DOI:http:dx.doi.org10.7314APJCP.2015.16.18.8579 Tissue Expression and Serum Levels of Angiopoietin-Like Protein 4 in Breast [602423]

DOI:http://dx.doi.org/10.7314/APJCP.2015.16.18.8579 Tissue Expression and Serum Levels of Angiopoietin-Like Protein 4 in Breast
Cancer Progression: Link to NF- κB /P65 Activity
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 8579

RESEARCH ARTICLE

Significance of Tissue Expression and Serum Levels of
Angiopoietin-like Protein 4 in Breast Cancer Progression: Link
to NF- κB /P65 Activity and Pro -Inflammatory Cytokines
Noha M Shafik1*, Dareen A Mohamed2, Asmaa E Bedder2, Ahmed M El-Gendy33
Abstract
Background: The molecular mechanisms linking breast cancer progression and inflammation still remain
obscure. The aim of the present study was to investigate the possible association of angiopoeitin like protein 4
(ANGPTL4) and its regulatory factor, hypoxia inducible factor- 1 α (HIF -1α), with the inflammatory markers
nuclear factor kappa B/p65 (NF- κB /P6 5) and interleukin-1 beta (IL- 1β) in order to evaluate their role in
inflammation associated breast cancer progression. Materia ls and Methods: Angiopoietin-like protein 4
(ANGPTL4) mRNA expressions were evaluated using quantitative real time PCR and its pr otein expression
by immunohistochemistry. DNA binding activity of NF- κB /P65 was evaluated by transcription factor binding
immunoassay. Serum levels of ANGPTL4, HIF- 1α and IL-1β were immunoassayed. Tumor clinico-pathological
features were investigated. Results: ANGPTL4 mRNA expressions and serum levels were significantly higher
in high grade breast carcinoma (1.47±0.31 and 184.98±18.18, respectively) compared to low grade carcinoma
(1.21±0.32 and 171.76±7.58, respectively) and controls (0.70±0.0 2 and 65.34±6.41, respectively), ( p<0.05). Also,
ANGPTL4 high/moderate protein expression was positiv ely correlated with tumor clinico-pathological featu res. In
addition, serum levels of HIF- 1α and IL-1β as well as NF-κB /P65 DNA binding activity were significantly higher
in high grade breast carcinoma (148.54±14.20, 0.79±0.03 and 247.1 3±44.35 respectively) than their values in low
grade carcinoma ( 139.14±5.83, 0.36±0.04 and 184.23±37.75, respectively) and controls (33.95±3.11, 0.13±0.03 and
7.83±0.92, respectively), ( p<0.001). Conclusion: ANGPTL4 high serum levels and tissue expressions in advanced
grade breast cancer, in addition to its positive correlation with tumor clinico-patholog ical features and HIF- 1α
could highlight its role as one of the signaling factors involved in breast cancer progres sion. Moreover, novel
correlations were found between ANGPTL4 and the inflammatory markers, IL- 1β and NF -κB/p65, in breast
cancer, which may emphasize the utility of these markers as potential too ls for understanding interactions for
axes of carcinogenesis and inflammation contributed for cancer progression. It is thus hoped that the findings
reported here would assist in the development of new breast cancer management strategies that would promote
patients’ quality of life and ultimately improve clinical outcomes. However, large-scale studies are needed to
verify these results.
Keywords: Breast cancer – angiopoietin –like protein -4 (ANGPTL4) – hypoxia inducible factor-1 alpha
Asian Pac J Cancer Prev, 16 (18) , 8579- 8587

Introduction
Breast carcinoma is the most common cancer in
females and is the principal cause of death among women
globally (Taghavi et al., 2012). Hypoxia and inflammation
associated cancer are important risk factors contributing
to the breast cancer pathogenesis (Philip et al., 2004).
Angiopoietin-like protein 4 (ANGPTL4) is a member
of the angiopoietin family of proteins that have a similar
structure. The native full-length ANGPTL4 exists in the
form of dimeric or tetrameric complexes that can undergo
proteolytic processing to generate the N-terminal coiled-
coil fragment (nANGPTL4) and the COOH-terminal
fibrinogen-like domain (cANGPTL4) (Feingold et al., 2012). It is highly expressed in adipose tissue, liver,
placental tissue, and ischemic tissues (Santulli, 2014).
The roles of ANGPTL4 in human cancers remain
controversial. However, a novel role of ANGPTL4 in
redox-mediated cancer progression has been postulated,
hypothesized to be exhibited through its COOH-terminal
fibrinogen-like domain (Feingold et al., 2012). Several
lines of evidence have indicated that ANGPTL4 can
be stimulated by inflammatory and hypoxic conditions
(Santulli, 2014). Hypoxia observed in tumors has been
noted to be the main cause of cell cycle arrest, apoptosis
and angiogenesis (Xia et al., 2014). Cellular oxygen-
signaling pathway requires the participation of hypoxia-
inducible factors (HIFs), which exist in two types,

1Department of Medical Biochemistry, Faculty of Medicine, 2Department of Pathology, Faculty of Medicine, 3Department of General
Surgery, Faculty of Medicine, Tanta University, Egypt *For correspondence: nohashafik2008@yahoo.com

Noha M Shafik et al
8580 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
oxygen-sensitive alpha subunit (HIF- α) and a constitutive
beta subunit (HIF- β). Both subunits are heterodimeric,
facilitating both oxygen delivery and adaptation to
oxygen deprivation (Li et al., 2011). HIF- 1α is a
transcription factor that binds to hypoxia responsive
element (HRE), which in turn enhances the transcription
of hypoxia-responsive genes. Thus, the survival of tumor
cells is increased under hypoxic conditions (Zhang et
al., 2013). A growing body of evidences highlights the
role of HIF- 1α as a regulator of ANGPTδ4 (Khong et
al., 2013). NF-kB is a sequence-specific transcription
factor that plays a crucial role in linking inflammation
and innate immunity to breast oncogenesis (Laere et al.,
2006). However, cytoplasmic NF- κB is deactivated by
binding of the inhibitory proteins IKB- α, IKB -β, IKB-İ,
p105, and p100. NF-κB activity occurs by its translocation
to the nucleus, binding to κB sites, and regulating target
genes as a result of the phosphorylation and subsequent
degradation of the inhibitory subunits (González-Ramos
et al., 2012). Its constitutive activation is one of the early
key events involved in breast cancer progression. Indeed,
extant studies indicate that NF- κB signaling stimulates
proliferation and prevents apoptosis (Biswas et al., 2003).
The p65 subunit of NF- κB (NF -κB/p65) is responsible
for most of NF-κB’s transcriptional activity (van Loo and
Beyaert, 2011). A cross-talk has been established between
the NF-κB and the HIF pathways, as HIF-1 redox-sensitive
induction might be due to binding at a distinct element
in NF- κB proximal promoter. εoreover, activation of
NF-κB pathway may be the main contributor of HIF -1
induction (Görlach and Bonello, 2008; Wang et al., 2015).
The expression of important molecules in tumorigenesis
such as chemokines, and inflammatory cytokines, all of
which promote tumor cell invasion and angiogenesis is
regulated by NF- κB (Biswas et al., 2003). Iδ -1β is a
member of IL-1 family that plays a central role in immune
and inflammatory response regulation (Rider et al., 2011).
IL-1β was found to be the primary mediator in context of
cancer-associated chronic inflammation (Katanov et al.,
2015). Interestingly, in cancer cells, HIF- 1α was found
to be activated by the proinflammatory cytokine IL-1β in
a NF-κB dependent manner (Tewari et al., 2012) . Thus it
is necessary to focus research efforts on critical molecular
phenomena that may link carcinogenesis and hypoxia to
inflammation in tumor microenvironment. The ultimate
goal of such studies is to propose new treatment strategies
that could improve survival rates and patients’ quality of
life. Thus, The aim of present study was to investigate
the possible association of angiopoeitin like protein 4
(ANGPTL4) and its regulatory factor, hypoxia inducible
factor- 1 α (HIF -1α) with nuclear factor kappa B/ p65
(NF- κB /P65) and interleukin -1 beta (IL- 1β) in order
to evaluate their role in inflammation associated breast
cancer progression.

Materials and Methods
Informed written consent was obtained from all patients
prior to commencing the study. The study protocol was
approved by the Local Research Ethics Committee, Tanta
University and was in accordance with the principles of the Declaration of Helsinki II. This study included 74 women
aged 40-56 years, who were admitted to the surgical
department of Tanta University Hospitals for breast
surgeries. All participants underwent clinical examination
and routine laboratory investigations prior to being divided
into two groups. Group I (n = 20) comprised of women
who underwent surgical removal of benign breast lesions.
As a part of this procedure, normal breast tissue samples
were taken in the vicinity of the lesions, and served as
controls. Group II (n = 54) consisted of female patients
that had primary invasive breast carcinoma (confirmed
by fine-needle aspiration) with no other primary cancers,
all of whom underwent modified radical mastectomy or
quadrantectomy with axillary clearance. Breast cancer
biopsies were taken from the apparent lesion, processed by
standard oncological procedures, studied and graded by a
specialized pathologist. According to the histopathological
grading, Group II was further subdivided into Group IIa
consisting of 10 patients with low-grade cancer (Grade
I) and Group IIb consisting of 44 patients with high-
grade cancer (Grade II & III). None of the patients with
invasive breast carcinoma had received any neoadjuvant
therapy. Patients with other malignancies, any endocrinal
disturbances, or systemic infections, those that received
neoadjuvant therapy and smokers were excluded from
the study. During the surgical procedures, tissue samples
were obtained and processed by standard oncological
procedures. These were subsequently divided into two
portions, whereby one portion was kept in liquid nitrogen
for ANGPTL4 mRNA gene expression investigations,
while the other was kept in 10% formalin solution for
histopathological and immunohistochemistry studies.

Biochemical and immunoassays:
All study participants followed overnight fasting
protocol prior to the morning surgery. Immediately before
the induction of general anesthesia, their early morning
venous blood samples (10 ml in plain vacutainer tubes)
were taken and transferred slowly into a dry sterile
centrifuge tube. The samples were allowed to clot at room
temperature, before being centrifuged at 2000 rpm for 10
minutes. Finally, serum was separated and stored at −70șC
for different estimations.
Enzyme-linked immunosorbent assays (ELISA)
were used to detect the levels of serum ANGPTL4
(Cat # DY3485, R&D Systems, ANGPTL4 Duo kit,
Minneapolis, USA), the levels of serum HIF- 1α (R&D
Systems, Minneapolis, MN 55413, USA) and serum
levels of IL- 1β (Cat # SEA563Hu, Cloud Clone Corp,
USA). All ELISA assays were performed according to
the manufacturer’s instructions.

Preparation o f peripheral blood mononuclear cells
(PBMCs):
PMNCs were prepared using Ficoll-Hypaque
(Pharmacia, Uppsala, Sweden) by means of density
gradient centrifugation. First, 5 ml of Heparinised blood
was layered on Ficoll and, after centrifugation for 30
minutes at 500 xg at room temperature, PBMC were
harvested from the white interphase before being washed
with phosphate buffered saline. The PBMCs samples

DOI:http://dx.doi.org/10.7314/APJCP.2015.16.18.8579 Tissue Expression and Serum Levels of Angiopoietin-Like Protein 4 in Breast
Cancer Progression: Link to NF- κB /P65 Activity
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 8581
were stored at −80oC until required for analysis of NFκB
DNA-binding activity.
NFκB activation was examined by using transcription
factor binding assay kit as described below:

1-Nuclear proteins isolation
Nuclear Extract kit (Cat # 40010 , Active Motif,
CA, USA) was used to isolate nuclear proteins from
PBMCs extract according to the manufacturer’s
instructions. Briefly, the kit provided ice-cold hypotonic
buffer containing, 10 mL KCL, 0.1 mmol/L ethylene
glycol tetraacetic acid, 10 mmol/L HEPES, 0.1 mmol/L
EDTA, 1mmol/L DTT; and protease inhibitors. 0.8 of
this buffer was used. 20 min incubation of homogenat es,
followed by adding 50µL of 10% Nonidet P-40, then
vortexed for 30 s and centrifuged for 2 min at 4°C in an
Eppendorf centrifuge. After decantation of supernatants,
the nuclear pellets were suspended in an ice-cold
hypertonic buffer, and then wash ed with hypotonic buffer
without Nonidet P- 40 (Gong et al., 2002). Next, they
were incubated on ice for 20 min at 4°C, mixed and
centrifuged for 12 min at 4°C. The supernatants were
collected as nuclear extracts and stored at −80 °C .
Concentrations of total proteins in the samples were
determined according to the method described by
Bradford (Bradford, 1976).

2-Determination of DNA-binding activity of NF-kB/p65
The ELISA-based NFκB /p65 transcription factor
assay kit was used to evaluate NFκB/p65 DNA-
binding activity (Cat # 40096, Active Motif, CA, USA),
according to the manufacturer’s instructions. Briefly,
the kit provided a duplexed NFκB oligonucleotide
containing ϰB consensus seq uence, which was attached
to the surface of 96-well plates. NFκB/p65 was
detected by incubation with specific primary antibod y
directed against the activated forms of NFκB/P65
contained in the nuclear extract . Then, incubation with
secondary antibody conjugated to horseradish
peroxidase was performed and used for colorimetric
scoring (Van Laere et al., 2006). The absorbance was
measured on an ELISA reader at 450 nm with a
reference wave length of 655 nm.

Estimation of ANGPTL4 mRNA expression levels by
real-time PCR
RNA extraction, cDNA synthesis and Real-time
quantitative PCR: Total RNA was extracted from breast
tissue samples using RN easy Mini Kit (Roche Diagnostics,
GmbH, Mannheim, Germany) according to manufacturer’s
instructions. RNA was eluted, its concentration was
measured spectrophotometrically and RNA samples were
subsequently stored at −80°C until required. Each RNA
sample was converted to cDNA using RT Superscript II
(Cat # K1632, Thermo Scientific Fermentas, St. Leon-Ro,
Germany), dNTP, and random primers (Roche, Mannheim,
Germany) according to the manufacturer’s instructions.
cDNA corresponding to the RNA was used as a template
for real-time PCR. PCR reactions were performed using Power SYBR Green PCR Master Mix and 7500 Fast
Real-Time PCR System (Applied Biosystems, CA, USA).
Sequence-specific primers were as follows: ANGPTL4:
forward5’ -ATTCTTTCCAGCGGCTTCTG- 3’, reverse:
5’-GAGGACTGGAGACGCGGAG- 3’ (According to
gene bank accession No: NM_001039667), β-actin:
forward: 5’- TGGCATTG CCGACAGGATGCAGAA- 3’,
reverse: 5’ -CTCGTCA TACTCCTGCTTGCTGAT- 3’
(According to gene bank accession No: NM_001101.3).
β-actin primers were used as an internal control. Real-
Time PCR was performed according to the manufacturer’s
instructions and comprised of denaturation at 95 ˚C for 10
s, followed by 40 cycles of 95 ˚C for 5 s and 60 ˚C for 20 s.
Presence of the expected amplification fragments without
unanticipated products and primers was confirmed by
melting curve analysis. Comparative Ct (threshold cycle)
method was used to determine relative product amounts,
according to the Applied Biosystems instructions. All
expression data were normalized by dividing the target
amount by the amount of β -actin for each sample.

Histochemical and immunohistochemistry studies:
For histopathological study, breast tissue biopsies
were embedded in paraffin. For this purpose 5 μm slices
were stained with hematoxylin & eosin (H & E). For
immunohistochemical staining, 4 mm thick sections
were formed. The tissue sections were deparaffinized
and rehydrated. Slides were incubated in 3% H2O2 for
10 minutes to reduce nonspecific background staining
arising due to endogenous peroxides. For antigen
retrieval, specimens were heated for 20 min in 10 mmol/l
citrate buffer (pH 6.0) in a microwave oven (700 W).
Following incubation with Ultra V Block (Lab Vision
Corporation, Fremont, California, USA) for 7 min at
room temperature to block background staining, slides
were incubated with ER (rabbit polyclonal, ab37438), PR
(rabbit monoclonal, ab16661), HER-2 (rabbit monoclonal,
ab134182), KI67 (mouse monoclonal, ab15580), CK5/6
(mouse monoclonal, ab86974), EGFR (rabbit monoclonal,
ab2430) and Mouse monoclonal to angiopoietin-like 4
(1:150 life science inc. Cat # MAB019Ra21) overnight at
room temperature in a humid chamber. Antibody binding
was detected using the Ultra Vision LP Detection System
(Lab Vision Corporation) according to the manufacturer’s
recommendations. Color development was performed
with 3, 30-diaminobenzidine and counterstained with
hematoxylin. Internal adipose breast tissue served as
positive control for ANGPTL4, whereas negative controls
were obtained by replacing the primary antibody with
non-immune immunoglobulin G. Immunostaining results
for ER, PR, HER2/new, EGFR and CK5/6 were used for
molecular subdivision of breast cancers (Holliday and
Speirs, 2011).
Immunostaining results pertaining to ANGPTL4 were
evaluated by image analysis (Q win Leica software). The
percentage of positive cells was rated by assigning the 0
score to 0-5%, 1 score to 6-25%, a score of 2 to 26-50%,
and 3 to more than 50%. Similarly, the staining intensity
was rated by assigning 0 to no staining, 1 to weak staining,
2 to moderate staining, and 3 to strong staining. The
percentage and intensity scores were added to an overall

Noha M Shafik et al
8582 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
score, whereby the ANGPTL4 protein expression with an
overall score of 0- 2 was designated as ‘low/neg ative and
that with an overall score of 3-6 was designated as ‘high/
moderate’ (Yi et al., 2013).

Statistical analysis:
The data were analyzed using statistical package for
the social science (SPSS) version 20.0 software (SPSS
Inc., Chicago, IL, USA). Quantitative data expressed as
mean and standard deviation. Categorical variables were
compared using Chi-square test. Multiple comparisons
were performed by one-way analysis of variance
(ANOVA) followed by Tukey’s post-hoc test. Correlations
were analyzed using the Pearson test. Receiver operating
characteristics (ROC) analysis was used to identify the
optimal threshold values of the studied parameters.

Results
A statistical comparison using ANOVA test followed
by Tukey’s test was performed between the studied groups
with respect to the age and laboratory biochemical findings,
as demonstrated in Table 1. No statistically significant
differences were detected between the studied groups
regarding age ( p >0.05). On the other hand, ANGPTL4
mRNA relative expression levels were significantly higher
in Group ІІb (1.47 ± 0.31) compared to Group ІІa (1.21
± 0.32). Once again, both groups had higher values than
those measured in the controls (Group І) (0.7 ± 0.02), ( p <
.001). In addition, in high grade breast carcinoma patients (Group ІІb), the serum levels of ANGPTL4, HIF- 1α, NF-
κβ/P65 and IL-1β (184.98 ± 18.18 ng/ml, 148.54 ± 14.20
μg/ml, 0.79 ± 0.03 and 247.13 ± 44.35 pg/ml, respectively)
were significantly higher than those in low-grade cancer
group (ІІa) (171.76 ± 7.58 ng/ml, 139.14 ± 5.83 μg/ml,
0.36 ± 0.04 and 184.23 ± 37.75 pg/ml, respectively) and
both were higher than those measured in the control
group (65.34 ± 6.41 ng/ml, 33.95 ± 3.11 μg/ml, 0.13
± 0.03 and 7.83 ± 0.92 pg/ml, respectively) ( p <0.001).
Table (2) presents the immunohistochemical findings
of cancer and control groups, by using chi-square test,
significant differences in ANGPTL4 protein expression
were revealed between control and study groups. More
specifically, high/moderate ANGPTL4 expression was
found in 86.4% (38/44) of the high-grade breast cancer
group (Group ІІb), and 40% (4/10) of low -grade breast
cancer group (Group ІІa), compared to only 25% (5/20) of
the control cases. When the studied parameters in breast

Table 2. Immunohistochemical Results of ANGPTL4
in Control and Cancer groups

Group І Group ІІa Group ІІb
(control) (low grade cancer) (high grade cancer)
(n= 20) (n= 10) (n=44)

Low/negative ANGPTL4 expression
15(75%) 6 (60%) 6 (13.6%)
High/moderate ANGPTL4 expression
5(25%) 4(40%) 38 (86.4%)

ANGPTL4: angiopoietin like protein 4; P value was cal culated by chi-
square test; P was considered significant at <0.001; *S ignificant

Table 1. Demographic and Laboratory Findings of the Studied Groups
Group І Group ІIa Group ІІb ANOVA
(control) (low grade cancer) (high grade cancer)
(n= 20) (n=10) (n= 44) f P-value
Age (years) 49.1±5.3 47.3±5.3 49.9±5.3 1.054 0.354
ANGPTL-4 mRNA relative expression 0.70±0.02*# 1.21±0.32# 1.4 7±0.31 55.862 <0.001*
Serum ANGPTL- 4 (ng/ml) 65.34±6.41*# 171.76±7.58# 184.98±18.18 462.2 82 <0.001*
Serum HIF- 1α (μg/δ) 33.95±3.11*# 139.14±5.83# 148.54±14.20 722.669 <0. 001*
NFKB/p65 binding activity 0.13±0.03*# 0.36±0.04# 0.79±0. 03 461.288 <0.001*
Serum IL-1β (pg/ml) 7.83±0.92*# 184.23±37.75# 247.13±44.35 287.3 45 <0.001*

ANGPTL4: angiopoietin like protein 4; HIF-1a: hypox ia inducible factor-1 alpha; NFKB/p65: nuclear fact or kappa B/p65 subunit ; IL-1 :
interleukin-1beta. *Data are presented as the mean ±SD; * statistically significant at p<0.05; * as compared to low grade breast carcinoma group ;
#as compared to high grade breast carcinoma group
Table 3. Pearson’s Correlations between the Studied Parameters
Age
(years) ANGPTL- 4
mRNA relative
expression Serum
ANGPTL- 4
(ng/ml) Serum
HIF- 1α
(μg/L) NFKB/p65
binding
activity
ANGPTL-4 mRNA relative expression

Serum ANGPTL-4 (ng/ml)

Serum HIF- 1α (μg/δ)

NFKB/p65 binding activity

Serum IL- 1β (pg/ml) r
P-value
r
P-value
r
P-value
r
P-value
r
P-value 0.047
0.738
0.103
0.459
0.235
0.087
0.034
0.807
0.04
0.772

0.297
0.020*
0.495
<0.001*
0.328
0.015*
0.492
<0.001*

0.495
<0.001*
0.41
0.002*
0.488
<0.001*

0.394
0.005*
0.657
<0.001*

0.786
<0.001*
ANGPTL4: angiopoietin like protein 4; HIF-1a: hypoxia induci ble factor-1 alpha ; NFKB/p65: nuclear factor kappa B/p65 subunit ; IL-1 β: interleukin-
1beta .P was considered significant at <0.05; *Significant

DOI:http://dx.doi.org/10.7314/APJCP.2015.16.18.8579 Tissue Expression and Serum Levels of Angiopoietin-Like Protein 4 in Breast
Cancer Progression: Link to NF- κB /P65 Activity
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 8583
Table 4. Association between ANGPTL4 Protein Expression and Clinico-pathological Features in Cancer Group

Group ІІ
(n=. 54) ANGPTL4
Total Low/negative expression ANGPTL4
High/moderate expression
P value
N % N % N %
Age (years) <45 20 37 2 10 18 90
>45 34 63 10 29.5 24 70.5 0.098
Tumor size T1 5 9.3 4 80 1 20
T2 21 38.9 3 14.3 18 85.7 0.011*
T3 19 35.2 4 21.1 15 78.9
T4 9 16.7 1 11.1 8 88.9
Lymph node metastasis N0 10 18.5 7 70 3 30
N1 30 55.6 3 10 27 90 <0.001*
N2 12 22.2 2 16.6 10 83.4
N3 2 3.7 0 0 2 100
Grade I 10 18.5 6 60 4 40
II 35 64.8 3 8.6 32 91.4 0.002*
III 9 16.7 3 33.3 6 66.7
Molecular type Non basal type 15 27.8 6 40 9 60
Basal type 39 72.2 6 15.4 33 84.6 0.047*
Proliferation index Negative 12 22.2 7 58.3 5 41.6 0.02*
Positive 42 77.8 5 11.9 37 88.1
ANGPTL4: angiopoietin like- 4; P value was calculated by chi-square test; P was considered significant at <0.05; *Significant

Figure 1.Immunohistochemical staining of ANGPTL4
in control case showed negative ANGPTL4
immunohistochemical expression in normal breast
tissue (X400)

Figure 2. Immunohistochemical Staining of ANGPTL4
in case of Invasive Breast Carcinoma Grade I Showed
Low ANGPTL4 Immunohistochemical Expression
(X200)
carcinoma were examined in table (3), no significant
correlations were found between age and other studied
parameters. Serum levels of ANGPTL4 exhibited a
significant positive correlation with HIF- 1α, NF -κβ/p65
DNA binding activity and IL-1β ( r = 0.495, r = 41 and r Figure 3. Immunohistochemical Staining of ANGPTL4
in Case of Invasive Breast Carcinoma Grade II Showed
High ANGPTL4 Immunohistochemical Expression
(X400)

Figure 4. Immunohistochemical staining of ANGPTL4
in case of invasive breast carcinoma grade III showed
moderate ANGPTL4 immunohistochemicalexpression
(X400)
= 0.488, respectively) ( p<0.05) and with relative mRNA
expression ( r =0.297) ( p <0.05). In addition, serum levels
of HIF- 1α showed positive correlations with NF -κβ/p65
DNA binding activity and IL-1β, as well as relative mRNA
expression ( r = 0.394, r =0.657 and r = 0.495, respectively)

Noha M Shafik et al
8584 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
(p < 0.05). NF- κβ/p65 DNA binding activity exhibited
positive correlations with IL- 1β and with ANGPTδ4
relative mRNA expression ( r = 0.786 and r = 0.328) ( p
<0.05). Finally, IL- 1β showed positive correlations with
ANGPTL4 relative mRNA expression ( r = 0.492). Table
(4) represents histopthological and immunohistochemical
findings. As can be seen from the data, an elevated
ANGPTL4 protein expression in breast carcinoma cases
was significantly associated with lymph node metastasis,
grade, tumor size and proliferation markers. However,
there was no correlation between ANGPTL4 protein
expression and patient’s age. As 54 cases were diagnosed
as invasive ductal carcinoma (Group ІІ), 10/54 (18.5%)
cases were classified as Grade I, 35/54 (64.8%) cases were
diagnosed as Grade II, and 9/54 (16.7%) cases were of
Grade III. Thus, patients in Group I were considered as
having low-grade cancer, whereas those in Group II and
III were considered as having high-grade cancer. With
respect to the tumor size, 5/54 (9.3%) cases were classified
as T1, 21/54 (38.9%) cases were T2, 19/54 (35.2%) cases
were T3, and 9/54 (16.7%) cases were classified as T4.
Moreover, 10/54 (18.5%) cases showed no lymph node
metastasis, while 30/54 (55.6%) cases were N1, 12/54
(22.2%) cases were N2 and 2/54(3.7%) cases were N3.
According to ER, PR, HER-2/new, CK5/6 and EGFR
expression and the breast cancer cases could be further
classified based on the molecular subtypes. According to
this grouping, 39/54 (72.2%) cases were basal subtypes (as
they showed ER-, PR-, HER2-, CK5/6+ and or EGFR+)
and 15/54(27.8%) cases were of non-basal subtype, which
were further divided into luminal A (ER+, PR+/-, HER2- )
or luminal B (ER+, PR+/-, HER2+). Finally, with respect
to KI-67 proliferation index, 12/54 (22.2%) cases were
KI-67 negative and 42/54(77.8%) were KI-67 positive.
These results are confirmed in Figures 1, 2 and 3.

Discussion
The novel adipocytokine, angiopoietin-like protein 4,
is posited to play an important role in cancer progression
and pathogenesis (Tan et al., 2012). Various types of
human cancers were found to have altered expression of
ANGPTL4 (Li et al., 2011; Kim et al., 2011). However,
its role in breast carcinoma as well as its relevance to
inflammation-related cancer progression remains
controversial. The present study revealed that ANGPTL4
serum levels as well as its protein and mRNA expressions
were significantly higher in cancerous breast tissue
compared to normal breast tissue samples. In addition,
increased tumor size, lymph node metastasis, high-grade
breast carcinoma and proliferation index were associated
with higher ANGPTL4 tissue expression, suggesting that
ANGPTL4 plays a role in tumor progression and cancer
cell differentiation. In line with these findings, increased
mRNA and protein expressions of ANGPTL4 have been
previously reported in other cancer types, such as oral
Kaposi sarcoma and ovarian carcinoma (Hu et al., 2011;
Brunckhorst et al., 2014). This phenomenon can be
explained by the fact that tumor-derived ANGPTL4
facilitated disruption of vascular endothelial cell-cell
junctions and tumor cell extravasation into other tissues, which ultimately leads to micro metastases (Padua et al.,
2008). The same finding was reported by Zhang et al.,
2012 who concluded that breast cancer metastasis was
promoted by ANGPTL4. Similarly, Yi et al., 2013 reported
that, in esophageal squamous cell carcinoma, higher
expression levels of ANGPTL4 were correlated with more
advanced tumor stages and more adverse clinical
outcomes. In the present study, significantly higher protein
expressions of ANGPTL4 in basal type of invasive duct
carcinoma were noted relative to the non-basal type. This
finding is in agreement with the results reported by
Yotsumoto et al., 2013 who found that ANGPTL4
expression was involved in aggressive tumor metastasis
to lungs and brain and in tumor development in triple
negative breast cancer xenograft model. In the current
study, there were significant differences between
ANGPTL4 and proliferation index of breast cancer, thus
concurring with Brunckhorst et al., 2014 who reported
that ANGPTL4 significantly increased the number of
proliferating ovarian cancer cells and KI-67. ANGPTL4-
induced breast carcinoma proliferation may be achieved
through tumor-derived ANGPTL4 interaction with
integrins, which stimulate the prosurvival pathways,
phosphoinositide 3-kinases/ protein kinase B (PI3K/
PKBα) and extracellular signal -regulated kinase (ERK)
through NADPH oxidase-dependent production of O2-
and proto-oncogene tyrosine-protein kinase Src, thus,
promoting anoikis and tumor growth (Zhu et al., 2011).
Further supporting the tumor induction role of ANGPTL4,
Tan et al., 2012 reported that ANGPTL4 functions as a
negative regulator of apoptosis. It is particularly
noteworthy that, under tumor hypoxic conditions,
ANGPTL4 is regulated by HIF- 1α (Wagner et al., 2011).
In addition, in microarray analysis, ANGPTL4 was found
to be the only gene bound and highly induced by the two
different regulators, peroxisome proliferators-activated
receptor family (PPARβ/į) and HIF -1α (Inoue et al.,
2014). Contrary to the results of the current study, Ng et
al., 2014 and Galaup et al., 2006 reported that ANGPTL4
suppressed growth, angiogenesis and metastasis in
hepatocellular carcinoma and melanoma. Tumor
progression and treatment response are highly affected by
hypoxia (Li et al., 2015). The current study revealed a
significant increase in serum HIF- 1α levels in high grade
breast cancer patients compared to low grade cancer and
control subjects. This finding can be explained by the fact
that protein translation and proteasome-dependent
degradation are impaired by hypoxia, which affects the
HIF- 1α levels (Anad et al., 2011). Furthermore, these
results are in line with the previous findings indicating
that mature and functional HIF- 1α are generated by cells
in response to hypoxia in solid tumors (Kafshdooz et al.,
2014) which plays a pivotal role in tumor progression,
infiltration and metastasis (Unwith et al., 2015). The
results yielded by this study also demonstrated that serum
HIF- 1α was significantly correlated with ANGPTL4
serum and expression levels. This finding is in keeping
with the notion of an existing crosstalk or interplay
between ANGPTL4 and HIF- 1α, particularly in cancer
progression. In agreement, Potente et al., 2011 reported
that tumor angiogenesis is orchestrated by angiogenic

DOI:http://dx.doi.org/10.7314/APJCP.2015.16.18.8579 Tissue Expression and Serum Levels of Angiopoietin-Like Protein 4 in Breast
Cancer Progression: Link to NF- κB /P65 Activity
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 8585
factors, including angiopoietin-like proteins and enhanced
by HIF- 1α. In addition, Inoue et al., 2014 found five HIF-
1α binding sites, identified under hypoxia in the
ANGPTL4 gene locus, suggesting that hypoxia is an
activator of ANGPTL4 gene. The authors further asserted
that pro-inflammatory cytokines in tumor microenvironment
and the subsequent activation of NF-kB transcription
factor play a role in tumor pathogenesis and progression.
In this context, we further investigated the serum levels
of IL- 1β and DNA -binding capacity of NF-kB/p65 in
breast cancer. The present study showed that the DNA
binding capacity of the p65 subunit of NF-κB significantly
increased in PMNCs from high-grade breast cancer
patients compar ed to those with low-grade cancer and the
control subjects. This finding is consistent with the results
reported by Wang et al., 2013 who noted that, in human
breast cancer cell line, NF-κB transcriptional activity was
increased secondary to overexpression of NF- κB/p65.
Consequently, cancer progression and metastasis can be
promoted by active NF- κB signaling through induction
of several cell cycle, anti-apoptotic and chemotactic
regulatory genes that enhance tumor (Xia et al., 2014). It
can thus be postulated that an existing interplay between
NF-κB and HIF- 1α particularly in carcinogenesis has been
established. The present study revealed that serum HIF- 1α
was significantly correlated with NF-κB/P65 activity and
that both played an important role in tumor progression.
HIF- 1α protein accumulation under hypoxia requires the
participation of NF-κB, which is a critical transcriptional
activator of HIF- 1α (Taylor and Cummins, 2009).
Moreover, authors of several studies focusing on different
cancer types reported that hypoxia-inducible factor- 1α is
up-regulated by a hypoxia-dependent transcription by
NF-κB (Yoshida et al., 2013), stimulating epithelial to
mesenchymal transition (Cheng et al., 2014), thus
promoting tumor progression and angiogenesis. NF-kB
transcription factor plays a crucial role in tumor
development through transcriptional regulation of genes
associated with tumor growth, invasion and metastasis,
including cytokines such as IL- 1β (Tewari et al., 2012).
The present study revealed that IL- 1β serum levels were
significantly increased in patients with high-grade breast
cancer compared to those with low-grade cancer and to
the control subjects. At the cellular level, IL-1β exerts its
effects through binding of IL-1 receptor I (IL-1RI) and
recruitment of the co-receptor, interleukin- 1 receptor
accessory protein, thus inducing cellular changes and
signal transduction (Aggarwal et al., 2006). In cancer cells,
several authors observed that IL-1β acts via autocrine and/
or paracrine mechanisms, positing that it may regulate the
expression of angiogenic factors, such as IL-8, thus
promoting angiogenesis and tumor progression (Katanov
et al., 2015). In addition, IL-1β contributes to the
progression of tumors through stimulation of cell growth
and differentiation and the inhibition of apoptosis of
altered cells at the inflammatory site (Snoussi et al., 2005).
The results yielded by the present study are in good
agreement with those reported by Saijo et al. 2002, who
demonstrated that interleukin IL-1β autocrine production
in pancreatic carcinoma cell lines induced tumor growth
and confers chemoresistance. With respect to the positive correlation between IL-1β and HIF- 1α, IL-1β and TNF- α
have been shown to increase HIF- 1α activity in breast
cancer through activation of NF- κβ pathway (Tewari et
al., 2012). In addition, IL- 1β mediated up -regulation of
HIF- 1α via an NF -kB/Cyclooxygenase-2 pathway was
reported to identify HIF- 1 as a critical link between
inflammation and oncogenesis progression (Jung et al.,
2003). Nevertheless, in this study, strong positive
correlations between ANGPTL4 levels and HIF- 1α, NF –
κB/p65 activity and Iδ -1β in breast cancer patients were
noted, reflecting ANGP TL4 involvement in inflammatory
pathways associated with breast cancer progression.
Collectively, serum levels of ANGPTL4, HIF- 1α, and
IL-1β and NF-κB/p65 activity were significantly
associated with breast carcinoma pathogenesis and
progression.
In conclusion, ANGPTL4 high serum levels and tissue
expressions in advanced grade breast cancer, in addition
to its positive correlation with tumor clinico-pathological
features and HIF- 1α could highlight its role as one of the
signaling factors involved in breast cancer progression.
Moreover, novel correlations were found between
ANGPTL4 and the inflammatory markers, IL- 1β and
NF-κB/p65, in breast cancer, which may emphasize the
utility of these markers as potential tools for understanding
interactions for axes of carcinogenesis and inflammation
contributed for cancer progression. It is thus hoped that
the findings reported here would assist in the development
of new breast cancer management strategies that would
promote patients’ quality of life and ultimat ely improve
clinical outcomes. However, large-scale studies are needed
to verify these results.

Disclosures
Funding: No grants or funding have been received
for this study.
Conflict of interest: the authors declare that they have
no conflict of interests .

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