Foxp3 is correlated with VEGF-C expression and lymphangiogenesis in cervical cancer
Tang et al. World Journal of Surgical Oncology
Foxp3 is correlated with VEGF-C expression and lymphangiogenesis in cervical cancer
Jiabu Tang 1 2
Zheng Yang 2 3
Zhuo Wang 2 3
Zhen Li 1 2
Hongmei Li 1 2
Jinbao Yin 1 2
Min Deng 0 2
Wei Zhu 1 2
Chao Zeng 1 2
0 Cancer Hospital and Cancer Research Institute, Guangzhou Medical University , Guangzhou 510095 , China
1 Department of Pathology, Guangdong Medical University , 1 Xincheng Road, Dongguan 523808 , China
2 Trial registration: Gunagdong Medical University , PJ2013049
3 Department of Pathology, The First Affiliated Hospital of Sun Yat-sen University , Zhongshan 2nd Road 74, Guangzhou 510080 , China
Background: Recent observations revealed Foxp3 participated in the development of cervical cancer. Furthermore, Foxp3 has a vital function in the lymphatic metastasis of cervical cancer. However, it is unclear whether Foxp3 is correlated with lymphangiogenesis of cervical cancer. Methods: In this experiment, expression of Foxp3 and VEGF-C was detected in 50 cervical cancer samples by immunohistochemistry. In addition, we evaluated the association between Foxp3 and VEGF-C expression and lymphangiogenesis of cervical cancer evaluated by lymphatic vessel density. Results: These data demonstrate Foxp3 is positively correlated with VEGF-C expression. Furthermore, Foxp3 is associated with lymphangiogenesis of cervical cancer. Conclusions: These results revealed Foxp3 play an important role in lymphangiogenesis of cervical cancer.
Foxp3; Cervical cancer; Lymphangiogenesis
Cervical cancer is the second common malignant tumor
in female [
]. As cervical cancer is characterized by few
clinical manifestations, it is hard to detect their existence
in early stage. Hence, the prognosis of cervical cancer in
advanced stage is unsatisfactory. A well-defined etiology
of cervical cancer is infected by high-risk human
papillomavirus (HPV). However, the underlying pathogenesis of
the disease requires further research.
FOXP3, a member of a forkhead box proteins family,
contained a winged helix DNA-binding domain. Human
Foxp3 gene is located at Xp1 1.23, and it plays as a
controller of the function of regulatory T-cells, which have a
vital role in the process of forming microenvironment
with immunosuppressive tumor [
]. Recent studies
report Foxp3 is not only presented in Treg cells but also
expressed in a variety of cancer cells [
example, Foxp3 is over-expressed in breast , gastric
], and thyroid cancer [
], and it also closely
correlated with progression and prognosis of these cancers.
Luo et al. revealed Foxp3 participated in advancement of
normal cervical tissues to cervical cancer [
Furthermore, Foxp3 had a significant role in facilitating
lymphatic metastasis of cervical cancer [
], and high
Foxp3 expression in the cervical cancer predicted a poor
Recently, Sasahira et al. revealed Foxc2 regulated
angiogenesis and lymphangiogenesis in oral squamous
cell carcinoma [
]. Foxc1 and Foxc2 are required for
lymphatic sprouting during vascular development [
Nevertheless, until now, no research has reported the
association of Foxp3 expression with lymphangiogenesis of
cervical cancer. In this experiment, we investigated the
association of Foxp3 with VEGF-C expression and the
role of Foxp3 in lymphangiogenesis of cervical cancer.
Patients and tissue samples
In the assay, 50 cervical cancer samples were obtained
from un-selected patients at Department of Pathology,
the First Affiliated hospital of Sun Yat-sen University.
More details of clinical and pathological information
about these patients are listed in Table 1. In these
samples, 42 cases were squamous cancer and 8 cases were
adenocancer. Thirty-four cases had no lymph node
metastasis, and 16 cases present lymph node metastasis.
The study was approved by Institutional Research Ethics
Board of Guangdong Medical University.
Paraffin block was cut into 4-μm sections and treated by
routine skill. After microwaved in citrate buffer for 5 min,
the slides were incubated with Foxp3 (ab10563, Abcam,
USA), VEGF-C (sc-374,628, Santa Cruz, CA), and D2-40
(MAB-0567, Maxim-Bio, Fuzhou, China) at room
temperature respectively. Then, the sections were
incubated with a secondary antibody (MaximBio Company,
Fuzhou, China). Labeling was monitored by
diaminobenzidine (Maxim-Bio Company). At last, hematoxylin was
used to stain the sections.
Foxp3 and VEGF-C expression was scored in accordance
with the intensity (0, no staining; 1, weak staining; 2,
moderate staining; 3, strong staining) and extent staining
of cervical cancer cells that were stained (0, no stained;
1, < 10% cervical cancer cells stained; 2, 10–50% cervical
cancer cells stained; 3, > 50% cervical cancer cells
stained; 4, > 75% cervical cancer cells stained). If the data
of multiplication between staining intensity and the
extent of positive cervical cancer cells is ≥ 2, it is regarded
as positive (+). Finally, in five unselected areas of a
1mm2 field, the number of lymphatic vessels was
accumulated, then calculated the average.
All statistical analyses were done by SPSS 13.0 (SPSS,
Inc., Chicago, IL). χ2 test was used to evaluate the
association of Foxp3 and VEGF-C with clinicopathological
parameters. The relation of Foxp3 with VEGF-C was
evaluated by Fisher’s exact. The difference was statistical
when the P value is < 0.05.
Expression of Foxp3 and VEGF-C in cervical cancer
Immunohistochemical staining of Foxp3 was performed
in 50 cervical cancer cases. After evaluating by two
pathologists, Foxp3 expression was observed in 66% (33/50)
cervical cancer tissues. As shown in Fig. 1a–c and Fig. 2a–d,
Foxp3 was found not only in nuclear of lymphocytes but
also in cytoplasm of cervical cancer cells. Similarly,
VEGFC was also immunostained in the cytoplasm of the
cervical cancer cells (Fig. 1d–f; Fig. 2e–h).
Table 1 demonstrates the association between Foxp3
expression and clinicopathological factors. Expression
of Foxp3 was positive in 87.5% (14/16) cases that had
lymph node metastasis, and the positive rate was
significantly higher than the samples with the absence of
lymph node metastasis (55.9%, 19/34). Furthermore,
statistical analysis demonstrates that Foxp3
immunoreactivity is associated with some clinicopathological
factors, such as clinical stage and lymph node
metastasis (all P < 0.05). However, there was no significant
relation between Foxp3 expression and the other
clinical parameters, including age, tumor size, and
histological type (all P > 0.05). On the other hand,
VEGFC immunoreactivity was observed in 76.0% samples
(38/50). As suggested in Table 1, VEGF-C expression
had a significant association with cases that presented
lymph node metastasis (P < 0.05).
Comparison of Foxp3 and VEGF-C expression
Both Foxp3 and VEGF-C had immunoreactivity in 31
samples. On the other hand, neither Foxp3 nor VEGF-C was
expressed in 10 samples. Only Foxp3 was expressed in 2
samples, whereas VEGF-C alone was expressed in 7 samples
(Table 2). These results indicated that Foxp3 expression was
significantly associated with VEGF-C expression (P < 0.05).
Expression of Foxp3 and VEGF-C predicts
lymphangiogenesis of cervical cancer
In this experiment, D2-40 was utilized to assess
lymphatic vessel density (LVD) in cervical cancer tissues. As
shown in Table 3, Foxp3-positive cases had more
lymphatic vessel than Foxp3-negative cases (11.25 ± 3.16 VS
4.14 ± 2.76). Similarly, cases with VEGF-C staining had
more lymphatic vessel compared with cases that had no
VEGF-C expression (14.67 ± 4.52 vs 5.32 ± 2.27).
Notably, samples that combined with Foxp3 expression and
VEGF-C expression had the highest lymph vessel
(16.83 ± 5.29; Fig. 2).
Foxp3, a member of the FOX protein family, is a
forkhead (FKH) box transcription factor. It contains a
DNA-binding FKH box domain which plays as a
transcriptional activator and repressor of specific genes. As
known to all, Foxp3 is widely known for its function in
the development of immunoregulatory T cells [
Recently, it has been discovered that abnormal Foxp3
expression is associated with a series of cancers, such as
], ovary [
], and breast cancer [
Extrinsic expression of Foxp3 inhibits proliferation and
induces apoptosis of gastric cancer cells by activating
ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9
]. Moreover, Tan et al. reported Foxp3 over-expression
significantly reduced the proliferation of melanoma cells
in vitro and in vivo [
]. These findings indicated that
Foxp3 might act as a tumor suppressor gene. Conversely,
high Foxp3 expression of the colorectal cancer cells was
correlated with unfavorable prognosis compared with
cases that had low Foxp3 expression [
]. Similarly, our
data suggested Foxp3 expression is correlated with higher
clinical stage and lymph node metastasis. This result
demonstrated Foxp3 might be involved in lymph node
metastasis of cervical cancer.
Interestingly, FOXP3 expression in breast cancer cells
was correlated with high Ki-67 index, indicating high
proliferative activity of FOXP3-positive tumors. Merlo et
al. also demonstrated increased Ki-67 staining in
FOXP3-mutated mammary cells. These results indicated
that FOXP3 expression could promote proliferation of
cancer cells. However, in our study, there was no
relation between Foxp3 expression and tumor size. This
discrepancy is attributed to different tumor types or
absence of cell experiment.
The current study, for the first time, revealed the role
of Foxp3 in lymphangiogenesis of cervical cancer. Firstly,
we found Foxp3-positive cases had more lymphatic
vessel than Foxp3-negative cases. VEGF-C is essential
for most lymphangiogenic processes by activating the
vascular endothelial growth factor receptors VEGFR-3
and VEGFR-2 [
]. Then, taking into account the critical
role of VEGF-C in lymphangiogenesis, we evaluated the
correlation between Foxp3 and VEGF-C expression. In
this study, our findings suggest Foxp3 had a significant
positive correlation with VEGF-C in cervical cancer.
The present assay shows that Foxp3 expression is
associated with advancement of cervical cancer and lymph
node metastasis. More importantly, Foxp3 might
promote lymphatic vessel formation in cervical cancer
correlated with VEGF-C expression. Although further
studies are needed to elucidate the molecular
mechanism of Foxp3 in lymphangiogenesis of cervical cancer,
the present assay will provide new insights into the
lymph node metastasis of cervical cancer.
HPV: High-risk human papillomavirus; LVD: Lymphatic vessel density
This work was supported by the National Natural Science Foundation of
China (grant no. 81302245).
Availability of data and materials
CZ, JBT, and ZW designed the experiments, analyzed data, and prepared the
manuscript. ZY, ZL, JBY, WZ, and HML performed the experiments. All authors
read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Medical Ethics Committee of Guangdong
Medical University, Guangdong, China.
Consent for publication
The authors declare that they have no competing interests.
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