br ends of actin filaments In addition to these functions

157 ends of SR11302 filaments.17–20 In addition to these functions,
158 we previously showed that CAPZA1 negatively regulates
159 autophagy by repressing expression of lysosomal-associated
160 membrane protein 1.21 Consequently, the autophagic 161 pathway is repressed because of inhibition of autolysosome
162 formation in CAPZA1-overexpressing gastric epithelial cells
163 infected with H pylori, resulting in accumulation of trans-
164 located CagA.21 Therefore, the presence of CAPZA1- 165 overexpressing cells in H pylori–infected gastric mucosa is
166 thought to increase the risk of gastric carcinogenesis.
167 However, it is uncertain how accumulation of CagA in
168 CAPZA1-overexpressing cells is associated with the devel-
169 opment of gastric cancer. Here, we show that CAPZA1 en-
170 hances expression of b-catenin, which is a transcription
171 factor for CD44, and epithelial splicing regulatory protein 1
172 (ESRP1), which increases alternative splicing of CD44 to
173 generate CD44v9. Consequently, CAPZA1-overexpressing
174 cells become CD44v9-positive cells after accumulation of
Results
CD44v9 Expression Is Induced Specifically in 177
CAPZA1-Overexpressing Gastric Epithelial Cells
on H pylori Infection
We previously reported that the H pylori–derived onco-
protein CagA, which usually is degraded by autophagy, is
stabilized in CD44v9-positive cancer stem-like cells in hu-
man gastric cancer tissues.16 In addition, we recently re-
ported that CagA evades autophagic degradation and
accumulates in CAPZA1-overexpressing cells in human
gastric cancer tissues, indicating that the presence of
CAPZA1-overexpressing cells in H pylori–infected gastric
mucosa increases the risk of gastric carcinogenesis.21 Based
on our previous reports, we hypothesized that
expression is enhanced in CAPZA1-overexpressing cells in H
pylori–infected gastric mucosa. To test this hypothesis, we
assessed whether CD44v9 is expressed in CAPZA1-
overexpressing cells in gastric cancer tissues obtained
from 5 cases of human gastric adenocarcinoma (cases 1, 2,
and 3 showed poorly differentiated adenocarcinoma; cases
4 and 5 showed well-differentiated adenocarcinoma).
CD44v9 expression was detected in cells strongly stained
for CAPZA1 (CAPZA1-overexpressing cells) (Figure 1A).
CAPZA1-overexpressing cells were detected consistently
across the gastric cancer tumors, and CD44v9-positive cells
selectively were detected among CAPZA1-overexpressing
cells (Figure 1A). In addition, the staining intensity of
CAPZA1 was significantly higher in CD44v9-positive cells
than in adjacent CD44v9-negative cells based on analysis of
These results suggest that CD44v9 and CAPZA1 are co-
expressed in human gastric cancer tissues. We subse-
quently examined whether CD44 expression
induces
CAPZA1 expression by transfecting MKN28 cells with an
expression vector harboring the CD44 standard
isoform 211
did not increase messenger RNA (mRNA) or
protein
expression of CAPZA1 (Figure 2A and B). Subsequently, to
examine the effect of overexpression of CAPZA1 on CD44v9
mRNA expression, we analyzed CD44v9 mRNA expression in
pCMA-ctrl–transfected AGS cells (AGS cells) and pCMV-
CAPZA1–transfected AGS cells (CAPZA1-overexpressing AGS
cells). Overexpression of CAPZA1 did not increase CD44v9
mRNA expression significantly (Figure 2C). We subse-
quently infected CAPZA1-overexpressing AGS cells with the
protein of muscle Z-line a subunit 1; CagA, cytotoxin-associated gene
A; cagPAI, cytotoxin-associated gene-pathogenicity island; CD44v9,
epithelial splicing regulatory protein 1; FBS, fetal bovine serum; KLF5, 228
Krüppel-like factor 5; mRNA, messenger RNA; qPCR, quantitative
polymerase chain reaction; SALL4, Sal-like protein 4; siRNA, small 229
© 2019 The Authors. Published by Elsevier Inc. on behalf of the AGA
Institute. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
web
270 Figure 1. CD44v9 expression is increased specifically in CAPZA1-overexpressing gastric epithelial cells upon H pylori 271 infection. (A) Immunostaining of CAPZA1 and CD44v9 in human gastric adenocarcinoma. Case 1: 80-year-old man, poorly