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Clinicopathologic and prognostic implications of progranulin
in breast carcinoma
LI Li-qin, HUANG Hui-lian, PING Jin-liang, WANG Xiao-hong, ZHONG Jing and
DAI Li-cheng1
Huzhou Key Laboratory of Molecular Medicine, Affiliated Central Hospital of
Huzhou Teachers College, Huzhou, Zhejiang 313000, China (Li LQ, Huang HL,
Zhong J and Dai LC)
Department of Pathology, Affiliated Central Hospital of Huzhou Teachers College,
Huzhou, Zhejiang 313000, China (Ping JL)
Department of Breast Surgery, Affiliated Central Hospital of Huzhou Teachers
College, Huzhou, Zhejiang 313000, China (Wang XH)
1
Correspondence to: DAI Li-cheng, Huzhou Key Laboratory of Molecular Medicine,
Affiliated Central Hospital of Huzhou Teachers College, Huzhou, Zhejiang 313000,
China (Tel: 86-572-2023301. Fax: 86-572-2025199. Email: [email protected])
Keywords: breast carcinoma; progranulin; endoglin; angiogenesis;
immunohistochemistry
Abstract
Background
Progranulin is a newly discovered 88-kDa glycoprotein originally
purified from the highly tumorigenic mouse teratoma-derived cell line P. Its
expression is closely correlated with the development and metastasis of several
cancers. However, no immunohistochemical evidence currently exists to correlate
progranulin expression with clinicopathologic features in breast carcinoma biopsies,
and the role of progranulin as a new marker of metastatic risk and prognosis in breast
cancer has not yet been studied. The aim of this study was to investigate the
clinicopathologic and prognostic implications of progranulin expression in breast
carcinoma and its correlation with tumor angiogenesis.
Methods
Progranulin expression was determined immunohistochemically in 183
surgical specimens from patients with breast cancer and 20 tissue samples from breast
fibroadenomas. The tumor angiogenesis-related biomarker vascular endothelial
growth factor was assayed and microvessel density was assessed by counting vascular
endothelial cells in tumor tissues labeled with endoglin antibody.
Results
The relationship between progranulin expression and the clinicopathologic
data were analyzed. Progranulin proteins were overexpressed in breast cancer. The
level of progranulin expression was significantly correlated with tumor size (P=0.004),
lymph node metastasis (P<0.001) and TNM staging (P<0.001). High progranulin
expression was associated with higher tumor angiogenesis, reflected by increased
vascular endothelial growth factor expression (P<0.001) and higher microvessel
density (P=0.002).
Conclusions These results suggest that progranulin may provide a valuable marker
for assessing the metastasis and prognosis of breast cancer, and could provide the
basis for new combination regimens with antiangiogenic activity.
Breast cancer is a major cause of morbidity and mortality in women worldwide. The
progression from normal breast epithelium to metastatic breast cancer is a complex
multistep process resulting from the uncoupling of the interactive systems controlling
cell proliferation and differentiation, thus leading to extensive cellular growth. 1
Numerous molecular markers such as cell-cycle regulators, cell-adhesion proteins and
growth factors have recently been investigated in relation to carcinoma metastasis.
Among these, the overexpression of growth factors and/or growth factor receptors has
been reported to play an important role in the process of metastasis. Progranulin is a
newly discovered 88-kDa glycoprotein originally purified from the highly
tumorigenic mouse teratoma-derived cell line P. Its expression is closely correlated
with the metastasis of breast carcinoma, in addition to stimulating proliferation of
tumor cell growth and development. 2
Tangkeangsirisin and Serrero suggested that progranulin could stimulate migration
and invasiveness in human breast cancer cells.2 Progranulin was highly expressed in
80% of pathologic biopsies of invasive ductal carcinomas, in correlation with
poor-prognosis markers such as tumor grade, p53 expression, and Ki67 index,
compared with 66% of ductal carcinoma in situ biopsies.3 Based on the results of
these studies, we hypothesized that progranulin might provide a novel biological
marker of breast carcinoma invasion and metastasis. However, no
immunohistochemical evidence is currently available to correlate progranulin
expression with clinicopathologic features in breast carcinoma pathologic biopsies, or
regarding the use of progranulin, as a newly discovered growth factor, as a novel
marker of metastatic risk and prognosis in breast cancer.
The aim of this study was to investigate the clinicopathologic and prognostic
implications of progranulin expression in breast carcinoma. The relationships among
microvessel density (MVD) (assessed by immunohistochemical detection of endoglin
(CD105)), vascular endothelial growth factor (VEGF), and progranulin in breast
carcinoma pathologic biopsies were also analyzed. The results of this study provide
histologic evidence for the involvement of progranulin in breast carcinoma
angiogenesis, and its potential clinical significance.
METHODS
Patient selection
On the basis of available archival pathology specimens and case-note reviews, a total
of 203 formalin-fixed, paraffin-embedded tissues from 2007 and 2008 were collected
from the archives of the Department of Pathology of the Huzhou Central Hospital
(Zhe Jiang, China). The 203 tissues included 31 ductal carcinomas in situ (DCIS), 152
invasive ductal carcinomas (IDC), and 20 breast fibroadenomas. No special-type
mammary carcinomas were included in this study. Clinicopathologic information
including age, tumor size, lymph node status, and clinical stage were also included in
the analysis. Twenty patients (all female, mean age 57.1 years) with benign fibromas
were selected as controls. No significant differences were found in age (t=1.53,
2
2
P=0.28), alcohol use (χ =0.16, P=0.89), or smoking habit (χ =0.06, P=0.81) between
the controls and the patients.
Immunohistochemistry
Sections (5 μm) were cut from each tumor and transferred onto glass slides. One
section from each sample was used for hematoxylin and eosin (HE) staining and three
others were used for immunohistochemical staining. The slides were immersed in 10
mmol/L sodium citrate buffer (pH 6.0) for 20 min at 100°C for antigen retrieval.
Sections were placed in 0.5% hydrogen peroxide in methanol for 20 min to block
endogenous peroxidase, and then rinsed in distilled water. They were then rinsed in
phosphate-buffered saline and incubated in blocking buffer for 30 min. The sections
were incubated overnight at 4°C in a mixture of s 1:50 dilution of primary mouse
monoclonal antibody to progranulin (Enzo Life Sciences, USA) and a 1:50 dilution of
primary rabbit polyclonal antibody to CD105 (Abcam, USA). VEGF was detected
using a mouse monoclonal antibody at 1:50 dilution (Dako, Denmark). Polymer
double staining assay kit (Zhongshan Goldbridge, China) was used for co-staining of
progranulin and CD105 (DAB and Ap-Red, separately). VEGF was stained separately,
using a DAB kit (Boster, China). Finally, the sections were counterstained with
hematoxylin. Negative controls were obtained by substituting the primary antibodies
with mouse or rabbit immunoglobulin G.
Evaluation of immunostaining
Progranulin expression was cytoplasmic and was analyzed semiquantitatively. Five
fields at 400× magnification were randomly selected for each section, and 200 cells
were counted in each field (total of 1,000 cells). The percentage of positive cells was
calculated. Staining was classified as negative (-) if < 5% of cells were stained, and
positive if >5% of cells were stained; positive staining was graded from weak/focal (+)
to moderate/focal or diffuse (++), to strong/diffuse (+++).4
The pattern of VEGF immunostaining was graded as: negative (-), if <10% of the
cells were reactive for VEGF; weak (+) if <25% of the cells were positive; moderate
(++) when <50% of the cells were positive; and strong (+++) when >50% of the cells
were positive. Five fields at 400× magnification were randomly selected for each
section, and 200 cells were counted in each field (total of 1,000 cells). The percentage
of positive cells was calculated.4
MVD was evaluated by immunohistochemical staining of tumor vessels for CD105 in
whole tissue sections. Any immunopositive single cell or cluster of cells, clearly
separate from adjacent clusters and from the background, with or without a lumen,
was considered to be an individual vessel. Microvessels in the five most vascularized
areas in a 200× magnification field (0.74 mm2) were counted simultaneously by two
observers, and the average value of the five fields was calculated.
Statistical analysis
The data were stored and analyzed using SPSS 13.0 for Windows software (SPSS.
Inc). The χ2 contingency test was used for categorical variables to determine
associations between groups. Continuous data were analyzed using t-tests. The
correlation between progranulin and VEGF expression was measured using
Spearman’s correlation coefficient. A P value <0.01 was considered to represent a
significant difference (α =0.01).
RESULTS
Expression of progranulin, VEGF, and CD105
Deep yellow or brown progranulin particles were confined to the cytoplasm. Strong
progranulin expression was observed in 111 of 183 carcinoma cases (61%), whereas
only two of the 20 benign tumor cases (10%) showed weak (+) progranulin staining
(Figure 1A). Sporadic progranulin expression was detected in the stroma (Figure 1B),
tumor cells of invasive carcinomas (Figure 1C), and in vascular endothelial cells
(Figure 1I). There was a striking difference in the levels of progranulin expression
between benign and malignant breast tumors. In addition, IDC showed higher levels
of progranulin expression than DCIS (Table 1).
In the benign breast tumor , four of the 20 benign breast tumors (20%) showed weak
VEGF staining (Figure 1D). VEGF expression was focal in the stroma of invasive
carcinomas (Figure 1E). In contrast, strong positive VEGF staining was present in a
diffuse cytosolic pattern in tumor cells from invasive carcinomas (Figure 1F).
Progranulin showed a similar expression profile. Whereas 97 of 152 cases of IDC
showed moderate or strong VEGF reactivity (2+ or 3+), only five of 31 cases of DCIS
showed moderate or strong VEGF staining (Table 1).
Table 1. Expression of progranulin and VEGF in breast tissues
Progranulin expression (%)
Cases
VEGF expression (%)
Tissue type
(n)
-
+
++
+++
-
+
++
+++
Benign tumor
20
18 (90)
2 (10)
0
0
16 (80)
4 (20)
0
0
DCIS*
31
8 (26)
11 (35)
7 (23)
5 (16)
11 (35)
15 (48)
4 (13)
1 (9)
IDC**
152
16 (11)
37 (24)
53 (35)
46 (30)
12 (8)
43 (28)
42 (28) 55 (36)
*DCIS:ductal carcinomas in situ
**IDC: invasive ductal carcinomas
Regarding endoglin expression, only four of 20 fibroadenoma breast tissues showed
weak positive staining (Figure 1G), while positive staining was observed in 180 of
183 (98%) breast carcinoma samples (Figure 1H). CD105-positivity was observed
principally in the endothelial cell membrane, and sporadically in the endothelial cell
cytoplasm within microvessels (Figure 1I). CD105 immunostaining was distributed
regularly as thin, linear deposits along the small vessels, which was suitable for vessel
counting. The MVDs were 10.31±4.74 and 23.5±10.6 in the DCIS and IDC,
respectively. There was a significant difference in MVDs between the DCIS and IDC
groups (P=0.002).
Correlation between progranulin expression and clinicopathologic
features
The relationships between progranulin expression in breast cancer biopsies and
clinicopathologic parameters in breast cancer patients are summarized in Table 2.
Higher progranulin expression in breast cancer was significantly correlated with
lymph node metastasis (χ2=52.864; degrees of freedom (df)=1; P<0.001), tumor size
(χ2=8.208; df=1; P=0.004), and TNM staging (χ2=28.81; df=1; P<0.001), but not with
age (χ2=2.16; df=1; P=0.142).
Table 2. Relationship between progranulin expression and clinicopathologic features
in patients with breast cancer
Pathologic features
Age (year)
≤45
＞45
Tumor size
≤2 cm
＞2 cm
TNM staging
0/I
II–IV
Lymph node metastasis
+
-
Progranulin expression
Cases
(n)
2
χ /P
-/+
++/+++
50
133
24
48
26
85
0.142
98
85
48
24
50
61
0.004
66
117
43
29
23
88
<0.001
89
94
11
61
78
33
<0.001
Progranulin expression and tumor angiogenesis
Tumors were divided into two groups, according to the levels of progranulin and
VEGF immunostaining: (1) negative/low progranulin reactivity (72 cases) and high
progranulin reactivity (111 cases); (2) negative/low VEGF reactivity (81 cases) and
high VEGF reactivity (102 cases). It was important to note that most of the cases with
high progranulin expression also had high VEGF expression (Table 3). Progranulin
protein expression in breast cancer cases was positively correlated with VEGF
expression (r=0.273; P<0.001, Table 3).
Table 2. Correlation between progranulin and VEGF expression in breast cancer
Progranulin
-/+
++/+++
VEGF
-/+
++/+++
44
37
28
74
P value
r
<0.001
0.273
The MVD count was higher in the high-progranulin-reactivity group than in the
negative/low-progranulin-reactivity group (t=3.542; df=181; P=0.002, Figure 2).
40
MVD counts
35
30
25
20
15
10
5
0
Progranulin-L (n=72)
Progranulin-H (n=111)
Figure 2. Student’s t-test showed that the MVD counts (mean±SD=28.3±10.3) in
high-level progranulin (progranulin-H) tumors were significantly higher than those in
low-level progranulin (progranulin-L) tumors (mean±SD=16.4±6.9) ( t=3.542;
df=181; P=0.002).
DISCUSSION
Granulins were originally described as transforming growth factors and epithelins,
based on their presence in epithelial cells and their autocrine ability to stimulate their
proliferation.5,6 These proteins are widely expressed and play roles in tissue repair,
host defense, carcinogenesis, and brain function.7 In mammals, they are synthesized as
larger proteins called progranulins, granulin/epithelin precursor, PC-derived growth
factor (PCDGF), or acrogranin. The growth factor proepithelin is a secreted
glycoprotein that functions as an important regulator of cell growth, migration, and
transformation. Increased expression of progranulin has been associated with tumor
progression and invasiveness in several cancers, including ovarian cancer,8-12 renal
carcinoma,13 hepatocellular carcinoma,14 myeloma,15 prostate cancer,16 endometrial
cancer,17 bladder cancer18 and lung cancer,19, 20 as well as in breast cancer, based on a
variety of experimental approaches.
Numerous in vivo and in vitro studies have demonstrated that overexpression of
progranulin can promote tumor invasion and metastasis, and that it thus plays a key
role in tumor development and progression. Monami et al reported that progranulin
promoted the migration of 5637 bladder cancer cells and stimulated in vitro wound
closure and invasion, supporting the hypothesis that it may play a critical role in the
establishment of the invasive phenotype.18 They further identified a significant
increase in progranulin mRNA expression levels in prostate cancers, compared with
non-neoplastic controls, and determined that progranulin promoted the migration of
both androgen-dependent and -independent human prostate cancer cells.16 Several
histologic studies have reached similar conclusions. Immunohistochemistry was used
to demonstrate increased progranulin expression in invasive ovarian cancer cells; the
progranulin level was high in invasive ovarian tumors, whereas it was undetectable in
serous tumors with low malignant potential.9 Inhibition of progranulin expression in
ovarian cancer cells by transfection of antisense cDNA led to reduced cell growth, a
decrease in the S-phase fraction, and loss of density-independent growth potential.21
Immunohistochemical studies demonstrated that high-grade renal cell carcinomas
expressed high levels of progranulin, compared with low-grade renal cell carcinomas
and normal tissue.13 In this study, we found that the level of progranulin expression
was higher in invasive ductal breast cancers than in benign breast tumors. In addition,
the most important results of the current study were the close correlations between
high levels of progranulin expression and tumor size (P=0.004), lymph node
metastasis (P<0.001), and TNM grading (P<0.001), which provided the first
histologic evidence for the clinicopathologic implications of progranulin in breast
carcinoma and suggested progranulin as a biomarker of malignant tumor as well as
previous reports.
We should specially focused on the conclusion that progranulin expression level was
significantly correlated with lymph node metastasis (P<0.001) in breast carcinoma
tissues. Metastasis is a hallmark of cancer transition from a noninvasive to an invasive
phenotype including multiple processes. However, progranulin is a relatively
little-studied growth factor, and only a few sporadic studies, mostly at the in-vitro
level, have investigated the mechanisms whereby progranulin promotes migration in a
variety of carcinomas. He et al reported that the level of progranulin expression was a
major determinant of the intrinsic activity of the mitogen-activated protein kinase,
phosphatidylinositol 3’-kinase in SW-13 cells, suggesting that the mitogen-activated
protein kinase and phosphatidylinositol 3’-kinase signaling pathways may be involved
in the promotion of tumor invasion and migration by progranulin.22
In addition, neovascularization is a required and critical step for the growth and
metastasis of tumors. Recent studies have suggested that progranulin may play a role
in carcinoma angiogenesis and have identified a close correlation between progranulin
expression and angiogenesis, which may provide an alternative explanation for its
high correlation with tumor metastasis progression. Progranulin was induced in the
capillary endothelium of wound granulation tissue and promoted the mitosis and
migration of adult dermal microvascular cells.23 In MCF-7 cells, progranulin
stimulated vascular endothelial growth factor (VEGF) expression, suggesting that
progranulin could promote angiogenesis in human breast cancer cells.24 Desmarais et
al found that progranulin expression in mink was localized to cytotrophoblasts and
fetal capillaries, as well as to the hypertrophied maternal endothelial cells in the
incipient labyrinth, and suggested that high level of progranulin expression
corresponded to active angiogenesis occurring during the establishment of the
placenta.25 Chen et al reported that the expression of both progranulin and VEGF
were higher in esophageal squamous cell carcinoma, indicating their close
relationship with angiogenesis.4 Tangkeangsirisin and Serrero found that the
expression of progranulin could affect the expression of VEGF in human breast
cancer cell lines, and that their expression levels changed in similar ways.2 VEGF is
one of the most important angiogenic factors. It is a homodimeric glycoprotein with a
molecular weight of 45 kDa that can act as an endothelial cell mitogen and a
modulator of changes in vascular permeability. In vitro and in vivo experiments have
shown that increased VEGF expression is associated with tumor growth and
metastasis, and it has been confirmed as an important target for solid tumor
treatment.26 In this study, progranulin protein expression in breast cancer cases was
positively correlated with VEGF expression (r=0.273; P<0.001), which suggested a
close relationship of progranulin and angiogensis in breast cancer tissues.
Similarly, the MVD count, assessed by CD105 expression, was higher in the
high-progranulin-reactivity group than in the negative/low-progranulin-reactivity
group (t=3.542; df=181; P=0.002). CD105 is a cell membrane glycoprotein that is
overexpressed in highly proliferative endothelial cells in culture, and which represents
a specific marker of neovascularization in various types of tumor. Dallas studied
CD105 expression by immunohistochemical assays in a series of 929 breast
carcinoma patients and correlated the findings with long-term follow-up data;
multivariate analysis indentified CD105 immunodetection as an independent
prognostic indicator.27 The histologic data for progranulin from the present study, and
its close association with CD105, may provide the basis for progranulin to act as a
valuable prognostic indicator in breast cancer, and more importantly, to provide the
foundation for new combination regimens with antiangiogenic activity.
Acknowledgments
This work was supported financially by the National Science Foundation of China
[30772534]
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Figure 1. Representative immunohistochemical staining for the expression of progranulin,
VEGF and CD105 in breast tissues. (A) Progranulin staining in benign tumors (DAB × 400). (B)
Positive progranulin and CD105 staining in stroma (DAB and AP-Red × 200). (C) Positive
progranulin staining in tumor cells (DAB × 400). (D) VEGF staining in benign tumors (DAB ×
400). (E) Positive VEGF staining in stroma (DAB × 200). (F) Positive VEGF staining in tumor
cells (DAB × 200). (G) CD105 staining in benign tumors (AP-Red × 200), arrows showed weak
CD105 staining. (H) Positive CD105 staining in vascular endothelial cells (AP-Red × 200). (F)
Positive CD105 and progranulin staining in vascular endothelial cells (AP-Red and DAB × 400).