CLCA1 suppresses colorectal cancer aggressiveness via inhibition of the Wnt/beta-catenin signaling pathway
Li et al. Cell Communication and Signaling
CLCA1 suppresses colorectal cancer aggressiveness via inhibition of the Wnt/ beta-catenin signaling pathway
Xiaofen Li 0 1 3
Wangxiong Hu 0 1
Jiaojiao Zhou 1
Yanqin Huang 1
Jiaping Peng 1
Ying Yuan 1 2
Jiekai Yu 1
Shu Zheng 1
0 Equal contributors
1 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, China), the Second Affiliated Hospital, Zhejiang University School of Medicine , Hangzhou, Zhejiang , China
2 Department of Medical Oncology, the Second Affiliated Hospital, Zhejiang University School of Medicine , Hangzhou, Zhejiang , China
3 Department of Abdominal Oncology, West China Hospital, Sichuan University , Chengdu, Sichuan , China
Background: Chloride channel accessory 1 (CLCA1) belongs to the calcium-sensitive chloride conductance protein family, which is mainly expressed in the colon, small intestine and appendix. This study was conducted to investigate the functions and mechanisms of CLCA1 in colorectal cancer (CRC). Methods: The CLCA1 protein expression level in CRC patients was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and western blotting analysis. Using CRISPR/Cas9 technology, CLCA1-upregulated (CLCA1-ACT) and CLCA1-knockout cells (CLCA1-KO), as well as their respective negative controls (CLCA1-ACT-NC and CLCA1-KO-NC), were constructed from the SW620 cell line. Cell growth and metastatic ability were assessed both in vitro and in vivo. The association of CLCA1 with epithelial-mesenchymal transition (EMT) and other signaling pathways was determined by western blotting assays. Results: The expression level of CLCA1 in CRC tissues was significantly decreased compared with that in adjacent normal tissue (P< 0.05). Meanwhile, the serum concentration of CLCA1 in CRC patients was also significantly lower when compared with that of healthy controls (1.48 ± 1.06 ng/mL vs 1.06 ± 0.73 ng/mL, P = 0.0018). In addition, CLCA1 serum concentration and mRNA expression level in CRC tissues were inversely correlated with CRC metastasis and tumor stage. Upregulated CLCA1 suppressed CRC growth and metastasis in vitro and in vivo, whereas inhibition of CLCA1 led to the opposite results. Increased expression levels of CLCA1 could repress Wnt signaling and the EMT process in CRC cells. Conclusions: Our findings suggest that increased expression levels of CLCA1 can suppress CRC aggressiveness. CLCA1 functions as a tumor suppressor possibly via inhibition of the Wnt/beta-catenin signaling pathway and the EMT process.
Chloride channel accessory 1; Colorectal cancer; Tumor suppressor; Early detection
The human chloride channel accessory proteins (CLCA)
are a family of secreted self-cleaving proteins that activate
calcium-dependent chloride currents [
]. The CLCA
family comprises three members, named CLCA1, 2 and 4
]. CLCA3 is a truncated pseudogene and does not
encode a protein [
]. All members of this protein family
share a high degree of homology in protein size, sequence,
and predicted structure but differ significantly in tissue
distribution and biological functions [
]. CLCA1, the first
reported human CLCA family member, is mainly expressed
in the large and small intestine, especially in crypt cells, and
can be secreted into the blood [
]. The second CLCA
isoform, CLCA2, is highly expressed in the trachea and
mammary glands [
], while CLCA4 is mostly expressed in
neural tissue (note that CLCA4 was misidentified as
CLCA2 in the study conducted by Agnel M, et al.) [
Upon their discovery in 1998, the functions of human
CLCA proteins were mostly thought to be associated with
calcium-dependent chloride conductance [
research continues, more interesting and valuable functions
of the CLCA family have been identified, such as
involvement in mucus secretion and tumor metastasis
and regulation of the cell cycle, apoptosis, and blood
The important role of certain ion channels in tumor
progression is well acknowledged [
]. These channels
influence cell volume, intracellular pH, the
concentration of signaling molecules, and the activity of protein
kinases and phosphatases by regulating ion currents
]. For example, voltage-dependent anion channel 1
(VDAC1) is overexpressed in many cancer types. In
addition, downregulation of VDAC1 expression inhibits
tumor development [
]. It has been reported that
potassium channel subfamily K member 9 (KCNK9) is
frequently overexpressed in breast cancer. In addition,
in vitro experiments indicate that the overexpression of
KCNK9 promotes tumor formation and is associated
with tumor resistance to hypoxia and serum deprivation
]. There is a growing body of evidence showing that
CLCA proteins, which act on calcium-dependent
chloride channels and facilitate chloride conductance, have a
close relationship with tumor progression [
For instance, studies have validated that the loss of
CLCA2 expression is closely associated with
tumorigenicity and the metastasis of breast cancer [
15, 20, 21
Secreted CLCA1 has been demonstrated to be a direct
modulator of TMEM16A, a member of the
calciumdependent chloride channel family  [
several studies have suggested that CLCA1 is
downregulated in tumors, and its repression predicts poor
]. In our previous proteogenomic study using
mass spectrometry and gene microarray, we determined
that CLCA1 protein and gene expression levels are
dramatically reduced in CRC tissue compared with adjacent
normal mucosa, suggesting that CLCA1 is a potential
biomarker of CRC [
]. However, the biological
functions and molecular mechanisms of CLCA1 in colorectal
cancer (CRC) remain to be elucidated.
By using a cohort of CRC blood and tissue samples
collected from the Second Affiliated Hospital of
Zhejiang University School of Medicine between 2015
and 2016, we found that the expression level of CLCA1
in CRC patient tissues/serum was markedly decreased
compared with that in adjacent normal tissues/healthy
controls. Moreover, CLCA1 serum concentration and
the CLCA1 mRNA expression level were inversely
correlated with CRC metastasis and tumor stage. To further
investigate the functions and mechanisms of CLCA1 in
CRC, we used CRISPR/Cas9 technology to construct
CLCA1-upregulated cells (CLCA1-ACT) and
CLCA1knockout cells (CLCA1-KO) in the SW620 cell line. In
vitro and in vivo experiments revealed that the increased
expression level of CLCA1 suppressed CRC proliferation
and metastasis, whereas inhibition of CLCA1 caused the
opposite effects. An increased expression of CLCA1 was
able to repress Wnt signaling and the EMT process in
CRC cells. These results suggest that CLCA1 may
function as a tumor suppressor in CRC by inhibiting the
Wnt/beta-catenin signaling pathway and EMT process.
As far as we know, this is the first study investigating
the role of CLCA1 in CRC in vivo.
Sample collection and patient characteristics
The serum samples used for enzyme-linked
immunosorbent assay (ELISA) were collected from 76 healthy
volunteers and 100 CRC patients prior to treatment,
who were admitted to the Second Affiliated Hospital,
Zhejiang University School of Medicine between 2015
and 2016. All serum samples were stored in a
refrigerator at −80 °C. For immunohistochemistry (IHC)
analysis, paired CRC and adjacent normal tissues were
surgically collected from 32 patients, fixed in 10%
buffered formalin and embedded in paraffin. For
western blotting analysis, 19 pairs of CRC and
adjacent normal tissues were surgically obtained and
frozen at −80 °C. Patient clinical characteristics such
as age, gender and TNM stage are listed in the
Additional file 1. Written informed consent was
obtained from each patient. The Ethics Committee of
the Second Affiliated Hospital, Zhejiang University
School of Medicine approved this study.
Enzyme-linked immunosorbent assay (ELISA)
Serum levels of CLCA1 in CRC patients and healthy
controls were measured using a commercially available
CLCA1 sandwich ELISA kit according to the
manufacturer’s protocol (USCN Life Science, SEG669Hu). All
samples and standards were detected in duplicate.
IHC staining and semi-quantitative analysis
IHC staining was performed with the
avidin–biotin–peroxidase complex method. Briefly, paraffin-embedded
blocks were sectioned at ~5-μm thickness. Slides were
baked at 60 °C overnight, deparaffinized with xylene and
rehydrated in a graded ethanol series. After the antigen
retrieval process, endogenous peroxidase inactivity and
preincubation in 10% normal goat serum, the sections
were incubated with the anti-CLCA1 antibody (1:1000
dilution, Abcam, ab180851) at room temperature for 2 h
and then with the peroxidase-polymer labeled secondary
antibody. Then, peroxidase activity was demonstrated
with diaminobenzidine. Finally, sections were
counterstained with hematoxylin.
Two independent pathologists evaluated the staining
expression based on both the intensity and distribution
of positive cells. Staining intensity was graded as follows:
0, absent; 1, weak staining; 2, moderate staining; and 3,
strong staining. Staining distribution was determined by the
percentage of positive cells (0, <5% positive cells; 1, 5–25%
positive cells; 2, 26–50% positive cells; and 3, >50% positive
cells). The two scores were summed and divided by 2 to
obtain the final score, which was categorized as negative
(−) for scores <2 or positive (+) for scores ≥2.
Western blotting analysis
Protein extracted from fresh-frozen tissues was loaded
and separated by 10% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Then, the proteins were transferred
onto polyvinylidene fluoride (PVDF) membranes by
electroblotting and incubated with primary antibodies.
Immunoreactive bands were detected by
chemiluminescence using corresponding horseradish peroxidase
(HRP)-conjugated secondary antibodies and enhanced
chemiluminescence (ECL) detection reagents. Gray
intensity analysis of the western blot images was
conducted by ImageJ software. Then, relative protein
abundance was determined.
Primary antibodies used for western blot include
anti-CLCA1 (1:1000 dilution, Abcam, ab180851),
antiGAPDH (1:1000, Cell Signaling Technology, #5174),
anti-beta-catenin (1:1000, Cell Signaling Technology,
#8480), anti-E-cadherin (1:1000, Cell Signaling
Technology, #3195), anti-N-cadherin (1:1000, Cell Signaling
Technology, #13116), anti-vimentin (1:1000, Cell Signaling
Technology, #5741), anti-slug (1:1000, Cell Signaling
Technology, #9585), anti-snail (1:1000, Cell Signaling
Technology, #3879), anti-p53 (1:1000, Cell Signaling
Technology, #2527), anti-Akt (1:1000, Cell Signaling
Technology, #4691), anti-Ras (1:1000, Cell Signaling
Technology, #14429), anti-NF-kappa B (1:1000, Cell
Signaling Technology, #8242) and anti-histone H3
(1:2000, Cell Signaling Technology, #4499).
The cancer genome atlas (TCGA) database analysis
All normal (n = 51) and CRC (n = 625) level 3 CLCA1
gene expression datasets were obtained from the TCGA
database (October 2015). To obtain a high-confidence
result, we only considered HiSeq samples for messenger
RNA (mRNA) (RNASeqV2). RSEM-normalized data for
CLCA1 was log2-transformed for better visualization.
The boxplot and statistical analysis were performed in
the R programming language (×64, version 3.0.2).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from fresh-frozen tissues or
CRC cells. The Takara PrimeScriptTM RT Master Mix
kit (Takara, RR036Q) was used for reverse transcription.
The SYBR Premix Ex Taq II kit (Takara, RR820A) and
Applied Biosystems 7500 Fast Real-Time PCR System
were applied for real time PCR analysis. Experiments
were carried out in triplicate, and GAPDH was used as
the loading control. The forward primer sequence of
CLCA1 was CGTCAAATACTCCCCATCGT (5′ to 3′),
and the reverse primer sequence was GCTGATGTTCT
GGTTGCTGA (5′ to 3′). The forward primer sequence
of GAPDH was ATCCCATCACCATCTTCCAG (5′ to 3′),
and the reverse primer sequence was
TGAGTCCTTCCACGATACCA (5′ to 3′). The ΔΔCt method was applied
to evaluate the mRNA relative expression level.
Cell culture and plasmid transfection
SW620 cells, which were bought from the American Type
Culture Collection, were cultured in Leibovitz L-15
medium at 37 °C in 5% CO2. Culture medium was
supplemented with 10% fetal bovine serum (FBS, Gibco,
10,100,139), 100 U/mL penicillin and 100 mg/mL
streptomycin. CLCA1 CRISPR/Cas9 (the Clustered Regularly
Interspaced Short Palindromic Repeats and
CRISPRassociated protein system) activation plasmid
(sc-402,998ACT) and CLCA1 CRISPR/Cas9 knockout plasmid (double
nickase plasmid, sc-402,998-NIC) were bought from Santa
Cruz Biotechnology and transfected into SW620 cells
according to the manufacturer’s protocols. To obtain stable
transfectants of the activation plasmid (CLCA1-ACT),
blasticidin, hygromycin and zeocin were used to select
successfully transfected SW620 cells. In addition, to obtain stable
transfectants of the knockout plasmid (CLCA1-KO),
puromycin was used to select cells. Meanwhile, non-targeting
plasmids were transfected in the same way for the negative
controls as for CLCA1-ACT and CLCA1-KO, i.e.,
CLCA1ACT-NC and CLCA1-KO-NC, respectively.
Cell proliferation analysis
Cell Counting Kit-8 (CCK-8, KeyGen) was applied to
evaluate cell proliferation. Experiments were performed
according to the manufacturer’s protocol. Briefly, 1 × 104 cells
were seeded in a 96-well plate containing 100 μL of
completed culture medium per well and incubated in a 37 °C
incubator. Culture medium was used as a blank control.
Cell proliferation was evaluated every day for approximately
1 week after plating. CCK-8 solution (10 μL) was added to
each well, and then, the plate was incubated with cells in
the 37 °C incubator for 2 h. An optimal density (OD) value
of 450 nm was used to measure cell proliferation. The
mean and SD were calculated from 3 independent assays.
counted by light microscopy, and the mean cell number of
five random visual fields at a magnification of 200× was
recorded. The assays were carried out in triplicate.
Cell colony formation assay
Cell colony formation experiments were performed to
reflect anchorage-independent cell growth. Approximately
1000 cells were seeded in a 6-well plate containing
complete culture medium and incubated in a 37 °C
incubator. Colonies consisting of more than 50 cells after
2 weeks were counted.
Cell migration and invasion assay
A transwell chamber with an 8-μm-pore filter membrane
(Corning Inc.) was used to evaluate cell migration. Cells
(2 × 105) in serum-free medium were seeded into the
upper chamber, while conditioned medium with 20% FBS
was added to the lower chamber. The chambers were
incubated for 48 h in a 37 °C incubator. Non-migrated cells
in the upper chamber were removed with cotton swabs.
Migrated cells on the underside of the filter membrane
were fixed in 4% (v/v) paraformaldehyde and stained with
crystal violet. The Matrigel-coated 8-μm-pore transwell
chamber (Corning Inc.) was used to evaluate cell invasion.
The procedures of the cell invasion assay were identical to
the cell migration assay. The migrated/invaded cells were
Construction of animal models
Animal experiments were conducted according to the
Animal Study Guidelines of Zhejiang University.
Fiveweek-old, female nude mice (BALB/C) were used for the
animal study. To construct the subcutaneous tumor
xenograft mouse model, 5 × 106 tumor cells were injected
subcutaneously at the costal margin. The size of the xenograft
tumor was measured every 3 days. The mice were killed
6 weeks later, and the subcutaneous xenograft tumors were
dissected and weighed. In the meantime, to construct a
liver metastasis mouse model, 2.5 × 106 tumor cells were
injected into the tail vein. After 8 weeks, the mice were
killed, and the number of liver metastases was enumerated
and fixed in 10% formalin.
SPSS Statistics 20.0 (IBM, Armonk, NY, USA) was used
to conduct statistical analysis. Statistical tests were
twosided, and P< 0.05 was considered statistically
significant. Chi-square tests were performed to compare
qualitative data; two-tailed Student’s t-tests were used to
compare quantitative data.
Decreased expression of CLCA1 in CRC
To evaluate the expression level of CLCA1 in CRC
tissue, we performed IHC and western blotting analysis in
pairs of CRC and adjacent normal tissues. Thirty-two
pairs of paraffin-embedded CRC and adjacent normal
mucosa were used for the IHC analysis. The results
showed that the IHC staining score in the normal group
was significantly higher than that in the CRC group
(2.94 ± 0.25 vs 0.67 ± 0.93, P < 0.0001, paired t-test, Fig. 1a
and b). In addition, the CLCA1-positive rate in the
normal group was 100% (32/32), which was in sharp
contrast with the mere 12.5% (4/32) in the CRC group
(P< 0.0001, chi-square test). Western blotting analysis of
the 19 pairs of fresh-frozen CRC and adjacent normal
mucosa confirmed the IHC findings, revealing markedly
decreased expression levels of CLCA1 in CRC tissue
compared to that in the adjacent normal mucosa
(P< 0.0001, paired t-test, Fig. 1c and d). We next analyzed
the CLCA1 serum expression level in 100 CRC patients
and 76 healthy controls by ELISA and found that the
CLCA1 serum expression level was significantly lower in
CRC patients than in healthy controls (1.48 ± 1.06 ng/mL
vs 1.06 ± 0.73 ng/mL, P = 0.0018, t-test, Fig. 2a). In addition,
compared to that in patients with early-stage CRC (TNM
stage I and II), the CLCA1 serum expression level was
significantly reduced in patients with local or distant
metastasis (TNM stage III and IV) (1.24 ± 0.86 ng/mL
vs 0.88 ± 0.53 ng/mL, P = 0.013, t-test, Fig. 2b).
To further validate the CLCA1 mRNA expression
level in CRC, we analyzed 51 normal intestinal mucosa
and 625 CRC samples from TCGA database. Similarly,
the CLCA1 mRNA expression level was significantly
lower in CRC tissue than in adjacent normal tissue
(P< 2.2e-16, t-test, Fig. 2c). Further analysis showed
that the expression level of CLCA1 was decreased
greatly in stage III/IV compared with stage I/II (P = 0.019,
t-test, Fig. 2c), which was in accordance with the results of
the ELISA, suggesting that CLCA1 is involved in CRC
These results demonstrated a decreased expression
level of CLCA1 in CRC tissues and patient serum. In
addition, the CLCA1 serum concentration and CLCA1
mRNA expression level were inversely correlated with
CRC metastasis and tumor stage.
Increased expression of CLCA1 suppresses CRC cell growth and metastasis in vitro
To clarify the role of CLCA1 action in CRC tumorigenesis
and metastasis, we constructed stable CLCA1-upregulated
(CLCA1-ACT) and CLCA1-knockout cells (CLCA1-KO)
in the SW620 cell line, as well as the respective negative
control cells (CLCA1-ACT-NC and CLCA1-KO-NC),
using CRISPR/Cas9 plasmids (Fig. 3a, b and c).
CCK-8 analysis was performed to evaluate cell
proliferation. The results showed that from day 2 to day 5, the mean
absorbance of the CLCA1-ACT group was dramatically
lower than the CLCA1-ACT-NC group (P< 0.05, t-test,
Fig. 3d). In contrast, on days 3 to 5, the mean absorbance
of the CLCA1-KO group was significantly higher than that
of the CLCA1-KO-NC group (P< 0.05, t-test, Fig. 3e). The
growth curves of CLCA1-ACT and CLCA1-KO cells
suggested that the increased expression of CLCA1
was able to suppress SW620 cell proliferation,
whereas inhibition of CLCA1 was able to promote
SW620 cell proliferation.
Consistently, the results of the colony formation assay
showed that the upregulation of CLCA1 retarded
anchorage-independent cell growth, while the inhibition
of CLCA1 promoted this ability (Fig. 4a and c).
To further evaluate the impacts of CLCA1 on CRC
metastasis, a transwell chamber with an 8-μm-pore filter
membrane was used to measure cell migration. Meanwhile,
Matrigel-coated 8-μm-pore transwell chambers were used
to evaluate cell invasion ability. Migrating/invading cells on
the underside of the filter membrane were stained and
counted. The results revealed that both the migration and
invasion properties of CLCA1-ACT cells were significantly
reduced compared with that of CLCA1-ACT-NC cells
(Fig. 5a). In contrast, both the migration and invasion
properties of CLCA1-KO cells were much more than that
of the negative control cells (Fig. 5b). These results
indicated that the increased expression level of CLCA1
was able to reduce cell migration and invasion, while
inhibition of CLCA1 was able to enhance cell migration
Collectively, our data suggested that the increased
expression level of CLCA1 probably suppressed CRC
cell growth and metastasis, whereas inhibition of
CLCA1 led to the opposite results.
Increased expression of CLCA1 inhibits CRC proliferation and metastasis in vivo
To further confirm the impact of CLCA1 on tumor growth
and metastasis in vivo, tumor xenograft mouse models
were constructed. CLCA1-ACT cells and negative controls
(5 × 106 cells per mouse) were injected subcutaneously into
mice. Tumor volume was measured every 3 days, and
thereby, the tumor growth curve was plotted. The mice
were killed 6 weeks after the injection, and the
subcutaneous xenograft tumors were dissected and weighed (Fig. 6a,
b, and c). Figure 6d and e show the xenograft tumor growth
curves and average tumor weight of the CLCA1-ACT and
CLCA1-ACT-NC group, suggesting that the increased
expression of CLCA1 inhibited CRC proliferation in vivo.
Additionally, liver metastasis mouse models were
constructed by injecting CLCA1-ACT and CLCA1-ACT-NC
cells into the tail veins (2.5 × 106 cells per mouse). Eight
weeks after the injection, the mice were killed, and liver
metastases were enumerated and fixed in 10% formalin.
We found that the average number of visible liver
metastatic nodules in the CLCA1-ACT group was markedly
smaller than that of the CLCA1-ACT-NC group (Fig. 7).
Taken together, these results revealed that the increased
expression of CLCA1 had a critical role in suppressing
CRC growth and metastasis in vivo.
The mechanism of CLCA1 inhibition of CRC aggressiveness
CLCA1 upregulation inhibits the epithelial-mesenchymal
To investigate the functional mechanism of CLCA1 in
CRC, we performed western blotting analysis of the
proteins related to EMT. The results revealed that the
increased expression level of CLCA1 increased the
Ecadherin expression level but repressed N-cadherin,
vimentin, slug, and snail expression levels (Fig. 8a).
Conversely, CLCA1 downregulation decreased the
Ecadherin expression level but enhanced N-cadherin,
vimentin, slug, and Snail expression levels (Fig. 8a). It is
well known that E-cadherin is an epithelial marker,
while N-cadherin, vimentin, slug and Snail are
mesenchymal markers. These results indicated that CLCA1
upregulation possibly inhibits EMT, which is a process
where polarized epithelial cells are converted into
nonpolarized mesenchymal cells; during this process,
migration and invasion abilities are improved.
CLCA1 upregulation inhibits the Wnt/beta-catenin signaling
Markers of several main signaling pathways such as the
p53, Wnt, PI3K, NF-kappa B and Ras/MAPK pathways
were detected by western blotting analysis (see
Additional file 2). The results showed that beta-catenin, which
is a marker of the Wnt pathway, was downregulated in the
CLCA1-ACT transfectants but upregulated in the
CLCA1KO transfectants. In addition, beta-catenin nuclear
translocation, which is a marker of Wnt signaling activation, was
decreased after CLCA1 was activated and increased after
CLCA1 was knocked down (Fig. 8b). These results strongly
suggested that the increased expression of CLCA1 might
inhibit the Wnt/beta-catenin signaling pathway.
To confirm this speculation, we treated CLCA1-KO
cells with the Wnt signaling pathway specific inhibitor,
XAV939. Dimethylsulfoxide (DMSO) was used as a
negative control. It was observed that, compared to that
in CLCA1-KO cells treated with DMSO, the expression
levels of beta-catenin and proteins associated with the
epithelial-mesenchymal transition (EMT) were
downregulated in CLCA1-KO cells treated with XAV939 (Fig. 8c).
We next assessed cell proliferation and metastasis again. As
described before, inhibition of CLCA1 promoted CRC
growth and metastasis, but we observed that these effects
could be abrogated when cells were co-treated with
XAV939 (Fig. 9a and b). These phenomena also confirmed
the association of CLCA1 with the Wnt pathway from the
CRC is the third most commonly diagnosed malignancy
worldwide and is the fourth leading cause of cancer-related
]. The five-year survival rates of CRC patients in
diagnosed at different stages vary dramatically, from more
than 70% in early stage-diagnosed patients to less than 10%
in those diagnosed at advanced stages [
]. More than half
of patients have regional or distant metastasis at the time of
]. Thus, early detection could significantly
improve CRC survival. Until now, there has not been a
highly sensitive or specific biomarker for CRC diagnosis or
In this study, we evaluated the association between
CLCA1 and CRC development and investigated the
biological functions and mechanisms of CLCA1 in CRC. Our
results demonstrated that the expression level of CLCA1 in
CRC tissues was markedly decreased compared with that in
adjacent normal mucosa. Compared with the healthy
controls, CLCA1 serum concentration in CRC patients was
dramatically reduced. In addition, both CLCA1 serum
concentration and CLCA1 mRNA expression level were
inversely correlated with CRC metastasis and tumor stage.
The in vitro and in vivo experiments suggested that the
increased expression of CLCA1 suppressed CRC cell
proliferation and metastasis, while the inhibition of CLCA1 caused
the reverse effects. Western blotting analysis indicated that
the Wnt signaling pathway was activated and EMT was
induced when CLCA1 was inhibited. Furthermore, these
effects were abrogated when cells were co-treated with the
Wnt signaling specific inhibitor, XAV939. In conclusion, we
believe that increased expression levels of CLCA1 can
suppress CRC aggressiveness. In addition, CLCA1 functions as
a tumor suppressor possibly by downregulating the Wnt/
beta-catenin signaling pathway and EMT.
Several studies have suggested that CLCA family proteins
play vital roles in tumor progression [
13, 15, 20–22, 25–27,
]. Early in 1999, Gruber A.D. and his colleagues 
determined that the expression of CLCA2 was frequently
lost in breast cancer and that the re-expression of CLCA2
repressed tumor metastasis in vitro and in vivo. It has been
concluded that CLCA2 may act as a tumor suppressor in
breast cancer. This was verified in later studies conducted
by Walia V. et al. [
]. Moreover, their results also
indicated that CLCA2 is a p53-inducible growth inhibitor 
and that the loss of CLCA2 promotes EMT in breast
]. Similarly, it has been reported that CLCA4 is
downregulated and promotes EMT in breast cancer, which
indicates a tumor-suppression function for CLCA4 [
2001, Bustin S.A. et al. [
] preliminarily observed that the
expression levels of the CLCA1 and CLCA2 genes were
significantly downregulated in CRC. The excellent work of
Yang B. and his team indicated that low expression of
CLCA1 predicts CRC recurrence and lower survival [
CLCA1 contributes to promoting spontaneous
differentiation and reducing CRC cell proliferation in vitro [
Similar conclusions have been obtained in murine and
porcine CLCA isoforms [
The conclusions of our study are in accordance with
previous reports [
]. Furthermore, for the first time,
we have investigated the functions of CLCA1 in vivo
using stably transfected cells and confirmed the
association of CLCA1 with EMT and the Wnt signaling
pathway. It is well-known that a great majority of CRC
patients carry mutations in the adenomatous polyposis
coli (APC) or beta-catenin (CTNNB1) gene and that
both genes are involved in the Wnt/beta-catenin
signaling pathway [
]. The aberrant activation of Wnt
signaling induces the cytoplasmic accumulation and
nuclear translocation of beta-catenin protein [
which is a vital mechanism involved in cancer cell
proliferation and metastasis. As reported, aberrant Wnt
signaling can trigger the EMT process, which also plays a
crucial role in cancer metastasis . During the EMT
process, epithelial cells convert to mesenchymal cells,
losing cell-cell adhesion and cell polarity and acquiring
migratory and invasive properties [
]. In our study,
the increased expression of CLCA1 reduced the
betacatenin expression level and repressed EMT, which
probably explains the tumor-inhibitory activity of CLCA1.
The outstanding work of Sala-Rabanal M. and her
colleagues demonstrates that CLCA1 can stabilize
TMEM16A on the cell surface and prevent its
internalization, thus activating chloride currents [
addition, several studies have indicated that TMEM16A
is overexpressed in certain cancers and closely
associated with tumor progression [
]. Therefore, we
hypothesize that the tumor-suppressor function of
CLCA1 might be related to TMEM16A stabilization
and thus reduce its tumor promotion ability, which
needs further investigation.
We do acknowledge that some limitations exist in our
study. For instance, we only included one CRC cell line for
our laboratory study, which may result in cell line bias. In
addition, the sample size of our clinical research was limited
by the difficulties of sample collection and preservation.
We are trying to collect more samples to validate our
conclusions. Furthermore, the precise mechanism of how
CLCA1 interacts with beta-catenin or other proteins
participating in the Wnt signaling pathway remains to be
determined. Related experiments, such as expression profile
sequencing and co-immunoprecipitation, are in progress.
In summary, we demonstrated that CLCA1 is
downregulated in CRC tissues and patient serum, suggesting
that CLCA1 may serve as a novel biomarker for the
early diagnosis of CRC. Both in vitro and in vivo
experiments revealed that the increased expression level of
CLCA1 was able to suppress CRC aggressiveness, which
is associated with inhibition of the Wnt signaling
pathway and EMT. These findings indicate that CLCA1 may
function as a tumor suppressor, but future efforts are
needed to elucidate the role of CLCA1 in the Wnt/
beta-catenin signaling network.
Additional file 1: Patients’ clinical characteristics in IHC, western blotting
analysis and ELISA. Table S1. listed patients’ clinical characteristics in IHC
experiment and IHC staining results. Table S2. and S3. descripted patients’
clinical characteristics in western blotting analysis and ELISA, respectively.
(DOC 62 kb)
Additional file 2: Western blotting images of markers of p53, Wnt, PI3K,
NF-kappa B and Ras/MAPK signaling pathway. The results showed that
beta-catenin, which is a marker of the Wnt pathway, was downregulated
in the CLCA1-ACT transfectants but upregulated in the CLCA1-KO
transfectants. (PNG 118 kb)
CCK-8: Cell Counting Kit-8; CLCA1: Chloride channel accessory 1;
CLCA1-ACT: CLCA1-upregulated cells; CLCA1-ACT-NC: Negative controls
of CLCA1-ACT; CLCA1-KO: CLCA1-knockout cells; CLCA1-KO-NC: Negative
controls of CLCA1-KO; CRC: Colorectal cancer; CRISPR/Cas9: Clustered regularly
interspaced short palindromic repeats and CRISPR-associated protein system;
DMSO: Dimethylsulfoxide; APC, adenomatous polyposis coli; ECL: Enhanced
chemiluminescence; ELISA: Enzyme-linked immunosorbent assay;
EMT: Epithelial-mesenchymal transition; GAPDH: Glyceraldehyde-3-phosphate
dehydrogenase; HRP: Horseradish peroxidase; IHC: Immunohistochemistry;
KCNK9: Potassium channel subfamily K member 9; MET: Mesenchymal-epithelial
transition; MRNA: Messenger RNA; OD: Optimal density; PVDF: Polyvinylidene
fluoride; qRT-PCR: Quantitative reverse transcription polymerase chain reaction;
SDS-PAGE: SDS-polyacrylamide gel electrophoresis; TCGA: The Cancer Genome
Atlas; VDAC1: Voltage-dependent anion channel 1
This work was supported by the Key Projects in the National Science &
Technology Pillar Program during the Twelfth Five-year Plan Period (Grant
No. 2014BAI09B07) and the National High Technology Research and
Development Program of China (Grant No. 2012AA02A506).
Availability of data and materials
The datasets used and analyzed during the current study are available from
the corresponding author upon reasonable request.
SZ and YK conceived of and designed the study. XL, WH and JZ
performed experiments and analyses. YH, JP and YY prepared all tables
and figures. XL and WH wrote the main manuscript. YY and SZ revised
the manuscript. All authors reviewed the manuscript and approved its
Ethics approval and consent to participate
The Ethics Committee of the Second Affiliated Hospital, Zhejiang University
School of Medicine approved this study. Written informed consent for
participation was obtained from each patient.
Consent for publication
Written informed consent for publication was obtained from each patient.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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