Open Access
Austin Journal of Clinical Pathology
Original Article
MMP13 and SERPINB2 as Novel Biomarkers for
Hypopharyngeal Cancer
Lv F1, Huang W2 and Ji X3*
1
Department of Oncology, Shengjing Hospital of China
Medical University, China
2
Department of Pathology, Shengjing Hospital of China
Medical University, People’s Republic of China
3
Department of Otorhinolaryngology Head and Neck
Surgery, the First Hospital of China Medical University,
People’s Republic of China
*Corresponding author: Xu Ji, Department of
Otorhinolaryngology Head and Neck Surgery, the First
Hospital of China Medical University, Shenyang 110001,
Liaoning, People’s Republic of China
Received: September 21, 2020; Accepted: October 13,
2020; Published: October 20, 2020
Abbreviations
AUC: Area Under ROC Curve; CI: Confidence Interval; DAB:
3, 3’-Diaminobenzidine tetrahydrochloride; DEGs: Differentially
Expressed Genes; ECM: Extracellular Matrix; FC: Fold-Change;
GEO: Gene Expression Omnibus; H&E: Hematoxylin and Eosin;
H3K4me3: Histone H3 trimethylation at lysine 4; HR: Hazard Ratio;
ING: Inhibitor of Growth; MMPs: Matrix Metalloproteinases; PAI:
Plasminogen Activator Inhibitor; TCGA: The Cancer Genome Atlas;
uPA: urokinase type Plasminogen Activator; WHO: World Health
Organization
Introduction
As an uncommon malignant tumor, hypopharyngeal cancer
accounts for 3–5% of head and neck tumors [1]. Most pathological
types of hypopharyngeal cancer are squamous cell carcinoma. Due
to the occult anatomical location of hypopharyngeal cancer and poor
surgical effect, local recurrence or distant metastasis often occurs in
patients with hypopharyngeal cancer following surgery. Pharyngeal
fistula also affects the quality of life of hypopharyngeal cancer
patients. Therefore, it is necessary to search forhypopharyngeal
cancer markers.
Tumor stroma is mainly composed of collagen fibers, vascular
vessels, and Extracellular Matrix (ECM) components. ECM
components are over-deposited in tumor tissues and function in
nourishing tumor cells. The system composed of urokinase type
Plasminogen Activator (uPA), plasmin, Plasminogen Activator
Inhibitor (PAI), and Matrix Metalloproteinases (MMPs) is one of
the main ways to regulate ECM degradation. MMP13, also known as
Collagenase 3, can degrade a variety of ECM components, including
tenascin C, fibronectin, and type I–IV collagen [2]. MMP13 has a
Austin J Clin Pathol - Volume 7 Issue 2 - 2020 Citation: Lv F, Huang W and Ji X. MMP13 and SERPINB2 as Novel Biomarkers for Hypopharyngeal Cancer.
ISSN : 2381-9170 | www.austinpublishinggroup.com Austin J Clin Pathol. 2020; 7(2): 1065.
Ji et al. © All rights are reserved
Abstract
Matrix Metalloproteinase (MMP)13 and serine peptidase inhibitor, clade B,
member 2 (SERPINB2) are important components of the extracellular matrix
and play a regulatory role in tumor stromal remodeling. The purpose of the study
is to evaluate the expression of MMP13 and SERPINB2 in hypopharyngeal
cancer. We utilized the public datasets to find the differentially expressed genes
of hypopharyngeal cancer, which were verified by immunohistochemistry. At the
same time, the role of differential expressed genes in prognosis was explored.
We found that MMP13 is upregulated and SERPINB2 is downregulated in
public datasets. We confirmed this conclusion by immunohistochemistry
of hypopharyngeal cancer tissues and found that MMP13 and SERPINB2
were related to some clinicopathological factors. Moreover, SERPINB2 is an
independent prognostic factor for hypopharyngeal cancer patients. MMP13 and
SERPINB2 may predict the early recurrence of hypopharyngeal cancer. MMP13
combined with SERPINB2 may be potential prognostic biomarkers and drug
targets in hypopharyngeal cancer.
Keywords: Hypopharyngeal neoplasms; Matrix metalloproteinase 13;
Plasminogen activator inhibitor 2; Immunohistochemistry
wide range of proteolytic functions, and MMP13 participates in
various physiological and pathological processes [3]. SERPINB2,
also known as PAI-2, is a member of the SERPIN superfamily of
serine protease inhibitors. SERPINB2 mainly inhibits uPA and tissue
plasminogen activator [4]. Previous studies have shown that MMP13
[5,6] or SERPINB2 [7,8] are associated with the occurrence or
prognosis of different tumors. However, until now, there has been no
report on the expression and regulation of MMP13 and SERPINB2 in
hypopharyngeal cancer. As a rare tumor, hypopharyngeal cancer also
lacks markers for diagnosis and prognosis.
This is the first study to investigate the expression and correlation
of MMP13 and SERPINB2 in hypopharyngeal carcinoma. Through
public datasets and immunohistochemistry, we showed the
expression changes and correlations of MMP13 and SERPINB2 in
hypopharyngeal cancer. MMP13 and SERPINB2 are related to the
survival of hypopharyngeal carcinoma patients and may predict
early recurrence. These findings suggest that MMP13 and SERPINB2
play an important role in hypopharyngeal cancer and may be a
potential prognostic marker and therapeutic targets for patients with
hypopharyngeal cancer.
Methods
Public datasets searching
From the National Center of Biotechnology Information
Gene Expression Omnibus (GEO) database (https://www.ncbi.
nlm.nih.gov/gds/), we searched raw gene expression data of the
hypopharyngeal cancer as the following key terms: (hypopharyngeal
or hypopharynx) and (cancer or tumor or carcinoma or neoplasm).
The publication time was not limited. Homo sapiens was selected in
the Organism and Expression profiling by array in the Type.
Ji X
GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) is an online
tool provided by GEO, which is based on the R language limma
package [9]. GEO2R was used to screen DEGs (differentially expressed
genes) between hypopharyngeal cancer and non-cancerous samples
in the GEO datasets and we further visualized DEGs by volcano plot
[10]. A P-value <0.05 and absolute log Fold-Change (FC) greater
than 2 for the DEGs were used as the cut-off criteria. We also used
the online tool Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/
index.html) to identify the overlapping DEGs that were up-/down-
regulated in the GEO datasets.
The Cancer Genome Atlas (TCGA) is a public funded project
that aims to catalogue and discover major cancer-causing genomic
alterations to create a comprehensive “atlas” of cancer genomic
profiles. We also obtained gene expression data in hypopharyngeal and
non-cancerous tissues from TCGA database (https://cancergenome.
nih.gov/). Both GEO and TCGA datasets’ non-cancerous tissues came
from keratinized epithelium of hypopharynx in healthy volunteers.
Patients and tissue samples
The research was in consent by the First Affiliated Hospital of
China Medical University Ethics Committee. We involved patients
who signed informed consent and collected samples according
to the informed consent. We collected 40 hypopharyngeal cancer
patients who underwent partial hypopharyngeal resection or total
laryngectomy and collected their cancer and paracancerous tissues.
Two independent pathologists examined all tissues. No distant
metastasis or preoperative chemoradiotherapy was observed. Besides,
we collected patients’ clinical information and followed up. Smokers
ware defined as those who smoke continuously or accumulatively
for six months or more in their lifetime [11], according to the World
Health Organization (WHO) guideline.
Immunohistochemistry
Formalin-fixed tissues were embedded in paraffin and cut into 5
μm–thick sections for Hematoxylin and Eosin (H&E) staining and
immunostaining. The expression of MMP13 and SERPINB2 was
assessed by immunohistochemical staining kit (Maixin, China). De-
waxed sections were washed in PBS and exposed to 3% H2O2 for 15
min at room temperature to quench endogenous peroxidase activity.
Then the tissues were blocked with normal goat serum for 20 min at
room temperature. After incubation overnight at 4 °C with primary
antibody (1:200) (MMP13: proteintech, 18165-1-AP; SERPINB2:
proteintech, 16035-1-AP), the tissues were incubated with the second
antibody and biotin-labeled horseradish peroxidase. Subsequently,
the antibody binding was visualized with a 3,3’-Diaminobenzidine
tetrahydrochloride (DAB) kit (Maixin, China) before brief
counterstaining with hematoxylin. Eventually, tissues were gradually
dehydrated, sealed with neutral gum, observed and photographed
with an inverted phase contrast microscope.
Immunoreactivity was semi-quantitatively evaluated and
evaluated by two pathologists. Five representative regions were
randomly selected from the 400-fold field of view of the microscope.
The score of each field of view was determined by the proportion
of positive cells and the color rendering intensity. According to the
proportion of positive cells: <5%, 5-25%, 25% - 50%, 50% - 75%,
75% - 100%, the scores of 0, 1, 2, 3 and 4 were obtained respectively.
According to the intensity of colouring, 0, 1 and 2 points were
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obtained for uncolored, yellow and brown respectively. The final
score was obtained by multiplying the scores of the two items and
taking the average score of the five visual fields. The total score of 0-4
was defined as Low-No expression, and more than 4 was defined as
Medium-High expression.
Statistical analysis
Each experiment was repeated at least three times and data was
analyzed by SPSS 21.0 (IBM Corp.). The expression of MMP13 and
SERPINB2 in cancer and non-cancerous groups was compared by
Student’s t-test (public data: unpaired t-test; specimen data: paired
t-test). Pearson’s X2 correlation was applied to test the correlations
between expression of MMP13 and SERPINB2. Univariate analysis
(Kaplain-Meier test) was used to determine the clinicopathological
characteristics related to survival, and multivariate analysis (Cox
regression) was used to further analyze the covariates (P <0.05) in
univariate analysis. Finally, the diagnostic value was evaluated by area
under ROC curve (R package ggplot2). Differences were considered
significant when p -values <0.05.
Results
Identification of Differentially Expressed Genes (DEGs)
The gene expression profiles of GEO datasets, including GSE686
(cancer samples: 9, non-cancerous samples: 3), GSE2379 (cancer
samples: 14, non-cancerous samples: 4), and GSE10774 (cancer
samples: 10, non-cancerous samples: 4) were downloaded from the
public GEO database by searching the terms mentioned. DEGs in
the GSE686 dataset were screened and we obtained 231 DEGs (36
upregulated and 172 downregulated), as shown in (Figure 1A).
In the GSE2379 and GSE10774 datasets, we identified 403 (204
upregulated and 199 downregulated) and 99 (23 upregulated and
76 downregulated) DEGs, respectively (Figure 1B- C). Among the
DEGs, we found two overlapped DEGs, one upregulated (MMP13)
and one downregulated (SERPINB2) (Figure 2A-B).
The Cancer Genome Atlas (TCGA) database contains normalized
RNA sequencing data of hypopharyngeal cancer patients using
the RNASeqV2 system. Thus, we verified our results in the TCGA
dataset. By analyzing MMP13 and SERPINB2 expression levels in
unpaired hypopharyngeal (n = 5) and non-cancerous tissues (n =
19) of the TCGA–HNSCC/hypopharyngeal cohort, we found that
MMP13 expression was significantly upregulated in hypopharyngeal
tissues (Figure 2C), while SERPINB2 was unchanged between
hypopharyngeal and non-cancerous tissues (Figure 2D).
Figure 1: Volcano plot of gene expression profile data in hypopharyngeal
cancer and non-cancerous samples. Volcano plot of GSE686 (a), GSE2379
(b), and GSE10774 (c). Red spots represent up-regulated genes, and blue
spots represent down-regulated genes.
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Ji X Austin Publishing Group

ranged from 46 to 77 and most were male patients. Approximately

one-third of patients had stage IV tumors, most of which were located
in the piriform fossa. This is consistent with the epidemiological
data of hypopharyngeal cancer reported in 2011 [12]. MMP13
expression was increased in 57.5% (23/40) of the patients and
SERPINB2 expression was decreased in 62.5% (25/40) of the patients.
MMP13 was expressed in the cytoplasm of hypopharyngeal cancer
cells and negative in squamous epithelial cells, while expression of
SERPINB2 was the opposite (Figure 3A-B). Expression of MMP13 in
hypopharyngeal cancer was correlated with N stage (P = 0.017) and
smoking history (P = 0.005); expression of SERPINB2 was correlated
with N stage (P = 0.005), but not with other clinicopathological
features (Tables 1 and 2). We also found a slight negative correlation
between MMP13 and SERPINB2 expression, but the difference was
not statistically significant (Pearson correlation coefficient = -0.274,
P = 0.083, (Figure 4)).
Prognostic significance of MMP13 and SERPINB2
Figure 2: Venn diagram of overlapping Differentially Expressed Genes
(DEGs) and expression of DEGs in the TCGA dataset. (a,b) Venn diagram Hypopharyngeal cancer patients were followed up for 2–18
of upregulated overlapping DEGs (a) and downregulated DEGs (b). (c,d) Dot months, with a median follow-up of 12 months. At the end of follow-
plot indicating expression level of MMP13 (c) and SERPINB2 (d) in TCGA–
HNNC/hypopharyngeal cancer. P value was determined using Student’s
up, 12 patients died as a result of disease progression and the common
t-test. Error bars represent mean ± standard deviation. sites of progression included the lungs and cervical lymph nodes.
Univariate analysis showed that MMP13 (hazard ratio [HR] = 3.691,
95% confidence interval [CI] = 1.157–11.78, P = 0.02, (Figure 5A) and
SERPINB2 (HR = 0.2305, 95% CI = 0.072–0.739, P = 0.01, (Figure
5B)) expression along with N stage, and smoking and alcohol history
were important factors for prognosis. Furthermore, multivariate
analysis showed SERPINB2 expression was an independent factor for
prognosis (HR = 0.120, 95% CI = 0.015–0.929, P = 0.04, (Table 3)).
Diagnostic significance of MMP13 and SERPINB2 for
early recurrence
Recurrence within one year after radical resection is called early
recurrence of hypopharyngeal cancer. To further detect whether
MMP13 and SERPINB2 expression has a role in the prediagnosis of
early recurrence of hypopharyngeal cancer, we calculated the area
under ROC curve (AUC) in 27 patients who can be diagnosed with
early recurrence or not. MMP13 and SERPINB2 levels were able to
differentiate between early recurrence and non-early recurrence
Figure 3: Immunohistochemistry of MMP13 and SERPINB2 protein Table 1: Correlation between expression levels of MMP13 and clinicopathological
expression in HNSCC tissues. (a) Expression of MMP13 in hypopharyngeal parameters of hypopharyngeal cancer patients.
cancer (upper) and adjacent normal squamous epithelium (lower). Upper Variable n MMP13 expression level P
left: Hematoxylin and Eosin (H&E) staining of hypopharyngeal cancer.
Upper middle and right: MMP13 of hypopharyngeal cancer (middle: 200×, Medium-High Low-No
right: 400×). Lower left: H&E staining of squamous epithelium. Lower middle <60 19 13 6
Age 0.184
and right: MMP13 of squamous epithelium (middle: 200×, right: 400×). (b) >=60 21 10 11
Expression of SERPINB2 in hypopharyngeal cancer (upper) and adjacent Male 39 23 16
Gender 0.239
normal squamous epithelium (lower). Upper left: H&E of hypopharyngeal female 1 0 1
cancer. Upper middle and right: SERPINB2 of hypopharyngeal cancer II III 25 17 8
T stage 0.083
(middle: 200×, right: 400×). Lower left: H&E staining of squamous epithelium. IV 15 6 9
Negative 11 3 8
Lower middle and right: SERPINB2 of squamous epithelium (middle: 200×, N stage 0.017
positive 29 20 9
right: 400×).
Yes 35 23 12
Smoke 0.005
no 5 0 5
Expression of MMP13 and SERPINB2 in hypopharyngeal Yes 33 21 12
Alcohol 0.088
cancer tissues no 7 2 5
>=4g/ml 21 15 6
To further test our conclusions predicted from public databases Fibrinogen
<4g/ml 19 8 11
0.061

at the mRNA level, we performed immunohistochemistry to detect Position

Pyriform sinus 33 18 15
0.412
others 7 5 2
the expression of MMP13 and SERPINB2 at the protein level. We Medium-High 15 6 9
detected the expression of MMP13 and SERPINB2 in paraffin- SERPINB2
Low-No 25 17 8
0.083

embedded tissues of 40 hypopharyngeal cancer patients. The ages all 40 23 (57.5%) 17 (42.5%)

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Table 2: Correlation between expression levels of SERPINB2 and

clinicopathological parameters of hypopharyngeal cancer patients.
Variable n MMP13 expression level P

Medium-High Low-No
<60 19 6 13
Age 0.462
>=60 21 9 12
Male 39 14 25
Gender 0.191
female 1 1 0
II III 25 8 17
T stage 0.354
IV 15 7 8
Negative 11 8 3
N stage 0.005
positive 29 7 22
Yes 35 13 22
Smoke 0.902
no 5 2 3
Yes 33 11 22
Alcohol 0.237
no 7 4 3
>=4g/ml 21 10 11 Figure 4: Representative images of differences in the expression of MMP13
Fibrinogen 0.165 and SERPINB2 in hypopharyngeal cancer tissues.
<4g/ml 19 5 14
Pyriform sinus 33 13 20
Position 0.591
others 7 2 5
Medium-High 23 6 17
SERPINB2 0.083
Low-No 17 9 8
all 40 15 (37.5%) 25 (62.5%)

Table 3: Univariate and multivariate analyses of factors associated with OS in

hypopharyngeal cancer patients.
Univariate analysis Multivariate analysis
Variables
HR (95% CI) P HR (95% CI) P
Age 0.643 (0.205-1.974) 0.44
Gender 3.062 (0.199-47.10) 0.42
T stage 0.913 (0.271-3.071) 0.88
N stage 6.060 (1.112-11.83) 0.04 Figure 5: Relationship between MMP13 (a) and SERPINB2 (b) expression
Smoke 4.181 (0.962-18.18) 0.05 and overall survival in hypopharyngeal cancer patients.
Alcohol 3.982 (1.008-15.74) 0.04
Fibrinogen 0.830 (0.261-2.640) 0.75
Position 1.193 (0.278-5.125) 0.81
MMP13 3.691 (1.157-11.78) 0.02
SERPINB2 0.2305 (0.072-0.739) 0.01 0.120 (0.015-0.929) 0.04

with an AUC of 0.650 (95% CI = 0.500–0.800, (Figure 6A)) and

0.750 (95% CI = 0.500–1.000, (Figure 6B)). Diagnostic accuracy
can be enhanced by combining biomarkers [13], so we explored
whether the combined expression levels of MMP13 and SERPINB2
could improve the diagnosis of early recurrence. First, we defined
increased MMP13 expression and decreased SERPINB2 expression
as the high-risk group and the rest as the low-risk group. The results
showed no significant change in AUC (0.744, 95% CI = 0.688–0.800,
(Figure 6C)). Then, another classification method was adopted to
test whether the diagnostic accuracy could be improved. We defined
the high-risk group as increased MMP13 expression and decreased
SERPINB2 expression, the low-risk group as decreased MMP13
Figure 6: ROC analysis for MMP13 (a), SERPINB2 (b), and combined
expression and increased SERPINB2 expression, and the rest as a
diagnostic markers (c, d) in early recurrence.
medium risk group. The results showed that the AUC was 0.775 (95%
CI = 0.688–0.800, (Figure 6D)). After the combined application of
of hypopharyngeal cancer are few. According to bioinformatics
markers, the diagnostic accuracy did not improve significantly and
prediction and immunohistochemical results, this study reported
the markers had only moderate predictive value for early recurrence.
the expression and correlation of MMP13 and SERPINB2 in
This may be due to the small sample size; in future studies, we will
hypopharyngeal cancer for the first time. MMP13 and SERPINB2 can
increase the sample size to look for additional markers suitable for
be used to evaluate prognosis and early recurrence of hypopharyngeal
predicting early recurrence.
cancer. This is consistent with a previous study of MMP13 and
Discussion SERPINB2 in osteosarcoma [14].
With the development of molecular biology and In the present study, MMP13 showed a weak negative
immunohistochemistry, the prognostic indicators of head and correlation with SERPINB2, but no statistical significance. MMP13
neck tumors are increasing, but studies of molecular markers and SERPINB2 are important regulatory components of the ECM

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Ji X
network and play an important role in tumor ECM remodeling.
SERPINB2 is an inhibitor of urokinase and ultimately plasmin, the
latter is an activator of pro-MMP13. But there is a lack of research on
the relationship between MMP13 and SERPINB2. As a member of
the serpin superfamily, SERPINB1 and SERPINB2 share 24% amino
acid sequence identity [15]. Several previous studies have reported
a correlation between MMP13 and SERPINB1. In liver fibrosis, Hu
et al found that by knocking down SERPINB1, MMP13 expression
was upregulated [16]. Ohkouchi et al found that Inhibitor of Growth
(ING)-2 combined with histone H3 trimethylation at lysine 4
(H3K4me3) participated in chromosome remodeling to activate
MMP13 and SERPINB1 was upregulated with overexpression of
ING2 [17]. Transforming growth factor (TGF)-β is an important
cytokine in ECM remodeling [18]. Studies have shown that TGF-β
regulates both MMP13 and SERPINB2 [19,20]. Other studies [21]
have shown that glucose stimulated mRNA expression of MMP13,
while SERPINB1 suppressed expression of the mRNAs induced
by glucose. Matrix components such as MMP13 and SERPINB2
have complex regulatory relationships, and their specific regulatory
mechanisms require further study.
In HNSCC, recurrence within 1 year following surgery is called
early recurrence, and the prognosis of patients with early recurrence
is very poor [22,23]. Currently, there are no markers to predict early
recurrence of HNSCC. In this study, we found that MMP13 and
SERPINB2 alone or in combination played a role in distinguishing
early and non-early recurrence of hypopharyngeal cancer, although
these results need to be confirmed with a larger sample size. We
expect that additional markers could be used for early diagnosis of
hypopharyngeal cancer.
This study has some limitations. First, this study was only based
on public databases and patient tissues; the role of MMP13 and
SERPINB2 needs to be further verified in vitro. Secondly, the sample
size of the study is small and all of our patients are from North-East
of China. We will further confirm our experimental conclusion with
a larger sample size.
Conclusion
Our study assessed the role of MMP13 and SERPINB2 in
hypopharyngeal cancer. The results showed that MMP13 may be
an oncogene while SERPINB2 may be an anti-oncogene and could
serve as a prognostic and diagnostic factor for hypopharyngeal cancer
patients. We believe MMP13 and SERPINB2 could be promising
biomarkers and drug targets for HNSCC.
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