Down-regulation of CD44 Inhibits Wnt/β-catenin Mediated Cancer Cell Migration and Invasion in

Gastric Cancer

Sunwoo Baek1, Woo Rin Lee2

1. The Hudson School, 601 Park Ave, Hoboken, NJ, 07030, United States
2. Department of Biological Science, University of Suwon, Wau-ri, Bongdam-eup, Hwaseong, Gyeonggi-
do, 16419, Republic of Korea

Summary

Gastric cancer is one of the leading causes of cancer-associated death. Recent studies indicated that CD44
is associated with tumorigenesis, tumor progression, and metastasis. However, the detailed mechanisms
by which downstream molecules of CD44 regulate gastric cancer metastasis remains poorly understood,
which limits the development of novel, effective therapeutic targets. Therefore, we aimed to determine
CD44 regulatory mechanisms on Wnt/β-catenin signaling pathway, which is a crucial factor to promote
cancer invasion and metastasis. The objective of the experiment was to test whether or not CD44
downregulation inhibits migration and invasion in gastric cancer cells through Wnt/β-catenin signaling
pathway. In this study, we found that CD44 was significantly related to poor prognosis in gastric cancer
patients. We demonstrated the CD44 down-regulation decreased β-catenin protein expression level. Our
result showed that CD44 down-regulation inhibits cell migration and invasion by downregulating β-
catenin expression level, which is further validated with rescue experiments. We identified not only the
CD44 regulates the expression level of β-catenin but also discovered CD44-β-catenin pathway regulates
cell migration and invasion in gastric cancer for the first time. Our result would support the development
of molecular therapies for gastric cancer patients targeting CD44-β-catenin pathway.

Keywords

Biology; Gastric Cancer; CD44; Wnt/β-catenin signaling; Migration; Invasion

Introduction

Gastric cancer is one of the top causes of human cancer deaths. Patients with lymph node metastasis and
liver metastasis often show decreased in surval rate in gastric cancer patients. As surgery is the only
curative treatment strategy and conventional chemotherapy has many limitations with low efficacy and
various side effects, molecular therapies are developed to overcome these limitations. Therefore,
understanding the molecular basis of gastric cancer in metastasis can be crucial to develop effective
molecular therapies to improve the survival rate of gastric cancer patients.

This investigation focused on CD44, which is known to be involved in cancer proliferation and metastasis
in gastric cancer. CD44 is known as a multifunctional cell surface molecule. A cancer initiating cell
marker named CD44 enriches Cancer Initiating Cells (CIC) that is composed of a minor population of
cells within the tumor, but often necessary for tumor maintenance and progression (1). CD44 has also
been identified as a major Hyaluronic Acid (HA) receptor that participates in uptake and intracellular
degradation of HA (2). In gastric cancer, CD44 is reported as one of the most consistently reported
markers and related to gastric cancer invasion, lymph node metastasis and poor outcomes (3). Moreover,
the increased expression of CD44 is found in gastrointestinal tumors and was associated with tumor
invasion, lymph node metastasis and patients’ survival (4).

The other key signaling pathway is Wnt/β-catenin, which is involved in cell adhesion and cancer
development. β-catenin is a protein present in many types of tissue and cells, mainly found at adherens
junctions that connect neighboring cells. β-catenin contributes to cell adhesion and communication
between cells and plays an efficient role in the Wnt signaling pathway (5). However, the abnormal
activity of Wnt/β-catenin signaling pathway is significantly connected to the development and
progression of gastric cancer (6).

In our investigation, we wanted to discover the effects of CD44 downregulation on both Wnt/β-catenin
signaling and gastric cancer cell migration and invasion. Previous studies have shown that CD44 is
known to be involved in Wnt/β-catenin signaling. A dose-dependent increase of Wnt signaling leads
overexpression of human CD44, suggesting that CD44 can positively regulate Wnt/β-catenin signaling
(7). N-myc downstream-regulated gene 1(NDRG1) inhibits cancer stem cell characteristics and
tumorigenesis of colorectal cancer through inactivation of β-catenin signaling and down-regulation of
CD44 (8). Thus, CD44 downregulation may induce impairment of Wnt/β-catenin signal resulting in
inhibition of cancer cell migration and invasion. We predicted that CD44 downregulation would change
β-catenin expression level, which is a key molecule for invasion phenotype of cancer cells.
Considering the close relationship between CD44 and β-catenin, we examined the effect of CD44 on cell
migration and invasion in MKN45 gastric cancer cells, which represents a high level of CD44 expression
level. In addition, we examined whether the variation of migration and invasion phenotype can be
influenced by β-catenin expression level.

Results

CD44 expression was significantly associated with overall survival of gastric patients

CD44 has been reported to be involved with tumor proliferation and metastasis in gastric cancer.
However, the prognostic value of CD44 has never been investigated in gastric cancer. To determine if the
expression levels of CD44 can be predictive for gastric cancer patient survival, a set of 945 patient
samples were analyzed. The analysis was performed using Kaplan-Meier Plotter (http://kmplot.com/). As
expected, patients with high expression of CD44 showed a decrease in overall survival that was
statistically significant (p=0.0034) (Figure 1). However, multiple types of CD44 isoforms have been
found in tumor cells (9). Therefore, it is important to investigate which specific type of CD44 isoform
affects gastric cancer patient survival rate.

CD44 down-regulation inhibits the cell migration and invasion of MKN45 cells

According to patient survival analysis, the level of CD44 expression may be associated with aggressive
tumor cell behaviors. Therefore, to confirm the function of CD44 on migration and invasion, CD44
siRNA (siCD44) was constructed and transfected into MKN45 cells, which express a high level of CD44
in gastric cancer cells (data not shown). After transfection, CD44 protein expression level in MKN45
significantly decreased compared with control (Figure 2).

β-catenin is the central molecule in Wnt signaling pathway, whose deregulation results in various
malignancies. Since many studies indicated that CD44 is involved in Wnt signaling, β-catenin protein
expression was also examined in this study. β-catenin protein expression level was decreased to 40% by
siCD44 (Figure 2). Since it was previously shown that CD44 can regulate cell migration and invasion in
various cancer cells, the effect of CD44 down-regulation was investigated on cell migration and invasion.
The CD44 down-regulation inhibited cell migration and invasion in MKN45 cells (Figure 3).

Rescue Experiments in MKN45 indicate that CD44 mediated migration and invasion depend on β-
catenin
To further test whether the CD44 inhibited cell migration and invasion of gastric cancer are directly
dependent on β-catenin, siCD44 and β-catenin over-expressing vector were cotransfected with siCD44 to
rescue β-catenin expression level in MKN45 cells (Figure 3). The result showed that the number of
migrated and invaded cells was reversed on cotransfecting the cells with both siCD44 and β-catenin over-
expressing vector. Taken together, these results demonstrate that CD44 regulates cell migration and
invasion by regulating β-catenin expression in gastric cancer cells.

Discussion

Many studies indicated that CD44 directly regulated Wnt/β-catenin signaling, which is involved
in tumor metastasis and progression. Down regulation of CD44 decreases Wntβ-catenin signaling in a
concentration-dependent manner. On the other hand, when CD44 is overexpressed, it increases Wnt/β-
catenin signaling in a concentration-dependent manner (7). Thus, we predicted that CD44
downregulation in gastric cancer cells would inhibit cancer cell migration and invasion through
impairment of Wnt/β-catenin signaling.
Previous studies supported that CD44 activates posttranscriptional modification of β-catenin.
CD44 activates acetylation of β-catenin by Hyaluronan (HA)-mediated CD44 interactions. As HA binds
to the CD44, it promotes p300 acetyltransferase activity. This process mediates acetylation of both β-
catenin and NFκB-p65. The HA/CD44-p300 pathway leads to β-catenin and NFκB signaling contribute to
cell survival and chemoresistance in breast cancer (10). These results may indicate that CD44 dependent
downregulation of acetylated β-catenin decreases cell survival and chemoresistance in gastric cancer.
Other studies showed that HA-mediated CD44 interacts with the neuronal Wiskott-Aldrich syndrome
protein (N-WASP) that leads to increased phosphorylated β-catenin. HA binding develops CD44-N-
WASP association with ErbB2 and activates ErbB2 kinase activity that in turn increases phosphorylation
of the cytoskeletal protein and β-catenin. After β-catenin is phosphorylated, it transports into the nucleus
and promotes TCF/LEF transcriptional coactivation as well as tumor cell migration. As a result, CD44
interaction with N-WASP and ErbB2 plays a pivotal role in developing β-catenin signaling and
cytoskeletal protein that leads to ovarian tumor progression (11). Our study showed that CD44
downregulation in gastric cancer cells decreased the total β-catenin protein expression level. Therefore,
CD44 dependent inhibition of cell migration and invasion would be mediated by decreased expression
level of both acetylated and phosphorylated β-catenin. Therefore, when CD44 is downregulated, the
expression level of both acetylated and phosphorylated β-catenin must be investigated in the future.
Results from this study were further validated with rescue experiments that upregulation of β-
catenin recovered the cell migration and invasion. The experiment clearly shows that CD44-dependent β-
catenin expression level is crucial for cancer cell migration and invasion. Therefore, we identified not
only the β-catenin expression was regulated by CD44 but also revealed that inhibition of CD44 and β-
catenin pathway could be a potential molecular target to treat gastric cancer patients. In conclusion, the
direct effect of CD44 downregulation on cell migration and invasion suggests that the inhibition of β-
catenin signaling may result in beneficial responses, especially in advanced stages of gastric cancer.
We would like to follow up on our findings by validating our results by using multiple types of
gastric cancer cell lines, as well measure the levels of CD44 and β-catenin. Since tumor invasion and
metastasis is a multi-step process that requires adaptation of cancer cells to various microenvironment
conditions, the mouse xenograft model implanted with CD44-downregulated MKN45 cells should be
investigated in future to experimentally evaluate metastasis.

Materials and Methods

Cell culture

The human gastric cancer cell line MKN45 was obtained from the Korean Cell Line Bank (Seoul, Korea).
MKN45 cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine
serum (Thermo Fisher Scientific) and 1% penicillin and streptomycin (Gibco) in a 5% CO2 atmosphere at
37 °C.

siRNA and vector transfection

Negative control scrambled siRNA med GC (12935–300) and CD44 siRNA (HSS101596) were
purchased from Thermo Fisher Scientific. pcDNA3 and pcDNA3- β-catenin vector was purchased from
Addgene. Cells were transfected with 20 nM siRNA or vector with lipofectamine RNAimax reagent
(Thermo Fisher Scientific) with 1:3 ratio of siRNA (μg) or vector (μg) to lipofectamine (μl) as described
by the manufacturer's protocol.

Western blot and antibodies

Whole-cell lysates were prepared with Passive Lysis Buffer (Promega). The cell extracts were load on a
15% sodium dodecyl sulfate-polyacrylamide (SDS) gel for electrophoresis. The proteins were transferred
to polyvinylidene fluoride (PVDF) membranes with a Mini Trans-Blot tank (Bio-Rad). The membrane
was probed with antibodies against CD44 (Cell signaling), β-catenin (Cell signaling), Actin (Santa
Cruz) . All secondary antibodies (Santa Cruz) were incubated for 1 h. Amersham ECL GST Western
Blotting Detection kit (GE Healthcare) was used to detect the chemiluminescence signal.

Cell migration and invasion

To assess cell migration in vitro, MKN45 cells (2.0 x 105) in RPMI supplemented with 1 % FBS were
placed in the top chamber of transwell migration chambers with floro-block inserts (Corning). The lower
wells were filled with RPMI supplemented with 10 % FBS. After 48 hours, the cells were stained with
CFDA (ThermoFisher Scientific). Unmigrated cells were removed from the upper surface of the transwell
membrane with a cotton swab. Migrated cells on the lower membrane were fixed with 4% formaldehyde,
and the remaining cells of the bottom of the insert were then visualized and counted by fluorescent
microscope (Nicon).

To assess cell invasion, in vitro invasion assays were performed under the same conditions as the
migration assays with Matrigel-coated transwells (BD Bioscience) followed by manufacturer’s guideline.

Statistical Analysis

Data are expressed as the mean values ± standard error and analyzed by Student’s t-test using Excel and
SPSS 11.5.

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Figure 1. Kaplan-Meier curves evaluating the difference in overall survival in gastric cancer
patients depending on CD44 expression. Significant differences in overall were observed for patients
with CD44 high (n = 712) and low (n = 233) expressions (p=0.0034).

Figure 2. Transfection with siRNA specific for CD44 decreased CD44 protein and β-catenin in
MKN45 cells. (A) MKN45 cells were transfected with CD44 short interfering RNA (siCD44) utilizing
siCon as a negative control. The protein expression of CD44, β-catenin, β-actin (loading control) were
blotted with the specific antibodies. (B) Densitometric analysis of CD44 and β-catenin. Mean ± S.D. is
plotted with statistical significance (t-test): **, p ≤ 0.01.

Figure 3. siCD44 inhibited β-catenin mediated migration and invasion of MKN45 cells. (A)
Represents the images of migrated and invaded cells on the bottom of transwell inserts (scale bar: 500
μm) (B) The cells on the bottom of transwell inserts were counted under the microscope. Mean ± S.D. is
plotted with statistical significance (t-test): **, p ≤ 0.01.