High brain acid soluble protein 1(BASP1) is a poor prognostic factor for cervical cancer and promotes tumor growth
Tang et al. Cancer Cell Int
High brain acid soluble protein 1(BASP1) is a poor prognostic factor for cervical cancer and promotes tumor growth
Huiru Tang 3 4 9
Yan Wang 2 9
Bing Zhang 8 9
Shiqiu Xiong 7 9
Liangshuai Liu 1 6 9
Wei Chen 1 6 9
Guosheng Tan 1 6 9
Heping Li 0 1 5 6 9
0 Department of Medical Oncology, The First Affiliated Hospital of Sun Yatsen University , 58 Zhongshan Road II, Yuexiu District, Guangzhou 510080 , People's Republic of China
1 Department of Interventional Radiology, The First Affiliated Hospital of Sun Yat-sen University , 58 Zhongshan Road II, Yuexiu District, Guangzhou 510080 , People's Republic of China
2 Department of Gynecology and Obstetrics, Nanfang Hospital, Southern Medical University , Guangzhou 510515 , People's Republic of China
3 Shenzhen Key Laboratory of Gynecological Diagnostic Technology Research , Shenzhen 518036 , People's Republic of China
4 Department of Gynecology & Obstetrics, Peking University Shenzhen Hospital , Shenzhen 518036 , People's Republic of China
5 Department of Medical Oncology , The First Affiliated
6 Department of Interventional Radiology, The First Affiliated Hospital of Sun Yat-sen University , 58 Zhongshan Road II, Yuexiu District, Guangzhou 510080 , People's Republic of China
7 Department of Biochemistry, University of Leicester , Leicester LE1 7RH , UK
8 Department of Nuclear Medicine, The First Affiliated Hospital of Sun Yat-sen University , Guangzhou 510080 , People's Republic of China
9 Hospital of Sun Yat-sen University , 58 Zhongshan Road II, Yuexiu District, Guangzhou 510080 , People's Republic of China
Background: The aim of this study was to determine whether brain abundant membrane attached signal protein 1 (BASP1) is a valuable prognostic biomarker for cervical cancer and whether BASP1 regulates the progression of cervical cancer. Methods: Quantitative real-time PCR, western blotting, and immunohistochemistry were used to determined BASP1 levels. Statistical analyses were used to examine whether BASP1 was a prognostic factor for patients with cervical cancer. The MTT assay, colony formation assay, cell cycle assay, anchorage-independent growth assay, and a tumor xenograft model were used to determine the role of BASP1 in the proliferation and tumorigenicity of cervical cancer. Results: Brain abundant membrane attached signal protein 1 was upregulated in cervical cancer tissues and cells, and BASP1 expression levels were higher in patients that had died during follow-up compared with those that survived. There was a positive correlation between BASP1 expression and clinical stage (p < 0.001), T classification (p < 0.001), N classification (p < 0.05), and survival or mortality (p < 0.05). Patients with higher BASP1 expression had a shorter overall survival time. Cox regression analysis shown BSAP1 was an unfavorable prognostic factor for patients with cervical cancer. Overexpression of BASP1 promoted the proliferation of cervical cancer and its colony formation ability, accelerated cell cycle progression, and enhanced tumorgenicity. BASP1 knockdown inhibited the proliferation of cervical cancer and its colony formation ability, suppressed cell cycle progression, and decreased tumorgenicity. Conclusions: The results showed that BASP1 not only is a novel prognostic factor for patients with cervical cancer, but also promotes the proliferation and tumorigenicity of cervical cancer.
BASP1; Cervical cancer; Prognosis; Tumor growth
In recent decades, certain prognostic factors and
therapeutic targets for cervical cancer have been found. For
example, weak pp125FAK expression correlates with
pelvic lymph node metastasis and recurrent disease,
and is a favorable factor for patients with cervical
cancer: the overall survival of patients with high and
moderate pp125FAK levels was longer than those with weak
PP125FAK expression [
]. Msi1 is upregulated in patients
with cervical cancer, and promotes the proliferation
of cervical cancer by directly inhibiting p21, p27, and
]. However, cervical cancer remains the fourth
most common cancer in women worldwide. Its
morbidity has decreased in some countries, probably because
of progress in early diagnosis and prevention. In China,
the morbidity of cervical cancer is 8.98/100,000 and the
mortality is 2.13/100,000, according to data from the
2012 Chinese Cancer Registry Annual Report [
suggested that new prognostic factors and therapeutic
approaches should be developed.
We used publically available gene expression profiles
of cervical cancer tissues and normal cervical tissues
(GSE9750) to screen for genes that regulate the
progression of cervical cancer, and found that BASP1 (encoding
brain abundant membrane attached signal protein 1) was
upregulated in cervical cancer tissues. BASP1, which is
also known as NAP-22 or CAP-23 [
], can interact with
Wilms tumor 1 (WT1). WT1 is a Wilms’ tumor
suppressor protein that plays an important role in nephrogenesis
and hematopoiesis [
]. BASP1 serves as a transcriptional
co-suppressor to inhibit transcriptional activity of WT1,
suggesting BASP1 regulates the function of WT1 in
]. Further analysis revealed that the
N-terminus of BASP1 could be myristoylated;
myristoylated BASP1 interacted with oleate-activated
transcription factor PIP2, which recruits histone deacetylase
histone deacetylase 1 (HDAC1) to the promoter regions
of WT1-dependent target genes, causing
transcriptional repression . Toska and colleagues observed that
BASP1 also interacted with Prohibitin to recruit BRG1 to
the promoter regions of WT1-dependent target genes,
causing coactivator p300/CBP to dissociate from the
promoter regions to inhibit target gene expression; the
interaction between BASP1 and Prohibitin is also
critical for the recruitment of PIP2 and HDAC1 to the target
genes of WT1 [
]. These findings suggested BASP1 plays
an important role in development. However, the role of
BASP1 in cervical cancer has not been reported.
In this study, we analyzed the relationship between
BASP1 expression and clinicopathological parameters
in patients with cervical cancer, and studied the role of
BASP1 in the cervical cancer growth. We found that
BASP1 is a new prognostic factor for cervical cancer, and
promotes tumor growth.
BASP1 is upregulated in cervical cancer tissues
To investigate whether BASP1 regulates the
development and progression of cervical cancer, we explored
BASP1 expression in cervical cancer tissues and normal
cervical epithelium tissues. Analysis of gene expression
profiles (GSE9750, Fig. 1a) showed that BASP1 was
significantly upregulated in cervical tissues compared with
normal cervical tissue. Six paired cervical cancer
tissues and matched adjacent normal cervical tissues were
used to examine BASP1 protein levels. Western blotting
showed that BASP1 levels were higher in cervical cancer
tissues than in matched adjacent normal cervical tissues
(Fig. 1b). Immunohistochemical analysis also revealed
that BASP1 was upregulated in cervical cancer tissues
(Fig. 1c). These results suggested that BASP1 is
upregulated in cervical cancer tissues.
BASP1 levels correlate with clinical aggressiveness of cervical cancer
To determine the relationship between BASP1 levels
and clinicopathological parameters, and whether BASP1
could serve as a new independent prognostic factor, we
determined BASP1 levels in a cohort of 136
paraffinembedded archived cervical cancer tissues using
immunohistochemistry. Positive staining for BASP1 was
observed in 98.5% (134/136) of samples, with low BASP1
staining in 59.6% (81/136) samples and high BASP1
staining in 40.4% (55/136) of the samples (Additional
file 1: Table S2). BASP1 levels were higher in patients
who had died during follow-up than in those had
survived during follow-up (Fig. 2a). BASP1 levels were
low in 67.3% (68/101) of surviving patients, and high
in 32.7% (33/101). However, BASP1 levels were low in
37.1% (13/35) of the patients who died and high in 62.9%
(22/35) (Fig. 2a). Kaplan–Meier survival curves
demonstrated that the overall survival of the patients with low
BASP1 expression was significantly longer than those
with high BASP1 expression (Fig. 2b, p = 0.001). These
results suggested high BASP1 levels might be an
unfavorable factor for patients with cervical cancer.
We further analyzed the correlation between BASP1
levels and the clinicopathological parameters of the
patients. As shown in Table 1, we found there was no
significant correlation between BASP1 levels and age or
pathological differentiation. BASP1 levels correlated
significantly with clinical stage (p < 0.001), T classification
(larger tumor size, p < 0.001), N classification (lymph
node involvement, p < 0.05) and survival or
mortality (p < 0.05). These results were confirmed by
Spearman’s correlation analysis, as summarized in Additional
file 2: Table S3, in which BASP1 levels correlated
significantly with clinical stage (p = 0.000), T classification
(p = 0.000), N classification (p = 0.023) and survival or
mortality (p = 0.002). Thus, the results demonstrated
that BASP1 levels correlated with clinical stage, T
classification, N classification, and survival or mortality. Our
findings suggested BASP1 levels correlated with the
clinical aggressiveness of cervical cancer.
To identify whether BASP1 could serve as a novel
prognostic factor, Cox regression analysis was performed,
which showed that a high BASP1 level, clinical stage, and
pathological differentiation were independent unfavorable
prognostic factors (Table 2). Thus, BASP1 abundance
correlated significantly with the prognosis of cervical cancer.
BASP1 regulates tumor growth of cervical cancer
Brain abundant membrane attached signal protein 1
could serve as a new prognostic factor; therefore, we
further determined whether BASP1 regulates tumor
growth of cervical cancer. BASP1 levels were
determined in cervical cancer cell lines and in two
papillomavirus-immortalized normal cervical cell lines.
BASP1 was upregulated in the cervical cancer cell lines
(Fig. 3a), which was consistent with our previous results
for BASP1 abundance in cervical cancer tissues (Fig. 1a,
b). We determined the functional role of BASP1 in
cervical cancer development by gain or loss of BASP1 in
ME-180 and HT-3 cells. Western blotting showed that
BASP1 levels were significantly upregulated when cells
were transfected with pMSCV-BASP1, and were
significantly downregulated when the cells were transfected
with a short interfering RNA (siRNA) targeting BASP1
(siBASP1; Fig. 3b). The MTT assay indicated that
overexpression of BASP1 promoted cervical cancer cell
proliferation. A colony formation assay also showed that the cell
number increased significantly when BASP1 was
overexpressed (Fig. 3d). These results suggested that BASP1
promotes proliferation of cervical cancer cells. To further
confirm that BASP1 regulates cell proliferation, we used
cell cycle assays to determine the effect of BASP1 on cell
cycle progression, overexpression of BASP1 increased the
proportion of cells in the S phase from 34.52 to 50.78%
in ME-180 cells, and from 31.31 to 52.58% in HT-3 cells,
with a concomitant decrease in the proportion of cells in
the G1/G2/M phases (Fig. 3e).
We confirmed these results by downregulating BASP1
in the indicated cervical cancer cell lines using siRNAs.
The MTT assay showed decreased proliferation of cells
transfected with siBASP1 compared to those transfected
with a scrambled siRNA (Additional file 3: Figure S1A).
The colony formation assay also showed that knockdown
of BASP1 inhibited cellular proliferation (Additional
file 3: Figure S1B). We also analyzed the effect of
knockdown of BASP1 on cell cycle progression: The proportion
of cells in the S phase decreased from 37.84 to 11.40% in
ME-180 cells, and from 35.79 to 12.81% in HT-3 cells,
with a concomitant increase in the proportion of cells in
the G1/G2/M phases (Additional file 3: Figure S1C).
To determine the role of BASP1 in tumorigenicity, we
used an anchorage-independent growth assay to
determine the effect of BASP1 on tumorigenicity in vitro.
BASP1 overexpression increased the
anchorage-independent growth ability of indicated cells significantly, as
shown by increased colony numbers and sizes (Fig. 4a).
When BASP1 expression as knocked down, we observed
a significant decrease the cells’ anchorage-independent
growth ability (Fig. 4a). This suggested that BASP1
promoted the tumorigenicity of cervical cancer.
To confirm that BASP1 regulates cell proliferation and
tumorigenicity, we determine the effect of BASP1 on
tumor growth in vivo. We transplanted the indicated cells
with BASP1 overexpression or knockdown into a
subcutaneous area of nude mice. Overexpression of BASP1
promoted cervical tumor growth in the nude mice, and
downregulation of BASP1 inhibited it (Fig. 4b, c). Ki67 is
a marker for cell proliferation [
]; therefore, we
determined Ki67 levels in the tumors grown in the nude mice,
and found that Ki67 levels were upregulated in tumors
overexpressing BASP1 and downregulated in BASP1
knockdown tumors (Fig. 4d). Together, these results
suggested that BASP1 promotes tumor growth. This
result also supported our clinical investigation, in which
BASP1 levels correlated with T classification.
In the present study, we demonstrated that BASP1
plays an important role in cervical cancer. BASP1 was
upregulated in cervical cancer, and is a novel
unfavorable prognostic factor for patients with cervical cancer.
High BASP1 levels correlated with poor clinical outcome.
BASP1 also regulates the proliferation and
tumorigenicity of cervical cancer; overexpression of BASP1 promoted
cellular proliferation and tumorigenicity and knockdown
of BASP1 had the opposite effect. These results suggested
that BASP1 not only serves as a prognostic factor, but
also can function as a target for cervical cancer therapy.
We found BASP1 levels were high in cervical cancer,
suggesting that BASP1 may be an oncogene. However,
previous reports have shown that BASP1 is
downregulated in v-myc-induced transformed cells, and that
overexpression of BASP1 inhibits transformation; further
analysis showed that BASP1 inhibits the target genes of
c-Myc, such as WS5, Q83 and BRAK, suggesting that
BASP1 could be a tumor suppressor [
]. Moribe and
colleagues used a gene microarray and pyrosequencing to
screen genes that are methylated specifically in
hepatocellular carcinoma (HCC), and found that BASP1 is
aberrantly methylated in HCC; its expression is low in HCC,
and it can function as a useful biomarker for the
diagnosis of HCC [
]. MicroRNA miR-191, an onco-miR, is
upregulated in transformed human bronchial epithelial
cells, and promotes epithelial-mesenchymal transition
(EMT) and self-renewal of cancer stem cells of
transformed cells. BASP1 is a direct target of miR-191; BASP1
inhibition by miR-191 leads to transactivation of WT1,
which activates the Wnt pathway to promote tumor
]. Many genes have been found to play
different roles in different kinds of tumors. For example,
inhibitor of DNA binding 2 (ID2) is downregulated in
breast cancer, in which it inhibits cellular invasion and is
a favorable prognostic factor for patients [
ID2 is upregulated in brain cancer, colon cancer,
pancreatic cancer, and prostate cancer, in which it promotes
tumor progression, making it an unfavorable prognostic
We found the opposite role of BASP1 in cervical
cancer. Gene set enrichment analyses (GSEA) demonstrated
that BASP1 expression correlated significantly with
progression and development of cervical cancer (Additional
file 4: Figure S2), revealing that BASP1 is an oncogene
in cervical cancer. We further studied whether BASP1 is
an unfavorable prognostic factor. Statistical analysis of
BASP1 levels in 136 paraffin-embedded cervical cancer
tissues suggested a positive correlation between BASP1
levels and clinical stage, T classification, N classification
and survival or mortality. Cox regression analysis
demonstrated that BASP1 is an independent prognostic factor
for patients with cervical cancer, and thus could be used
to predict their prognosis.
We also determined the role of BASP1 in the
proliferation and tumorigenicity of cervical cancer;
overexpression of BASP1 promoted proliferation, colony
formation, cell cycle progression, and tumorigenicity.
Knockdown of BASP1 had the opposite effects. These
results suggested that BASP1 regulates the
proliferation and tumorigenicity of cervical cancer, making a
potential therapeutic target. However, the mechanism
by which BASP1 promotes the proliferation and
tumorigenicity of cervical cancer requires further study; for
example, a chromatin immunoprecipitation assay could
identify the target genes of BASP1 associated with
We demonstrated that BASP1 is an independent
prognostic factor for patients with cervical cancer that
promotes cervical cancer growth.
Methods and materials
Cell culture and transfection
Human cervical cancer cell lines Ca Ski, MS751, ME-180,
SiHa, HT-3, HeLa, C4-I and SW756 were cultured in
Dulbecco’s modified Eagle’s medium (DMEM)
(Natocor) supplemented with 10% fetal bovine serum (FBS)
(GIBCO). Ectocervical Ect1/E6E7 and endocervical
End1/E6E7 cells were cultured in Keratinocyte-Serum
Free medium (GIBCO-BRL, 17005-042, USA)
supplemented with 0.1 ng/mL human recombinant EGF,
0.05 mg/mL bovine pituitary extract (BPE, Sigma) and
0.4 mM CaCl2 (Natocor). The cells were maintained in
5% CO2 at 37 °C.
The full-length BASP1 cDNA was cloned into vector
pMSCV-puro; pMSCV-empty vector was used as the
negative control. pMSCV-BASP1 and pMSCV-empty
vector were cotransfected with the IK packaging plasmid
into 293T cells using the calcium phosphate transfection
method. Supernatants were collected at 48 h after
transfection, and infected with cervical cells for 12 h in the
presence of polybrene (2.5 μg/mL). Puromycin was used
to select the transfected cell lines.
For BASP1 knockdown experiments, two siRNAs
for BASP1 and one scrambled siRNA were
synthesized by Guangzhou RiboBio Co (Guangzhou, China).
The sequences used to downregulate BASP1 were:
siBASP1#1: 5′CGGGATCCATGGGA3′, siBASP1#2:
5′CGGAATTCTCACTCT3′. 50 nM of the siRNA
was transfected into ME-180 and HT-3 cells using the
Lipofectamine RNAiMAX Transfection Reagent (Life
Patients and tissue samples
Six cervical cancer tissues and matched adjacent normal
cervical epithelium tissues were obtained from the First
Affiliated Hospital of Sun Yat-sen University.
Guangzhou. These samples were snap-frozen immediately and
stored in liquid nitrogen until use. To further analyze
the relationship between BASP1 expression and the
clinicopathological parameters, a cohort of 136
paraffinembedded cervical cancer tissues was used. These tissues
were diagnosed histopathologically and clinically at the
First Affiliated Hospital of Sun Yat-sen University. For
the use of above clinical samples for research purposes,
prior patient’s consent and approval from the Institute
Research Ethics Committee of the First Affiliated
Hospital of Sun Yat-sen University were obtained. The detailed
clinicopathological parameters are shown in Additional
file 5: Table S1.
Western blotting and immunohistochemistry (IHC)
Western blotting was performed according to standard
methods, as described previously [
], using anti-BASP1
(ab101855, Abcam), anti-Ki67 (sc-7846, Santa Cruz)
antibodies. The membranes were stripped and reprobed with
anti-β-actin antibodies as a loading control. The band
intensity was analyzed using Image J software.
IHC was performed as described previously [
the anti-BASP1 antibody (ab101855, Abcam). The results
of staining were scored independently by two
pathologists blinded to the clinical outcome, and were based on
both the proportion of positively stained tumors cells and
the intensity of staining. The proportion of stained tumor
cells was scored as follows: score 0, no positive cells;
score 1, up to 25% positive cells; score 2, 26–50%
positive cells; score 3, 51–75% positive cells; score 4, over 75%
positive cells. The intensity of staining was determined
as: 0 (no staining), 1 (light yellow = weak staining), 2
(yellow brown = moderate staining), and 3 (brown = strong
staining). The staining index (SI) was determined as the
product of staining intensity × percentage of positive
tumor cells. Cutoff values for high and low expression
of BASP1 were chosen based on a measurement of
heterogeneity using the log-rank test with respect to
overall survival. An SI score of greater than or equal to 6 was
considered to be high expression, and an SI score of less
than 6 was considered low expression.
Cell proliferation and cell cycle assay
To examine the role of BASP1 in the proliferation of
cervical cancer cells, an MTT assay and colony formation
assay were performed using previous described methods
Cell cycle analysis was performed using a previously
described method [
]. Briefly, cells were harvested and
washed in cold PBS followed by fixation in 70% alcohol
overnight at 4 °C. After washing with cold PBS three
times, the cells were resuspended in PBS solution with
20 μg/mL propidium iodide (Sigma) and μg/mL RNase A
for 30 min at 37 °C. Samples were then analyzed using a
FACSCalibur cytometer (Becton–Dickinson).
Anchorage‑independent growth assay
An anchorage-independent growth assay was performed
according to a previously described method [
Briefly, 500 cells were suspended in 2 mL of complete
medium containing 0.3% agar (Sigma). The agar–cell
mixture was plated on top of a solid bottom layer
containing 1% complete medium agar mixture. After 10 days,
colonies that contained more than 50 cells or were larger
than 0.1 mm in diameter were counted. The experiment
was performed in triplicate.
Growth of tumor xenografts in nude mice
Animal studies were performed according to institutional
guidelines. BALB/c nude mice (4–5 weeks old) were
used to make a xenograft model using ME-180 cervical
cancer cell lines. Five mice were assigned randomly to
each group and injected with ME-180 transfected with
pMSCV-BASP1, pMSCV-empty, scramble siRNA, or
BASP1 siRNA, and used to determine the role of BASP1
in tumor progression. 1 × 106 cells were injected into the
subcutaneous sites of nude mice. The tumor volume was
calculated every 2 days for 1 month. The tumors were
excised and subjected to protein extraction to determine
Ki67 levels using western blotting.
All statistical analyses were performed using the SPSS
19.0 statistical software package. Results are presented
as the mean ± standard deviation (SD) for at last three
repeated individual experiments for each group. Chi
square and Fisher’s exact tests were used to analyze the
relationship between BASP1 levels and
clinicopathological parameters. Bivariate correlations between variables
were calculated using Spearman’s rank correlation
coefficients. The survival curve was plotted using Kaplan–
Meier survival analysis and compared using a log-rank
test. Univariate and multivariate Cox regression analyses
were used to estimate the significance of various
variables for survival. A value of p < 0.05 was considered
significant in all cases.
Additional file 1: Table S2. The expression of BASP1 in cervical
Additional file 2: Table S3. Spearman correlation analysis between
BASP1 and clinicopathological factors.
Additional file 3: Figure S1. Knockdown of BASP1 inhibited the
proliferation of cervical cancer. (A) MTT assay showing the effect of BASP1
knockdown on cell proliferation. (B) Colony formation assay showing the
effect of BASP1 knockdown on cell proliferation, Representative
micrographs (left) and quantification (right) of crystal violet stained cell colonies.
(C) Cell cycle analysis indicating the effect of BASP1 knockdown on cell
proliferation. *p < 0.05, error bars represent mean ± SD.
Additional file 4: Figure S2. Gene set enrichment analyses (GSEA)
demonstrating that BASP1 expression correlates significantly with progression
and development of cervical cancer. ES: enrichment score, NES:
normalized enrichment score.
Additional file 5: Table S1. Clinicopathological characteristics of cervical
carcinoma patient samples.
HRT, GST, and HPL designed the experiments. HRT and BZ collected clinical
data. SQX, LSL, and WC performed experiments and data collection. HRT and
HPL analyzed the data. HRT, GST, and HPL interpreted of the data and drafted
the manuscript. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. All authors read
and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Please contact author for data requests.
Consent for publication
Ethics approval and consent to participate
The study protocol was approved by the Institutional Review Board of the First
Affiliated Hospital of Sun Yat-sen University according to the Ethics Committee
of the First Affiliated Hospital of Sun Yat-sen University. Informed consent was
obtained from patients or their guardians.
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