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  • br ends of actin filaments In addition to these functions

    2020-08-28


    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