinant LOXL2s. In addition, we detected a set of four alternative disulfide bonds in low abundance that is not associated with the mature LOXL2. These results suggest that the major set of five disulfide bonds is retained post-LTQ formation.
... By contrast, ADAMTS2 overexpression has been linked to an increased incidence of some forms of gastric [50] and oral cancers [51,52], possibly suggesting that longer LOX-PP isoforms containing sulfated tyrosine residues resulting from ADAMTS 2 or 14 cleavage only, should they accumulate, may reduce the tumor suppressor functions of LOX-PP and/or increase LOX enzyme tumor promoting effects. One possibility is that the 25 kDa enzyme generated after ADAMTS2/14 cleavage would have increased substrate specificity for a tumor promoting target rather than collagen, such as PDGFRβ, increasing PDGF signaling and cancer progression [53]. ...
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Lysyl oxidases are multifunctional proteins derived from five lysyl oxidase paralogues (LOX) and lysyl oxidase-like 1 through lysyl oxidase-like 4 (LOXL1–LOXL4). All participate in the biosynthesis of and maturation of connective tissues by catalyzing the oxidative deamination of lysine residues in collagens and elastin, which ultimately results in the development of cross-links required to function. In addition, the five LOX genes have been linked to fibrosis and cancer when overexpressed, while tumor suppression by the propeptide derived from pro-LOX has been documented. Similarly, in diabetic retinopathy, LOX overexpression, activity, and elevated LOX propeptide have been documented. The proteolytic processing of pro-forms of the respective proteins is beginning to draw attention as the resultant peptides appear to exhibit their own biological activities. In this review we focus on the LOX paralogue, and what is known regarding its extracellular biosynthetic processing and the still incomplete knowledge regarding the activities and mechanisms of the released lysyl oxidase propeptide (LOX-PP). In addition, a summary of the roles of both LOX and LOX-PP in diabetic retinopathy, and brief mentions of the roles for LOX and closely related LOXL1 in glaucoma, and keratoconus, respectively, are included.
... Within the past two decades, researchers have also acknowledged the interconnection of the nervous system with cancer progression and metastasis [43][44][45][46][47][48]. This cancer-nerve crosstalk occurs in cancers of highly innervated areas, including the pancreas [49][50][51][52], head and neck [53][54][55], colon and rectum [56][57][58], breasts [59][60][61][62], prostate [63][64][65], cervix [66][67][68], lungs [69], etc. There are two established subtypes of cancer-nerve crosstalk: perineural invasion (PNI) occurs when cancer cells migrate into the perineural layer of adjacent nerves [70][71][72] while its relative opposite, tumor innervation (TI), describes the phenomenon in which neurites extend into solid tumors [73,74]. ...
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Since Otto Warburg’s first report on the increased uptake of glucose and lactate release by cancer cells, dysregulated metabolism has been acknowledged as a hallmark of cancer that promotes proliferation and metastasis. Over the last century, studies have shown that cancer metabolism is complex, and by-products of glucose and glutamine catabolism induce a cascade of both pro- and antitumorigenic processes. Some vitamins, which have traditionally been praised for preventing and inhibiting the proliferation of cancer cells, have also been proven to cause cancer progression in a dose-dependent manner. Importantly, recent findings have shown that the nervous system is a key player in tumor growth and metastasis via perineural invasion and tumor innervation. However, the link between cancer–nerve crosstalk and tumor metabolism remains unclear. Here, we discuss the roles of relatively underappreciated metabolites in cancer–nerve crosstalk, including lactate, vitamins, and amino acids, and propose the investigation of nutrients in cancer–nerve crosstalk based on their tumorigenicity and neuroregulatory capabilities. Continued research into the metabolic regulation of cancer–nerve crosstalk will provide a more comprehensive understanding of tumor mechanisms and may lead to the identification of potential targets for future cancer therapies.
... LOXL2 is involved in fibrogenesis of various diseases, and modulates the process of matrix remodeling and EMT by cross-linking of collagen I [19]. A variety of studies have reported that dysregulation of LOXL2 is strongly associated with cancer progression [20] and fibrosis-related diseases [21]. Moreover, LOXL2 expression was also detected in serums of idiopathic pulmonary fibrosis disease [22], rheumatoid arthritis-associated interstitial lung disease [23] and was positively correlated with atrial fibrosis in patients with atrial fibrillation [24]. ...
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Background Lysyl oxidase-like 2 (LOXL2) belongs to a family of the LOX secretory enzyme, which involves the cross-linkage of extracellular matrix (ECM) proteins. Here, we aimed to analyze the correlation between serum LOXL2 and pelvic adhesion in chronic pelvic inflammatory disease (PID). Methods A total of 143 patients with PID and 130 healthy controls were included in this study. The serum levels of LOXL2 were measured using enzyme-linked immunosorbent assay (ELISA) kits. The patients were divided into non-adhesion group (102 cases) and adhesion group (41 cases). Results It was found that the serum level of LOXL2 expression was elevated in PID patients compared with healthy controls, and was elevated in PID patients with pelvic adhesion compared to patients without adhesion. In all PID patients, serum LOXL2 level was positively correlated with matrix metalloproteinases-9 (MMP-9), transforming growth factor-β (TGF-β1), whole blood viscosity (WBV) at low shear rate (LSR), WBV at high shear rate (HSR), and hematocrit (HcT). Multivariate logistic regression analysis showed that serum LOXL2 level was an independent risk factor for pelvic adhesion in PID patients (OR = 1.058; 95% CI = 1.030–1.086, P < 0.001). Conclusions Serum LOXL2 level not only predicts the presence of PID, but serum LOXL2 concentration is also associated with the presence of pelvic adhesions.
... All the experiments were performed with prior approval of the Institutional Animal Care and Use Committee, Boston University Medical Campus, Boston University. C57BL/6J mice were fed with 4NQO (100 mg/mL in propylene glycol and water) in their drinking water for 16 weeks, followed by regular water for the remainder of the experiment using a well-established protocol (16). The progressive changes at different stages of OSCC were determined at specific time points. ...
Article
Lysine-specific demethylase 1 (LSD1) is a histone demethylase that contributes to the etiology of oral squamous cell carcinoma (OSCC) in part by promoting cancer stem cell phenotypes. The molecular signals regulated by LSD1, or acting with LSD1, are poorly understood, particularly in the development of OSSC. In this study, we show that conditional deletion of the Lsd1 gene or pharmacologic inhibition of LSD1 in the tongue epithelium leads to reduced development of OSCC following exposure to the tobacco carcinogen 4NQO. LSD1 inhibition attenuated proliferation and clonogenic survival and showed an additive effect when combined with the YAP inhibitor Verteporfin. Interestingly, LSD1 inhibition upregulated the expression of PD-L1, leading to immune checkpoint inhibitor therapy responses. Implications: Collectively, our studies reveal a critical role for LSD1 in OSCC development and identification of tumor growth targeting strategies that can be combined with LSD1 inhibition for improved therapeutic application.
... But it has not yet been tested in a clinical (Smithen et al., 2020). XS-5382A, an oral LOXL2 inhibitor, has been shown to slow tumor growth and reduce collagen accumulation in LY2 oral cancer models and is currently being investigated in Phase 1 clinical trials in healthy adults (Clinical trial identifier: NCT04183517) (Mahjour et al., 2019). Although no inhibitors of the LOX family have currently been approved for routine clinical practice, the developing LOX family inhibitors have shown high specificity and low toxicity. ...
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Background: Gastric cancer (GC) remains the fifth most commonly diagnosed malignancy worldwide, with a poor prognosis. The lysyl oxidase (LOX) family, a type of secreted copper-dependent amine oxidases, is comprised of LOX and four LOX-like (LOXL) 1–4 isoforms and has been reported to be dysregulated in a number of different type cancers. However, the diverse expression patterns and prognostic values of LOX family in GC have yet to be systematically analyzed. Methods: ONCOMINE, GEPIA, UALCAN, Kaplan–Meier Plotter, LOGpc, cBioPortal, GeneMANIA and Metascape databases were utilized in this study to analyze the expression, prognostic values, mutations and functional networks of LOX family in GC. Results: The mRNA expression levels of LOX, LOXL1 and LOXL2 were significantly higher in GC, the expression level of LOXL3 was contrary in different databases, while the expression level of LOXL4 made no difference; the expression levels of LOX, LOXL1 and LOXL3 were higher in stages 2–4 than that of normal tissues and stage 1, while the mRNA level of LOXL2 in stage 1–4 was higher than normal tissues; patients with high expression of LOX and LOXL 2-4 had poor OS; the genes correlated with LOX and LOXL2 were enriched in extracellular matrix organization, vasculature development and skeletal system development. Conclusion: Our results indicated that the LOX family, especially LOX and LOXL2, might play an important role in GC oncogenesis, and they may become biomarkers for predicting tumor prognosis and potential targets for tumor therapy.
... Oral squamous cell carcinoma (OSCC), a malignancy arising from the surface epithelium of oral cavity, accounts for more than 90% of all oral cancers [1]. As the estimated 5-year survival rate for OSCC significantly decreases from approximately 85% if detected at early stages (I and II) to 40% if detected at advanced stages (III and IV) [2], its early detection is crucial. ...
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Introduction Moldova, Belarus, and Armenia are post-Soviet countries with a high rate of heavy smokers and a relatively high age-standardized incidence of oral cancer. However, to our knowledge, there is lack of available information on dentists’ knowledge on prevention of oral cancer in the countries in question. Accordingly, this study aimed to assess the knowledge, opinions, and practices related to oral cancer prevention and oral mucosal examination among dentists in Moldova, Belarus, and Armenia. Methods This was a multi-country, cross-sectional study based on a self-administered questionnaire. A structured questionnaire was distributed to 3534 dentists (797 in Chisinau, Moldova, 1349 in Minsk, Belarus, and 1388 in Yerevan, Armenia). Dentists' knowledge about risk factors for oral cancer development and its clinical picture, current practices and opinions with regard to oral mucosal screening and oral cancer prevention, and their consistency to perform oral mucosal examination were assessed. A knowledge score ranging from 0 to 14 points was generated based on each dentist’s answer to the questionnaire. Results A total of 1316 dentists responded, achieving an overall response rate of 37.2% (34.5% in Moldova; 52.3% in Belarus; 24.2% in Armenia). Most dentists in the three countries correctly identified tobacco (83.8–98.2%) and prior oral cancer lesions (84.0–96.3%) as risk factors for oral cancer. Most dentists correctly identified leukoplakia as a lesion with malignant potential (68.7% in Moldova; 88.5% in Belarus; 69.9% in Armenia), while erythroplakia was identified by much fewer in all three countries. Less than 52% of dentists identified the tongue, rim of tongue, and floor of mouth as the most common sites for oral cancer. The mean knowledge score for all countries combined was 7.5 ± 2.7. The most commonly reported barriers to perform oral mucosal examination were lack of training, knowledge, and experience. Conclusions This study highlights the need for improved oral cancer-related education and training on oral mucosal examination for dentists in Moldova, Belarus, and Armenia. Such skills are essential to enhance oral cancer prevention and to improve the prognostic outcome by early detection.
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Lysyl oxidase-like 2 (LOXL2) is a member of the lysine oxidase (LOX) family. Although its overexpression is known to play pivotal roles in carcinogenesis, its involvement in cervical cancer remains undefined. Here, we comprehensively explored the expression level and functional mechanism of LOXL2 in cervical cancer using bioinformatics and experimental methods. Bioinformatics analysis revealed that LOXL2 was significantly upregulated in cervical cancer compared to normal tissues. Enrichment analysis showed that most positively or negatively correlated genes of LOXL2 were correlated with extracellular matrix (ECM) formation and epithelial-mesenchymal transition (EMT). Further experiments confirmed that overexpression of LOXL2 greatly enhanced the malignant transformation abilities (e.g., proliferation, invasion, and migration) of cervical cancer cells via mediation of EMT. Furthermore, the small molecule inhibitor of LOXL2 ((2-Chloropyridin-4-yl) methanamine hydrochloride) significantly decreased the invasive ability of cervical cancer by reversing the process of LOXL2-induced EMT. In summary, LOXL2 may be a promising diagnostic and therapeutic biomarker for cervical cancer, and its small molecule inhibitor may be an effective anti-tumor drug.
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Decades of research have concluded that disruptions to Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) have profound effects on cancer progression. However, as our understanding of the tumor stroma has evolved, we can appreciate that disruptions to tumor suppressors such as PTEN should not be studied solely in an epithelial context. Inactivation of PTEN in the stroma is associated with worse outcomes in human cancers, therefore, it is important to understand activities regulated downstream of PTEN in stromal compartments. Studies reviewed herein provide evidence for important mechanistic targets downstream of PTEN signaling in cancer-associated fibroblasts (CAFs), a major component of the tumor stroma. We also discuss the potential clinical implications for these findings.
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It has been reported that one of the neurotrophin receptors, tropomyosin receptor kinase B (TRKB), is frequently overexpressed in various tumor tissues including oral squamous cell carcinoma (OSCC), and that its upregulation promotes tumor progression in human cancers. However, the correlation between TRKB overexpression and clinicopathological characteristics is not fully elucidated. Here, we present the correlation between the expression levels of TRKB and/or its secreted ligand, brainderived neurotrophic factor (BDNF), and clinicopathological characteristics, especially regarding tumor differentiation, tissue invasion, and disease-free survival in patients with OSCC. The results obtained through immunohistochemical analysis of human OSCC tumor specimens showed that the expression levels of TRKB and/or BDNF, were significantly higher in moderately and poorly differentiated OSCC (MD/PD-OSCC) tumor cells than in well differentiated cells (WD-OSCC). Moreover, the OSCC tumors highly expressing TRKB and/or BDNF exhibited promotion in tissue invasion and reduction in disease-free survival in the patients. In an orthotopic transplantation mouse model of human OSCC cell lines, administration of a TRKB-specific inhibitor significantly suppressed the tumor growth and invasion in PD-OSCC-derived tumor cells, but not in WD-OSCC-derived tumor cells. Moreover, the TRKB inhibitor selectively blocked BDNF-induced tumor cell proliferation and migration accompanied with the suppression of TRKB phosphorylation in PD-OSCC but not in WD-OSCC in vitro. Taken together, these data suggest that the BDNF/TRKB signaling pathway may regulate tumor progression in poorly differentiated OSCC. Expression levels of signal molecules may be an accurate prognosis marker for tumor aggressiveness, and the molecules may be an attractive target for new OSCC therapies.
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Lysyl oxidase (LOX) is an extracellular copper-dependent monoamine oxidase that catalyzes crosslinking of soluble collagen and elastin into insoluble, mature fibers. Lysyl oxidase-like proteins (LOXL), LOX isozymes with partial structural homology, exhibit similar catalytic activities. This review summarizes recent findings describing the roles of LOX family members in urological cancers and fibrosis. LOX/ LOXL play key roles in extracellular matrix stability and integrity, which is essential for normal female pelvic floor function. LOX/LOXL inhibition may reverse kidney fibrosis and ischemic priapism. LOX and LOXL2 reportedly promote kidney carcinoma tumorigenesis, while LOX, LOXL1 and LOXL4 suppress bladder cancer growth. Multiple studies agree that the LOX propeptide may suppress tumor growth, but the role of LOX in prostate cancer remains controversial. Further studies are needed to clarify the exact effects and mechanism of LOX/LOXL on urological malignancies.
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Oral potentially malignant disorders (OPMDs) have a statistically increased risk of progressing to cancer, but the risk varies according to a range of patient- or lesion-related factors. It is difficult to predict the risk of progression in any individual patient, and the clinician must make a judgment based on assessment of each case. The most commonly encountered OPMD is leukoplakia, but others, including lichen planus, oral submucous fibrosis, and erythroplakia, may also be seen. Factors associated with an increased risk of malignant transformation include sex; site and type of lesion; habits, such as smoking and alcohol consumption; and the presence of epithelial dysplasia on histologic examination. In this review, we attempt to identify important risk factors and present a simple algorithm that can be used as a guide for risk assessment at each stage of the clinical evaluation of a patient.
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Lysyl oxidase like 2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. In spite of its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65 kDa form lacking the first two Scavenger Receptors Cysteine Rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro towards a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV.
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Gingival overgrowth is a side effect of certain medications, including calcium channel blockers, cyclosporin A, and phenytoin. Phenytoin-induced gingival overgrowth is fibrotic. Lysyl oxidases are extracellular enzymes that are required for biosynthetic cross-linking of collagens, and members of this enzyme family are upregulated in fibrosis. Previous studies in humans and in a mouse model of phenytoin-induced gingival overgrowth have shown that LOXL2 is elevated in the epithelium and connective tissue in gingival overgrowth tissues and not in normal tissues. Here, using a novel LOXL2 isoform-selective inhibitor and knockdown studies in loss- and gain-of-function studies, we investigated roles for LOXL2 in promoting cultures of human gingival fibroblasts to proliferate and to accumulate collagen. Data indicate that LOXL2 stimulates gingival fibroblast proliferation, likely by a platelet-derived growth factor B receptor-mediated mechanism. Moreover, collagen accumulation was stimulated by LOXL2 enzyme and inhibited by LOXL2 inhibitor or gene knockdown. These studies suggest that LOXL2 could serve as a potential therapeutic target to address oral fibrotic conditions.
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In the present study, we investigated the role of lysyl oxidase‑like 2 (LOXL2), the correlation between LOXL2 and epithelial to mesenchymal transition (EMT) and the effects of using β‑aminopropionitrile (BAPN) to inhibit LOXL2 with the aim of reducing tumor progression in hepatocellular carcinoma (HCC). The expression level of LOXL2 was evaluated in HCC and adjacent non‑cancerous tissues using quantitative reverse transcription polymerase chain reaction and clinicopathological analyses. The effects of BAPN on cell proliferation, migration and invasion were investigated in vitro. Additionally, LOXL2 expression was assessed in the culture supernatants of HCC cell lines. Our results revealed that LOXL2 expression was higher in HCC cell lines and tissues. There was a significant correlation between EMT status and LOXL2 levels (P=0.004). BAPN reduced migration and invasion in HCC cells. HCC patients with high levels of LOXL2 expression had relatively shorter disease‑free survival (P=0.009) and overall survival (P=0.035). The expression level of LOXL2 was similar between cell supernatants and HCC cell lines. A multivariate analysis demonstrated that portal vein invasion (P=0.015), venous invasion (P=0.026), serum AFP (α‑fetoprotein) levels (P=0.019) and LOXL2 expression (P=0.009) were independent prognostic factors. Our results indicated that a higher level of LOXL2 may contribute to tumor progression, indicating that LOXL2 has clinical value as a therapeutic target in HCC.
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The lysyl oxidase-like protein LOXL2 has been suggested to contribute to tumor progression and metastasis, but in vivo evidence has been lacking. Here we provide functional evidence that LOXL2 is a key driver of breast cancer metastasis in two conditional transgenic mouse models of PyMT-induced breast cancer. LOXL2 ablation in mammary tumor cells dramatically decreased lung metastasis, whereas LOXL2 overexpression promoted metastatic tumor growth. LOXL2 depletion or overexpression in tumor cells does not affect extracellular matrix stiffness or organization in primary and metastatic tumors, implying a function for LOXL2 independent of its conventional role in extracellular matrix remodeling. In support of this likelihood, cellular and molecular analyses revealed an association of LOXL2 action with elevated levels of the EMT regulatory transcription factor Snail1 and expression of several cytokines that promote pre-metastatic niche formation. Taken together, our findings established a pathophysiological role and new function for LOXL2 in breast cancer metastasis.